key: cord- -ikni acz authors: li, zengbin; zou, zixiao; jiang, zeju; huang, xiaotian; liu, qiong title: biological function and application of picornaviral b protein: a new target for antiviral drug development date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ikni acz picornaviruses are associated with acute and chronic diseases. the clinical manifestations of infections are often mild, but infections may also lead to respiratory symptoms, gastroenteritis, myocarditis, meningitis, hepatitis, and poliomyelitis, with serious impacts on human health and economic losses in animal husbandry. thus far, research on picornaviruses has mainly focused on structural proteins such as vp , whereas the non-structural protein b, which plays vital roles in the life cycle of the viruses and exhibits a viroporin or viroporin-like activity, has been overlooked. viroporins are viral proteins containing at least one amphipathic α-helical structure, which oligomerizes to form transmembrane hydrophilic pores. in this review, we mainly summarize recent research data on the viroporin or viroporin-like activity of b proteins, which affects the biological function of the membrane, regulates cell death, and affects the host immune response. considering these mechanisms, the potential application of the b protein as a candidate target for antiviral drug development is discussed, along with research challenges and prospects toward realizing a novel treatment strategy for picornavirus infections. the picornaviridae family consists of genera and species, mainly including enterovirus, hepatovirus, cardiovirus, aphthovirus, and rhinovirus [ ] . to date, research on picornaviruses has mainly focused on enterovirus (ev) , coxsackievirus (cv), poliovirus (pv), encephalomyocarditis virus (emcv), foot-and-mouth disease virus (fmdv), human rhinovirus (hrv), and hepatitis a virus (hav). picornavirus infections can cause enormous damage in humans and animals. the ev , cva , and cva cause hand, foot, and mouth disease in millions of children in asia-pacific region each year and can cause more serious clinical symptoms such as aseptic meningitis, acute flaccid paralysis, and neurological respiratory syndrome [ ] [ ] [ ] . picornaviruses are non-enveloped spherical viruses with an icosahedral-structured viral capsid. the picornaviral genome consists of a single-stranded positive-sense rna, which is approximately . - . kilobases in length, with a highly conserved structure, including a -noncoding region , an open reading frame, a -ncr, and a -end polya tail [ ] . the -ncr contains multiple rna secondary structural elements, including the internal ribosome entry site. the open reading frame of the viral genome consists of three regions: p , p , and p . the p region is translated and processed to form the structural proteins vp , vp , vp , and vp , which compose the capsid structure of a picornavirus. the p and p regions are separately translated to the non-structural proteins a, b, and c and a, b, c, and d, respectively. the majority of related research has focused on structural proteins of picornaviruses, such as vp , whereas the importance of the non-structural protein b has been relatively overlooked. viroporins are proteins found in a variety of viruses and are generally comprised of to amino acids. each viroporin contains a highly hydrophobic domain capable of forming at least one amphipathic α-helical structure, which oligomerizes to form transmembrane hydrophilic pores [ , ] . the b protein is a crucial component of picornaviruses that exhibits viroporin or viroporin-like activity, plays a key role in the picornavirus life cycle by inducing a series of cytotoxic reactions to promote picornaviral replication and release [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the b protein has a highly conserved sequence, which can be exploited for viral detection [ ] [ ] [ ] , vaccine development [ ] [ ] [ ] , and rna interference [ ] [ ] [ ] [ ] [ ] . in addition, the b protein exhibits a viroporin or viroporin-like activity, and thus, targeted drugs against viroporin could potentially target b protein as a novel strategy to treat or prevent picornavirus infections. however, the detailed mechanism of action of the b protein has not been elucidated to date. therefore, here, we review the recent research data on the role of the b protein in the picornaviral life cycle and discuss its possible application in antiviral therapy. the picornaviral b protein is a relatively short molecule, containing a maximum of two predicted putative transmembrane hydrophobic helices, along with n-and c-terminal domains, which are connected by a short stretch of amino acid residues. the α-helix-turn-α-helix sequence of the b protein is the basis for forming a transmembrane pore through homo-multimerization and the major determinant of the b protein function [ , , [ ] [ ] [ ] [ ] [ ] . a computational approach has demonstrated that the ev b protein is a tetramer, and b proteins with different orientations have different activities [ , ] . the b protein belongs to the type ii family of viroporins, which can be further divided into different types according to the number and orientation of the membrane-spanning domains ( figure ). in type iia viroporins, the n-and c-termini stretch to the organelle lumen, such as in the b protein of cvb [ , , , ] , whereas the n-and c-termini of type iib viroporins face the cytoplasmic matrix, such as in pv [ , , ] and fmdv [ , , ] . in addition, the c-terminus of the hav b protein has a viroporin-like activity [ ] . picornaviral b proteins target the membrane and form pores mainly through their transmembrane regions. the protein molecules are first inserted into the membrane individually and then self-interact and homo-oligomerize to form higher-order structures, which are important for the pore-forming activity, determined by the specific sequence and structure [ , , ] . the majority of b proteins are localized to organelles, with predominant co-localization with the golgi apparatus and the endoplasmic reticulum (er) in cvb , pv, and hrv ( figure ) [ ] . the hrv and fmdv b proteins are mainly localized to the er, whereas the emcv b protein is not localized to either the golgi complex or the er [ , , ] . seggewiss et al. [ ] found that the hav b protein was not localized to the er either but was involved in the amendment of the er-golgi apparatus intermediate compartment. since the protein structure determines its ultimate function and b proteins belonging to different viroporin species show both similarities and differences in their functions, much insight can be gained from research on the same viroporin b protein and on different viroporins. picornavirus infects the host cell, and then viral gene encodes b proteins. the b protein belongs to the type ii family of viroporins, including two transmembrane hydrophobic helices which are the basis for forming a transmembrane pore that results in the changes in cell membrane permeability. in type iia viroporins, the n-and c-termini stretch to the organelle lumen, whereas the n-and c-termini of type ii b viroporins face the cytoplasmic matrix. the majority of b proteins are localized to organelles, with predominant co-localization with the golgi apparatus and the endoplasmic reticulum (er), resulting in an obvious decrease in ca + of the er and golgi complex, along with a decrease in calcium uptake by the mitochondrion, and causing an influx of extracellular ca + . the b protein can induce many cellular reactions, such as changing membrane permeability, regulating apoptosis and autophagy, and affecting host immune responses. these functions are all related to changes in ion concentrations, especially of calcium ions (ca + ). therefore, here, we mostly focus on the role of ca + in these b protein activities. a common feature of infection by animal viruses is the damage to the ion balance in host cells. the picornaviral b protein may change the membrane permeability of target cells, disturbing the ion balance, especially that of ca + , in organelles, such as the er and the golgi apparatus ( figure ) [ , , ] . the changes in membrane permeability, caused by the b protein, have also been suggested to be regulated by the content of specific membrane phospholipids [ , ] . the ca + are involved in the activation of enzymes in cells and play a crucial role in viral replication and other viral biological processes [ ] [ ] [ ] . however, the role of the b protein in ca + homeostasis remains unclear. initial studies only indicated that host cells had elevated the ca + levels, owing to the expression of the b protein [ , , , , [ ] [ ] [ ] , but the mechanism was not clarified. since then, some researchers have proposed that the decrease in the concentration of ca + stored in organelles triggers the opening of specific calcium ion channels on the plasma membrane of cells, causing an influx of extracellular ca + [ ] . this idea was supported by the findings that expression of the cvb and pv b proteins resulted in an obvious decrease in the ca + concentration in the er and golgi complex, along with a decrease in calcium uptake by the mitochondria. meanwhile, the increased ca + level in the cytoplasm was suggested to be mainly due to the influx of extracellular ca + [ , , ] . similarly, the expression of the hrv b protein was shown to decrease the ca + concentrations in the er and golgi apparatus, whereas the emcv b protein only significantly reduced the ca + concentration in the er [ ] . in contrast, other studies showed that expression of the hav and fmdv b proteins elevated the cytoplasmic ca + level but did not alter the level of stored ca + in organelles, such as the er and golgi complex [ , ] . taken together, these studies suggest that there are different mechanisms by which b proteins affect the ca + concentrations, depending on the virus type. furthermore, it is unknown whether ca + directly pass through the channel formed by the b protein. pham et al. [ ] demonstrated, using a planar lipid bilayer and liposome patch-clamp electrophysiological technique, that the rotavirus non-structural protein (nsp ) viroporin region acts as a ca + conduction channel. although there is currently no direct evidence that the b protein can directly induce the observed changes in the ca + concentration in host cells upon infection, the above-reviewed studies suggest an association, and the mechanism requires further investigation. given the importance of ca + signaling for numerous cellular processes, further studies on picornaviral b protein function should include determination of the ca + concentration, which may provide more insight into the detailed function of the b protein. in particular, the b protein may change the ca + concentration to regulate autophagy and apoptosiswhich are distinct cell death mechanisms controlled by the virus to effectively evade the host immunity, thereby promoting viral replication and release [ ] [ ] [ ] [ ] [ ] . picornaviruses can form new cytoplasmic vesicles by inducing membrane remodeling, thereby promoting their own proliferation [ , , ] . the b protein is capable of binding to the membrane and inducing target membrane remodeling to form a unique membrane structure that can serve as a viral replication site. this site, known as the viroplasm, is generated from the er to accumulate all of the cellular components required for viral replication ( figure ) [ , , [ ] [ ] [ ] . the viroplasm is also the main membrane source of autophagy [ , ] . the cvb b protein is dependent on its transmembrane hydrophobic region to induce autophagy [ ] , which may be related to alterations in membrane permeability, especially with regard to the ca + concentration. moreover, at an early stage of fmdv cell infection, the virus specifically recognizes and binds to the cell surface receptors, and the b protein rapidly upregulates the autophagy pathway, leading to punctate aggregation of a large number of autophagy marker proteins, such as the microtubule-associated protein light chain (map -lc ) [ , ] . in addition, rotavirus encodes the nsp viroporin, which releases the er-stored ca + into the cytoplasm, thereby activating the ca + /calmodulin-dependent kinase kinase-β (camkk-β) signaling pathway, leading to autophagy ( figure ) [ ] . further, cvb induces autophagy in a calpain-dependent manner, causing an accumulation of lc lipids and autophagosomes [ ] . considering the ability of the b protein to alter cellular calcium homeostasis, along with its viroporin-like activity, it is feasible that the b protein may regulate autophagy mainly by changing the ca + concentration. the b protein has also been shown to regulate apoptosis through the endogenous pathway, which can be divided into er stress and the mitochondrial pathway, providing another potential mechanism of bypassing the host immune response to facilitate infection [ , , , , ] . the ca + plays a pivotal role in er stress-dependent apoptosis by regulating the flow between the er and the mitochondria [ , ] . excessive mitochondrial uptake of ca + exerts a cytotoxic effect because a high ca + concentration can open numerous mitochondrial transition pores, increase mitochondrial permeability, and destroy the mitochondrial outer membrane; consequently, cytochrome c and other proapoptotic factors are released, leading to apoptosis ( figure ) [ , , , , ] . the cvb b protein was shown to inhibit caspase activation and cell death induced by actinomycin d and cycloheximide by regulating the intracellular ca + concentration [ , ] . additionally, the b protein of hrv induced an er stress response, accompanied by an increased expression of cleaved caspase- and ccaat-enhancer-binding protein homologous protein (chop), which might have also involved a change in the ca + level [ ] . collectively, these results suggest that the b protein may regulate apoptosis by altering calcium homeostasis. furthermore, the b protein can regulate apoptosis through the mitochondrial pathway. madan et al. [ ] showed that the pv b protein interacted with the mitochondria and altered the mitochondrial morphology, in addition to the release of cytochrome c after the pv b gene expression. cong et al. [ ] reported that the ev b protein was localized to the mitochondria and induced apoptosis by directly activating the proapoptotic b-cell lymphoma -associated x (bax) protein, without a significant uptake of ca + by the mitochondria. therefore, the activation of the mitochondrial apoptotic pathway and subsequent apoptosis, induced by the ev b protein, may not involve ca + signaling. collectively, picornaviral b proteins can induce cell death in a variety of ways, with ca + playing an important role in most of these mechanisms. figure . b protein regulates autophagy and apoptosis. the b protein induces target membrane remodeling to form the viroplasm, which is generated from the endoplasmic reticulum (er). the isolation membrane is produced by the viroplasm. activation of the ca + /calmodulin-dependent kinase kinase-β (camkk-β) signal pathway is due to an increased intracellular calcium concentration. furthermore, mitochondrion takes up ca + from the er, thereby cytochrome cis released, leading to apoptosis. the host immune system is an important line of defense against pathogens, and pathogens can affect the immune system in a variety of ways. the b protein mainly affects the host immune response through inflammasome activation and by direct antagonism of the host immune response. recognition of pathogens by the immune system is mainly mediated by pathogen-associated molecular pattern receptors, known as pattern recognition receptors, including nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), retinoic acid-inducible gene-i (rig-i)-like helicases, and pyrin domain-containing (nlrp ) [ ] [ ] [ ] . activation of nlrp inflammasome occurs during a period of changes in ion concentrations [ , ] . the nlrp belongs to the nlr family of inflammasomes and causes interleukin (il)- β and il- secretion via caspase- activation [ ] . the emcv, pv, ev , and hrv b proteins all activate the nlrp inflammasome but use distinct mechanisms [ , ] . the hrv and emcv b proteins can stimulate the nlrp inflammasome pathway to activate caspase- , which catalyzes the proteolysis of pro-il- β to il- β, leading to its secretion from across the plasma membrane by inducing a ca + efflux from intracellular storage ( figure ) [ , ] . wang et al. [ ] found that cvb -infected cells induced the nlrp activation in association with a k + efflux. the influenza virus m protein, which is also a viroporin, is capable of transporting na + and k + , resulting in activation of the nlrp inflammasome [ , ] . since the cvb b protein acts as a viroporin and can disrupt the intracellular ion balance [ , ] , it has been speculated that the induction of nlrp activation in cvb -infected cells may be related to the b protein. in addition to activating the inflammasome, the b protein also antagonizes the host immune response. both in vitro and in vivo studies have suggested that inhibition of protein trafficking would effectively allow viral evasion of the host immune response (figure ) [ ] [ ] [ ] . moreover, inhibition of protein transport may be related to changes in the ca + concentration [ ] . similar to the cvb b protein, the b proteins of pv, hrv and hrv , were shown to significantly inhibit the protein transport through the golgi complex, whereas the hav, fmdv, and emcv b proteins did not inhibit the protein transport [ , , ] . the fmdv b and c proteins did not block protein secretion, whereas the transport of proteins from the er to the golgi complex were blocked by the fmdv bc protein, and this effect was reproduced upon co-expression of the c and b genes [ , ] . collectively, these findings suggest that the b protein may participate in a viral evasion of the host immune response, mainly by inhibiting protein transport. figure . b protein affects the host immune response. the b protein can stimulate the nlrp inflammasome pathway to activate caspase- , which catalyzes the proteolysis of pro-il- β to il- β, which leads to their secretion across the plasma membrane by inducing a ca + efflux from intracellular storage. moreover, the b protein inhibits protein transport through the golgi protein which may be effective to evade the host immune response. the b protein has also been suggested to facilitate the viral evasion of the host immune response through other means. thus, the b protein antagonizes rig-i-mediated antiviral responses by inhibiting the expression of rig-i as an fmdv-specific reaction [ ] . the rna helicase lgp (also known as dexh-box helicase , dhx ) is a crucial factor involved in the host antiviral immune response [ ] . the fmdv leader protein (lpro), c protein, and the b protein have the ability to induce a decrease in lgp protein expression [ ] . in addition, pv b variants were shown to inhibit the antiviral interferon (ifn) system [ ] , whereas the hav b protein inhibited the synthesis of ifn-β by affecting the mitochondrial antiviral signaling protein activity, thereby antagonizing the host immune response [ ] . collectively, these evidences indicate that picornaviral b proteins can affect the host immune response, thereby promoting viral amplification or the release of viral particles. as discussed above, picornaviral b proteins have a viroporin or viroporin-like activity and play an important role in the picornaviral life cycle. therefore, many common applications targeting viroporins may be translatable to those targeting b proteins. in addition, the b protein may serve as a new target for the development of antiviral drugs. thus, further studies on the structure and function of the b protein might open up new avenues for the prevention and control of picornaviruses. owing to its highly conserved sequence, the use of the b gene as a marker could effectively improve the accuracy of virus detection. li et al. [ ] designed primers and taqman probes, based on the b and d regions, which were successfully used in real-time polymerase chain reaction to accurately detect and quantify fmdv during infection and replication. in addition, wang et al. [ ] developed a lateral-flow detection system, which could rapidly and easily detect fmdv using the b gene. in addition to gene-based detection, the virus could be detected using a b antibody. biswal et al. [ ] used an indirect enzyme-linked immunoassay based on a recombinant b protein to detect antibodies specific for fmdv. this method can be applied not only to fmdv but also to other picornaviruses, including cvb and ev . given the significant threat that picornaviruses pose to humans and animals, resulting in enormous economic damage to the livestock industry, development of picornavirus vaccines is of great significance. although inactivated virus vaccines can offer effective prevention, there are associated residual risk issues, including incomplete virus inactivation and escape during the vaccine production process [ ] [ ] [ ] . therefore, genetically engineered vaccines are considered more suitable options to overcome these shortcomings of inactivated viral vaccines. the ev vp protein is located outside the viral membrane and is thus exposed to the greatest amount of immune stress. accordingly, vp shows an extreme serological variability, thus providing the most reliable molecular epidemiological information. consequently, the vp region of ev has become a focus of vaccine research for picornavirus infections [ ] . however, dna constructs containing the vp gene of ev showed low levels of antigenicity. therefore, there is still a need to develop an effective adjuvant strategy to increase the antigenicity. one possibility in this regard is the use of recombinant vaccines incorporating the b gene to enhance the efficacy of vaccines. at present, applications of the b gene in recombinant vaccines have mainly concentrated on fmdv. the addition of a b fragment to a vaccine designed with vp as the core has been shown to effectively enhance the vaccine efficacy [ ] [ ] [ ] and reduce the dose and side effects [ ] . these effects may be similar to those leading to a greater efficacy of the adenoviral vector vaccine fused to the fmdv b protein against serotype o, which is associated with the induction of specific cd + and cd + protective t cell responses [ ] . therefore, future designs of other picornaviral genetically engineered vaccines would benefit from the addition of the b gene to increase the vaccine efficacy, including the addition of the b gene to a genetically engineered ev vaccine with the vp gene as the core. since viroporin plays an important role in all life stages of the virus, it is an attractive antiviral therapeutic target, and there have been great breakthroughs in this regard. by contrast, research and development of drugs targeting the b protein are relatively delayed. since b proteins have a viroporin or viroporin-like activity, screening for anti-picornavirus drugs among existing viroporin-targeting drugs may be a viable approach. there are four main types of inhibitors of viroporin activity, including adamantane, amiloride, alkyl iminosugar, and spirane amine [ ] . adamantane (amantadine and rimantadine) inhibits the m channel of influenza a virus by destroying the transmembrane network of hydrogen-bonded water molecules, thereby inhibiting the viral amplification [ ] . in bhk- cells infected with fmdv, the virus titer gradually decreased with an increase in amantadine concentration, which may have been due to abrogation of the pore-forming activity of the b protein and ultimate inhibition of fmdv replication [ ] . however, clinical trials showed that amantadine was not only selective for specific resistance mutations in hepatitis c virus (hcv) p [ ] , but also caused a rapid emergence of amantadine-resistant variants of influenza a virus during monotherapy for influenza [ ] . amiloride is a composite of two drugs, -(n,n-hexamethylene) amiloride and a novel inhibitor, bit , targeting hcv p and hiv- vpu, which can together block the viroporin ion channel activity or prevent ion channel formation, resulting in a potent antiviral effect [ ] [ ] [ ] [ ] . the alkyl iminosugar inhibits the formation of ion channels by targeting the hcv p viroporin [ ] . finally, spirane amines, such as bl- , also inhibit the influenza a virus m protein, with an antiviral mechanism similar to that of amantadine [ ] . there are also other drugs that act as viroporin inhibitors, including , -dibenzyl- ( h- , , , tetraazol- -yl) hexahydropyrimidine (cd), n-( -phenylethyl)- -[ -(phenylsulfonyl)- -piperazinyl]- quinazolinamine (lds ), and -methyl- , , -trihydroxyanth-raquinone (emodin), among others [ , , ] . the mechanism of action of these viroporin inhibitors is based on the inhibition of the viroporin channel activity. therefore, these drugs may have the potential to be applied for the treatment of picornavirus infections by targeting the b gene. however, this application will require further detailed investigations and drug screening. nevertheless, the b protein has the potential to widen the range of antiviral treatment strategies. furthermore, specific degradation of complementary mrna can be triggered by small interfering rnas (sirnas) or folded short hairpin rnas (shrnas) [ ] , which can be explored as an rna interference strategy, a relatively novel technology that has already been applied to treat many important pathogens, including hiv- , hepatitis b virus, and herpes simplex virus [ ] [ ] [ ] . currently, shrnas targeting the highly conserved b gene sequence are widely used in picornavirus research, including fmdv [ , ] , emcv [ ] , and cvb [ ] , and significant experimental viral suppression has been achieved. basically, rna interference against b gene affects the stability and integrity of the whole viral genome. the high nucleotide sequence conservation makes the b gene an attractive target for rna interference, which may potentially be effective against multiple picornavirus types, and open the door for additional sirna drugs. to date, there have been few studies specifically focusing on inhibitors of the b protein. xie et al. [ ] found that , -diisothiocyano- , -stilbenedisulfonic acid (dids) blocked a chloridedependent current, mediated by the ev b protein, and suppressed viral amplification. however, further research is needed to uncover the underlying mechanism. despite the many challenges in drug development, new technologies such as fourier-transform infrared spectroscopy and design of molecular dynamics analogs, as well as cryo-electron microscopy and spectroscopy, are expected to greatly contribute to the development of antiviral drugs. recent studies have gradually clarified the function and the potential of the b protein, along with increasingly recognizing its importance in the viral life cycle. however, there are still some challenges to overcome in investigations of the picornaviral b protein. in particular, its strong hydrophobicity makes it difficult to achieve soluble expression. ao et al. [ ] conjugated the small ubiquitin-like modifier (sumo) protein to the n-terminus of the fmdv b protein and successfully achieved soluble expression. therefore, this method can be tested for other picornaviral b proteins. moreover, the detailed molecular mechanism of the action of the b protein requires further study, along with the identification of interactions of b protein with host proteins, to better understand the role of the b protein in the pathogenesis of picornaviruses. in murine cells, the b protein was suggested to react with host proteins to promote rhinovirus proliferation [ ] . using a yeast two-hybrid system, the fmdv b protein was found to interact with the host elongation factor γ (eef g), and mislocalization of eef g demonstrated that the eef g deletion affected the synthesis of membrane proteins [ , ] . although a yeast two-hybrid system is a common laboratory protein-screening technique, it has a low success rate and is time-consuming. alternatively, affinity purification-mass spectrometry can be used to overcome these shortcomings, which has already been widely used in studies on dengue, zika, and ebola viruses [ , ] . at present, the development of antiviral drugs against viroporins is focused on three aspects, including viroporin and membrane fusion inhibitors, ion channel inhibitors, and targeted viroporin antibodies [ ] . with respect to the biological function of the b protein, antiviral drugs targeting the b protein could be designed based on the following three approaches: broad-spectrum screening for anti-picornavirus drugs among existing viroporin inhibitors, screening for b protein and membrane fusion inhibitors, and screening for b protein pore activity inhibitors. as discussed herein, the most important basis for the function of the b protein is that it can be polymerized into pores, thereby changing the permeability of the membrane. therefore, the design of drugs targeting b protein should be based on inhibiting polymerization of the b protein into pores, thereby reducing its effects on cellular ion homeostasis. however, these designs first require detailed determination of the refined atomic structure of the b protein, along with the expansion of screening techniques and applications of meticulous medicinal chemistry. furthermore, to develop better antiviral drugs, it will be necessary to elucidate the exact role of the b protein channel in the viral life cycle. thus, the main points of focus for research on the structure and function of the b protein toward ultimate drug development are: ( ) mechanism of increasing membrane permeability to disturb the ion balance, ( ) regulation of autophagy and apoptosis, ( ) inhibition of the host immune response, and ( ) promotion of viral replication and release. taken together, as research aimed at further elucidation of the role of the b protein progresses, along with the adoption of new technologies, it is expected that more strategies will come to light for 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with foot-and-mouth disease virus nonstructural protein b: identification by yeast two-hybrid system protein interaction mapping identifies rbbp as a negative regulator of ebola virus replication comparative flavivirus-host protein interaction mapping reveals mechanisms of dengue and zika virus pathogenesis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -qg pb authors: lavi, ehud; wang, qian; stieber, anna; gonatas, nicholas k. title: polarity of processes with golgi apparatus in a subpopulation of type i astrocytes date: - - journal: brain res doi: . / - ( ) - sha: doc_id: cord_uid: qg pb the golgi apparatus-complex (ga), is a key organelle involved in several posttranslational modifications of polypeptides destined for lysosomes, plasma membranes and secretion. as reported from this laboratory, certain astrocytes in rat brain contain cisternae of the ga not only in perikarya, but also in processes. in order to further investigate which type of astrocytes contain ga in processes we conducted the present study using primary cultures of rat astrocytes and organelle specific antibodies against the ga and the rough endoplasmic reticulum (rer). while the perikarya of all cells contained elements of the ga, only a single process of a subset of type i astrocytes, negative to antibodies a b and hnk- , contained ga. in contrast, elements of the rer were found within perikarya and all processes. in order to confirm that the immunostained structures in processes indeed represent the ga, we exposed cultures to brefeldin a (bfa), a secretion blocker which disperses the ga and redistributes it to the rer. we observed that bfa disrupted the ga of both perikarya and processes. however, astrocytes were resistant to prolonged incubations with bfa, while a similar treatment killed cultured fibroblasts and pc- cells. furthermore, in astrocytes exposed to bfa for several days, the delicate network of glial fibrillary acidic protein (gfap), was replaced by large perinuclear masses of the protein. these observations demonstrate that a subset of type i astrocytes have a single process with elements of the ga. we suggest that this specialization of the ga may be related to yet unrecognized secretory or protein processing functions of these cells. the resistance of astrocytes to bfa and the striking changes in their cytoskeleton induced by the drug, may contribute to studies on the mechanism(s) of action of bfa. the ga plays a pivotal role in secretion, plasma membrane biosynthesis and targeting of lysosomal enzymes [ ] . the organelle is involved in numerous posttranslational modifications and in the sorting or targetting of processed polypeptides. in addition to the general properties of the organelle, cell specific features of the ga, such as selective production of certain enzymes (e.g. high paps cerebroside sulfontransferase in neuronal ga), have been identified [ , , , , , , ] . astrocytes are actively involved in numerous secretory functions such as production of cytokines and growth factors [ , , ] . it is therefore reasonable to speculate that the ga of astrocytes would express function-related properties of the ga. astrocytes, recognized by their specific cytoskeleton protein gfap, are not a homogeneous population of cells [ , ] . in tissue sections, astrocytes can be divided by morphological criteria into 'fibrous' (mostly in the white matter) and 'protoplasmic' (mostly in the gray matter). additional markers such as the monoclonal antibody a b [ ] and tetanus toxin are positive in cultures of astrocytes with numerous delicate branching processes (type ii) and negative in flat astrocytes (type i) [ ] . additional evidence suggests a dual lineage of the two types of astrocytes in some culture systems [ , ] . type i astrocytes and their processes envelop blood vessels, suggesting a function related to the blood brain barrier (bbb); also, type i astrocytes proliferate in response to injury, while type ii astrocytes are associated with the nodes of ranvier and die in response to axonal injury [ ] . there is no clear parallel between the in vivo and in vitro classifications of astrocytes. moreover, there is some question as to whether or not type ii astrocytes have an in vivo analogue [ ] . astrocyte heterogeneity has been reported even within a morphologically distinct group, particularly for type i astrocytes cultures [ ] . this includes heterogeneity in ion channel, expression of neurotransmitter receptor and glycoprotein, neuropeptide and cytoskeletal content, prostaglandin and peptide secretion, influence on neuronal differentiation, as well as morphology [ , , , , ] . the use of organelle-specific markers is central to this study. since the conceptualization of the secretory pathway [ ] , several groups have developed organelle-specific antibodies as a tool to study the ga and other organelles of the secretory-endocytic pathways [ ] [ ] [ ] [ ] [ ] , , , , , ] . in this laboratory an intrinsic membrane sia[oglycoprotein (mg- ) of medial cisternae of the ga, was purified from rat brains [ ] . the original monoclonal antibody, a , used in the isolation of mg- from rat brains, reacts only with rat tissues. however, a polyclonal antiserum raised against immunoaffinity purified mg- reacts with several species and cell types, including human [ ] , suggesting that mg- or closely related molecules are conserved polypeptides of the ga. in a previous ultrastructural, immunocytochemical study from this laboratory, using affinity-purified polyclonal sera specific for the ga, elements of the organelle were detected in peripheral segments of some astrocytic processes in sections of rat brains [ ] . other glial cells did not display extensions of the ga in processes. this localization of the ga in processes of astrocytes, and the investigation of other structural properties of the ga of astrocytes is the subject of the present study. in this study in order to further investigate the properties of elements of a putative ga in astrocytic processes, we used primary cultures of astrocytes and the organelle specific antisera against the ga and the rer [ , , ] . the primary culture model offers the opportunity of experimental manipulations and better resolution of immunocytochemical staining, not easily obtainable in in vivo studies. to further ascertain whether immunostained elements indeed represented the ga, we exposed cultures to bfa, a secretion blocker, which disrupts the ga and induces its redistribution into the rer [ , ] . results of this study indicate that processes of a subpopulation of astrocytes display elements of the ga in a polarized fashion. primary glial cell cultures were prepared from newborn lewis rats (charles river laboratories) as previously described [ , , , ] . the method for the preparation of the glial cultures was adapted from the original method of mccarthy and de vellis [ ] . careful removal of meninges resulted in minimal contamination of cultures by fibroblasts. over % of the cultured cells stained with the monoclonal antibody against gfap [ ] , an astrocyte specific intermediate filament protein. the cell lines of pc- cells (rat pheochromocytoma cells), and l- cells (rat fibroblasts) were originally obtained from american type culture collection -atcc (rockville md). the preparations of the anti-mg- monoclonal antibody ( a ), and the immunoaffinity purified anti-mg- polyclonal antibodies, were described in previous publications [ , , ] . the preparation of the monoclonal antibody h , a rer marker, has been previously described [ ] . cells, grown on poly-d-lysine treated coverslips, were fixed with % paraformaldehyde for min at room temperature, washed times in pbs, incubated for min in . % saponin/ % goat serum (gs) in pbs, washed times in % gs in pbs, incubated with primary antibody ( : dilution in pbs of immunoaffinity purified rabbit anti-mg- antibodies, or supernatant from the anti mg- hybridoma a ) overnight at room temperature. cultures were then washed, incubated with a biotinylated goat anti-rabbit igg antibody, incubated with the avidin-biotin complex (abc), and stained with diaminobenzidine tetrahydrochloride (dab) ( mg dab/ ml tris-saline containing mm imidazole and . % h ), according to standard methods [ , ] . cells, plated on poly-d-lysine treated coverslips, were rinsed with mm phosphate buffer ph . / . m naci (solution b), and fixed in % paraformaldehyde ( rain), incubated ( min) with mm phosphate buffer ph . / % saponin/ . naci/ . % fish gelatin (solution a), rinsed with solution b, incubated with blocking solution made with % serum of the same species used for the secondary antibody in solution b ( min), incubated with primary antibody in solution a, washed in solution b, and incubated with secondary antibody linked to fitc or ritc in solution a ( min), washed with solution b ( min), washed in : dilution of solution b to remove non specific binding of antibodies in high salt, and mounted in mowiol (polysciences, inc.). for the detection of antigens expressed on cell surfaces, cells were incubated with antibodies before fixation (supernatants from a b hybridoma and hnk- hybridoma, obtained from american type culture collection, rockville, md) [ , ] . double staining was done by incubations with primary antibodies followed by secondary fitc-conjugated antibody, followed by a second primary antibody and then an ritc-conjugated secondary antibody. cells were viewed with phase, rhodamine, and fluoresceine optics with a zeiss microscope equipped with a filter system selective for rltc and fitc excitation and observation. in each experiment, negative controls of normal preimmune serum and supernatants from a non-productive myeloma cell line (sp / ) were used. cells grown on therminox were fixed overnight at °c with . % glutaraldehyde+l% paraformaldehyde in . % cacodylate buffer ph . + . % caci . subsequently cultures were postfixed in % osmium tetroxide+l. % potassium ferrocyanide, dehydrated in ethanol, and embedded in araldite. sections ( - nm thick) were stained with lead and uranyl salts and viewed in a transmission electron microscope (jeol cx) at kv [ , ] . bfa (sigma) stock solution diluted in ethanol, was diluted in pbs and applied to cultures at a concentration of /~g/ml for periods of min, or as specifically indicated in the text. control experiments included incubation of cultures for the same time periods with the same dilutions of ethanol used to dissolve bfa. to confirm and extend a previous ultrastructural immunocytochemical observation from this laboratory on the presence of ga in astrocytic processes in rat brain sections [ ] , we stained by the indirect immunoperoxidase method, using immuno-affinity purified rabbit polyclonal antibodies against mg- , formalin-fixed, paraffin embedded, sections of rat brain and spinal cord. immunostaining showed perinuclear ga staining in all cells. as expected, proximal neuronal dendritic processes contained short extensions of ga. in addition, areas of white matter (devoid of neuronal dendrites) also exhibited linear staining of occasional processes, consistent with those of astrocytes ( fig. ). the monoclonal ( a ) and polyclonal antibodies against mg- detected the ga in rat astrocytic cell cultures in virtually % of cells. the stain was cytoplasmic, mostly perinuclear and in a granular or reticular (network) form, consistent with the morphology and distribution of the ga (fig. ) . staining of mg- in processes in rat astrocyte enriched cultures was detected by immunohistochemistry and immunofluorescence with both polyclonal and monoclonal antibodies against mg- . staining of mg- in processes appeared in approximately - % of the cells (in cultures ranging from % to % of gfap-positive cells). staining of ga in processes was also granular and in some cells in continuity with the perinuclear ga (fig. ) . to confirm that cells which expressed mg- in processes were astrocytes, double-labeling immunofluorescence with anti-mg- and astrocytic-specific intermediate filament marker (gfap) antibodies were performed. all cells with staining of the ga in processes were gfap positive (fig. a,b ). the morphology of ceils expressing mg- in their processes was consistently of type i astrocytes. in mixed glial cultures these cells were flat and constituted part of the adherent lower layer. these cells contained abundant cytoplasm and a small number of wide flat processes. the staining of the ga in perikarya of type i astrocytes was more abundant and in the form of a network while in type ii astrocytes the staining of the ga was punctate (figs. - ) . mg- was detected only in wide, non-branching processes of type i astrocytes and not in delicate branching processes of type ii astrocytes. to further characterize the astrocytic cell type expressing mg- , the differential adhesion ('shake-off') technique was used, adapted from mc-carthy and de vellis [ ] . loosely attached cells were shaken off during incubation in an orbital shaker at °c/ rpm for h and then plated on coverslips. in cultures of adherent cells, enriched in type i astrocytes, % of cells were positive for gfap and negative for a b antigens. on average, about % of type i astrocytes contained extensions of the ga in processes. following 'shake off' the loosely adherent layer of mixed glial cultures was transferred to coverslips and incubated with % fetal bovine serum to promote development of type ii astrocytes instead of oligodendrocytes which require serum-free medium [ ] . these cultures were enriched for type ii astrocytes and had over % of a b -positive, gfap-positive cells. type li enriched astrocytic cultures had only a small perinuclear ga staining with anti-mg- antibodies. in contrast to type i astrocytes, in type ii astrocytes there was no evidence for extensions of the ga into cell processes. only rare cells in type li enriched cultures displayed ga staining in processes, however, these cells had the distinct morphology of contaminating type i flat astrocytes. since cultures were not % pure for any cell type we wished to further confirm that ga in cell processes was present only in type i astrocytes. cells were double labeled with a b and anti-mg- in both mixed glial and type-enriched cultures. in astrocytic cultures, a b is considered to be a marker that identifies type ii astrocytes and their precursors [ ] . only a b negative cells (type i astrocytes) exhibited mg- staining in processes. these cells were gfap positive when double stained with gfap and anti-mg- (fig. a,b) . a b -positive cells which were gfap positive had only a small perinuclear ga staining with anti-mg- antibodies (fig. a-d) . to further confirm that type ii astrocytes did not contain ga in processes, another astrocyte type ii marker, hnk- , was used with essentially the same results (fig. e,f) . hnk- belongs to a group of markers (l- /hnk- /j /elec- ) against surface adhesion molecules such as myelin associated glycoprotein (mag) which are present in a subtype of astrocytes (type ii) associated with axons in perinodal locations [ , , , ] . in tissue sections and culture, astrocytes have processes which do not display distinct patterns of polarization. therefore, we asked whether an analysis of the orientation of the ga may reveal possible internal polarization. in type i astrocytes with either several or only two processes (bipolar, or spindle cells), mg- immunoreactivity was detected only in one process per cell (figs. , and a) . we then asked whether the polarity of the ga in one process of certain type i astrocytes was also displayed by other cell organelles. the most closely functionally and structurally related organelle to the ga is the rer. in order to examine the differential presence of gavs rer in these cells we performed double labeling by immunofluorescence, using a polyclonal antiserum against mg- as a marker for the ga, and a monoclonal antibody against a rer antigen h . astrocytes expressing linear staining of mg- in one process exhibited rer staining in the entire cytoplasm and in all other processes including the one stained for mg- (fig. ) . in unstained preparations the unique cell process containing the ga could not be distinguished from other cell processes not containing the organelle. in many mammalian cells, brefeldin a, a secretion blocker, disperses the cisternae of the stack of the ga, which subsequently fuse with membranes of the rer. in bfa-treated ceils, the cisternae and vesicles of the trans golgi network (tgn) aggregate in the form of tubules and vesicles around the centriole. previous studies from other laboratories showed that enzymes of cisternae of the golgi stack are redistributed to the rer [ , ] . in contrast to the golgi stacks, which under the exposure of cells to bfa fuse with the rer, cisternae of the tgn under bfa treatment collapse around the microtubule organizing center [ , ] . in order to investigate whether the immunostained (mg- ) elements of astrocytic processes are indeed part of the ga, we treated cultures of astrocytes with bfa. following treatment of astrocytic cultures with increasing amounts of bfa, a change in mg- distribution was observed ( table ) . instead of the usual perinuclear staining of the ga which is in the form of coarse granules or network, bfa treatment caused a fine granular staining of mg- , dispersed through the entire cytoplasm. a plateau of effect related to the dose of bfa occurred with amounts greater then /xg/ml (table ) . a complete dispersion of the mg- immunostaining occurred with concentrations of or /zg/ml of bfa (fig. ) . since bfa was dissolved in ethanol we conducted control experiments in which cultures were incubated with the same concentrations of ethanol used to dissolve the bfa. in these experiments, mg- staining was not affected by the ethanol treatment. the kinetics of ga dispersion were compared between astrocytes and other cells. dispersion of the ga in astrocytes, pc- cells (rat pheochromocytoma cell line) and l ceils (mouse transformed fibroblasts cell line) was complete - min after the beginning of treatment. staining of the astrocytic cultures for the r e r with monoclonal antibodiy h reacting with a - kda polypeptide [ ] revealed that the same concentrations of bfa which disrupted the ga did not affect the structure of the r e r (table ). both ga in astrocytic perikarya and the putative elements of the ga in processes dispersed with bfa treatment. this observation confirms that astrocytic processes indeed contain elements of the ga. since the effect of bfa is reversible, we examined the recovery of astrocytic and pc- cell cultures following bfa treatment. after a -min treatment with bfa, which caused complete dissociation of the ga as judged by the mg- stain, cells were washed and incubated with fresh medium. reassembly of mg- was gradually observed and was complete within - min. the kinetics of bfa effect and recovery were similar in both astrocytes and pc- ceils. the pattern of ga staining in astrocytes after recovery from a -min bfa treatment was identical to the ga staining before bfa treatment. since process formation in astrocytes can be a dynamic event modulated by various chemicals [ , ] we examined the effect of bfa on astrocytic processes after a longer exposure to bfa. astrocyte cultures exposed to / x g / m l of bfa once and examined h later, or cultures exposed to the same amount of bfa table dose response of brefeldin a (bfa) on the ga in astrocytes dose * effect . ~g / m l . /j.g/ml . ~t g / m l . / z g / m l . / z g / m l no effect on the ga partial dissociation of the ga complete dissociation of the ga complete dissociation of the ga no effect on the r e r * primary newborn rat astrocytic cultures were incubated with each of the above doses of bfa for min, then fixed in % g a a / % ethanol and the effect of bfa was analyzed by indirect immunoperoxidase staining using rabbit-anti-mg- antibodies or h , a monoclonal antibody against a r e r protein. for four -h periods and examined at the end of the -h period, did not reveal any change in the number of processes, as judged by immunoperoxidase reaction against mg- , an organelle specific marker of the ga. the effect of bfa on the redistribution of ga elements into the rer, although a reversible phenomenon when applied for short periods, has eventually detrimental consequences on the vitality and function of cells. when cultured l- ceils or pc- cells were exposed to continuous p~g/ml of bfa treatment for h, the majority of cells exhibited degenerative changes consisting of shrinkage and hyperchromasia of nuclei, pyknosis of cytoplasm, and kariorhexis, and ceils completely disappeared by h. in contrast, primary glial cell cultures survived the p~g/ml of bfa treatment, and numerous cells were present after h in bfa. immunoperoxidase staining for mg- of cultures maintained in bfa for h revealed that the effect of the drug on the dispersion of the ga was still present in all surviving cells similar to the effect after h (fig. ) . to analyze the nature of surviving cells under bfa, double-labeling by immunofluorescence was performed h after the initial introduction of bfa ( /zg/ml) into the medium. all cells showed the bfa effect on the ga as expressed by a diffuse granular mg- staining. the majority of cells were gfap positive, although occasional gfap negative flat cells with abundant cytoplasm (presumably macrophage/microglia cells) were also present. occasional hnk- positive cells were present. thus both type i and type ii astrocytes resisted the prolonged bfa treatment. to test whether bfa is partially metabolized and 'detoxified' in the astrocytic culture, we compared primary glial cultures treated with /.~g/ml of bfa only once and kept for h, to cultures that were washed every h and replenished with the same dose of fresh bfa. cultures with one bfa treatment survived during the -h observation period, while cultures which were supplied with new bfa died between - h. in both cultures the ga was dispersed as shown by staining for mg- . control experiments using similar dilutions of ethanol and pbs (which were used to dissolve and dilute bfa) did not affect the pattern of ga staining or the concentration of cells on the cover- fig. . electron micrograph of astrocyte cultures demonstrating a type i astrocyte with a flat broad process extending from the cell body (a) (× ). a higher magnification of a distal segment of the cell process (delineated by arrowheads) shows a linear ga within the astrocytic process in addition to bundles of glial filaments (b) (x ). inset shows higher magnification of the ga in astrocytic process. cells exposed to bfa (one treatment of p~g/ml) for - days exhibited somewhat shorter processes. to test whether prolonged exposure to bfa affects the cytoskeleton of astrocytes, gfap staining was performed at and h after an initial exposure of cultures to ~g/ml of bfa. all cells showed the bfa effect on the ga as expressed by a diffuse granular staining for mg- . after h, double staining with gfap and mg- showed a coarser network of gfap staining in ceils with dispersed ga, and the beginning of perinuclear condensation. after h in bfa, the effect was more pronounced and occasional cells exhibited collapse of gfap to a form of a perinuclear 'ball' while gfap stain was absent in the rest of the cytoplasm and processes (fig. c,d) . both the ga and gfap network were reconstituted in cultures treated with bfa for h, washed, placed in fresh media and observed h later. astrocytic cultures were examined by transmission electron microscopy. perinuclear ga with the characteristic membrane-bounded stack of flat cisternae was detected in all cells. in addition, flat cells with broad processes, exhibited characteristic stacks of cisternae of the ga which were parallel to the long axis of the process, and were approximately perpendicular to the orientation of the ga in the perikarya (fig. ) . only one process per cell showed evidence of ga elements. thus, electron microscopy corroborated the immuno° histochemical findings of polarity of the ga in type i astrocytic processes. moreover, the ultrastructure of the ga in processes appeared identical to that in the perinuclear areas. cells containing ga in processes were identified as astrocytes by the presence of bundles of intracytoplasmic intermediate filaments. only broad processes of some cells (which belong to type i astrocytes) contained elements of the ga, while delicate branching processes (which belong to type ii astrocytes) did not. considerable differences exist among the ga of different cell types. for example, in contrast to liver golgi and similar to renal golgi, neuronal golgi contains high paps cerebroside sulfontransferase [ , ] . cell-specific distributions of enzymes (glycosyltrans-ferases) of the ga have been shown in intestinal cells [ ] . also, the ga has polarized distributions in a variety of cell types. in certain epithelial cells the intracellular position of the organelle correlates with the direction of the vectorial transport and release of secretory products [ ] . in pancreatic islet b cells, the ga is located in the juxtacapillary face of the cell [ ] . in natural killer cells (nk) and in cytotoxic t cells there is adaptation of the polarization of the ga, in an orientation necessary for secretion of cytotoxic factors [ , ] . in sertoli cells, polarization of the ga has been seen in cyclic fluctuations during the spermatogenic cycle [ ] . thus, the ga of several cells displays structure-function relationships which are specific to cell types. the ga localization in polarized ceils can be related to their function [ ] . in morphologically nonpolar cells, the location and orientation of the ga can serve as an indication of permanent or transient cell polarity [ ] . it has been shown that polarity determined by the location of the ga is related to specific membrane recycling functions in the adjacent cytoplasmic membrane of the cell, and this has important role in cell motility [ ] . the present study demonstrates several unique structural features of the ga of astrocytes. there is a unique polarization and distribution of the ga in processes of a subset of type i astrocytes. thus, a subpopulation of type i astrocytes exhibit a polarized cell process similar to the axons and dendrites in neurons. these astrocytic processes contain elements of the ga as dendrites do. however, unlike the polarized nature of neuronal processes, which may be recognized with a nissl stain and do not require organelle specific stains, the recognition of the polarized nature of astrocytic processes requires immunohistochemical staining for the ga. additional support for the existence of polarity in astrocytic processes is derived from the report of specific distribution of sodium ion channels in astrocytic processes. astrocyte processes forming the glial limitans and surrounding blood vessels display reduced immunoreactivity for a sodium channel specific protein, compared to longitudinally oriented or perinodal astrocytic processes, while focal regions of the glial limitans exhibit robust immunostaining [ ] . the finding of increased density of potassium ion channals in the endfeet of astrocytic processes [ ] provides further support for the possibility that astrocytic processes are actively involved in specialized functions. the subset of astrocytes with a polarized process may be involved in specialized secretory functions requiring rapid exchange of secretory products with the environment adjacent to this specialized cell process. it has been suggested that the ga plays a major role in cell motility [ ] , and that polarization of the ga in motile cells serves as a steering mechanism in the direction of cell migration [ ] . therefore type i astrocytes with polarized ga may belong to a subpopulation of astrocytes that are motile and capable of migration in response to injury. taken together the above evidence strongly suggests that the polarization of the ga in certain cells is not a random and insignificant phenomenon. however, the functional significance of the polarization of the ga in astrocytes remains to be investigated. the differential expression of ga in processes in the two distinct astrocytic types further emphasizes the hypothesis that functional, anatomical, and morphological differences separate these two cells which express the same intermediate filaments (gfap). furthermore it provides further evidence of the heterogeneity that exists among type i astrocytes. the experiments comparing the bfa effect among various cells reveal that dissociation and reassembly of the ga in astrocytes, as revealed by the immunostaining against mg- , is similar to that of other cells. these experiments suggest that the ga in processes has the same rate of dissociation as the perinuclear ga. the experiments of prolonged exposures of astrocytes to bfa show unique properties of these cells. unlike other cells in culture tested in this study, and with the possible exception of macrophages, astrocytes do not succumb to ga disruption, and continue to survive in culture despite the dispersion of their ga. this observation is consistent with studies which showed that several golgi enzymes redistributed into the er during bfa treatment continue their functions [ ] . cell lines resistant to bfa have been described [ , ] ; however, these cell lines do not exhibit dissociation of the golgi back into the er. studies of astrocytic functions are now necessary to further explore the effect of bfa on astrocytes. the observation of the resistance of astrocytes to bfa may be also useful for future studies on the mechanism of the effect of bfa on these cells, and of its specific interaction with the ga. the cytoskeleton which is intimately connected with the ga also shows changes under bfa effect. bfa treatment for - h did not destabilize the cytoskeleton (detyrosinated microtubule network) in gh cells derived from rat pituitary tumors [ ] ; however, this may be due to the fact that the effect of bfa on gfap is evident after h, at a time most other cells do not survive this treatment. bfa has also been shown to induce disassembly of actin microfilaments in bfasensitive cells, but not in bfa-resistant cells [ ] . our finding that bfa at /.lg/ml does not kill astrocytes hut still affects the ga and the cytoskeleton may be useful in future studies of the interaction between ga and cytoskeleton. in that regard, it would be interesting to study whether astrocytes contain / -cop and whether under bfa this protein dissociates from the ga within seconds as in other cell types [ , ] . traditionally astrocytes have been considered as supporting cells. astrocytes presumably fulfill functions such as regulation of cell migration and differentiation during development, support of neurons by regulating the extracellular environment for ion and neurotransmitter exchange, lipid metabolism, maintenance of the blood brain barrier and various interactions with endothelial cells and blood vessels [ ] . astrocytes are also known to be the major cells in the cns that respond to various pathologic conditions including trauma, ischemia, inflammation, and degeneration. more recent studies define the changes that occur in reactive astrocytes: ( ) proliferation and migration [ , ] , ( ) hypertrophy with increased cytoplasm and gfap content [ , ] , ( ) changes in surface expression of mhc class i and ii, and icam- molecules [ ] , [ ] , ( ) production of cytokines and growth factors including il- , il- , il- , tnf, ifn, complement components, mip- , gm-csf in response to various stimulations [ , , ] . cytokines are traditionally considered the product of cells 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nondiffusible, dominant factor that makes the golgi apparatus resistant to brefeldin a expression and localization in the developing cerebellum of the carbohydrate epitopes revealed by elec- , an igm monoclonal antibody related to hnk- polarization of the golgi apparatus and the microtubule-organizing center within cloned natural killer cells bound to their targets a critical role for the polarization of membrane recycling in cell motility the polarization of the golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound on the mechanism of unidirectional killing in mixtures of two cytotoxic t lymphocytes. unidirectional polarization of cytoplasmic organelles and the membrane associated cytoskeleton in the effector cell non random positioning of golgi apparatus in pancreatic b cells coronavirus mhv-a causes a persistent, productive infection in glial cells induction of mhc class i antigens on glial cells is dependent on persistent mouse hepatitis virus infection monoclonal antibodies to gel excised glial filament protein and their reactivities with other intermediate filament proteins bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct surface features and 'neuron-like' gamma-aminobutyric acid transport astrocyte origins production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er antibodies to the golgi complex and the rough endoplasmic reticulum, the in vivo and in vitro synthesis of sulphatides during development preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue pharmacologically-distinct subsets of astroglia can be identified by their calcium response to neuroligands ls reactive gliosis a property of a distinct subpopulation of astrocytes? the macroglial cells of the rat optic nerve fibrous and protoplasmic astrocytes are biochemically and developmentally distinct fragmentation of the golgi apparatus of motor neurons in amyotrophic lateral sclerosis revealed by organelle-specific antibodies inward-rectifying potassium channels in retinal glial (muller) cells lntracellular aspects of the process of protein transport glial cell diversification in the rat optic nerve a glial progenitor cell that develops in vitro into an astrocyte depending on culture medium perturbation of the morphology of the trans golgi network following brefeldin a treatment: redistribution of a tgn-specific integral membrane protein, tgn reactive astrocyte and axonal overgrowth in the injured cns: is gliosis really an impediment to regeneration? differential subcompartmentation of terminal glycosylation in the golgi apparatus of intestinal absorptive and goblet cells binding properties of liposomes containing the myelin-associated glycoprotein mag to neural cell cultures antibodies to rat pancreas golgi subfractions: identification of a -kd cis-golgi protein identification by monoclonal antibody of a -kd protein associated with the tubulo-vesicular compartment at the cis side of the golgi apparatus morphological modulation of cultured rat brain astroglial cells: antagonism by ganglioside gm intercellular adhesion molecule (icam- ) in cellular immune reactions in the central nervous system the golgi apparatus-complex of neurons and astrocytes studied with an anti-organelle antibody staphylococcal adpribosyltransferase-sensitive small g protein is involved in brefeldin a action induction of glial cell mhc antigen expression in neurotropic coronavirus infection: characterization of the h- inducing soluble factor elaborated by infected brain cells coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes development of knowledge about astrocytes since virchow multiple sclerosis: relevance of class i and class it mhc expressing cells to lesion development astrocyte heterogeneity two integral membrane proteins located in the cis-medial ant trans-part of the golgi system aquire sialated n-linked carbohydrates and display different turnovers and sensitivity to camp dependent phosphorylation the authors thank jun chen for providing the h monoclonal antibodies. key: cord- - t s fr authors: marie, m.; sannerud, r.; avsnes dale, h.; saraste, j. title: membrane traffic in the secretory pathway: take the ’a’ train: on fast tracks to the cell surface date: - - journal: cell mol life sci doi: . /s - - - sha: doc_id: cord_uid: t s fr cholesterol, certain lipids, membrane-bound and soluble proteins, as well as viruses that are synthesized in the endoplasmic reticulum (er), reach the plasma membrane (pm) via non-classical pathway(s) that remain poorly understood. typical for this transport is (i) its insensitivity to brefeldin a (bfa), which dissociates selected coat complexes from membranes, resulting in the disassembly of the golgi apparatus; (ii) its rapid kinetics as compared to the classical secretory pathway; and (iii) its role in the trafficking of lipid raft components. based on results showing that the intermediate compartment (ic) at the er-golgi boundary constitutes a stable tubular network that maintains its dynamics in the presence of bfa, we propose that two bidirectional golgi-bypass pathways to the pm exist, a direct route from early ic elements, and another, reminescent of the yeast secretory pathway, from late ic elements via the endosomal system. these pathways have implications for the organization of the secretory processes in different cell types. (part of a multi-author review) it can be estimated that about one-third of the proteins encoded by the eukaryotic genome are synthesized on er-bound ribosomes. the er is also the major site of cellular sterol and lipid synthesis. the secretory pathway is primarily responsible for distribution of the newly synthesized products among the endomembrane compartments, as well as delivery to the exterior of the cells. the prevailing view is that soluble and membrane-bound cargo is transferred vectorially from the er to the cell surface [ ] . cargo molecules are first transported from er exit sites to the entry (cis) side of the golgi apparatus by mobile elements of the intermediate compartment (ic) [ , ] , and then pass through the polarized golgi stacks, consisting of cis-, medial-and trans-cisternae, via cisternal maturation (progression), vesicular and tubular traffic, or a combination of both [ , ] . the golgi apparatus is a key organelle of the classical secretory pathway due to its central positioning and numerous functions. most proteins and many lipids undergo post-translational modifications in the golgi system, including glycosylation (attachment or terminal modification of sugars), proteolytic processing, sulfation and phosphorylation. in the case of proteins these modifications are required for their maturation, oligomerization and/or trafficking [ ] . sequential modification of the itinerant cargo molecules can be determined by the polarized organization of the golgi stacks or the specificities of the golgi enzymes themselves [ ] . the golgi apparatus is also regarded as the central station for the sorting of cargo molecules; however, the topology of the sorting processes in this system is still not completely understood [ ] . a common idea is that two main sorting compartments, referred to as cis-and trans-golgi networks (cgn and tgn), operate at the entry and exit faces of the organelle, respectively. an important function of the cgn is to recycle molecules back to the er to maintain the functional integrity of this organelle. the tgn again is considered as the principal distribution center, where the lipids and proteins reaching the trans-golgi are sorted to pleiomorphic carriers for further transport to various cellular destinations, including different domains of the pm, the endosomal system, as well as earlier golgi compartments [ , , a] . the tgn has also been suggested as the site of formation of cholesterol and sphingolipid-rich membrane microdomains (rafts), which participate in membrane traffic [ - ] . however, recent studies of yeast and mammalian cells provide evidence that raft assembly also takes place in pre-golgi compartments and endosomes [ - ] . membrane-bound coat proteins (cops) play important roles in protein sorting at the er-golgi boundary, in the golgi apparatus and endosomes, as well as at the pm [ ] . in addition to concentrating cargo into vesicular and tubular transport carriers, cops also organize membrane domains in large-sized transport intermediates and create organelle architecture [ ] . copii coats function at er exit sites in the initial export of cargo from the er [ , ] , copi coats operate in forward transport and recycling in the ic and cis-golgi membranes [ ] , while clathrin and associated adaptor proteins (ap , ap , ap , gga) localize variably to trans-golgi/tgn and endosomes [ ] . notably, whereas the copii and copi coat complexes have been well characterized [ , ] , our current knowledge of the multiple coat machineries that operate in the trans-golgi/tgn remains limited [ ] . it has been known for some time that the export of proteins from cells can also occur via mechanisms that are distinct from the above-outlined classical secretory pathway. the term non-classical secretion is often linked to proteins (e.g. growth factors, cytokines, and extracellular matrix components) that lack signal sequences and remain in the cytosol, but nevertheless can be released to the extracellular space [ ] . it appears that the export, as well as import [ ] , of some of these proteins also involves membrane traffic [ ] . however, here we focus on another type of nonclassical secretion, namely, the trafficking of molecules that enter the secretory pathway in the er, but instead of taking the classical route to the pm, follow pathway(s) that bypass the golgi apparatus. this type of transport is characterized by its insensitivity to the fungal metabolite brefeldin a (bfa), which blocks the activation of a subset of adp-ribosylation factors (arfs). these gtpases regulate the binding of cops -such as copi and clathrin-ap -to ic, golgi and endosomal membranes [ ] . the release of the cops by bfa results in rapid disassembly of the golgi apparatus and the redistribution of golgi proteins to the er and the endosomal system [ , ] . early studies with bfa showed that this compound blocks the transport of many proteins, such as the vesicular stomatitis virus g glycoprotein (vsv-g) and secretory proteins in hepatocytes [ , ] . however, subsequent work revealed that the effects of bfa are often variable, depending on the cell type, protein examined and concentration of the drug [ , ] . moreover, bfa-resistant cell types were identified, such as the epithelial mdck cells and ptk cells, in which the golgi apparently remains intact [ - ] . cop-free subways? the first evidence for bfa-insensitive pathways that bypass the golgi apparatus was obtained by urbani and simoni, who compared the biosynthetic transport of cholesterol and vsv-g [ ] . they showed that the transport of both molecules is inhibited at o c, a temperature that blocks the transport of newly synthesized proteins from the ic to the golgi stacks [ ] . moreover, at o c cholesterol and vsv-g were found in the same low-density vesicle fraction, suggesting that they both accumulate in the ic. however, in contrast to vsv-g, which is transported via the golgi [ ] , the transport of cholesterol to the pm after shift from o c to o c was not inhibited by bfa, suggesting that it employs a non-conventional pathway [ ] . the rate of transport of cholesterol to the pm (~ min) is considerably faster than that of vsv-g (~ min), and resistant to monensin, an ionophore that inhibits golgi function and blocks vsv-g transport [ ] . more recently, heino et al. followed the movement of cholesterol from er to the pm using a high-resolution transport assay, and obtained similar results regarding its rate of transport, as well as the differential effects of bfa on cholesterol and protein (influenza virus hemagglutinin) transport. importantly, like biosynthetic protein trafficking, cholesterol transport was shown to be dependent on an intact microtubule (mt) system [ ] . we carried out a literature search resulting in an interesting list of molecules that, like cholesterol, are transported to the pm in a bfa-insensitive manner (table ) . although certainly not complete, even this short list reveals the diversity of molecules that display this type of non-classical trafficking. in addition to cholesterol, a number of lipids are included, such as sphingolipids (sphingomyelin), glycolipids (ganglioside gd ), and phosphatidylethanolamine. the situation with lipids is more complex since their intracellular movements also involve non-vesicular transport through the cytosol [ ] , possibly occurring at specialized membrane contact sites [ , , ] . the proteins display different membrane topologies, including lumenal (calsequestrin), integral (e.g. cd ), peripheral (flotillin- ), and gpi-anchored (f /contactin) proteins. they fall into different functional categories: cell adhesion proteins (e-cadherin, integrin ps , metalloproteinase), channel proteins (connexin- ) and signalling proteins (heterotrimeric g-protein, ras). interestingly, in addition to golgiindependent trafficking, a common denominator between the membrane-bound molecules is their association with lipid rafts. the biochemical and topological diversity of the molecules listed in table , as well as their typical association with lipid rafts, indicate that their unconventional trafficking does not involve pathways that are artificially created by bfa. this conclusion is supported by the results obtained with the two glycoforms of protein-tyrosine phosphatase cd . these variants of the same polypeptide are transported to the pm with different kinetics displaying differential sensitivities to bfa. moreover, the drug does not affect the transport rate of the bfa-insensitive, highmannose form of cd , which reaches the pm more rapidly than the bfa-sensitive, terminally glycosylated form of the protein [ ] . there also exists other types of evidence for nonconventional secretory pathways. the ae anion exchanger is a channel protein that first reaches the pm in high-mannose form, apparently bypassing the golgi, and then is endocytosed and receives terminal modifications on its sugars [ ] . similarly, the subunits of the sodium channel (enac) located at the apical surface of epithelial cells contain predominantly highmannose sugars, which could be explained by golgiindependent trafficking [ ] . evidence for non-classical transport of the cystic fibrosis transmembrane conductance regulator (cftr), a chloride channel, was obtained in biochemical and immunolocalization studies. the latter showed the presence of the protein in the er, ic, and peripheral cis-and and trans-golgi tubules, but not in the golgi stacks [ ] . it has also been proposed that the transfer of cftr to the pm takes place via an endosomal intermediate [ , ] . further, immuno-em localization of fibronectin, an extracellular matrix protein, showed its absence from the golgi cisternae, but presence in golgi vacuoles that frequently display an acidic luminal ph [ , ] , correlating with results showing that the secretion of this protein is partially resistant to monensin [ ] . recently, a golgi-bypass pathway was implicated in the secretion of integrins (aps ) that link cells via fibronectin to their extracellular matrix [ ] . moreover, rotavirus is released from the apical surface of intestinal cells via a non-conventional pathway that is insensitive to monensin [ ] . virus assembly begins in the er, whereafter immature particles transit through the ic [ ] and reach the trans-golgi region where their assembly is completed. one of the virus coat proteins, vp , is not detected in the er or the golgi stacks [ ] . it interacts with flotillin and mediates the association of mature virus particles with lipid rafts, which appear to facilitate their movement from the trans-golgi to the outside of the cell [ ] . finally, results from knockdown of the arf guanidine nucleotide exchange factors (gefs), gbf and big, that control the binding of copi and clathrin-aps to ic/cis-golgi and trans-golgi membranes, respectively, are compatible with the existence of the golgi-bypass pathway(s) discussed here. accordingly, knockdown of gbf (like bfa) results in copi release, but allows the transport of vsv-g from the er to the ic, where it becomes arrested [ ] . by contrast, these cells are still capable of secreting soluble proteins [ ] . unexpectedly, knockdown of big does not affect the transport of vsv-g to the pm, indicating that this process is not dependent on a functional tgn [ ] . different possibilities have been suggested to explain the trafficking of molecules in the presence of bfa. either the er -most likely its smooth domainmakes direct contact with the cell surface or, alternatively, the transport involves a golgi-independent, post-er vesicular pathway to the pm [ , ] . so far, the latter possibility has been regarded as unlikely since the ic, an obligatory way station in post-er trafficking [ , ] , is expected to become non-functional under these conditions. namely, the binding of copi coats to the ic elements [ - , ] has been considered as a prerequisite for their formation and/or mt-dependent movement to the cis-golgi region [ ] . although earlier studies on the cycling ic protein p / ergic- suggested that this compartment, unlike the golgi, persists in bfa-treated cells [ - ] , later work showed that the protein accumulates at the er exit sites, which remain operational in the absence of copi binding [ , ] . however, recent results on the dynamic organization of the ic are changing this picture. it turns out that this compartment consists of structurally and functionally distinct vacuolar and tubular subdomains, which play different roles in trafficking at the er-golgi boundary. the vacuolar domain contains anterograde cargo (e.g. vsv-g), the cargo receptor p and membrane-bound copi coats. by contrast, the tub- vol. , review article bfa-insensitive (erc to pm) (synthesized in erc?) [ ] bfa-insensitive mt-dependent [ ] fast transport to pm (half-time~ min) [ ] glucosylceramide membrane constituent lipid rafts bfa-independent temperature-insensitive ( o c) [ ] bfa-insensitive mt-dependent [ ] ganglioside [ ] , suggesting that it is involved in the bfa-resistant, temperature ( o c)sensitive and mt-dependent transport of cholesterol to the cell surface [ , ] . unexpectedly, live cell imaging revealed that the ic is a long-lived compartment, that maintains its dynamic properties in the presence of bfa. the rab -mediated pathway from the ic to the growth cones remains operational in bfa-treated, polarized pc cells [ ] . moreover, the topological organization of the early and late ic elements is maintained, and bidirectional communication between them via dynamic tubules continues in the drug-treated cells [marie et al., submitted] . by removing copi coats bfa most likely influences the function of the copi-containing ic vacuoles in er-golgi trafficking, whereas the operation of the tubules remains unaffected. since copi has been implicated in both forward and retrograde trafficking at the er-golgi boundary [ ] , copi-independent pathways in both directions should remain operational in the presence of bfa. evidence for such pathways has been obtained both in mammalian cells [ , ] and the yeast saccharomyces cerevisiae [ , ] . the response of the ic to bfa therefore turns out to be similar to that of the endocytic system. despite causing extensive tubulation of endosomes, bfa does not dramatically affect the function of the endocytic pathway, since both internalization at the pm, mem-brane recycling, and communication between endosomes and lysosomes continues in the drug-treated cells [ , - ] . regarding post-golgi trafficking, bfa inhibits both constitutive and regulated transport from trans-golgi compartments to the pm [ ] . notably, the membrane protein tgn that cycles between trans-golgi/tgn membranes and endosomes accumulates in a pericentrosomal location in bfa-treated cells [ ] , either due to the collapse of the tgn membranes around the centrosomes or the arrest of tgn in an endocytic compartment. how to circumvent the central station? based on the above results showing that the ic maintains its dynamic properties in the presence of bfa, we propose that different routes that circumvent the golgi apparatus connect topographically distinct domains (early and late) of this compartment and the pm (see fig. ). [since it may help to think about the proposed routes by drawing an analogy to traffic in a metropolitan area (see also acknowledgments), some practical instructions to drivers and eventual passengers are given in square brackets.] route a -a . this pathway involves mt-dependent movements of vacuolar and tubular ic elements from er exit sites to the cell center and direct communication of late ic elements with endocytic compartment(s). [drive uptown on a , but be careful, the road might be slippery. make a stop before the central station and take the bypass to a . it may be a good idea to change your vehicle to one of the e models.] route a . a direct connection from early ic to the pm. this pathway, which could also exists in yeast cells [ ] , may -depending on the cell type -be more rapid and less sensitive to temperature reduction and depolymerization of mts. from a geometric point of view, the narrow tubules observed [ ] would appear as optimal carriers for membrane constituents. [take based on the suggested routes, it is possible to reexamine the pathways that some of the molecules listed in table utilize in their trafficking. in fact, some could follow several routes, depending on the cell type and the prevailing physiological conditions. in light of previous studies [ , ] cholesterol could move on route a to the late ic and then take the bypass route to the endosomal system ( fig. ) , where rab and rab are known to participate in cholesterol transport [ , ] . caveolin, which is transported from the er to late ic or golgi, where it oligomerizes [ ] , has been implicated in cholesterol transport to the cell surface [ ] . direct delivery from the ic to endosomes could explain an observed cholesterol gradient (cis-to-trans) across the golgi stacks [ ] . since raft formation is expected to mainly take place in the golgi region, many of the raft proteins listed in table could also follow route a to the cell center. as mentioned above, rotavirus vp protein, which localizes to the ic and participates in virus maturation in the trans-golgi region [ ] , interacts with flotillin, suggesting (indirectly) that flotillin and caveolin could utilize similar pathways. ras proteins, such as h-ras and k-ras, associate with er membranes and seem to employ both classical (er-golgi-pm) and non-classical pathways during their trafficking to the pm [ ] , correlating with the lipid modification(s) (farnesylation, palmitylation) that they receive [ ] . however, recent studies indicate that h-ras associates with the ic, but not with golgi membranes, and moves to the pm via pathways that are bfa-insensitive, but show variable requirement for intact mts [ ] . it is possible that the trafficking of h-ras involves alternative vesicular routes (fig. , a -a and a ) , which may differ in their sensitivity to depolymerization of mts. the recently described bfa-insensitive and syntaxin -independent secretory pathway of integrin aps from er/ic directly to the basolateral surface of drosophila follicular epithelial cells [ ] -resembles the route a proposed here. finally, in the presence of bfa connexin- is transported to the pm, where it forms functional gap junctions [ ] . since the assem-bly of connexins takes place in post-er compartments between the ic and trans-golgi [ , ] , it is possible that connexin- uses the golgi-bypass route a -a (fig. ) . the existence of a putative golgi-bypass route a -a (fig. ) is also of interest regarding the comparison of the secretory pathways that operate in yeast and mammalian cells. although these pathways share many similarities, there are also considerable differences. morphologically, the secretory system of the bakers yeast s. cerevisiae, consisting of continuous tubular networks which connect with the er and form secretory granules, appears simpler than its mammalian counterpart [ , ] . whereas s. cerevisiae does not contain cisternal golgi stacks, such structures are present in the fission yeast schizosaccharomyces pombe, correlating with the fact that secretion of model proteins in these unicellular eukaryotes is differentially affected by bfa, continuing in the former but blocked in the latter [ , ] . biochemically, the high-mannose structures added to yeast and mammalian glycoproteins are very similar, but subsequent processing steps differ considerably. s. cerevisiae builds extensive mannose-based structures but lacks glycosyltransferases of the kind that function in terminal processing of glycoproteins (addition of sialic acid, galactose and fucose) in the mammalian golgi apparatus [ ] . by contrast, n-linked oligosaccrarides are modified by addition of galactose in s. pombe [ ] . regarding the transport machineries, s. cerevisiae gets along with ypt/rab gtpases, of which three -ypt (in humans: ic/cis-golgi rab ), ypt / (trans-golgi/endosomal rab ) and sec (trans-golgi/endosomal rab ) -function in the secretory pathway, whereas rabs are encountered in different mammalian cells [ - ] . in spite of the above differences, a common idea is that s. cerevisiae, like human cells, contains a subcompartmentalized golgi apparatus. the presence of the ic in yeast has remained an enigma, although it has been considered on the basis of the similar phenotypes of copi and copii mutants [ ] . notably, the proposed golgi-bypass route a -a (fig. ) shares striking similarity with the secretory pathway of s. cerevisiae. thus, it could represent a basic secretory system that is shared by the two cell types, whereas additional cop machineries have developed in mammalian cells to enhance protein sorting and to build up a more versatile glycosylation apparatus. first, the rate of transport along this bfa-insensitive route in mammalian cells is comparable to the rapid secretion in yeast cells, where glycoproteins such as invertase traverse the secretory pathway in~ min [ ] . second, the functions of ypt (rab ) and ypt / (rab ) in yeast are linked at the molecular level by the two switchable forms of the conserved trapp complex, which act as gefs to successively activate these gtpases [ ] . finally, the secretory pathway in yeast seems to consists of two branches, of which one transits through endosomes [ , ] , acyl-tripeptides containing the asn-tyr-ser sequence have previously been used to measure the rate of bulk flow from the er via the golgi apparatus to the pm [ ] . these tripeptides are taken up by cells by an unknown mechanism -which, however, may be related to the transport routes utilized by certain protein toxins (see below) -and apparently reach the er, where they become n-glycosylated. depending on the cell type, the half-time of subsequent secretion of these glycopeptides was found to be - min, i.e. similar to that of cholesterol and sphingomyelin [ , , ] . their secretion from cells is blocked at o c and by gtp-g-s, indicating that it involves vesicular trafficking [ ] . depending on the cell type, up to % of the glycopeptides are modified by golgi glycosyltransferases, including addition of terminal galactose and sialic acid by trans-golgi enzymes [ ] . since the secretion of the glycopeptides is not affected by bfa [ ] , it is possible that they utilize the proposed golgi-bypass route a -a (fig. ). these small peptides are not expected to contain sorting information, and thus may become initially concentrated in the vacuolar parts of the ic [ , ] . their golgi-specific processing in control cells could be explained in two (or more) ways. either only~ % of the molecules pass through the golgi stacks and become modified, or the peptides are transferred from late ic to endosomal compartments. by receiving newly synthesized enzymes from the er, and communicating with the golgi stacks, these peri-golgi compartments may contain sufficient amounts of the golgi enzymes to support the observed terminal glycosylation [ - ] . in the presence of bfa the enzymes are redistributed, explaining the inhibition of the terminal processing of the tripeptides [ ] . like highways and subways, membrane traffic routes are bidirectional. for example, the tubules containing fluorescent rab a not only move from centrally located ic elements to the cell periphery, but also in the opposite direction [ ] . studies of bacterial toxins lead to the discovery that the secretory pathway is reversible [ ] . the cellular uptake of these protein toxins from the pm involves different endocytic mechanisms. subsequently, certain toxins (such as shiga and cholera toxin, as well as ricin) are transported to the golgi region, and further to the er from which they (or their subunits) are translocated to the cytosol to exert their toxic effects [ , ] . the golgi-to-er transfer of these toxins involves different mechanisms, but can also occur in a copi-independent fashion, suggesting the involvement of golgibypass pathways [ , - ] . the comprehensive endocytic tracer wheat germ agglutinin (wga) also reaches the er in bfa-treated cells; however, only when the drug is added during the later stages of endocytic uptake, when the lectin has accumulated in the trans-golgi region of hepg hepatoma cells [ ] . in addition, caveolar endocytosis of polyoma virus sv [ ] and the autocrine motility factor receptor [ ] results in their delivery to smooth er, providing further examples of endocytic processes that may involve the golgi-bypass routes discussed here [ ] . the ideas concerning transport routes that bypass the golgi apparatus go back in time. based on their studies on the secretory process in pancreatic exocrine cells, jamieson and palade concluded that the transfer of secretory proteins from tubulo-vesicular membranes at the cis-side of the golgi apparatus to condensing vacuoles at its trans-side occurs without apparent involvement of the cisternal stacks [ ] . similarly, morrØ and ovtracht used the term boulevard pØriphØrique to describe the flow of secretory material in the plant golgi apparatus [ ) . previously, alex novikoffs gerl (golgi-er-lysosome) concept had postulated that newly synthesized lysosomal enzymes (and possibly also secretory proteins) are sequestered into specialized er tubules that make direct contact with the trans-golgi cisternae, suggesting that the biogenesis of lysosomes involves a pathway that bypasses the golgi stacks [ ] . two decades later, griffiths and simons introduced the tgn as a trans-golgi sorting compartment, which also functions in the routing of lysosomal enzymes, contributing to the replacement of the gerl concept [ ] . largely based on biochemical and immuno-em studies on the intracellular site of accumulation of newly synthesized virus membrane proteins during the o c temperature block [ , , ] , the tgn was originally defined as a tubular reticulum extending from the last trans-golgi cisterna [ ] . however, since different cargo proteins display variable localization in the o c-treated cells, it remained unclear whether they are arrested in a specialized trans-golgi secretory organelle, or within a complex membrane system located at the crossroads of the exocytic and endocytic pathways [ , , a] . supporting the latter possibility, the extensive em studies by rambourg and colleagues showed that the tgn is variably expressed in different cell types correlating with their secretory and endocytic activities. cells that are active in endocytosis generally contain well-developed trans-tubular networks, whereas certain professional secretory cells seem to lack these structures altogether but, instead, display procounced cis-tubular networks [ , ] . the structure and size of the tgn also varies in individual cell types, and in cells subjected to different experimental manipulations that affect membrane traffic [ , - ] . whereas resting hepg cells do not contain a detectable tgn, endocytic uptake of wga results in the formation of a golgi-associated membrane network consisting of vacuolar and tubulovesicular domains. this endocytic tgn remains initially a separate structure located at variable distance from the golgi apparatus, but is subsequently incorporated into the cisternal stacks and decreases in size [ , ] . in addition, the appearance of the tgn varies depending on the extent of flow of secretory cargo through the golgi system [ ] . for example, recent em tomographic studies showed that whereas the tgn is poorly developed in cells where ic-to-golgi transfer of secretory proteins is prevented during incubation at o c, it expands considerably as a consequence of the shift of cells to a higher temperature ( o c) that triggers trafficking across the golgi stacks [ ] . em tomography of the golgi apparatus has been instrumental in revealing important structural features of the trans-golgi/tgn and contacts between these membranes and other organelles [ , - ] . studies of nrk cells kept at o c showed that the predominant effect of the low-temperature incubation is the bulging of the penultimate trans-golgi cisternae and the disappearence of their tubular extensions, but did not provide evidence for a tubular network connecting with the trans-most cisterna [ ] . notably, only the latter contains clathrin coats, suggesting that it specifically communicates with endosomes [ ] . -d reconstructions have also provided evidence for the close association between trans-golgi membranes and specific er domains [ ] , as first proposed by novikoff. however, the current idea is that these direct er-trans-golgi contacts correspond to the sites where intercompartmental transport of ceramide (the precursor of sphingolipids) and oxysterol via the cytosolic proteins cert and osbp, respectively, takes place [ , ] , bypassing the more proximal golgi compartments. however, the precise non-vesicular pathways in which these transfer proteins operate remain unknown [ , ] . if membrane contact sites are indispensable for the trafficking of certain lipids and sterols, they are expected to persist when the golgi is disrupted by bfa, explaining the ongoing delivery of these membrane constituents to the surface of the drug-treated cells (table ). in conclusion, a number of studies suggest that the tgn is not an autonomous, stable secretory organelle and emphasize its inducible nature at the cell center, where multiple membrane traffic pathways converge [ a] . also, it has been well established that molecular sorting along the secretory pathway occurs at multiple locations [ - ] . pre-golgi sorting takes place both at er exit sites and in the ic [ , , , , ] , whereas post-golgi sorting involves endosomal compartments [ , , , ] . in particular, studies of both polarized and non-polarized cells have shown that the endocytic recycling compartment (erc) operates as a way station during the transport of e.g. vsv-g and e-cadherin to the pm [ - ] , emerging as a major sorting site for both biosynthetic and endocytic trafficking. if the tgn is not the principal recipient of er-derived secretory cargo and the major site of its sorting in the golgi apparatus, how is this system then organized? the studies discussed below may give additional clues to address this question. first, orci, rothman and coworkers generated megavesicles, i.e. vacuoles with a diameter of~ nm, by depositing aggregates of an exogenous, non-glycosylated protein into the er and golgi lumen and then employed the o and o c temperature blocks to regulate the transport of these membrane-enclosed aggregates across the golgi stacks [ ] . they concluded that transport through the stacks (cis-to-trans) takes about min. during their journey, the aggregates were mostly (~ %) observed to associate with the dilated ends of the cisternae, but a smaller fraction was present in free vacuoles at the periphery of the stacks. however, since low-temperature treatment can exert a major effect on the morphology of the golgi apparatus [ , ] , including extensive tubulation [ ] , it is possible that part of the connections between these peripheral vacuoles and golgi cisternae could not be detected by the serial section analysis that was carried out. [ , ] . at both temperatures the proteins were localized by immuno-em to vacuoles ( - nm) at one side of the golgi stacks. these vacuoles were partly continuous with the golgi cisternae, but mostly devoid of any apparent attachment. again, the serial section analysis could have missed possible, narrow tubular connections between the vacuoles and cisternae, or these were broken due to the mild chemical fixation employed. in cells shifted from o to o c (or o c), the apparent transport of the proteins into the golgi stacks via tubular parts of the ic elements was accompanied by a considerable loss of label from the vacuoles [ ] . based on their studies on the transport of vsv-g in nocodazole-induced golgi ministacks, employing em tomography and quantitative immuno-em, luini and co-workers arrived at a similar conclusion on the role of tubules in the transfer of cargo from the ic into the cis-golgi cisternae. moreover, they reported that the synchronized forward movement of cargo across the golgi stacks (via cisternal progression) is coupled to the formation of intercisternal tubular connections, which mediate retrograde transport of golgi enzymes [ ] . third, the transport pathways taken by the mainly unglycosylated secretory proteins in resting and stimulated pancreatic acinar cells seem to differ. as mentioned above, in resting cells the proteins move directly from peripheral cis-golgi elements to the trans-golgi condensing vacuoles [ ] , whereas in stimulated cells they are more readily detected in the dilated rims of the cisternae and also can occupy more central regions of the golgi stacks. notably, the kinetics of secretion is not affected by stimulation [ ] . collectively, the above studies suggest that the peripheral, dilated rims and the central cisternal stacks correspond to different subdomains of a single functional unit, having specialized roles in protein transport across the golgi apparatus. in addition, recycling endocytic traffic from the pm can be directed to the dilated ends of golgi stacks [ , ] . accordingly, the following mechanisms could be discussed in the transfer of different molecules in the golgi system (fig. ). to begin with, centrally located ic elements could generate golgi organization in a process where their vacuolar domains, containing concentrated lumenal cargo [ ] , give raise to the dilated cisternal ends. integral membrane components are sorted from the vacuoles into the central regions of the golgi stack, whereas soluble cargo is mainly restricted to the vacuolar (saccular) parts. the latter, however, possibly depending on the type of cargo they contain, also move to more central regions of the stacks [ ] . vesicles could shuttle certain components from one cisternae to another, possibly operating together with cisternal progression [ ] , and tubular connections provide retrograde pathways for the intercisternal transfer of golgi enzymes [ ] . however, the model in figure proposes that the golgi stacks do not operate in a vectorial manner regarding the transfer of all cargo molecules. bidirectional pathways bypassing the golgi stacks would figure . a map of the proposed golgi-bypass pathways connecting the ic with the pm. bidirectional pre-and post-golgi pathways are indicated by arrows. a and a indicate bidirectional routes between early and late ic elements, and early (ee) and late (le) endosomes, respectively. a designates a direct route from early ic to the plasma membrane (pm) [ ] . the function of these pathways depends on various coat proteins (cops) [ , ] , or possible scaffold proteins [ , ] , which differ in their sensitivity to brefeldin a (bfa). the pre-golgi pathways that are sensitive or insensitive to bfa, including the proposed route from late ic elements to the endosomal system, are indicated by red and green colour, respectively (see the text for further discussion). for simplicity, the biosynthetic [ , ] and endocytic recycling compartments (brc and erc) extending from ic and endosomal vacuoles, respectively, are shown as short tubules, although they constitute extensive tubular networks [ , ] . similarly, the tubular networks at the cis-and trans-sides of the golgi stacks (cgn and tgn) are not included in this planar diagram. eres = er exit site. cell. mol. life sci. vol. , review article make it possible that entry and exit of molecules can take place at the two poles of the organelle. for example, certain golgi membrane proteins could bypass the cis-and medial-golgi compartments and enter the trans-golgi from endosome-derived tubules, resulting in their polarized distribution within this organelle. novel live-cell imaging techniques and model systems will hopefully make it possible in the future to study and compare the transport routes of different cargo molecules in the golgi system at high resolution. the biosynthetic and endocytic pathways could exert opposing forces on golgi dynamics (fig. ) , giving rise to different traffic patterns observed in different cells. this could clarify some previous findings and discrepancies, for example, the variable localization of the cation-independent mannose- -phosphate receptor (m pr) that functions in the sorting of lysosomal enzymes. in professional secretory cells, such as pancreatic acinar cells, the m pr is localized to cis-golgi membranes [ ] , whereas in other cell types it can be present throughout the stacks, or con-centrate in the trans-golgi/tgn [ ] . different cell types, depending on their relative secretory and endocytic activities, could variably allocate their sorting processes either at the cis-or trans-side of the golgi stacks [ ] . as another example, in differentiating leucocytes the formation of different secretory granules shifts from one pole of the golgi to the other [ ] . in the case of many newly synthesized proteins the transfer time through the golgi apparatus is propably in the range of - min [ , ] ; however, some proteins require even longer times for their passage [ ] . the golgi-bypass pathways considered here, by granting direct transport to the cell surface, could have an important role in cell physiology. for example, they could participate in phagocytosis and the delivery of membrane components to the leading edge of migrating cells, such as fibroblasts [ - ] . although it has been reported that long-term treatment with bfa prevents cell polarization and motility [ ] , golgi disassembly by this drug has no immediate effect on these events [h. a. d, m. m. and j. s., unpublished data]. in addition, rapid transport to the cell surface in incompletely processed form, followed by endocytosis and golgi-specific modification, may be a life style that is shared by many pm proteins, including receptors, channels and pumps. the notion that these pathways constitute a basic transport system that involves the intracellular assembly of membrane microdomains (lipid rafts) implies that they function in signalling events and the coordination of the biosynthetic and endocytic functions of the cell. finally, these routes may have a role in antigen presentation [ ] and intracellular replication of viruses [ - ] . the existence of golgi-bypass routes in plant cells [ ] , and in cells equipped with a relatively simple secretory apparatus, such as plasmodium and giardia [ - ] , suggests that these pathways belong to the secretory repertoire of a variety of eukaryotic cells. the finding that fibroblasts can express the apical and basolateral routes defined in polarized epithelial cells [ ] further suggests that eukaryotic cells share a basic, adjustable secretory system that can adopt various functional forms in specialized cell types. in neurons a pathway that circumvents the golgi apparatus could give rise to the satellite pathway that functions in local protein synthesis and adaptation of dendritic spines to allow long term information storage in the brain [ - ] . however, a number of questions regarding the operation of the golgibypass routes in different cell types remain unanswered. for example, do they involve the new types of coats (membrane-shaping proteins) that recently have been suggested to operate in organelle formation and membrane traffic [ , ] ? or, what are the fusion factors (e.g. snares) that function in the required intercompartmental transport events? the center of a cell is a crowded place, and the golgi apparatus has many near neighbours. also, since the residents of many of the packed organelles pay frequent visits to adjacent compartments, it is not always easy to tell how many compartments there are, and who lives where. a cell biologist hoping to map the arrangement and interactions of endomembrane compartments, for example, exosomes [ ] and endosomes, could copy a cytogeneticist, who takes a dipteran chromosome and spreads it on a glass slide or em grid to reveal its puffs and nucleosomes. indeed, preparations like this can provide important information, but as is the case with chromosomes, the -d picture is definitely lost. fortunately, a living cell, an epithelial cell or a neuron, can help with the mission. the golgi is in some cells more widespread than in others, but still too compact to reveal its secrets. to understand the traffic plan of this organelle, perhaps one should take another look at the periphery of the cell instead of just looking at the center. the road taken: past and future foundations of membrane traffic er-to-golgi transport visualized in living cells visualization of er-to-golgi transport in living cells reveals a sequential mode of action for copii and copi the debate about transport in the golgi -two sides of the same coin? the golgi apparatus: years of progress and controversy what can yeast tell us about n-linked glycosylation in the golgi apparatus? the golgi apparatus: balancing new with old the golgi complex: in vitro veritas? trans-golgi network sorting exiting the golgi complex robert feulgen lecture . lipid microdomains and membrane trafficking in mammalian cells functions of lipid rafts in biological membranes lipid rafts and membrane traffic lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface intestinal dipeptidyl peptidase iv is efficiently sorted to the apical membrane through the concerted action of n-and o-glycans as well as association with lipid microdomains differential sorting and fate of endocytosed gpi-anchored proteins sorting gpi-anchored proteins erlin- and erlin- are novel members of the prohibitin family of proteins that define lipid-raft-like domains of the er diversity of raft-like domains in late endosomes coat proteins regulating membrane traffic coat proteins: shaping membrane transport bi-directional protein transport between the er and golgi copii and exit from the endoplasmic reticulum regulation of membrane traffic in animal cells by copi retrograde transport from endosomes to the trans-golgi network the copii cage: unifying principles of vesicle coat assembly the formation of tgn-toplasma-membrane transport carriers the mystery of nonclassical protein secretion. a current view on cargo proteins and potential export routes transport of exogenous growth factors and cytokines to the cytosol and to the nucleus arf proteins: roles in membrane traffic and beyond brefeldin a and the endocytic pathway. possible implications for membrane traffic and sorting brefeldin a: insights into the control of membrane traffic and organelle structure brefeldin a, a specific inhibitor of intracellular translocation of vesicular stomatitis virus g protein: intracellular accumulation of high-mannose type g protein and inhibition of its cell surface expression novel blockade by brefeldin a of intracellular transport of secretory proteins in cultured rat hepatocytes selective inhibition of transcytosis by brefeldin a in mdck cells ptk cells contain a nondiffusible, dominant factor that makes the golgi apparatus resistant to brefeldin a ricin transport in brefeldin a-treated cells: correlation between golgi structure and toxic effect cholesterol and vesicular stomatitis virus g protein take separate routes from the endoplasmic reticulum to the plasma membrane pre-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the golgi apparatus en route to the plasma membrane transport of cholesterol from the endoplasmic reticulum to the plasma membrane how proteins move lipids and lipids move proteins the proteintyrosine phosphatase cd reaches the cell surface via golgidependent and -independent pathways chicken erythroid ae anion exchangers associate with the cytoskeleton during recycling to the golgi non-coordinate regulation of enac: paradigm lost? traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway bafilomycin a -sensitive pathway is required for the maturation of cystic fibrosis transmembrane conductance regulator intracellular localization of fibronectin using immunoperoxidase cytochemistry in light and electron microscopy vesicles and cisternae in the trans golgi apparatus of human fibroblasts are acidic compartments abnormal glycosylation of human fibronectin secreted in the presence of monensin rotavirus is released from the apical surface of cultured human intestinal cells through non-conventional vesicular transport that bypasses the golgi apparatus dissecting rotavirus particle-raft interaction with small interfering rnas: insights into rotavirus transit through the secretory pathway different ways to reach the top of a cell. analysis of rotavirus assembly and targeting in human intestinal cells reveals an original raftdependent, golgi-independent apical targeting pathway rotavirus assembly: an alternative model that utilizes an atypical trafficking pathway distinct functions for arf nucleotide exchange factors at the golgi complex: gbf and bigs are required for assembly and maintenance of the golgi stack and tgn, respectively dissecting the role of the arf guanine nucleotide exchange factor gbf in golgi biogenesis and protein trafficking pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the golgi complex the er-golgi intermediate compartment (ergic): in search of its identity and function ) b-cop localizes mainly to the cis-golgi side in exocrine pancreas vesicular tubular clusters between the er and golgi mediate concentration of soluble secretory proteins by exclusion from copi-coated vesicles dissection of copi and arf dynamics in vivo and role in golgi membrane transport er-to-golgi transport: form and formation of vesicular and tubular carriers microtubule-dependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway distribution of the intermediate elements operating in er to golgi transport brefeldin a induced dose-dependent changes to golgi structure and function in the rat exocrine pancreas maintenance of golgi structure and function depends on the integrity of er export molecular basis for golgi maintenance and biogenesis retrograde transport from the pre-golgi intermediate compartment and the golgi complex is affected by the vacuolar h+-atpase inhibitor bafilomycin a rab defines a novel pathway connecting the pre-golgi intermediate compartment with the cell periphery evidence for a copi-independent transport route from the golgi complex to the endoplasmic reticulum imaging of procollagen transport reveals copi-dependent cargo sorting during er-to-golgi transport in mammalian cells two redundant systems maintain levels of resident proteins within the yeast endoplasmic reticulum copi-independent anterograde transport: cargo-selective er to golgi protein transport in yeast copi mutants brefeldin as effects on endosomes, lysosomes, and the tgn suggest a general mechanism for regulating organelle structure and membrane traffic brefeldin a causes a microtubule-mediated fusion of the trans-golgi network and early endosomes the morphology but not the function of endosomes and lysosomes is altered by brefeldin a post-golgi membrane traffic: brefeldin a inhibits export from distal bypassing the golgi apparatus golgi compartments to the cell surface but not recycling tgn and its orthologues: roles in post-tgn vesicle formation and maintenance of tgn morphology the yeast secretory pathway is perturbed by mutations in pmr , a member of a ca + atpase family modulation of cellular cholesterol transport and homeostasis by rab rab -dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells a new paradigm for membrane-organizing and -shaping scaffolds caveolins and membrane cholesterol heterogeneous distribution of filipin-cholesterol complexes across the cisternae of the golgi apparatus h-ras but not k-ras traffics to the plasma membrane through the exocytic pathway palmitoylation: policing protein stability and traffic h-ras does not need copi-or copii-dependent vesicular transport to reach the plasma membrane multiple pathways in trafficking and assembly of connexin , and into gap junction intercellular communication channels multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin- , occurs after exit from the er pathways and control of connexin oligomerization functional morphology of the secretory pathway organelles in yeast morphodynamics of the secretory pathway effect of brefeldin a on biosynthesis of cellular components in candida albicans brefeldin a sensitivity and resistance in schizosaccharomyces pombe. isolation of multiple genes conferring resistance overview of n-and o-linked oligosaccharide structures found in various yeast species vesicular transport: how many ypt/rab-gtpases make a eukaryotic cell? ypt/rab gtpases: regulators of protein trafficking rabs and their effectors: achieving specificity in membrane traffic the role of coat proteins in the biosynthesis of secretory proteins order of events in the yeast secretory pathway trappii subunits are required for the specificity switch of a ypt-rab gef parallel secretory pathways to the cell surface in yeast a subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway the rate of bulk flow from the endoplasmic reticulum to the cell surface the rate of bulk flow from the golgi to the plasma membrane reconstitution of steps in the constitutive secretory pathway in permeabilized cells. secretion of glycosylated tripeptide and truncated sphingomyelin complete golgi passage of glycotripeptides generated in the endoplasmic reticulum of mammalian cells demonstration of an extensive transtubular network continuous with the golgi apparatus stack that may function in glycosylation er-golgi intermediates acquire golgi enzymes by brefeldin a-sensitive retrograde transport in vitro the gm and grasp golgi proteins cycle through and define a subdomain of the intermediate compartment ectopic localizations of golgi glycosyltransferases modulation of galt and sialt sub-golgi localization by sialt expression reveals an organellar level of glycolipid synthesis control membrane traffic exploited by protein toxins retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol surfing on a retrograde wave: how does shiga toxin reach the endoplasmic reticulum? retrograde transport of protein toxins under conditions of copi dysfunction evidence that the transport of ricin to the cytoplasm is independent of both rab a and copi induction of direct endosome to endoplasmic reticulum review article with a temperature-sensitive defect in e-coatomer protein (e-cop) brefeldin a-regulated retrograde transport into the endoplasmic reticulum of internalized wheat germ agglutinin caveolar endocytosis of simian virus reveals a new twostep vesicular-transport pathway to the er localization of autocrine motility factor receptor to caveolae and clathrinindependent internalization of its ligand to smooth endoplasmic reticulum distinct caveolae-mediated endocytic path-ways target the golgi apparatus and the endoplasmic reticulum intracellular transport of secretory proteins in the pancreatic exocrine cell. i. role of the peripheral elements of the golgi complex dynamics of the golgi apparatus: membrane differentiation and membrane flow the endoplasmic reticulum: a cytochemists view (a review) the trans-golgi network: sorting at the exit site of the golgi complex reduced temperature prevents transfer of a membrane glycoprotein to the cell surface, but does not prevent terminal glycosylation exit of newly synthesized membrane proteins from the trans cisterna of the golgi complex to the plasma membrane low temperatureinduced transport blocks as tools to manipulate membrane traffic threedimensional architecture of the golgi apparatus in sertoli cells of the rat three-dimensional structure of the golgi apparatus in mammalian cells golgi structure in three dimensions: functional insights from the normal rat kidney cell golgi apparatus and tgn during endocytosis. histochem retrograde traffic in the biosynthetic-secretory route secretory traffic triggers the formation of tubular continuities across golgi sub-compartments predicting function from structure: -d structure studies of the mammalian golgi complex structure of the golgi and distribution of reporter molecules at degrees c reveals the complexity of the exit compartments inter-organelle membrane contact sites: through a glass, darkly cert and intracellular trafficking of ceramide oxysterol-binding protein and vamp-associated membrane protein are required for sterol-dependent activation of the ceramide transport protein protein sorting upon exit from the endoplasmic reticulum protein sorting in the golgi complex: shifting paradigms polarized biosynthetic traffic in renal epithelial cells: sorting, sorting, everywhere how many ways through the golgi maze newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface recycling endosomes can serve as intermediates during transport from the golgi to the plasma membrane of mdck cells rab in recycling endosomes regulates the sorting and basolateral transport of e-cadherin antibody to ap b adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes megavesicles implicated in the rapid transport of intracisternal aggregates across the golgi stack structure and dynamics of the golgi complex at degrees c: low temperature induces the formation of golgi-derived tubules synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells recovery of surface membrane in anterior pituitary cells. variations in traffic detected with anionic and cationic ferritin the golgi apparatus (complex)-( - )-from artifact to center stage the er to golgi interface is the major concentration site of secretory proteins in the exocrine pancreatic cell procollagen traverses the golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation the mannose- -phosphate receptor for lysosomal enzymes is concentrated in cis-golgi cisternae bypassing the golgi apparatus possible pathways for lysosomal enzyme delivery the distribution of -kilodalton mannose -phosphate receptors within cis (heavy) and trans (light) golgi subfractions varies in different cell types origin of granules in polymorphonuclear leukocytes. two types derived from opposite faces of the golgi complex in developing granulocytes moving membrane up to the front of migrating cells the polarization of the motile cell endocytic recycling pathways: emerging regulators of cell migration functional symmetry of endomembranes disruption of the golgi apparatus by brefeldin a blocks cell polarization and inhibits directed cell migration lonely mhc molecules seeking immunogenic peptides for meaningful relationships immunocytochemical analysis of uukuniemi virus budding compartments: role of the intermediate compartment and the golgi stack in virus maturation structural maturation of the transmissible gastroenteritis coronavirus assembly of vaccinia virus revisited: de novo membrane synthesis or acquisition from the host? effects of picornavirus a proteins on protein transport and gbf -dependent cop-i recruitment poliovirus infection blocks ergic-to-golgi trafficking and induces microtubule-dependent disruption of the golgi complex cell biology of the plant golgi apparatus export of plasmodium proteins via a novel secretory pathway bfa-sensitive and insensitive exocytic pathways in entamoeba histolytica trophozoites: their relationship to pathogenesis the secretory apparatus of an ancient eukaryote: protein sorting to separate export pathways occurs before formation of transient golgi-like compartments different biosynthetic transport routes to the plasma membrane in bhk and cho cells dendritic and postsynaptic protein synthetic machinery evidence for a satellite secretory pathway in neuronal dendritic spines secrets of the secretory pathway in dendrite growth growing dendrites and axons differ in their reliance on the secretory pathway rough sheets and smooth tubules brefeldin a does not inhibit the movement of phosphatidylethanolamine from its sites for synthesis to the cell surface sphingomyelin transport to the cell surface occurs independently of protein secretion in rat hepatocytes transport of newly synthesized glucosylceramide to the plasma membrane by a non-golgi pathway ganglioside gd traffics from the trans-golgi network to plasma membrane by a rab -independent and brefeldin a-insensitive exocytic pathway mechanisms of cx and cx transport to the plasma membrane and gap junction regeneration selective inhibition of protein targeting to the apical domain of mdck cells by brefeldin a the paranodal complex of f /contactin and caspr/paranodin traffics to the cell surface via a non-conventional pathway analysis of transport and targeting of syndecan- : effect of cytoplasmic tail deletions prointegrin maturation follows rapid trafficking and processing of mt -mmp in furin-negative colon carcinoma lovo cells exocytic pathwayindependent plasma membrane targeting of heterotrimeric g proteins the exocytotic trafficking of tc occurs through both classical and non-classical secretory transport pathways in t l adipocytes flotillin- /reggie- traffics to surface raft domains via a novel golgiindependent pathway. identification of a novel membrane targeting domain and a role for palmitoylation low temperature induces the delivery of mature and immature cftr to the plasma membrane vesicle budding from endoplasmic reticulum is involved in calsequestrin routing to sarcoplasmic reticulum of skeletal muscles synthesis of surface sphingomyelin in the plasma membrane recycling pathway of bhk cells epithelial sphingolipid sorting is insensitive to reorganization of the golgi by nocodazole, but is abolished by monensin in mdck cells and brefeldin a in caco- cells immunocytochemical localization of b-cop to the er-golgi boundary and the tgn dgrasp-mediated non-canonical integrin secretion is required for drosophila epithelial remodeling bypassing the golgi apparatus we are grateful to joao barroso, michael key: cord- - heg iql authors: armstrong, john; mccrae, malcolm; colman, alan title: expression of coronavirus e and rotavirus vp membrane proteins from synthetic rna date: - - journal: j cell biochem doi: . /jcb. sha: doc_id: cord_uid: heg iql some viruses acquire their envelopes by budding through internal membranes of their host cell. we have expressed the cloned cdna for glycoproteins from two such viruses, the e protein of coronavirus, which buds in the golgi region, and vp protein of rotavirus, which assembles in the endoplasmic reticulum. messenger rna was prepared from both cdnas by using sp polymerase and either translated in vitro or injected into cultured cv cells or xenopus oocytes. in cv cells, the el protein was localised to the golgi region and vp protein to the endoplasmic reticulum. in xenopus oocytes, the e protein acquired post‐translational modifications indistinguishable from the sialylated, o‐linked sugars found on viral protein, while the vp protein acquired endoglycosidase‐h‐sensitive n‐linked sugars, consistent with their localisation to the golgi complex and endoplasmic reticulum, respectively. thus the two proteins provide models with which to study targeting to each of these intracellular compartments. when the rnas were expressed in matured, meiotic oocytes, the vp protein was modified as before, but the e protein was processed to a much lesser extent than in interphase oocytes, consistent with a cessation of vesicular transport during cell division. in the eukaryotic cell, the rough endoplasmic reticulum is the site of synthesis for both secretory proteins and integral proteins of the plasma membrane. to reach the cell surface, both types of protein must traverse the golgi complex. during this transport process, a series of post-translational modifications may occur. since the modifying enzymes are localised along the secretory pathway, the endoplasmic reticulum and golgi complex constitute a series of membrane-limited compartments, each apparently with a distinct complement of proteins [reviewed in . how are these proteins confined to their appropriate destinations, rather than migrating onward to the plasma membrane? we are investigating two viral model proteins, one for the endoplasmic reticulum and one for the golgi complex, with a view to determining the features of each molecule responsible for its correct localisation. rotaviruses are a class of animal viruses which characteristically assemble at the endoplasmic reticulum [ ] . although assembly appears to be a budding process, the matured virion surprisingly lacks a visible envelope . the bovine uk strain encodes two glycoproteins: vp , which forms part of the virion, and vpio, which does not [ , ] . the latter is an integral membrane glycoprotein [ , ] bearing "immature" n-linked sugars which imply a failure to leave the endoplasmic reticulum [ w . by contrast, with coronaviruses the envelope is acquired by budding initially at smooth membranes close to the golgi complex but continuous with the endoplasmic reticulum; as infection progresses budding may also occur in the golgi cisternae or the rough endoplasmic reticulum [ , . the strain mhv-a bears two glycoproteins, the smaller of which, e l , is restricted within the cell and does not appear to reach the plasma membrane except as part of a budded virion , . thus we have adopted the proteins vplo and e l as potential models for the class of membrane proteins which localise within compartments of the secretory pathway. previously we have reported the cloning and sequence analysis of both proteins [ , ] . here we show that both cdnas can be expressed via artificial mrna prepared using sp polymerase [ , . the mrna may be translated either in vitro or after injection into xenopus oocytes or cultured cvi cells. expressed in this way, vplo and e l prove to be valid model proteins for the endoplasmic reticulum and golgi complex, respectively. in addition, we have exploited the fact that xenopus oocytes can be matured into a meiotic state [ , to investigate the transport of the two proteins to their destinations during cell division. all restrictions, ligations, and transformations were carried out according to standard methods . full-length cdna for bovine rotavirus vplo (assembled and kindly provided by h. baybutt) was excised with the enzymes ahaiu and sali, to give a fragment corresponding to nucleotides - [in figure of . this was inserted into the bglii site of the transcription vector psp t [ . e l cdna [ , was excised with aha and fom, representing nucleotides - [in , plus the following bases of the adjacent nucleocapsid gene [ ] and inserted into the bglii site of psp t . both plasmids were linearised with ecori and rna transcribed with sp polymerase in the presence of . mm m g ( ')ppp( ')g exactly as described [ ] . since the vector psp t contains a poly-a tract between the bglii and ecori sites, this method results in the synthesis of capped, polyadenylated mrna in a single reaction. translation in vitro in reticulocyte lysates, injection of xenopus oocytes, immunoprecipitation of proteins, microinjection of cv cells, and immunofluorescence analysis were all performed as before [ ] . for analysis of vplo protein, rabbit antiserum to a fusion protein of bacterial -galactosidase and vpio, whose prepara-tion will be described in detail elsewhere, was used. to detect el, we used a rabbit serum to e l purified by detergent extraction of virus [ ] , kindly provided by s. tooze, embl, heidelberg, who also provided stocks of coronavirus mhv-a and its host, sac-cells. radiolabelled virus was produced by infection of cells at a ratio of pfukell, incubation for hr in methionine-free medium to which was added pci/ml s-methionine (amersham) , and collection of the culture supernatant. endoglycosidase h digestions were carried out as before [ . xenupus oocytes were matured into second meiotic metaphase by incubation in p g / d progesterone as described [ . previously we showed that rna prepared with sp polymerase could be translated in cultured cells, provided it has both a ' cap structure, and a ' poly-a tract added using poly-a polymerase [ ] . e l rna prepared in this way was found to be translated in both cv cells and xenopus oocytes (not shown). we have further simplified this method by using a vector which includes a sequence of a's in the transcribed region [ , eliminating the requirement for a second reaction. rnas prepared by this method for vplo and e l were translated efficiencly in reticulocyte lysates, xenopus oocytes, and cultured cvl cells (figs. , ). rough endoplasmic reticulum; these structures may be cleaved by endoglycosidase h [ ] . vplo protein which had been immunoprecipitated from injected oxcytes was found to be completely sensitive to endoglycosidase h, yielding a species which approximately comigrated with the unprocessed form produced in the reticulocyte lysate (fig. , lanes a-d) . thus the vplo protein is likely to be localised within the endoplasmic reticulum of the oocyte. e l protein was synthesized in oocytes as a spectrum of forms, the most mobile of which comigrated with the unmodified protein from the reticulocyte lysate (fig. , lanes e,f). the oocyte proteins were very similar in both mobilities and relative abundance to the species of e l found in virus particles (fig. , lane ) . these have been shown to contain -linked sugars as their only posttranslational modification [ , ] . since at least the later stages of -linked glycosylation are thought to occur in the golgi complex , most or all of the el protein would appear to have reached this organelle in the oocyte. with both antibodies, no significant labeled proteins were precipitated from oocytes in the absence of the appropriate mrna. sp rnas for vplo and el were microinjected into cvl monkey kidney cells, and the resulting proteins were detected by immunofluorescence. vplo proteins was found in an elaborate pattern around the nucleus and throughout the cytoplasm, characteristic of the rough endoplasmic reticulum (fig. a) . by changing the plane of focus, it became clear that all of the nuclear envelope was labeled (not shown). this was expected as the outer nuclear membrane is continuous with, and usually considered as part of, the endoplasmic reticulum. in contrast, e l protein showed a much more localised fluorescence pattern (fig. b) . labeling was concentrated in a perinuclear area corresponding to the golgi region and coincident with the intracellular pattern shown by fluorescent wheat germ agluttinin, a golgi marker (not shown). the pattern also closely resembled the distribution of e l protein in virally infected cells , . if injected cells were not permeabilised with detergent, no labeling of the cell surface was detected with either protein (not shown). incubation of xenopus oocytes in progesterone causes them to mature from their initial stage, first meiotic prophase, to second meiotic metaphase [ ] . in this state, the golgi apparatus is broken down and protein secretion blocked [ . we investigated the synthesis of vplo and e l in such matured oocytes. vplo was electrophoretically indistinguishable from the form produced in nonmatured oocytes (fig. , lanes a,b) . e l , in contrast, showed a striking reduction in the extent to which it was processed in comparison to nonmatured oocytes (fig. , lanes c,d) , implying that most of the protein is denied access to the modifying enzymes of the golgi complex under these conditions. transcription of rna with sp polymerase has been used as a route for expression of cloned cdnas either in vitro or in the xenopus oocyte [ . if the rna incorporates a ' cap structure and a ' poiy-a tract, it may also be expressed in a b c d fig. . expression of vplo (a,b) and e l (c,d) in normal (a,c) or progesterone-matured (b,d) oocytes. proteins were analysed as for figure , and the fluorograph was exposed for wk. cultured cells [ . by using the vector psp t [ , such rna can be prepared in a single reaction. we assume that the presence of an encoded poly-a sequence in the vector is the critical factor in allowing expression of the rna in cultured cells ; however, the transcribed region also contains ' and ' untranslated regions from a globin cdna, and a sequence of c bases at the extreme ' end [ , and any of these elements may contribute to the stability of the mrna or the efficiency with which it is translated. whichever feature is important, the system provides a quick and versatile approach to the expression of cloned dnas. its principal limitation, at present, is the lack of a technique for the efficient introduction of rna into large populations of cultured cells. it appears that both of the proteins we have studied, vplo and el, are localised in a similar fashion whether they are expressed in isolation in cell types as diverse as oocytes and cvl cells, or in the course of viral infection. thus each is likely to be a legitimate model with which to study targeting of proteins to its respective intracellular destination. in the case of vpio, this is the rough endoplasmic reticulum, as judged by fluorescence microscopy and sensitivity of its n-linked oligosaccharides to endoglycosidase h (figs. , ) . the corresponding protein of rotavirus sall , termed ncvps, bears oligosaccharides of the form man glcnac [lo] . in contrast, the principal location of the e l protein appears to be the golgi complex. as the bulk of the protein made in oocytes has been modified to give forms of similar mobilities to the viral protein (fig. , lanes f,g; see also [ ] ), it has presumably acquired -linked oligosaccharides, an indication of having reached the golgi complex [ ] . most of the oligosaccharides on viral el contain terminal sialic acid [ ] , whose presence is detectable by changes in electrophoretic mobility of the glycoprotein after neuraminidase digestion. however, we have repeatedly failed to observe any effect on mobility of oocyte el by treatment with neuroaminidases from various sources and under various conditions (not shown). the explanation for this is not clear; perhaps the oocyte adds exotic sialic acids which are not susceptible to cleavage. immunofluorescence of cultured cells expressing e l clearly showed a very localised perinuclear distribution (fig. b ) characteristic of the golgi region and similar to the pattern observed during viral infection [ ] . however, not all of the viral protein is restricted to the flattened cisternae of the golgi complex; some at least is found in smooth membranes which are in the same region of the cell but are in fact continuous with the rough endoplasmic reticulum [ , . these membranes are reminiscent of the transitional elements of specialised secretory cells [ ] and are the earliest site of budding during viral infection [ ] . at the resolution of light microscopy it is impossible to say whether the pattern in figure b includes this compartment. it will be of interest to resolve this point by immunoelectron microscopy, which should also allow us to determine how many of the golgi cisternae are labelled. nevertheless, it is probably safe to conclude that the behaviour of the e l protein is largely responsible for determining the intracellular budding site of coronavirus. the localisation of vplo might argue for a similar function in rotavirus morphogenesis. however, this protein's precise role in viral infection remains enigmatic. the other rotavirus glycoprotein, vp , also appears to be restricted to the endoplasmic reticulum when it is expressed in the absence of other viral proteins [ . thus both proteins may be involved in determining the viral assembly site, but the subsequent fate of vplo is unclear, since it is not detectable in purified virions [ ] . perhaps the simplest hypothesis is that the newly budded virus has a lipid envelope including vplo and vp , which is somehow shed at a later stage to leave vp connected directly to components of the capsid [ ] . an aspect of the biogenesis of the endoplasmic reticulum and golgi complex which has recently attracted attention is their fate during cell division. in mammalian cells, both organelles are thought to break up into vesicles which then partition randomly between the daughter cells and fuse together; concomitant with this disruption is a cessation of traffic of secretory and plasma mebmrane proteins to and through the golgi complex [reviewed in . the same phenomena appear to occur in the xenopus oocyte, which has the experimental advantage that its cell cycle state may be manipulated with hormones [mi. the results presented in figure are entirely consistent with this model; vplo is synthesized and processed normally in meiotic oocytes, but e l is processed to a far lesser extent than in interphase oocytes, and thus has probably failed to traverse what is assumed to be the first vesicle-mediated step of membrane traffic, from the endoplasmic reticulum to the golgi complex. it might be of interest to study the vesicles in which e l becomes trapped under these conditions. what features of each protein might be involved in determining its intracellular destination, and what are the mechanisms involved in each case? the two proteins share a superficial topological similarity, in having a very short domain on the lumenal side of the membrane which is n-terminal but does not arise from cleavage of a signal sequence; e has the additional unusual feature of hydrophobic regions large enough to span the membrane three times [ , , , , ] . two mechanisms have been proposed for retention of proteins in the endoplasmic reticulum. one is that a species variously known as bip or grp attaches to improperly folded or assembled molecules and prevents their exit to the golgi complex [ - . the second is that all of the membrane proteins of the endoplasmic reticulum form a continuous interacting network and are unable to diffuse in the lipid bilayer , . in respect of either model, we have never observed any host species which coprecipitates from oocytes with vplo, although admittedly our labelling schedule is perhaps not optimal for detection. (note that the unidentified band in figure , lanes b-d is made only in response to vplo mrna.) neither have we observed such species with our golgi model protein, e l , whose sorting mechanism is even more obscure. however, with the availability of cloned cdna and a reliable expression system, it is now possible to dissect each molecule and identify the characteristics involved in its correct targeting within the cell. molecular cloning: a laboratory manual molecular biology and pathogenesis of coronaviruses this work was supported by grants from the cancer research campaign (to a. colman) and the wellcome trust (to a. colman and m. mccrae). key: cord- -b fgb wf authors: murakami, kazuya; mihara, katsuyosi; omura, tsuneo title: the transmembrane region of microsomal cytochrome p identified as the endoplasmic reticulum retention signal( ) date: - - journal: j biochem doi: . /oxfordjournals.jbchem.a sha: doc_id: cord_uid: b fgb wf microsomal-type cytochrome p s are integral membrane proteins bound to the membrane through their n-terminal transmembrane hydrophobic segment, the signal anchor sequence. to elucidate the determinants that enable the p s to be located in the er, we constructed cdnas encoding chimeric proteins in which a secretory form of carboxyesterase, carboxyesterase sec, was connected to the n-terminus of the full-length or truncated forms of a microsomal-type p , p o(m ), and the constructed plasmids were expressed in cos cells. since carboxyesterase sec is an iv-glycosylated secretory protein, endo h treatment could be used to determine whether these chimeric proteins were located in the er or not. carboxyesterase sec with the n-terminal amino acids, containing the transmembrane region, of p o(m ), was located in the er, as determined from the endo h sensitivity of the expressed protein and immunofluorescence staining of the cells. as the expressed protein exhibited carboxyesterase activity, it was not retained in the er through the bip-dependent quality control system recognizing unfolded proteins. another chimeric protein construct in which carboxyesterase sec was connected to the c-terminal region of rat udp-glucuronosyltransferase (udp-gt), that contained a double-lysin er retention motif, was also located in the er, as determined from the endo h sensitivity and immunofluorescence staining. on the other hand, the sugar moiety of the carboxyesterase sec connected to the transmembrane segment of udp-gt, sec/gtd, was partially resistant to the endo h treatment. from the results of immunofluorescent staining and cell fractiona-tion, it was concluded that the sec/gtd product was located in the golgi apparatus. these observations indicated that the n-terminal hydrophobic segment of p o(m ) is sufficient for the er membrane retention, whereas the transmembrane segment of udp-gt is not. to determine whether microsomal p s are recycled between the er and golgi compartments or not, a dna construct encoding cathepsin d connected to the n-terminus of p o(m ) was prepared and expressed in cos cells. the fusion protein was phosphorylated, but the phosphorylation was sensitive to alkaline phosphatase. as a control, authentic cathepsin d was subjected to phosphorylation of its oligosaccharide chain that was resistant to the alkaline phosphatase treatment. since glcnac-p-transferase, which forms the alkaline phosphatase-resistant phosphodiester in the sugar chains of lysosome-targeting proteins, is located in the golgi apparatus, it was concluded that the oligosaccharide chain of the cathepsin d portion of the fusion protein was not phosphorylated, and that the chimeric protein did not go to the golgi apparatus. these results indicate that p o(m ) is not recycled from the golgi compartments to the er in cells. membrane proteins of the er depends on the presence of the c-terminal kdel sequence. these proteins exit the er but they are captured by the kdel receptors in the golgi apparatus or in the er-golgi intermediate compartment ( , ) . much less is known, however, about the retention mechanism for the other er membrane proteins. the er residency of a few membrane proteins with the type i orientation, such as udp-glucronosyltransferase, is guaranteed by the presence of the c-terminal kkxx or kxkxx motif, the double lysine motif, in their short cytoplasmic tail ( ) . they exit the er to be retrieved from the subsequent compartments probably through a receptormediated retrieval process ( ) . however, a number of type i er membrane proteins have no double-lysine motif in their c-terminus. the retention mechanism for these proteins and the proteins with the type h orientation or with multiple transmembrane segments remain largely unknown. to-study the retention mechanism for the er membrane proteins without the kdel or double-lysine motif, we chose microsomal cytochrome p s (p s). they are integral membrane proteins localized in the er, and carry the type i signal-anchor sequence at their n-terminus, which functions both as a classical signal sequence and as a stop-transfer sequence ( ) ( ) ( ) . the concerted action of this signal-anchor sequence and the signal recognition particle targets p s to the er, and the p s are inserted into the membranes in the type i (nin-ccyt) orientation { ). although the insertion of p s into the er has been studied extensively, the mechanism by which p s become er residents is not yet clear. although p s assume the type i orientation in the er membrane, no double-lysine motif is present in their c-terminus. to understand the retention of p s in the er membrane, in this study we defined the region of the p o(m ) molecule that determines p s' er residency. we constructed chimeric proteins, in which the whole or portions of the p o(m ) molecule were attached to the c-terminus of carboxyesterase sec, a secretory protein used as the reporter. as a control, other types of fusion proteins were constructed in which the c-terminal portion of udpglucronosyltransferase, which contains the double-lysine motif for the er retention, was attached to carboxyesterase sec. the fusion proteins were expressed in cos cells, and the subcellular localization of the fusion proteins was determined. to determine whether p s are retained permanently in the er or recycled between the er and post-er compartments through the "export and retrieval" process, we chose a lysosomal enzyme, cathepsin d, as a reporter, which was fused to the n-terminus of p at the dna level and the fusion protein was expressed in cos cells. the results with the fusion protein suggested that the transmembrane segment of p o(m ) was sufficient for the er retention, which diverted p s from the export and recycling pathways to be retained in the er. plasmids-the cos cell expression vectors were based on plasmid psg , which was kindly provided by dr. y. ikehara of fukuoka university. this plasmid contains the sv origin and sv early promoter. a cdna fragment carrying the entire coding sequence of carboxyesterase sec was excised from a clone ( - / - ) ( ) by ecori-huuhh digestion. the fragment was blunt-ended and ligated with .ecori linkers, and then inserted into the ecori site of psg to obtain sec. to construct sec/ml, the cdna fragment of p o(m ) was excised by bamhi digestion from pd ml ( ) , and then the fragment was inserted into the bctl site of sec. to construct sec/mi-pro, sec/ ml-tm, sec/ml-h, and sec/ml-q, the fragments encoding p o(m ) amino acids - , - , - , and - were amplified from pml ( ) by the polymerase chain reaction (pcr) using a common ' pcr primer, '-gactctaga-ggatccagtcctagt- ' (a bamhi site is underlined), and ' pcr primers, ' -cctctcccataatgatcaaac-aacctt- ' for the - fragment, '-tcactctggtaa-tgatcatttgggaga- ' for the - fragment, '-ctc-actctctaatgatcacttctcctc- ' for the - fragment, and ^ctcactctctaatgatcactctcctct- ' for the - fragment (bell site is underlined). each resulting fragment was digested with bamhi and bell, and then inserted into the bell site of sec. to construct sec/gt and sec/gtd, the fragments containing udp-glucronosyltransferase amino acids - and - were amplified from pygt (a gift from dr. yuasa of rakuno gakuen university), which contains the cdna sequence encoding the c-terminal amino acids of rat udp-gt, using a ' pcr primer for both fragments, '-acctcccctgggatccgtaccact- ', and a ' pcr primer, '-cattgtcatg^atccctactcattctt- ' for the - fragment, and '-cttttgcttttagga-tccgaatcggta- ' for the - fragment (bamhi site is underlined). each resulting fragment was digested with bamhi and then inserted into the bell site of sec. to construct ml and cathd, p o(m ) and cathepsin d cdna fragments were excised by .ecori digestion from pd ml and pcat- , and then inserted into the ecori site of psg . to construct cathd/ml, the bamhi site of pcat- was shifted from bp position to by in vitro site-directed mutagenesis. a cdna fragment coding the n-terminal amino acids of cathepsin d was excised from the mutated pcat- by digestion with ecori and bamhi. the fragment was then ligated into psg digested with ecori and bamhi. the resultant plasmid was digested with bamhi, and then ligated with the dna fragment containing p o(m ) that had been obtained from pd ml by bamhi digestion. antibodies-rabbit anti-carboxyesterase (anti-est) antibodies were prepared as described previously with purified rat liver microsomal carboxyesterase el as the antigen ( ) . anti-est antibodies recognize carboxyesterase sec as well as other microsomal-type carboxyesterases, including carboxyesterase el. rabbit anti-p (ml) antibodies were previously described ( ) . rabbit anti-cathepsin d antibodies were a gift from dr. himeno of kyushu university. transfections, labeling, and immunoprecipitation-cos- cells were seeded on the day before the transfection at a density of . x * cells per cm culture dish in dulbecco's modified eagle medium (gibco, gent, belgium), which contained % fetal calf serum. the dish was incubated at "c under an atmosphere of constant humidity ( %) comprising % co in air. the cells were approximately % confluent after h. the plasmid dna ( pig) in . ml of phosphate-buffered saline (pbs) was mixed with . ml of a cationic liposome solution in pbs ( mg/ml) prepared with , 'didodecyl-iv-[p-( -trimethylarnmoniumethoxyl)benzoyl] -lglutamate bromide ( ) . the dna-liposome complex, which was formed by mixing, was added to the cell culture. the cells were incubated at *c for h, and then ml of dmem supplemented with % fetal calf serum was added. forty hours later, the cells were washed with a methionine-free medium and then incubated in the same medium for h. [ s]methionine ( ^ci/ml) was then added and the cells were incubated for h. for pulse and chase studies, the cells were labeled as above, and then washed with fresh culture medium and further incubated in fresh medium containing mm non-radioactive methionine for h. for s p labeling, the cells were pre-incubated for h in a phosphate-free medium, and then labeled in the same medium with mci po per dish for h. for immunoprecipitation, the culture medium was separated from the cells by decantation. the cells were washed with pbs, and then scraped off with a rubber policeman into ml of pbs. a . volume of % sds was added to the culture medium, and the mixture was heated at 'c for min. it was then diluted with volumes of dilution buffer [ . % triton x- , mm nacl, mm tris-hcl (ph . ), and mm edta}. the cells were homogenized with ml of pbs containing % sds by strokes through a syringe needle, and then diluted with volumes of the dilution buffer. equivalent amounts of the culture medium and cell extracts were incubated with the appropriate antibodies at 'c for h. the immunocomplexes were recovered using protein a-sepharose as described previously ( ) . cell fractionation and alkali extraction of the membrane fraction-cells were washed with pbs and harvested in ice-cold ste buffer [ . m sucrose, mm tris-hcl buffer (ph . ), mm edta, and //g/ml each of leupeptin and pepstatin]. the suspension was centrifuged at x g for min and the pellet was suspended in a hypotonic buffer [ . m sucrose, mm tris-hcl buffer (ph . ), mm edta, and //g/ml each of leupeptin and pepstatin]. the suspension was kept on ice for min and then centrifuged. the precipitated cells were suspended in ml of ste buffer and then homogenized with a dounce homogenizer. the homogenate was centrifuged at x g for min to precipitate the nuclei and undisrupted cells. the supernatant (crude membrane fraction) was layered on a discontinuous sucrose gradient consisting of ml of m sucrose, . ml of . m sucrose, . ml of m sucrose, and . ml of . m sucrose ( ) , and then centrifuged for h at , rpm in an rps- t rotor (hitachi). fractions were collected from the top of the tube. for alkali extraction, the crude membrane fraction was treated with mm na co on ice for min ( ) . western blotting-sds-page was carried out using % polyacrylamide gels containing . % sds. the gels were blotted onto nitrocellulose niters as described by towbin et al. ( ) , and the filters were incubated in pbs containing % skim milk powder and . % tween- , and then treated with appropriate antibodies for h. the niters were washed with pbs and then treated with horseradish peroxida e-conjugated goat anti-rabbit igg antibodies in pbs for h. after washing times for min each, the immunoreacted bands were visualized with an ecl western blotting analysis system (amersham, u.k.). enzyme digestion-for endoglycosidase h and iv-glycosidase f digestions, protein samples were precipitated with volumes of ice-cold acetone. after centrifugation, the pellets were dissolved in mm sodium citrate buffer (ph . ) containing % sds, and then the solutions were heated at 'c for min. nine volumes of mm sodium citrate buffer (ph . ) containing % triton x- was then added to each sample. endo h ( . mu/ml) (boehringer mannheim gmbh, germany) was added to the samples, which were then incubated at 'c for h. iv-glycosidase f (boehringer mannheim gmbh) digestion was performed as described for endo h digestion except for the following modifications; sodium phosphate buffer (ph . ) was used instead of sodium citrate buffer (ph . ), and the enzyme concentration was mu/ml. for alkaline phosphatase digestion, the protein samples were heated at 'c in . % sds for min and then incubated with calf intestinal phosphatase ( u/ml) (boehringer mannheim gmbh) at 'c for . h. immunofluorescence microscopy-immunofluorescence microscopy was performed by the method described by munro and pelham ( ) . five micrograms each of the plasmid psg harboring various cdna constructs was transfected into cos cells in a . cm dish. after incubation for h, the cells were fixed with % paraformaldehyde and . % glutaraldehyde in pbs for min, and then permeabilized with % triton x- for min. the fixed cells were treated with appropriate antibodies, and then fitc-conjugated goat anti-rabbit igg antibodies (cappel england) in pbs containing % bovine serum albumin. in vitro translation-in vitro transcription and translation were performed as described ( ) . messenger rnas transcribed with t polymerase from the psg plasmids harboring various cdna constructs were translated in a wheat germ cell-free system in the presence of dog pancreas rough micro omes for min at 'c. analytical methods-esterase activity was measured spectrophotometrically using p-nitrophenyl acetate in mm tris-hcl buffer (ph . ) at nm according to krisch ( ) . nadph-cytochrome c reductase activity was measured as described ( ) . a-mannosidase ii activity was measured as described ( ) . for the retention to er-to identify the regions that are responsible for the retention of p (ml) in the er, we used carboxyesterase sec as a reporter protein. carboxyesterase sec, an isozyme of liver microsomal carboxyesterases, contains no kdel-type sequence at the c-terminus and is secreted into the blood as a glycoprotein with complex type sugar chains. thus, the type of the carbohydrate modification occurring during the transit through the secretory pathway is a good diagnostic clue as to the localization of the enzyme. precarboxyesterase sec was fused at the cdna level to the amino terminus of intact p o(m ) or modified p o(m ), with various deletions from the c-terminal region, into the transmembrane segment ( fig. ) . the chimeric cdnas were transfected into cos cells. forty hours later, the cells were labeled with [ s]meth-ionine for h and then incubated in the chase medium containing non-radioactive methionine for a further h. the labeled proteins in the cells and medium were immunoprecipitated with anti-est antibodies, which cross-reacted with carboxyesterase sec, and then analyzed by sds-page. when sec dna was transfected into cos cells, a single immunoprecipitable product was detected in the cells after h pulse-labeling ( fig. a, lane ) , and a significant amount of another sec product with lower mobility appeared in the medium ( fig. a, lane ) . after a h chase, the amount of the sec product in the cells decreased and that in the medium increased significantly ( fig. a, lanes and ) . the sec product in the cells was sensitive to endoglycosidase h treatment ( fig. a , lanes and ) and migrated with slightly lower mobility in the gel than the unglycosylated sec product synthesized in the presence of tunicamycin ( fig. a, lanes , , , and ). the slight difference in the mobility between these products was probably due to the fact that endo h cleaved the glcnac-glcnac bond of the high mannose type sugar chains leaving glcnac-asn in the substrate molecule. on the other hand, the sec product in the medium was more resistant to endo h digestion than that in the cells, although its resistancy was not complete ( fig. a, lanes and ) . the partial resistancy of the sugar chains of the secreted sec product to the endo h treatment was considered to be evidence for its transit through the golgi apparatus, where the complex type sugar modifications occur. taking advantage of this difference in the endo h sensitivity of the reporter protein, we examined the subcellular localization of the expression products of sec/ml, sec/ mi-pro, and sec/ml-tm cdnas in cos cells. when the cdna coding for the carboxyesterase sec-entire p o(m ) fusion protein (sec/ml) was transfected into cos cells and pulse-labeled with [ s]methionine for h, the radiolabeled product was detected in the cells (fig. b, lane ) , but not in the medium (fig. b, lane ) . the product remained stably in the cells even after a h chase (fig. b, lane ) and was not secreted into the medium (fig. b, lane ) . the chimeric protein in the cells remained in an entirely endo h-sensitive form during the pulse and chase periods (fig. b, lanes and ) . these results indicated that the p o(m ) molecule, when fused to the c-terminus of the reporter protein, exhibited the ability of retaining the reporter protein in the er. to identify the region that is responsible for the er retention of the reporter protein, we constructed cdnas in which the c-terminal cytoplasmic portion of p o(m ) in the fusion protein was deleted, leaving either the transmembrane domain or both the transmembrane and proline-rich (pr) domains, sec/ml-tm (fig. d) and sec/ml-pro (fig. c ), respectively. the transfection products of these chimeric cdnas were detected only in the cells, i.e., not in the medium (fig. , c and d, lanes , , , and ). both the products detected in the cells were sensitive to endo h digestion throughout the pulse and chase periods (fig. , c and d, lanes and ). these results indicated that the transmembrane segment of p o(m ) was sufficient for retention of the chimeric proteins in the er. the c-terminal flanking region of the transmembrane segment of microsomal p s contains clustered multiple basic amino acid residues followed by proline and glycine clusters, the pr region. the present a. however, there remained a possibility that the oligosaccharide chains of the reporter protein portion of the chimeric proteins had lost the potential to undergo the complex type sugar modifications. to rule out this possibility we performed brefeldin a (bfa) treatment of the transfected cells. bfa shuts off the anterograde membrane traffic of the golgi complex while enhancing the retrograde pathway to the er and, as a result, causes disassembly of the golgi apparatus and its mixing with the er. therefore, the high mannose type glycoproteins retained in the er are rapidly processed by the cis/medial golgi enzymes into complex type glycoproteins. as shown in fig. , the transfection products of sec/ml, sec/mi-pro, and sec/ml-tm cdnas gained partial resistancy to the endo h treatment when bfa was present during the pulse period (fig. , lanes , , and ), excluding the above possibility. to define the retention signal in the transmembrane segment of p o(m ) further, we constructed sec/ml-h and sec/ml-q cdnas (see fig. ), in which either the n-terminal one-half or one-quarter of the transmembrane segment of p o(m ) was fused to the c-terminus of carboxyesterase sec. the transfection products of these cdnas were detected only in the cells, i.e., not in the medium, after the pulse or the pulse and chase periods (fig. , e and f, lanes , , , and ) . the products in the cells were sensitive to endo h digestion (fig. , e and f, lanes and ), again indicating that both chimeric proteins are retained in the er. it should be noted that the intracellular stability of sec/ml -q seemed to be lower than that of other sec constructs, since the recovery of the immunoprecipitation products decreased significantly after a h chase (compare lanes and in fig. f) . assessment of the correct folding of the sec portion of the chimeric proteins, as monitored as carboxyesterase activity-it is well known that correct folding and assembly are necessary for the efficient export of proteins from the er. unfolded or malfolded proteins tend to bind to the chaperones in the er or to form aggregates and thereby are retained in the er to be degraded. it is, therefore, necessary to know if the carboxyesterase sec fusion proteins expressed in the cos cells described above attained the correct conformations or not. for this purpose, we assayed the carboxyesterase activity of the reporter molecule, carboxyesterase sec, and the results are shown in table i . the basal activity of the carboxyesterase of the cos cells was about equal to the activity of the expressed sec fusion proteins. the expressed products of the cdnas of sec/ml, sec/mi-pro, and sec/ml-tm exhibited significant carboxyesterase activities. in contrast, however, no activity was detected for the sec/ml-h or sec/ml-q protein. these results indicate that the sec/ml, sec/ml-pro, and sec/ml-tm proteins had attained the correct conformations in the cos cells, whereas the sec/ml-h and sec/ ml-q proteins were in malfolded states and therefore retained in the er, probably through interaction with the chaperones in the er or formation of aggregates. we wanted to know whether the sec/ml-h and sec/ml-q proteins were firmly anchored to the er membranes or translocated the membranes to bind loosely to the luminal surface, since the sec/ml-h and sec/ml-q proteins carried the shorter truncated transmembrane segments of p o(m ). the whole cells expressing the sec, sec/ml, sec/ml-h, and sec/ml-q proteins were treated with mm sodium carbonate (ph . ), which extracts both peripheral membrane proteins and luminal proteins, and the alkali-soluble and -insoluble fractions were examined by western blotting with anti-est antibodies. as shown in fig. , the original sec protein was efficiently recovered in the alkali-soluble fraction (lane ), whereas other fusion proteins were mainly recovered in the alkali-insoluble membrane fractions (fig. , lanes , , and ) . these results indicated that the sec/ml-h and sec/ml-q proteins were both stably anchored to the er membrane, even though their c-terminal hydrophobic domains seemed to be too short for stable anchoring to the membrane. 'double lysine motif-dependent retention of a carboxyesterase sec-gt fusion protein-the above described data strongly suggested that the transmembrane segment of p o(m ) was sufficient to retain the reporter protein as well as p o(m ) itself in the er. however, there remains the possibility that the carboxyesterase sec portion of the fusion proteins, when expressed as the membrane bound form, somehow inhibits the exit of the proteins from the er. to exclude this possibility, we constructed cdnas in which the p -derived transmembrane domain of the fusion protein, sec/ml-tm, was replaced either with the transmembrane domain and the cytoplasmic tail of udpglucronosyltransferase (sec/gt in fig. ) or with the same region from which the c-terminal "double lysine motif," the er retention signal, had been deleted (sec/gtd in fig. ). cos cells were transfected with these cdnas, and then pulse and chase experiments were performed. as shown in fig. g , the sec/gt protein was detected exclusively in the cells (fig. g, lanes , , , and ) and the carbohydrate moiety of the pulse-labeled chimeric protein was sensitive to endo h digestion (fig. g, lane ) , and the sensitivity was not altered even after the chase (fig. g, lane ) , indicating that the double-lysine motif functioned to retain the sec/gt protein in the er. on the other hand, transfection with sec/gtd cdna gave heterogeneous products shortly after the pulse-labeling or after the chase (fig. h, lanes and ) , whose mobilities in the gel were obviously lower than that of the in vitro synthesized sec/gtd protein (fig. h, lanes and ) . of particular significance was the finding that the carbohydrate chains of the sec/gtd protein gained partial resistance to endo h digestion (fig. h , lanes and ), clearly indicating that the fusion protein was exported from the er and its carbohydrate moiety underwent the complex type sugar modifications during the transit through the golgi apparatus. these results indicate that the sec portion of the fusion proteins did not prevent the proteins from being transported out of the er. to determine the subcellular localization of the sec/gt and sec/gtd proteins in the transfected cells, we fractionated the cell lysate by sucrose density gradient centrifugation, followed by immunochemical detection of the fusion proteins by western blotting (fig. , b and c) . each of the fractions was also assayed for nadph-cytochrome c reductase and a -mannosidase ii activities to locate the er and golgi membranes, respectively (fig. a) . the sec/gt protein was detected in fractions and , which was coincident with nadph-cytochrome c reductase activity, but not with that of a -mannosidase ii (fig. b) . in marked contrast, sec/gtd exhibited a diffuse distribution, but was mainly detected in fractions , , and , where the golgi marker enzyme, a -mannosidase ii, was present, although a small amount was present in the er fractions (fig. c) . these results confirmed that the sec portion of the fusion proteins did not prevent the export of the proteins from the er, providing further support for the conclusion that the transmembrane segment of p (ml) was sufficient for the retention in the er. intracellular localization of carboxyesterase sec fusion proteins detected by immunofluorescence microscopy-in order to confirm the above biochemical observations morphologically, we next compared the intracellular localization of p o(m ) and various carboxyesterase sec fusion proteins by immunofluorescence microscopy. p o(m ) expressed in cos cells showed a tubular network extending throughout the cytoplasm, the typical er pattern (fig. a) . when sec cdna was expressed in cos cells, the intracellular sec protein also exhibited an er distribution (fig. b) , although it is a secretory protein. the exit of the sec protein from the er was probably rate-limiting due to the high expression of the protein ( ) . similar fluorescence patterns were observed with the cells expressing sec/ml, sec/mi-pro, sec/ml-tm, sec/ tm-h, sec/tm-q, and sec/ml-gt (fig. , c-h) . in contrast, the cells expressing sec/gtd showed intensely stained golgi-like juxtanuclear structures (fig. ) , supporting the above observations that the sugar chains of the sec/gtd protein were resistant to endo h digestion and the expressed sec/gtd protein cofractionated with a golgi marker enzyme on sucrose density gradient centrifugation of the cell lysates. expression and localization of cathepsin d fusion proteins-to determine whether or not p (ml) is recycled from the golgi compartments, we used cathepsin d as the reporter protein. the carbohydrate side chains of cathepsin d are subject to phosphorylation of the mannose residues at the th position, this modification targeting lysosomal enzymes to their destination ( , ) . the man-no e- -phosphate of the sugar chains is formed through two steps, which occur in separate subcellular compartments. as the first step, glcnac-p is linked to mannose to give a phosphodiester. the glcnac residue is subsequently removed by the second enzyme to form a mannose- -phosphate residue, whose phosphomonoester bond is sensitive to alkaline phosphatase. the first enzyme is located in the cis-golgi compartment, whereas the second step occurs in the medial golgi compartment ( ) ( ) ( ) ( ) . investigations on the formation of the mannose- -phosphate residue of the sugar chains of the fusion protein, p (ml)-cathepsin d, will show us whether the fusion protein goes to a golgi compartment or not. the fusion protein (cathd/ml in fig. ) was expressed in cos cells. to confirm the expression of cathepsin d, p o(m ) and the cathd/ml protein, the cells were labeled with [ s]methionine for h, and then the labeled proteins in the cells and the medium were analyzed by immunoprecipitation with appropriate antibodies and sds-page of the immunoprecipitates (fig. ) . since no detectable band of cathepsin d or p o(m ) was observed with the mock-transfected cells, the antibodies against rat cathepsin d and p o(m ) used in this experiment did not cross-react with the endogenous cathepsin or p of the cos cells (fig. , lanes - ) . two bands with close mobilities were observed with the intracellular products of the transfected cathd cdna (fig. , lane ) . a band with the same mobility as the lower band was also detected for the medium (fig. , lane ) . the lower band was probably the pro-form of cathepsin d, but the nature of the upper band was not clear. the secretion of the pro form of cathepsin d was possibly caused by the overexpression of the enzyme in the cos cells. the ml (fig. , lanes and ) and cathd/ml (fig. , lanes - ) products accumulated in the cells, but were not detected in the medium. when examined morphologically by means of immunofluorescence microscopy with the antibodies to cathepsin d and p (ml), the cells expressing the original cathepsin d showed intensely stained lysosomes. the golgi apparatus near the nucleus was also stained (fig. a) . on the other hand, the cells expressing cathd/ml showed a typical er pattern of staining with no detectable fluorescence in the golgi region (fig. b) . this confirms the er localization of the fusion protein. to determine whether or not the cathd/ml fusion protein was recycled from a post-er compartment, cells transfected with cathd, ml or cathd/ml were labeled with s po for h. the expressed proteins were immunoprecipitated with anti-mi for ml, and with anti-cathd for the cathd and cathd/ml products. the cathd, ml, and cathd/ml products were all phosphorylated (fig. , lanes , , and ) . to define the structures of the phosphorylated products, they were treated with alkaline phosphatase. mannose- -p-glcnac was resistant to the alkaline phosphatase treatment whereas mannose- -phosphate was sensitive ( , ) . the cathd product was partially resistant to the alkaline phosphatase treatment (fig. , lane ) . in contrast, the radioactive phosphate incorporated into the cathd/ml product was completely removed by the alkaline phosphatase treatment (fig- , lan e ). this result apparently indicated that the fusion protein did not undergo the glcnac-p modification of the mannose in the sugar chain: the fusion protein did not reach the cis-golgi compartment. however, there is a possibility that the conversion of the glcnac-p moiety of the fusion protein to m p was very rapid in the transfected cells, resulting in the undetectable amount of the glcnac-p intermediate. however, such a possibility seems to be very low since the expressed cathepsin d carried a detectable amount of the glcnac-p intermediate. incidentally, the radioactive phosphate incorporated into the ml product was also removed by the alkaline phosphatase treatment. it is known that some p s, including p o(m ), are phosphorylated by camp dependent protein kinase ( ) . therefore, the phosphate incorporation into the cathd/ml and ml products was probably due to the phosphorylation of the protein moiety by the protein kinase. the retention mechanism for the er resident membrane proteins is insufficiently understood. as a step to elucidate the retention mechanism, we examined microsomal p -(ml) to locate the region in the polypeptide chain responsible for its er retention, and found that the amino acidlong transmembrane signal-anchor region of the membrane-bound hemoprotein was sufficient for the retention. this conclusion was based on the expression of the fusion proteins consisting of carboxyesterase sec, a secretory protein used as a reporter, and various portions of p -(ml) in cos cells. the reporter portion of the expressed fusion proteins attained the correct conformation judging from the fact that the fusion proteins were enzymatically active. it was therefore concluded that the fusion proteins were retained in the er by the retention signal in the fusion proteins and not through the action of the quality control system for unfolded proteins in the er lumen. several highly conserved structural motifs have been noted in microsomal p s; the basic amino acid-rich region at the c-terminus of the signal-anchor sequence, the proline-rich region following the basic amino acid-rich region, the heme binding region containing a conserved cysteine residue, and the threonine-serine cluster in the middle portion of the molecule. however, these common structural motifs seem to have nothing to do with the er retention of the p s. when carboxyesterase sec was fused with the carboxyterminal portion of microsomal udp-glucuronosyltransferase (gt) consisting of a transmembrane segment and a short cytoplasmic tail, the fusion protein expressed in cos cells was retained in the er, indicating that the doublelysine motif in the cytoplasmic tail functioned as the er retention signal. in contrast, another fusion protein in which the double lysine motif was deleted, sec/gtd, was transported from the er and retained in the golgi apparatus. it is known that some er transmembrane proteins retained in the er by the double lysine motif are transported to the plasma membrane on deletion of the retention motif ( ) . the reason why the sec/gtd product was not transported to the plasma membrane is not clear. anyhow, these results indicate that the carboxyesterase sec portion of the fusion proteins did not prevent the proteins from being transported out of the er. it is well documented that the er luminal proteins are retained in the er through the "export and retrieval" mechanism ( , ) . a similar mechanism is probably operative in the er retention of type i integral membrane proteins with the c-terminal double lysine motif, although the detailed mechanism remains obscure in the latter cases. we addressed this point for microsomal p s using lysosomal cathepsin d as the reporter protein to monitor the er to golgi traffic in the cell. the carbohydrate moiety of cathepsin d underwent mannose- -phosphate modification in the golgi compartments, but the cathepsin d-p o(m ) fusion protein expressed in cos cells was retained in the er and showed no golgi-type modification of the sugar chains, providing evidence that the fusion protein, and probably p s too, is not recycled through the golgi compartments. these results seem to be consistent with the previous observation that p (pb) was not detected in the golgi membrane immunoelectronmicroscopically, even after maximal induction of the cytochrome by phenobarbital administration ( ) . recently, szczesna-skorupa and kemper ( ) reported that p c , which was localized in the er when expressed in cos cells, did not show any shift in intracellular distribution when the cells were incubated at low temperatures, at which some membrane bound er proteins have been shown to shift to the intermediate or cis-golgi apparatus. they also showed that a modified p c with an artificially added iv-glycosylation site at its n-terminus received a sugar chain which was sensitive to treatment with endo h or endo d, which indicated that the sugar chain had not been processed by the golgi-associated enzymes. their observations are consistent with our conclusion that microsomal p s are not recycled between the er and golgi compartments. thus, the er residency of p s seems to be attained through a unique transmembrane segment-mediated mechanism, indicating the functioning of the transmembrane segment as the er retention signal. there have been several reports that the retention of membrane proteins in the golgi compartments is dependent on the transmembrane segments ( ) ( ) ( ) ( ) ( ) and the charged residues flunking the segments ( ) ( ) ( ) . the signal-anchor region of p -(ml) has no conserved sequence, as compared with the corresponding proteins of all known microsomal p s. the n-terminal transmembrane region of microsomal p- and the transmembrane region of golgi membrane proteins comprise similar hydrophobic stretches, but the former retains the proteins in the er whereas the latter keeps the proteins in the golgi membrane. these two types of transmembrane segments must contain unique information for the retention of membrane proteins in different intracellular membranes, and do not show any homology to each other. the transmembrane region of microsomal p s may associate together in the membrane to form homo-oligomers or interact with other membrane proteins to assemble into a network of membrane proteins, which cannot enter the transport vesicles exiting from the er. the function of the transmembrane region in the er retention of p s has to be clarified in the future. after completion of this study, ann et al. ( ) reported that the amino-terminal amino acids of cytochrome p ( c ) are sufficient for its retention in the er. biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi the rate of bulk flow from the endoplasmic reticulum to the cell surface assembly of asparaginelinked oligosaccharides lysosomal enzymes and their receptors a c-terminal signal prevents secretion of luminal er proteins evidence that luminal er proteins are sorted from secreted proteins in a poat-er compartment identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum retrieval of transmembrane proteins to the endoplasmic reticulum signals for the incorporation and orientation of cytochrome p in the endoplasmic reticulum membrane positive charges at the nh, terminus convert the membrane-anchor signal peptide of cytochrome p- to a secretory signal peptide a short aminoterminal segment of microsomal cytochrome p- functions both as an insertion signal and as a stop-transfer sequence core glycosylation of cytochrome p- (arom) molecular cloning and nucleotide sequence of cdna of microsomal carboxyesterase el of rat liver expression of a rat liver microsomal cytochrome p- catalyzing testosterone or-hydroxylation in saccharomyces cerevisiae: vitamin d -hydroxylase and testosterone o--hydroxylase are distinct forms of cytochrome p- importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome p- s purification and characterization of three male-specific and one female-specific forms of cytochrome p- from rat liver microsomes mitochondrial targeting signal of rat liver monoamine oxidase b is located at its carboxy terminus an hsp -like protein in the er: identity with the kd glucose-regulated protein and immunoglobulin heavy chain binding protein posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications reaction of a microsomal esterase from hog-liver with diethyl p-nitrophenyl phosphate a new method for simultaneous purification of cytochrome ih and nadph-cytochrome c reductase from rat liver microsomes isolation of subcellular organelles a soluble secretory protein is first concentrated in the endoplasmic reticulum before transfer to the golgi apparatus subcellular location of two enzymes involved in the synthesis of phosphorylated recognition markers in lysosomal enzymes synthesis of phosphorylated recognition marker in lysosomal enzymes is located in the cis part of golgi apparatus evidence for extensive subcellular organization of asparagine-linked oligosaccharide processing and lysosomal enzyme phosphorylation subfractionation of rat liver golgi apparatus: separation of enzyme activities involved in the biosynthesis of the phosphomannosyl recognition marker in lysosomal enzymes phosphorylation of hepatic phenobarbital-inducible cytochrome p- recycling of proteins from the golgi compartment to the er in yeast control of protein exit from the endoplasmic reticulum iscytochrome p- transported from the endoplasmic reticulum to the golgi apparatus in rat hepatocytes? an n-tenninal glycosylation signal on cytochrome p is restricted to the endoplasmic reticulum in a luminal orientation the membrane spanning domain of y -l, -galflctosyltransferase specifies trans golgi localization a golgi retention signal in a membrane-spanning domain of coronavirus el protein golgi retention of a trans-golgi membrane protein, galactosyl-transferase, requires cysteine and histidine residues within the membrane-anchoring domain ) ysl, -galactosyltransferase: a short nh,-terminal fragment that includes the cytoplasmic and tranamembrane domain is sufficient for golgi retention the signal for golgi retention of bovine / , -galactosyltransferase is in the tranbmembrane domain sequences within and adjacent to the transmembrane segment of <*- , -sialyltran fera e specify golgi retention the signal anchor and stem regions of the /j-galactoaide <* , -sialyltranbferase may each act to localize the enzyme to the golgi apparatus the transmembrane and flanung sequences of / , -jv-acetylglucosaminyltransferase i specify medial-golgi localization the amino-terminal amino acids of cytochrome p c are sufficient for retention in the endoplasmic reticulum key: cord- -xivnsldf authors: tartakoff, alan m. title: the confined function model of the golgi complex: center for ordered processing of biosynthetic products of the rough endoplasmic reticulum date: - - journal: int rev cytol doi: . /s - ( ) - sha: doc_id: cord_uid: xivnsldf the organized and characteristic elements of the golgi complex (gc) are the stacked smooth-surfaced cisternae, which are found in the centrosphere of all eukaryotic cells. these cisternae, in conjunction with other associated smooth-surfaced membranes, are responsible for executing net unidirectional intracellular transport (ict) from the rough endoplasmic reticulum (rer) toward more distally located structures. this chapter focuses on the broad range of accessory activities that occur during transport, the family of “posttranslational modifications.” these events are, in all likelihood, not essential for the “primary” function of the gc yet they are crucial in allowing the cell to tailor its biosynthetic products for its own needs and the needs of the organism as a whole. in addition to modifying products of the rough endoplasmic reticulum, the gc may be involved in processing events because of its participation in other routes of vesicular traffic—for example, centripetal traffic from the cell surface. various nonequivalent criteria have been used to ascribe processing events to the gc-autoradiography, preparative or analytic subcellular fractionation, interruption by ict inhibitors, and delay in the impact of cycloheximide. the organized and characteristic elements of the golgi complex (gc)' are the stacked smooth-surfaced cisternae which are found in the centrosphere of all eukaryotic cells. these cisternae, in conjunction with other associated smoothsurfaced membranes, are responsible for executing net unidirectional intracellular transport (ict) from the rough endoplasmic reticulum (rer) toward more distally located structures, e.g., lysosomes and the cell surface ( , , , , , , ) . the present article is not centered on this apparently most primary of golgi functions, but rather on the broad range of accessory activities which occur during transport, the family of "posttranslational modifications. " t k s e i . the pharmacologic approach which makes use of monensin to interrupt ict. as has been discussed elsewhere ( ) ( ) ( ) ( ) , the carboxylic ionophore monensin causes partial equilibration of intra-and extracellular na+ and k + , rapid (within seconds) dilation of all golgi cisternae, and an impressive slowing of ict. the agent is highly selective, at least after brief treatment ( pj monensin, hour, °c): atp levels and intracellular ph are little changed, many biosynthetic events continue [protein synthesis, lipid addition to glycoproteins ( ) hyaluronic acid synthesis ( ) etc.], and the effects of the ionophore are reversible. * since in the presence of monensin newly synthesized proteins continue to exit from the rough endoplasmic reticulum (rer), and gain access to but do not traverse the gc, one may inquire whether given posttranslational modifications occur. if they do, they may be assigned to a relatively early subcompartment. if they do not, given appropriate control experiments, they may be assigned to relatively late sub compartment^.^ the first indications that the site of interruption of ict by monensin lies only part way across the gc came from study of the progress of n-linked oligosaccharide maturation of ig ( ) ; however, a recent use of monensin should be mentioned since it adds independent support for this idea ( ) . when bhk cells are infected with sindbis or semliki forest virus, viral budding is normally observed at the cell surface. if monensin is present, transport of the glycoproteins is interrupted in the gc ( , , ) and viral nucleocapsids are found adhering to and budding across the membranes of dilated vacuoles ("icbms") in the golgi region. these nucleocapsids are presum-*in the context of the later discussion, the study of hyaluronic acid biosynthesis is especially important since it strongly suggests that sugar nucleotide metabolism is little perturbed and that the effects of monensin on sugar incorporation (e.g., into glycoproteins) are directly related to intermption of ict. hyaluronic acid, since it lacks a protein core, would be free from such effects. the appropriate controls vary according to the event being studied. for example, in the case of ig carbohydrate maturation although monensin blocks i h]gal and [ h]fuc labeling of ig, the two isotopes continue to be incorporated into other acid-insoluble material ( ) . in the case of the inhibition of acquisition of endo h resistence by transfenin in vsv-infected hepatoma cells, an internal control is provided by the g protein, which does become endo h resistant ( ) . were posttranslation modifications due to enzymes with acid ph optima, inhibition by monensin might be due to alkalinization of the cistemal content resulting from h+-na+ (or k + ) exchange. however, all oligosaccharide processing enzymes studied have roughly neutral ph optima. furthermore, there are no indications that they are sensitive to relative n a + / k + levels. to date the investigators who use monensin unfortunately do not all conform to the rather short-term protocols first used. comparison of data is therefore often difficult. the extent to which ict is perturbed by monensin is thought to depend on the alterations of cytoplasmic ion levels which it provokes. the impact of the ionophore may therefore reflect the activity of the na-k-atpase of the cell under study. ably signaling the presence of and adhering to an overaccumulation of the viral glycoproteins. since other nearby golgi-derived elements are histochemically positive for thiamine pyrophosphatase (a histochemical marker of distal cisternae) but icbms are negative one can conclude that the viral glycoprotein has only partly traversed the golgi stack. (the author's interpretation of these data is in fact that the site of monensin arrest is within "medial" or "intermediary" cisternae since the icbms bear ricin binding sites.) both the nucleocapsid-laden icbms and the nucleocapsid-free vacuoles can be partially separated from each other ( ) (vide infra). the use of colchicine is more limited than that of monensin, since in a number of cell types ict proceeds in the presence of the drug. studies of the liver suggest that the site of arrest of ict by colchicine may be later than the monensin site ( , ). . the approach of analyric subcellular fractionation ( , a, , , , ) in which microsomal fractions or subfractions are spread, for example, on continuous density gradients. the goal of this method is to be all inclusive rather than to enrich for selected markers which might prove nonrepresentative of the whole. at present the cytologic origin(s) of such subfractions cannot be systematically identified. . the in virro reconsrirurion approach ( , ) in which an attempt is made to simulate selected steps of transport by mixing appropriate subcellular fractions or cell extracts. the read-out from such experiments may involve the monitoring of posttranslational modifications which are considered to be diagnostic of transport. this is a method which may provide proof of the adequacy of subfractionation procedures and lead toward elucidation of the mechanisms of transport. . the approach of cyrochernisrry ( , , , ) making use of antisera which recognize processing enzymes or lectin conjugates. this method cannot indicate at which site processing enzymes are active, but it should locate the enzymes in a cytologic context. the lectin conjugates localize products of sugar transferase activity, with the reservation that it may prove difficult to establish whether a given sugar residue is borne by an n-or -linked or possibly lipid carrier. the presence of lipoprotein particles within the golgi cisternae of the hepatocyte has major implications for liver golgi subfractionation. preparative golgi fractions can be obtained free from smooth er and plasma membrane because of the density perturbation which results from this buoyant content. for analytic isopycnic fractionation one would anticipate a dispersion of density in proportion to variability in the number of lipoprotein particles contained within golgi fragments produced by homogenization. moreover, the relative isopycnic density of different subregions of the gc would reflect the degree of maturation of the lipoproteins. if the lipid content-and as a result the density shift-increases monotonically during ict then the relative densities of golgi subfractions might correspond inversely to their position along the time axis of transport; however, there is no proof that such a monotonic relation applies. confined function model of the golgi complex . a possible means of localizing a golgi component relative to selected processing enzymes is to inquire whether or not it has undergone proteolytic cleavage, terminal sugar addition to n-linked oligosaccharides, initiation or completion of any o-linked oligosaccharides etc. the degree to which it has undergone posttranslational modifications should reflect its ontogeny and site of residence. . a kinetic ordering of processing events can be obtained by performing, for example, an amino acid labeling pulse-chase experiment and inquiring as a function of time, when the product in question undergoes one or another maturation step. distinct kinetics need not, of course, imply distinct compartmentalization. the resolution of this approach is limited by any dispersion of transport rates of the pulse-labeled molecules. the exploitation of these approaches has by no means been sufficiently extensive to allow assignment of the multitude of processing events to subregions of the gc. moreover, the present lack of detailed knowledge of the gc is such that it is the analysis of numerous processing events which may actually serve to enumerate the number of subcompartments which exist. judging from histochemical data ( , , , ) , at least three classes of cisternae can be distinguished: the most proximal (overstained by oso,), medial cisternae (reactive for nicotinamide adenine dinucleotide phosphatase at ph in certain cells) and the most distal (reactive for nucleoside diphosphatase = uridine diphosphatase = thiamine pyrophosphatase). in addition, the structurally somewhat removed tubules and cisternae of "gerl" are histochemically reactive for acid phosphatase. present studies employing lectins to stain the golgi cisternae of various cells have also distinguished two or three distinct staining regions along the proximal-to-distal axis ( , , , ) (table i) . the idea of subcompartmentalization of the gc is discussed here in the context of distinct varieties of posttranslational processing several of which are illustrated, in abbreviated form, in figs. - . to a certain extent, these events might all be found within a single cell; however, since the biosynthetic repertoire (e.g., of exported glycoconjugates) varies among cell types, there is every reason to anticipate significant enzymologic variability of the gc as well. in fact, it is clear that selected sugar transferases are highly enriched only in certain tissues. there also must exist a set of altogether constant golgi components-those which are responsible for its characteristic structure and endow it with the ability to conduct transport. no such components have yet been identified. a secondary task in this mapping of golgi functions is to position the processing paths one relative to the next. for example, is the compartment responsible for proteoglycan sulfation the same as that responsible for galactose addition to asparagine-linked oligosaccharides? the ideal systems to investigate to obtain such information are those in which a single polypeptide undergoes several numerous secretory and membrane proteins are selectively cleaved during their passage through the gc to the cell surface. the best studied examples are proalbumin, the spike glycoproteins of many enveloped virus, and the family of precursors of polypeptide hormones [pro-insulin, -glucagon, -parathyroid hormone, -gastrin, -enkephalin ( ), -opiomelanocortin ( ), vasopressin-neurophysin precursor ( ) ]. many of the data implicating the gc are in fact only indirect. the approaches used involved study of the effect of agents which block secretory protein exit from the rer (e.g., uncouplers) ( ), correlation between the kinetics of ict and cleavage, or, best of all, analysis of smooth microsomal or golgi-enriched subcellular fractions [e.g., of hepatocytes ( , ) or virally infected cells ( ) ]. in the case of sindbis and semliki forest virus it has been suggested that cleavage occurs at the moment of arrival at the cell surface since extracellular antisera directed against their glycoproteins can block cleavage and since precursor species are not detected by surface-labeling procedures ( , ). however, the cytologic site of cleavage may depend on both the virus and the host cell-for several paramyxovirus, influenza virus and certain rna tumor virus cleavage has been reported to occur intracellularly ( ) . for rous sarcoma virus the suggested site of cleavage ranges from the gc to the virion itself ( a). moreover, certain host cells do not cleave the viral glycoproteins and therefore may yield noninfectious virus. the covalent site of proteolysis is strikingly uniform ( , ) . in all the cases mentioned above, cleavage occurs adjacent to one to four basic amino acids and results in the release of lysine or arginine. such amino acid excision and the generation, for example, of insulin from proinsulin, have been ascribed to the concerted action of both trypsin-like and carboxypeptidase b-like activities ( ) , however, none of the claims of attribution of these activities to identifiable enzymes is unequivocally accepted. such protease activities have yet to be used as golgi markers. an outstanding example of differential cleavage is the case of proopiomelanocortin. the same precursor is cleaved (always adjacent to basic amino acids) to yield different products by cells of the anterior or intermediary lobe of the pituitary ( , ) . moreover, in other endocrine cells minor amounts of unorthodox or incomplete cleavage products may be detectable ( ) . the result is a variety of microheterogeneity reminiscent of the microheterogeneity of processed carbohydrate structures of glycoproteins. it has been suggested that the proteolysis of proinsulin to insulin may occur both within the gc and in secretion granules ( , ) . the experiments on which this idea is based involve study of isolated granule fractions derived from biosynthetically labeled tissue. these data suggest that protease(s) of the gc may be transported along with the secretory product. in the case of proopiomelanocortin processing, successive cleavage events may occur within different compartments-p-lipotropin and acth are generated during transport, while further conversion to p-endorphin occurs in granules ( a, ) . there are no indications that cleavage is a prerequisite for transport. in the case of proalbumin, proinsulin, and the precursors of viral proteins, altered polypeptides which resist cleavage (mutant species or the products of incorporation of amino acid analogs) have been shown to arrive at the cell surface ( , ) . amino acid labeling pulse-chase experiments show that monensin interrupts the cleavage of viral surface glycoproteins, proalbumin, and proopiomelanocortin. (table i) . therefore, one can ascribe cleavage to a late subcompartnient. this conclusion is consistent with the liver golgi subfractionation studies which show that proalbumin-albumin conversion occurs upon entry into ''postcisternal" golgi elements-those which carry the greatest load of lipoprotein ( , ) . experiments on the influence of colchicine on n-linked oligosaccharide maturation of secretory glycoproteins show that it interrupts ict at a very late site ( , ) . it is therefore not surprising that it causes an accumulation of postcisternal elements containing an excess of both albumin and proalbumin ( , ) . when taxol is used to stabilize microtubules cleavage to albumin proceeds, and discharge is much reduced ( ). in the case of glycoproteins which undergo proteolytic cleavage prior to arrival at the cell surface there are insufficient data to generalize with respect to the relative order of the events of carbohydrate maturation and proteolysis. nevertheless, pulse-chase studies of proopiomelanocortin biosynthesis employing monensin suggest that the acquisition of mature carbohydrate units precedes proteolysis ( ) . in this study the inhibition by monensin may reflect neutralization of intragranular ph since the granule protease has a ph optimum at ph ( ) . chondroitin sulfate, dermatan sulfate, heparin, and heraran sulfate all contain the same internal tetrasaccharide structure (xyl-gal-gal-glcua) linked to multiple serine residues of a protein core. the four corresponding monosaccharide transferases have been characterized and substantially purified, and a limited amount of information is available concerning the polypeptide recognized by the initiator xylosyl transferase ( ) . there are provisional indications that at least the xylosyl and first galactosyl transferases might be physically associated with each other-they are both recovered in immunoprecipitates prepared with monospecific anti-xylosyl transferase antisera ( i). the literature is equivocal with respect to the cytologic site(s) of addition of these internal sugar residues. attempts at subcellular fractionation of embryonic cartilage suggest that the four corresponding enzyme activities belong to both rough and smooth microsomal fractions; however, the enzyme assays employed are in several respects not ideal ( ). part of the fascination with proteoglycan biosynthesis comes from recognition of their immense size [beautifully visualized by electron microscopy ( )] relative to the dimensions of the machinery of ict. a monomer of cartilage proteoglycan, for example, consists of a core protein (- nm long when spread) which bears on the order of lateral sulfated chains (internal tetrasaccharide and repeating sulfated galnac, glcua disaccharide) each of which is about residues long. recent experiments making use of antisera directed against protein determinants of the proteoglycan monomer indicate that there is a substantial intracellular pool of core protein lacking the repeating disaccharides ( , ). in amino acid pulse-chase experiments, this pool, which is presumably in the rer, can be chased into mature proteoglycan with a half-time of - minutes, i.e., several noncovalent assembly steps result in the ultimate extracellular product: monomeric units of derivatized core protein firmly associated with the hydrophobic "link" glycoprotein and hyaluronic acid. a set of ingenious competition experiments has shown that hyaluronic acid-which is synthesized by the same cells which synthesize the monomer-is bound only after discharge ( ). although intracellular monomer can bind the link protein after solubilization, it is not known whether this event or the association with collagen ( , ) normally occurs prior to discharge. a word should be said about the biosynthesis of hyaluronic acid. it is at no time covalently bound to protein and cycloheximide does not interrupt its biosynthesis ( ). its repeating disaccharide (glcnac, glcua) can be assembled in vitro by incubating cell extracts with udp-activated substrates ( ) . growth is at the reducing end of the chain, which is transiently membrane bound, possibly via udp ( b). autoradiographic study of synovial cells proves that hyaluronic acid synthesis does indeed occur within the gc ( ). the demonstration that its synthesis is not influenced by doses of monensin which block sulfation of chondroitin sulfate by the same cells is consistant with its not having a protein core-sugar addition can occur in situ without ongoing ict ( ) . in order to assign its synthesis to a subcompartment it will be important to learn whether monensin blocks its secretion. the most complex of the proteoglycan biosynthetic pathways is that of heparin, which has been in large part elucidated with the help of mast cell tumors. following the internal tetrasaccharide addition and elongation of the repeating (glcnac, glcua) disaccharide units, five further enzymes intervene ( , ) : ( ) a deacylase which removes most acetyl groups from glcnac (this activity is low in cells synthesizing heparan sulfate and is stimulated by nsulfation at other residues), ( ) an n-sulfotransferase which acts on most of the sites exposed by ( i ) , ( ) the epimerase which converts - % of glucuronic acid residues of the polymer to iduronic acid, ( ) an -sulfotransferase which acts at c- of many iduronic acid residues, and ( ) an -sulfotransferase which acts at c- of glucosamine. there are no data indicating whether some or all of these activities are expressed in the same cytologic subcompartment in which disaccharide elongation occurs. recent studies have indicated the existence of two classes of heparan sulfate. one is released from the cell (e.g., hepatocyte); the other is hydrophobic and membrane-associated. it is thought to have a hydophobic core protein which serves as anchor ( ). study of the biosynthesis of chondroitin sulfate is potentially of unique interest for defining the subcompartmentalization of the gc since the same core protein which bears xylose-linked repeating disaccharides also bears - n-linked complex oligosaccharides, - short -linked galnac-containing oligosaccharides, - single galnac residues, and (in many cases) keratan sulfate chains ( , , ) . such multiple derivatization of a single polypeptide provides ideal material for establishing the relative sub-golgi sites and rates of processing of such divergent structures. the presently available kinetic data shown that the acquisition of endoglycosaminidase h (endo h) resistance by the n-linked oligosaccharides of chondroitin sulfate is roughly coincident with the addition of the repeating disaccharides ( ). study of the incorporation of labeled glucosamine into the repeating disaccharides and the internal residues of the short linked units suggest that these two events occur simultaneously ( ). other data bearing on the sub-golgi localization of proteoglycan synthesis come from use of monensin. both the addition of repeating disaccharides and sulfation of chondroitin sulfate are markedly reduced in the presence of monensin though the pools of atp and phosphoadenosine phosphosulfate are unchanged ( , , , ). one can provisionally conclude that these events take place relatively late during transit through the gc. the limited amount of chondroitin sulfate which is secreted in the presence of monensin is undersulfated and may serve as a model for certain achondroplasias. c. tailoring of asparagine-linked oligosaccharides glycoproteins ( , , - , , ) with the realization that preassembled dolichol-linked oligosaccharides are cotranslationally added to selected asparagine residues, the further processing of the common precursor at the top left can be trimmed and further processed toward complex or hybrid structures, structures characteristic of lysosomal enzymes ("unblocked unit"), or keratan sulfate. as is extensively discussed elsewhere ( , ) along the "complex pathway" several options and control points exist. the activities of glcnac transferases (ti-tiv) are of key importance. tiii, for example. generates a "bisected" (bis) structure by addition of glcnac to the most internal (plinked) mannose and as a result blocks the activity of tii, tiv. fucosyl transferase, and mannosidase . ti action is a prerequisite for action of several further transferases. the structure bearing glcnac-phosphate, which can give rise to an unblocked unit, indicates only one of several possible phosphorylated mannose residues. the actual degree of concomitant mannose trimming is progressive but not uniform. tr, terminal sugar (fucose, gal, sialyl) transferases; g , glucosidase; m, mannosidase. such moieties has been rapidly elucidated. the recent reviews of these investigations are too thorough to warrant an exposition of this now well-known sequence of steps which involves both sugar trimming and terminal sugar addition. the processing to a triantennary complex unit, e.g., of the g protein of vesicular stomatitis viruses (vsv) in infected cho cells, involves nine enzymes and of these only the first two glucosidases and possibly the first mannosidase act at the level of the rer ( , , ) . the rest have been localized to preparative golgi fractions. the p( - ) galactosyltransferase responsible for subterminal gal addition is, at present, the best characterized golgi enzyme. not only have its enzymological properties been investigated in several tissues, but in addition first studies of its trans-membrane orientation ( ) and biosynthesis ( ) have been reported. in hela cells the transferase bears both -linked and endo h-resistant n-linked oligosaccharides (g. strous, personal communication). several laboratories have monitored attempts at sucrose gradient golgi subfractionation with the use of this set of enzyme activities. the observations to date are fragmentary and the separations only partial. the first mannosidase has been reported to have an isopycnic density in sucrose gradients which is higher than that of galactosyltransferase [cho cells ( ) and liver (loo)]. such a difference is not observed in bhk cells even when they are infected with semliki forest virus and treated with monensin so as to increase the density of medial golgi elements ( ). in the case of cho cells the density differences are small but intriguing since the mannosidase activity is coisopycnic with those vesicles in which the g protein acquires covalently bound lipid (vide infra). attempts to separate subfractions differentially enriched in the terminal sugar transferases (glcnac, gal, sialyl) by sucrose gradient analysis have failed ( , ) or provided only slight differential enrichment ( a). both affinity methods and countercurrent distribution studies do discriminate between liver golgi subfractions differentially enriched in galactosyltransferase vs "er-like" enzyme activities ( , ). an outstanding only partly resolved question is what governs the particular path and degree of processing at a given glycosylation site. among the key considerations is the repertoire of processing activities of a given tissue, possible competition between transferases of overlapping specicificity ( , ) , and the steric environment of the oligosaccharides. in the case of thyroglobulin ( ), certain igm myeloma proteins ( i ) and certain viral envelope glycoproteins the situation is particularly intriguing since different sites along a single polypeptide ungergo radically different degrees of processing. in this last-mentioned case, it has been observed that when sindbis virus infects different host cells different sites are processed to different extents ( ). additional variables in terminal sugar addition concern ( ) the degree to which the transferases may be present at the cell surface as well as in the gc, and ( ) the apparent intracellular pool size of secretory glycoproteins which have already accepted their terminal sugars. both enzymologic ( ) and, less equivocally, immunocytochemical studies ( ) show that in selected cells galactosyltransferase may be exposed at the (apical) cell membrane. presumably this is a reflection of some structural change in the enzyme or other determinative golgi elements which abrogates the normal affinity of the enzyme for the gc. with respect to ( ), comparative studies of immunoglobulin synthesis by several mouse myelomas indicate that the intracellular pool of ig which can be labeled with terminal sugars may be either undetectably small or quite substantial ( , ) . these variations may parallel the degree of differentiation of the myeloma in question. a variable location of the terminal sugar transferases might explain such observations, i.e., in some cases they might act later along the path of ict than in others. several cytologic studies pertain to this pathway. an immunocytochemical study has localized p( - ) galactosyltransferase to the distal (thiamine pyrophosphatase-positive) cisternae of the gc of hela cells ( ). the presumed products of terminal sugar transferases have also been localized. concanavalin a-gold conjugates (which interact primarily with immature asparagine-linked oligosaccharides) stain the cisternal space of the rer and all golgi cisternae, whereas ricin (which interacts with nonreducing galactosyl residues) stains only the more distal cisternae of bhk cells ( , ) . in a methodologically quite different study of igm-secreting myeloma cells and an ig-negative mutant, concanavalin a-peroxidase has been shown to stain the cisternal space of both the rer and proximal cisternae while a wheat germ agglutinin-peroxidase conjugate (specific for clustered sialic acid) stains the cisternal space of more distal cisternae and associated vesicles ( ). any such studies must be interpreted in light of what is known of the principle glycoproteins and glycolipids of the cells studied. especially in the case of the igm myeloma (and to a lesser extent with the bhk cells when they are infected with semliki forest virus) one has reason to consider much of the staining to be due to a single glycoprotein. nevertheless, the study of the myeloma mutant suggests that the oligosaccharides of underlying golgi components distribute in roughly the same way as the ig oligosaccharides. taken as a whole, the cytologic observations argue strongly that the direction of transport is indeed proximal-todistal and that the terminal sugars are acquired during passage across the stack of golgi cisternae. these conclusions are also compatible with the available monensin data. in short-term experiments with igm-secreting plasma cells and myeloma cells, monensin interrupts ict within the gc at a site where the ig heavy chains are still sensitive to endo h and have acquired neither galactose, fucose, nor sialic acid ( , ) . terminal sugar addition to other glycoproteins continues and ig ( ) . similar results have been reported for the effect of monensin on maturation of the oligosaccharides of transfernin ( ) and of the surface glycoproteins of semliki forest virus ( ) . in other cells and employing somewhat different protocols the major intra-golgi site of accumulation of membrane proteins in the presence of monensin may be such as to have allowed somewhat further oligosaccharide maturation (table i) . nevertheless, transport to the cell surface is not observed. there is a line of experimentation indicating the existence of two sequential subcompartments traversed by the g protein of vsv. the ultimate interpretation of the data remains to be determined, but is thought to pertain to the ability of the membranes of golgi subfractions to undergo fusion with the membrane of a relatively late subcompartment. in the protocols employed ( , a, ) vsvinfected mutant cho cells (lacking glcnac transferase i) are pulse-labeled with [ s]methionine and chased for increasing intervals. at each time point a cell hornogenate is mixed with a wild-type homogenate, briefly incubated, and assayed for the proportion of s-labeled g (initially sensitive to endo h and perpetually so in the mutant cho cell) which has acquired resistance to endo h. the in virro maturation is manifest only after a brief chase interval, suggesting that at later times g has gained access to a subcompartment which will no longer participate in such membrane-membrane fusion. numerous yeast glycoproteins, for example, secretory invertase and acid phosphatase, bear both asparagine-linked and -linked polymannosyl oligosaccharides. the asparagine-linked units are donated from the same dolichol-linked intermediates as in mammalian cells, trimmed to a structure containing glcnac and man, and elongated by massive further addition of mannose and phosphate ( ) . studies of yeast temperature-sensitive ict mutants which overaccumulate rer, golgi-like cisternae, or vesicles suggest that the polymannosyl elongation of the n-linked oligosaccharides of invertase occurs within the gc ( ). the phosphorylation of the secretory protein also occurs within this compartment ( ). ( , , ) in cultured fibroblasts, the proper intracellular targeting of lysosomal enzymes depends on the presence of mannose -phosphate on high mannose asparaginelinked oligosaccharides. the phosphorylated structure is the result of the posttranslational addition of one or several glcnac- -phosphate units to several oligosaccharides per polypeptide, followed by removal of much of the glcnac. biosynthetic labeling studies show that the "unblocked" phosphate structure is achieved within hour after a [ h]mannose pulse and that phosphoryl groups are ultimately lost, presumably upon entry into lysosomes ( ) . the enzymes responsible both for addition of the blocked phosphate and subsequent removal of the glcnac are both enriched in preparative liver golgi fractions. in attempts at liver golgi subfractionation, the two activities have been reported to be coisopycnic with mannosidase i, i.e., denser than galactosyltransferase ( ) . sucrose gradient subfractionation of a mouse lymphoma and macrophage cell line has partially resolved the phosphotransferase, phosphodiesterase, and gal transferase ( a) . there is also a further suggestion that mannosidase i and the phosphotransferase are localized to the same golgi subcompartment-in h-man pulse-chase studies the degree of trimming to a man, structure correlates with the progressive addition of blocked phosphates ( ) . since lysosomal enzymes also bear complex oligosaccharides ( , , ) , they must pass via the terminal sugar transferases (i.e., anatomically distal cisternae), and indeed they can be visualized within all golgi cisternae by histochemical methods. this route of passage is acutely manifest in the i-cell syndrome in which glcnac- -phosphotransferase activity is missing and many lysosomal enzymes are released from cells. such molecules contain more sialic acid and are bound to insolubilized rich to a greater extent than in the wild type ( , a) . a somewhat analogous situation is also observed for a cho cell mutant in which lysosomal enzymes bear only complex oligosaccharides ( a). also consistent with this line of reasoning is a report that the mannose-phosphate receptor-which is thought to mediate the ict of newly synthesized lysosomal enzymes-bears sialic acid; however, the receptor molecules isolated may largely originate from the cell surface rather than from the gc ( ) . how does the cell determine whether or not to add the blocked phosphate to a given oligosaccharide? two points should be made. ( ) the signals have not been identified, but when the glcnac-phosphotransferase is presented with any of several high mannose secretory proteins or lysosomal enzymes, transfer is overwhelmingly more active toward the lysosomal enzymes ( , ) , and ( ) the studies on processing of lysosomal enzymes once again indicate the existence of microheterogeneity : the degree of addition of blocked phosphate, of unblocking, and of addition of terminal sugars is variable. in yeast, where lysosomal enzyme oligosaccharides are not essential for targeting to the lysosome ( , lysosomal carboxypeptidase y is phosphorylated while in the rer ( ) . its carbohydrate is processed in two stages while tranversing the gc-temperature-sensitive ict mutants blocked at the level of the gc accumulate one of two endoglucosamindiase h-sensitive species ( ) . data on the influence of monensin on lysosomal enzyme transport are suggestive. in i-cell fibroblasts monensin reduces hexosaminidase secretion ( b). in normal cells it may actually stimulate their release ( a). it is striking that chloroquine, which also raises intralysosomal ph, also causes release of newly synthesized lysosomal enzymes ( ) . this effect can be attributed to neutralization of lysosomal content by the ionophore and a consequent tying up of the mannose-phosphate receptor: however, the situation has not yet been fully analyzed. the secreted lysosomal enzymes are phosphorylated ( a). proteins ( , ) the presence of covalently bound lipid is not restricted to proteins which traverse the secretory pathway; however, a number of plasma membrane glycoproteins have been shown to acquire fatty acids posttranslationally. the best studied cases are the envelope glycoproteins of vesicular stomatitis virus, semliki forest virus, and sindbis virus, the proteolipid of myelin, the transfemn receptor, hla antigens, and t , a lymphocyte surface antigen. the covalent sites of fatty acid linkage are suspected to be cysteine or serine residues. the corresponding enzymes and activated fatty acid precursors have not been identified. four lines of experimentation suggest that an early golgi subcompartment is the site of palmitate addition in virally infected cells. ( ) pulse-chase studies employing labeled palmitic acid show that the half-time required by cycloheximide to shut off acylation of viral proteins is minutes. hence acylation is certainly not a cotranslational event ( i ) . ( ) within only to minutes after acylation these proteins become resistent to endo h (vsv) or undergo proteolytic cleavage (sindbis), whereas when amino acids are used to label the same glycoproteins, the half-time for acquisition of endo h resistance is - minutes ( ). ( ) subcellular fractionation of vsv-infected cho cells pulse-labeled with [ h]palmitate shows that acylated g is coisopycnic with mannosidase i activity ( ). in a closely related system, however, this isopycnic coincidence is eliminated when the density of "medial" golgi elements containing the acylated viral proteins is increased by infecting the cells with semliki forest virus and then treating with monensin ( ) . in this situation mannosidose i is unperturbed and remains coisopycnic with galactosyl transferase. ( ) the acylation of these glycoproteins is not blocked by monensin ( ). it is curious that in yeast ict mutants acylation has been assigned to the rer (m. schlesinger, personal communication). since the phosphorylation of yeast lysosomal enzymes also occurs in such rer-blocked mutants ( ), but has been localized to preparative liver golgi fractions ( ), one wonders whether the conventional anatomic boundary between rer and gc corresponds to the mutant phenotypes. in higher organisms a partial enzymologic overlap has been noticed between the rer membranes and the gc ( , , ) and electron microscopic lectin-binding studies ( , , ) suggest that it is the anatomically proximal golgi cisternae that resemble the rer. moreover, as already mentioned, monensin experiments have assigned both acylation and lysosomal enzyme phosphorylation to an early subcompartment. perhaps in the yeast ict mutants in question the accumulated unit includes what is conventionally considered to be the proximal face of the gc as well as the rer. an additional modification of asparagine-linked oligosaccharides occurs in the cornea where they may be processed to corneal keratan sulfate, a rather small ( kilodalton) proteoglycan characterized by a repeating sulfated (glcnac, gal) disaccharide. since keratan sulfate i biosynthesis can be blocked by tunicamycin ( ), these units appear to be the result of an alternate derivatization of the same trimmed oligosaccharide which can be processed to a sialic acid-containing complex oligosaccharide. indeed, the linkage region is identical to that of the complex sites-only three mannose residues remain and these bear glcnac and gal followed by the repeating disaccharide ( ). present data are largely in favor of the notion that the addition of the most internal galnac to serine or threonine takes place in the gc, long after polypeptide chain termination. the data derive from the distribution of galnac transferase activity in subcellular fractions of intestinal mucosa ( ), and oviduct ( ), from study of the impact of cycloheximide on galnac addition to mammary glycoproteins ( ), and from the comparative evaluation of the kinetics of galnac and glcnac addition to o-linked and n-linked units, respectively ( ). the result of glycosylation is most impressive for mucins-hundreds of oligosaccharides are added and the product may be greater than % carbohydrate ( ). the enzymes responsible for chain elongation have been studied in sufficient detail as to rationalize the set of structures observed. it has been reported that neuraminic acid of mucins can be acetylated and that acetyl groups can be replaced by glycolyl groups after their addition to mucin ( ). two approaches localize mucin-specific transferase activities to golgi subcompartments: ( ) an autoradiographic study of [ h]gal pulse-labeled mucus secreting cells which indicates that incorporation takes place over distal cisternae and associated vesicles ( ), and ( ) a cytochemical study of thin sections of goblet cells stained with lectin-gold conjugates to detect nonreducing terminal galnac ( ). proximal cisternae are stained and medial cisternae negative. only in blood group a + material (where terminal galnac is present) arc: the distal cisternae and secretion granules also stained. both studies are, therefore, consistent with the idea of progressive sugar addition during proximal-to-distal transport across the golgi stack. a single investigation indicates that the subcellular site of incorporation of the the best characterized surface glycoprotein of the human red blood cell, glycophorin, bears -linked tetrasaccharides and one n-linked oligosac-charide. the o-linked units, which begin with galnac, should serve as a model for step-by-step analysis of chain elongation, as should the o-linked oligosaccharides of the envelope glycoproteins of murine corona virus. the incomplete biosynthetic data suggest, paradoxically, that o-linked oligosaccharides are added when the glycophorin message is translated in the presence of dog pancrease microsomes ( ) . the only published data on the corona virus show that very long-term monensin treatment blocks the addition of o-linked sugars ( ) . a similar observation has been made in the study of the influence of monensin on the biosynthesis of the o-linked oligosaccharides of herpes virus glycoproteins ( a) . skeletal keratan sulfate (keratan sulfate ) is produced by chrondrocytes of mature cartilage and contains the same repeating disaccharide as the n-linked variety (keratan sulfate i). in this case, the saccharide is linked via galnac to serine or threonine residues of the core protein. by analogy with data discussed above, one would expect both chain initiation and elongation to occur in the gc. since the same protein core contains short mucin-like o-linked oligosaccharides and since the distribution of these units is not random there must be signals indicating which serine or threonine residues are to receive the repeating disaccharide chains ( , ) . the abundant and diverse classes of glycolipids have received little attention in the context of ict, although glycolipids are enriched in plasma membrane and golgi-rich fractions. nevertheless, a unique elegant biosynthetic study of cultured neuroblastoma cells shows that several gangliosides are transported from an intracellular site (presumably the gc) to the cell surface with a half-time of minutes ( ) . following the addition of the most internal glucose or galactose to ceramide, a broad range of further processing pathways may be available, according to the tissue in question. the sugar transferases involved are thought to be largely distinct from the transferases involved in glycoprotein synthesis. glycolipid sugar transferases responsible for synthesis of gm , gm , gml, and gd have been found enriched in preparative golgi fractions of rat liver, kidney, and bovine thyroid ( , , ) ; however, it is striking that the activity responsible for the conversion of gm to gd is not especially enriched in the thyroid fractions (fig. ) . recent [ h]gal pulse-chase studies of rat glioma cells show a precursor-product relation between glucosylceramide and more complex glycosphingolipids. this conversion is effectively blocked by monensin, suggesting that it involves transport from the early part of the gc (or rer) to a later region. in strict analogy with studies of secretion of [ h]gal-labeled lg ( ) studies of the distribution of sulfotransferases involved in sulfatide synthesis have also localized these enzymes to preparative golgi fractions of testes and kidney ( ) ( ) ( ) ) . a single study demonstrates transport of sulfatides from gc to the cell surface ( ) . since the methods for detailed analysis of glycolipids are now relatively streamlined and since the biological importance of glycolipids is increasingly apparent, further data on their ict should be forthcoming. in the hepatocyte secretory lipoprotein particles are visible within the smooth endoplasmic reticulum and become conspicuous within the gc, especially after ethanol intoxication ( ). the secreted particles contain noncovalently bound triglycerides, phospholipids, and free and esterified cholesterol. recent studies of isolated golgi fractions suggest that the phospholipids of the contained lipoproteins are in rapid equilibrium with the phospholipids of the membranes of golgi cisternae ( ) . it therefore appears likely that it is within the gc that they acquire most of their lipids. nevertheless, since lipoprotein particles recovered from rat and chicken liver golgi fractions do not have the same lipid composition as in serum, further intra-or extracellular maturation events must be involved ( , ). in murine macrophages the binding of apolipoprotein e to cholesterol occurs only after discharge from the cell ( ). secretory cells may be divided into two classes: the nonregulated class (e.g., plasma cells, fibroblasts) and the regulated class (e.g., pancreatic and pituitary cell types). only members of the regulated class store their secretory products in granules. this content is released by exocytosis upon physiologic stimulation. the genesis of such secretory granules and lysosomes takes place either within distal golgi cisternae or in vacuoles (e.g., "condensing vacuoles") in their immediate vicinity, according to cell type and physiologic conditions ( , ). such impressive condensation of content is in many cases thought to reflect the electrostatic interaction between secretory products, for example, between secretory proteins and proteoglycans ( , , ). presumably the site of visible condensation corresponds to the region of the gc within which the proteoglycan is synthesized (e.g., sulfated) or to a later region. as already mentioned, experiments employing monensin indicate that sulfation of chondroitin sulfate is a rather late golgi event. the proteoglycans of secretory granules have been reported to be heparin, heparan, dermatan, and chondroitin sulfate ( , , , ) . a fundamental question pertaining to the organization of the gc should also be considered. a wealth of data demonstrates that the terminal sugar transferases, for example, are restricted to a subregion of the gc. why do these proteins, synthesized in the rer, exit only as far as this portion of the gc and migrate, if at all, only much more slowly to the cell surface? several explanations might be invoked: ( ) the ionic content of the gc is unlike that the the rer and as a consequence a conformational change is induced in such membrane proteins which reduces their mobility-the possibility that the golgi content may have unique ionic properties has recently been discussed ( ), or ( ) the protein encounters, within the membrane of golgi cisternae, certain stable residents (proteins?) for which it has a high affinity, or ( ) a posttranslational covalent modification executed within the gc is responsible. to date, no experimental means have been identified which cause mislocalization of such golgi markers and no information is available to indicate whether one of the three proposed explanations might be correct. the point of departure for this article was the supposition that the primary function of the gc is to accomplish net unidirectional ict. it is presumably through evolution that the gc has acquired an impressive array of other responsibilities. these activities are the subject of the preceding pages and are summarized in table i . what is the impact of housing these processing events on the gc, and what are some of the dividends for the cell? . the gc has become such an exceedingly active center for sugar addition that the content of the cisternae must be a seething bath of activated sugar nucleotides. these species, to the extent that they have been investigated, gain access to the cisternal space by facilitated transport through specific sites ( , , , ) . this set of transporters may be considered as additional set of golgi markers. . the gc must deal with the side products of these reactions-the mono-and dinucleosides which are produced by the transferases-especially since udp inhibits galnac and gal transferase hyaluronate synthetase ( , , b). both the udp and gdp and to a lesser extent cmp are broken down within the cisternal space by nucleoside diphosphatase ( , ) . furthermore, the end-products of hydrolysis must exit from the cisternae in order to function in repetitive cycles. only a single report pertains to this second class of carriers ( ) . in the same vein, the ability of the gc to synthesize lactose (in the mammary gland) and the presence of both mannosidase and phosphodiesterase activities suggests that golgi membranes are impermeable to disaccharides yet permeable to monosaccharides. experimental observations on the mammary gland and myeloma cells suggest that this is the case ( , ). . the ability to assemble proteoglycans may be considered a prerequisite for the existence of the regulated secretory cell phenotype. . the addition of both neutral and negatively charged sugars to intrisic glycoproteins and glycolipids of the gc obviously modifies the environment in which other golgi activities occur. for example, the local membrane potential may be perturbed at sites where sialic acid has been added, and the presence of ceramide derivatives may result in membrane rigidification ( ) . the assembly of membrane-bound oligosaccharides clearly allows the cell to endow its surface with negative charge and an array of specific structural sites beyond those simply encoded in its polypeptides. . the generation of mannose phosphate groups, in many cells, is essential for targeting of lysosomal enzymes. such considerations indicate that the existence of posttranslational processing events is an integral part of the cells repertoire of biosynthetic equipment. given this realization, it would not be altogether suprising to learn that nucleoside diphosphatase, for example, had subsumed certain functions at the level or overall golgi structrue and organization. other proteins involved in more specialized processing events, e.g., select glycolipid sugar transferases, found only in the gc of certain cells, might not be expected to have acquired such dual responsibilities. other common features of processing deserve comment, for example, the microheterogeneity which is commonplace among glycoproteins and may also be characteristic of proteolytic cleavage. no experimental studies have attempted to manipulate the degree of microheterogeneity , e .g., by reducing the temperature so as to slow transport through the gc. nevertheless, in addition to the enzymologic considerations mentioned above (overlapping transferase specificities, etc.), one wonders whether dispersion in the rates of entry and exit and duration of residence within a given subcompartment may not be responsible. in this sense, the length of an oligosaccharide chain, e.g., of chondroitin sulfate, might be considered an indirect measure of the kinetics of transport. for the moment, knowledge of the sequences of posttranslational processing events can explain the final structures of many macromolecules which pass through the gc. this article has summarized the incomplete data now available which points to subcompartmentalization of these events. it is to be anticipated that as the assignment of specific steps to subcompartments is made with greater certainty, this information will lead to an increased appreciation and understanding of the dynamics of membrane-membrane interactions which underly the operation of the organelle as a whole. plenum, transport of macromolecules in cellular systems cell surface carbohydrate chemistry i n "structural carbohydrates in the liver proc. narl. acad. sci biochemistry of glycoproteins and proteoglycans proc. nut/. acad. sci biochemistry of glycoproteins and proteoglvcans biochemistry of glycoproteins and proteoglycans mucus in health and disease proc. int. svmp. gl.vcoconj. th eiochem merhods a. smith, c. ( ) proc. narl. acad. sci. u.s.a. , . thonar key: cord- -h ttoo z authors: bowman, grant r.; cowan, andrew t.; turkewitz, aaron p. title: biogenesis of dense-core secretory granules date: - - journal: trafficking inside cells doi: . / - - - - _ sha: doc_id: cord_uid: h ttoo z dense core granules (dcgs) are vesicular organelles derived from outbound traffic through the eukaryotic secretory pathway. as dcgs are formed, the secretory pathway can also give rise to other types of vesicles, such as those bound for endosomes, lysosomes, and the cell surface. dcgs differ from these other vesicular carriers in both content and function, storing highly concentrated cores’ of condensed cargo in vesicles that are stably maintained within the cell until a specific extracellular stimulus causes their fusion with the plasma membrane. these unique features are imparted by the activities of membrane and lumenal proteins that are specifically delivered to the vesicles during synthesis. this chapter will describe the dcg biogenesis pathway, beginning with the sorting of dcg proteins from proteins that are destined for other types of vesicle carriers. in the trans-golgi network (tgn), sorting occurs as dcg proteins aggregate, causing physical separation from non-dcg proteins. recent work addresses the nature of interactions that produce these aggregates, as well as potentially important interactions with membranes and membrane proteins. dcg proteins are released from the tgn in vesicles called immature secretory granules (isgs). the mechanism of isg formation is largely unclear but is not believed to rely on the assembly of vesicle coats like those observed in other secretory pathways. the required cytosolic factors are now beginning to be identified using in vitro systems with purified cellular components. isg transformation into a mature fusion-competent, stimulus-dependent dcg occurs as endoproteolytic processing of many dcg proteins causes continued condensation of the lumenal contents. at the same time, proteins that fail to be incorporated into the condensing core are removed by a coat-mediated budding mechanism, which also serves to remove excess membrane and membrane proteins from the maturing vesicle. this chapter will summarize the work leading to our current view of granule synthesis, and will discuss questions that need to be addressed in order to gain a more complete understanding of the pathway. in eukaryotes, newly-synthesized proteins destined for secretion are first transferred from the cytoplasm to the lumen ofthe endoplasmic reticulum, and then progress through the golgi apparatus to the trans-golgi network (tgn). at the tgn, the choice of secretory pathways broadens. one route, which appears to be present in all cells, is constitutive in the sense that secretion does not depend on extracellular signals. such secretion involves the budding of vesicles or tubular elements from the tgn and their subsequent transport to and fusion with the plasma membrane, and is essential for cell growth since, among other functions, it provides new material to expand the cell surface. in addition to a constitutive route , many cells maintain a secretory mode that is adapted for the tight coupling of protein release to extracellular stimuli. for such regulated exocytosis, the vesicles that carry newly-synthesized protein from the tgn accumulate in the cytoplasm until specific extracellular events trigger their fusion with the plasma membrane, resulting in the release of vesicle contents.f the vesicles involved are called dense-core granules (dcgs), the name reflecting the fact that the contents are so highly condensed that they form a large electron-dense plug in the vesicle lumen. a large amount ofprotein, as well as other molecular cargo, is thus efficiently stored in vesicular reservoirs and later released on demand. this pathway therefore permits larger and more rapid secretory responses than can be generated via constitutive secretion. classical dcgs in endocrine, exocrine and neuroendocrine cells are responsible for storage of a wide array of signaling molecules (e.g., peptide hormones) and secreted enzymes, and related vesiclesare found in metazoan cells of other lineages as well as in numerous unicellular organisms. the secreted proteins and macromolecules playa vast range of functions, from tissue coordination in metazoans to cyst formation in protists . regulated secretion also depends upon mechanisms for controlling the timely release of dcg contents, and this is accomplished by regulating the fusion of the vesicle membrane with the plasma membrane. much of the progress in understanding the mechanisms that mediate this step has been preceded or aided by studies of synaptic vesicles (reviewed in ref ) , which undergo regulated fusion with the plasma membrane, but differ from dcgs in their biogenesis and acquisition of contents. comparable work in dcg secretion has shown that many of the molecular components involved in regulating exoeytosis and achieving membrane fusion are shared by these two vesicle types. in addition to proteins that appear to be specific for regulated fusion with the plasma membrane, the mechanisms include factors, such as snare s and rab proteins, which are members of families of proteins that are of central importance to vesicular trafficking at multiple stages in the eukaryotic secretory pathway. thus, regulated exocytosis appears to be accomplished by the coupling of a regulatory mechanism to a universal core of membrane trafficking machinery. although many of the protein components have been identified, and a more complete understanding of the process remains an important goal for ongoing research. the mechanistic studies of regulated membrane fusion are too extensive to be included in this chapter, but have been covered in many reviews, - and above. figure .at least pathways diverge at the tgn in neuronal, endocrineand exocrinecells. a) a subsetof proteinsare destinedfor densecoresecretorygranules for release via regulatedexocytosis, these are found asaggregates in distendedareasofthe cisterna. b)proteinsdestinedforconstitutivesecretionaretransported via vesicles or tubules. c) proteinsdestinedfor lysosomes are concentrated,via the mannose- -phosphate receptor, into clathrin coated pits and vesicles. darkly-shaded squaresand circlesrepresentproteins that tend to coaggregate under tgn conditions, and that are subsequentlystored in dcgs. lightly-shaded formsrepresentproteinsthat primarilyexitthetgn viaother pathways.arrowheads representproteinsthat areligandsforthe mannose- -phosphatereceptor. crescents representproteinsthat arefound in a relatively evendistribution throughout the lumen. the extent to whichsomeconstitutively secretedproteinsmaybe concentrated within specific regionsof individualcisternaeis not incorporated into this model. this chapter will instead focus on a part of the pathway that precedes regulated exocytosis, namely the synthesis steps that lead to the formation of dcgs, beginning at the tgn. the tgn is a complex compartment that gives rise not only to the regulated and constitutively released classes of secretory vesicles but also to vesicles that carry hydrolytic enzymes to lysesomes. - therefore, we seek an understanding ofthe signals that guide outbound proteins in a -way tgn sorting problem (fig. ) . dcg protein sorting also continues in a post-tgn compartment, where additional factors come into play. this pathway has been the subject of numerous valuable reviews, - with a particularly thorough treatment by arvan and cascle. the anatomy ofdcg formation a number of important insights into the pathway of dcg formation have come from electron microscopy, providing a context for molecular and genetic studies. first , the fact that dcgs appear dense implies the existence of mechanisms to drive a degree of macromolecular aggregation that is unusual within the secretory pathway. many lines of research have led to the conclusion that protein sorting and concentration are intimately linked in this pathway, both relating to the self-aggregating tendency of dcg proteins that will be discussed below. rambourg and colleagues have investigated the localization of protein aggre:fiates, using serial thin sections to reconstruct the golgi apparatus during granule formation. - in cells producing mucous-containing dcgs, the cis and medial golgi appear as flat cisternae, and secretory proteins are evenly distributed in their lumina. in contrast, cisterna in trans regions are marked by multiple perforations and are dilated in regions that accumulate aggregates of secretory material. those dilations grow progressively larger in the more distal regions, while the nondilated portions take on a tubular appearance. at the trans-most cisterna, the dilated regions with their concentrated secretory cargo appear to exist as independent bodies, separate from a residual network oftubular membranes. several points were established or reinforced by these images. the first is that the visible concentration of dcg proteins begins within golgi cisternae . a second point is that the tgn, the vesicle donor, appears to be undergoing large-scale changes itself. the images also indicate that the vesicles do not bud conventionally in the manner that is well-established for coat (e.g., clarhrinl-mediared steps, since no coats are seen. electron microscopy also suggested that the aggregates undergo progressive changes, and are therefore likelyto be dynamic in nature. in pancreatic cellsthat are synthesizing insulin-storage granules, the proteinaceous cores seen in golgi dilations appeared less dense than the cores of insulin granules in the cell cyroplasm. since the latter are derived from the former, this implied that proteins reorganize during an organellar maturation process. an important conclusion is that dcg formation should be considered as a multi-step process that plays out in sequential compartments. an early phase occurs at the trans face ofthe golgi and results in the production of vesicles bearing concentrated secretory proteins. these are called immature secretory granules (isgs). subsequently those vesiclesare remodeled, as reflected morphologically by cargo condensation and biochemically by changes in protein composition, to become mature dcgs. . for simplicity, we will refer to the first process as budding, and the second as maturation. central issues to be considered in this chapter are the mechanisms responsible for protein sorting during those successivesteps. again for simplicity, we will largely confine our discussion to the dcgs found in neuronal and endocrine cells. many of the same mechanisms are likely to apply to other classes of dcgs, among which are those in hematopoetic cells; for example, see references and . genetics frequently offers a natural complement to morphological studies for developing an overview ofa pathway. unfortunately, a weakness in current approaches to analyzing dcg biogenesis is the absence of developed genetic models, although several systems show promise. no human diseases are known to stem from an inability to synthesize neuroendocrine dcgs. presumably, strong defects in dcg formation would result in embryonic lethality in a complex multicellular organism, since such a defecr would preclude regulated secretion of many peptides involved in tissue coordination. however, this has not prevented the generation of regulated exocytosis mutants in more simple systems, such as drosophila. in flies, the null phenotype of a gene called dcaps (calcium-activated protein for secretion), is embyronic lethal, but analysis of the larva has shown that the gene product is necessary for dcg exocytosis, as predicted from earlier work in mammalian chromaffin cells. . although mutations affecting earlier stages in the pathway (i.e., dcg synthesis) have not been characterized in this organism, the characterization of caps mutants in this system provides hope that the earlier steps will be accessible by further mutational analysis. c elegans offers another potentially useful system for the genetic analysis of dcg synthesis, and several mutations affecting regulated exocytosis have been identified in this organism (reviewed in ref. ) . currently, the only examples of dcg synthesis mutants are found in single-cell systems: the unicellular ciliates tetrahymena thermophila and paramecium tetraurelia, in which the mutations were chemically induced, and spontaneously-arising clones of the rat pheochromocytoma line pci . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the viability ofthese mutants substantiates the idea that regulated exocytosis, unlike constitutive secretion, is not involved in basal cell growth . that is, dcgs are essential for organismal survival in metazoans, but not for individual cell viability. in the pcl lines, some mutations appear to disrupt the transcription ofnumerous granule protein genes. , in the ciliate mutants, which appear to be due to single recessivealleles, the cargo genes are still expressed though no granules are synthesized. in one tetrahymena line, normal granule cargo appears to be shunted to the constitutive secretory pathway. this phenotype indicates that dcg cargo proteins are not sufficient to direct granule formation, a result which was particularly interesting in the context of experiments in which mammalian dcg cargo proteins were expressed in tissue culture cells that do not normally make dcgs. - such cells make vesicles with dense cores, presumably because cargo proteins expressed in nonspecialized cells can induce the formation oftheir own carriers from the tgn. these results implied that the capacity to make dcgs was inherent in the basic organization of the golgi/tgn since it could also occur in such nonspecialized cells. since this capacity appears to have been lost in the tetrahymena mutant, the defect in that line may point to an aspect of goigi/tgn function that is critical for regulated but not constitutive secretion. the full relevance of the ciliate or pc cell mutants to dcg biosynthesis will only be known when the mutations themselves havebeen identified. such geneticapproaches provide an unbiasedmethod for the identification of novel genes, and mayprovecritical in broadening our understanding of the granule synthesis pathway. although many of the dcg cargoproteins themselves have been cloned and characterized, much less is known about the mechanismsthat control protein sorting and condensation. geneticsystems may help to identifythe regulatory factors that are involved in theseprocesses. proteinsortingtakes place in thetgn and duringmaturation. in eachcase, a single compartment gives rise to multiple pathways, and the challenge is in understanding how dcg proteins, both in the lumen and the membrane, are cosorted from a larger cohort that includes proteins destined for other pathways. the relevant contributions oftgn vs, isg sortingarelikely to be cell-type specific and aregenerally difficult to quantify experimentally. however, the mechanisms for controlling sorting at both stages may be fundamentally similar. in particular, the considerationsthat arise from protein aggregation are relevant for both compartments. a long-standing issue is whether the primary mode of dcg protein sorting is active or passive. the model of active sorting was initially inspired by the paradigmof sorting to lysosornes, in which sorting derives from recognition of a set of soluble lumenal proteins by a transmembranereceptor. extendingthis to dcg biogenesis, the modelpositedthat a subsetof proteinshavepositive sorting signals for inclusionin isgs. • in this scheme, proteinsin the tgn lumen that lack targetingsignals are presumedto follow an alternative, default pathway of constitutive secretion. this model has been called "sorting for entry" (fig. la) . an alternative model posits that newly synthesized proteins can be targeted to isgs by default , even in the absence of specific targeting signals, if the flux of bulk membrane traffic toward isgs is greater than that to constitutive or lysosomal carriers. this may indeed be the case for cells that are highly committed to regulated exoeytosis. , in this case, the major sorting events occur in the isg, which becomes a functional extension of the tgn. proteins that are retained as isgs undergo maturation end up as the contents of mature granules. nongranule proteins can be selectivelywithdrawn from isgs during this period, and this model is termed "sorting by retention" (fig. b) . in evaluating either model, the sorting of dcg proteins cannot be considered in precisely the same terms that apply in other pathways, because the tendency of such proteins to self-aggregate facilitates a unique mode of targeting. among other things, it allows a large group of proteins to be sorted together in a single step. one implication is that sorting receptors, if present, could presumably function at concentrations that are dramatically sub-stoichiometric to their dcg protein ligands. furthermore, such receptors would only have to recognize some subset ofdcg proteins , since the remainder could be sorted indirectly via aggregation. in fact, no receptor has ever been unambiguously identified in this pathway. this does not by itself eliminate a "sorting for entry" model, because a second unusual feature of many dcg proteins is a tendency to bind to membranes. this has implications for sorting that will be discussed in a later section. many isolated dcg proteins will self-associate under in vitro conditions believed to approximate the tgn; namely, a slightly acidic flh and high calcium concentration relative to earlier compartments in the secretory pathway. , this can serve as a mechanism for sorting because it is selective: proteins that are constitutively secreted tend to remain soluble under conditions that promote dcg protein aggregation. this first sorting step can therefore be imagined as the evolutionary version of ammonium sulfate precipitation, with the collective behavior based on the proteins' individual biophysical properties, for example their surface charge. while the ability of individual proteins to aggregate is variable, mixtures of proteins may show cooperativiry in vitro, thereby increasing the efficiency of the step (fig. a) . efficient protein aggregation might be expected to show concentration-dependence, and indeed isolated dcg proteins only self-associate above a threshold concentration. this in turn suggests that minor constituents of dcgs may depend for their efficient sorting on coassociation with more abundant species, whose concentrations must be sufficiently high to drive their independent self-aggregation. the sorting efficiency of individual proteins can be experimentally measured as the fraction that is stored in dcgs as opposed to being mistargeted to the constitutive pathway. asexpected from coassociation models, the sorting efficiency of a protein may vary widely between different cell lines. one would also predict that the sorting efficiency ofa protein could be boosted by increasing the expression levelof other proteins with which it coaggregates, particularly those which are most abundant. chiefamong the abundant metazoan dcg proteins are the chromogranin/secretogranins, a group of proteins with shared physical characteristics despite their very limited sequence similariry. , indeed, the overexpression ofchromogranin b (cgb) in the att- neuroendocrine cell line increased the sorting efficiency of a second dcg protein, pro-opiomelanocortin (pomc). nonetheless , it is inherently difficult to test the proposition that self-or coaggregation is a primary sorting determinant using conventional structurefunction analysis, since aggregation is thought to be directed by gross biophysical properties of dcg proteins , and there are no clear "aggregation signals" at the amino acid sequence level. however, recent studies have shown that sorting efficiency can be increased by providing an artificial aggregation signal. heterologous expression of a his-tagged secretory protein enhanced the aggregation and dcg storage, in a calcium-dependent fashion, of cga. , the authors speculate that the tag functions as an "aggregation chaperone" by providing a local site for the binding of divalent cations, thereby nucleating the aggregation process. curiously stably incorporated into the aggregates, suggesting that dcg proteins in their aggregated form interact more strongly with other dcg proteins than with the his tagged peptide. whether endogenous proteins have similar nucleation-promoting properties remains to be determined. identifying the role of any single protein or protein domain in dcg sorting is complicated by the high degree ofcooperativity that is hypothesized to exist within dcg protein aggregates. colo mer et al took advantage of the observation that two exocrine dcg proteins, amylase and gp , do not coaggregate with neuroendocrine dcg proteins in solution, to study the sorting of dcg proteins the absence of coaggregation. when expressed in the neuroendocrine cells, the exocrine proteins were not stored in dcgs but instead secreted constitutively. in similar experiments, an endothelial dcg protein, von willebrand factor, was expressed in neuroendocrine att cells. this resulted, however, not in the constitutive secretion ofvon willebrand factor but instead in the formation of two morphologically-distinct classes of granules. one contained endogenous chromogranins, while the other contained von willebrand factor. a possibility is that two sets ofproteins aggregate independently in the tgn, which could be determined by a number of factors. for example, the two sets could precipitate at relatively distinct ph and/or calcium concentrations and thus be spatially or temporally separated. specific aggregate formation can also arise from conventional protein-protein interactions. in pituitary and pancreatic islet cells, for example, efficient sorting ofcga to dcgs depends on its association with secretogranin iii, and an essential targeting sequence in cga has been determined by gene truncation. cga sorting in pc cells also depends on a specific sequence in the protein, which overlaps with, but is not identical to, that region which is required in pituitary cells. this difference suggests that cga may be interacting with a different partner in pc cells, and indeed these cells do not express secretogranin iii. one possibility is that different surfaces of a cga domain can interact specifically with a range of partners, like a good host at a cocktail party. in summary, the data indicate that the aggregation of a particular protein depends on a number offactors, including its attraction to other potential binding partners within the aggregate, and the physiologic qualities of the lumenal environment, such as ph and calcium concentration, which affect the strengths of those interactions. the expression of proteins that are differentially sensitive to lumenal conditions or that form exclusive sets of protein-protein interactions can potentially result in the formation of multiple distinct aggregates in the same tgn compartment, each comprised of different proteins . these mechanisms could underlie the natural ability ofsome cell types to produce more than one classofdcgs, as is observed in aplysia bag cell neurons, bovine pituitary cells, as well as some protozoa. o-n though the model of sorring-by-aggregation is well established, the actual nature of the molecular interactions within such aggregates is difficult to define. the process of aggregation must be reversible so that the contents can be released into solution following exocyrosis, and moreover, it must be dynamic enough to permit the reorganization oftheir substituents during maturation.v' the latter isparticularly clear in pancreatic~-cells, in which the insulin-containing dcgs exhibit a crystalline ultrastructure, observed by electron microscopy, that is not found in isgs. in comparison to the production of insulin crystals, which involves the assembly of a single protein, the formation of dcg ultrastructure in protozoa may be significantly more complex. in these cells, the lumen ofmature dcgs is filled by a crystalline core that consists of multiple varieties ofproteins,? , indeed , the localization of different proteins within the cores of paramecium dcgs has revealed that the crystals contain at least two distinct layers, each with a different set of protein componenrs. f" images ofisgs reveal that the components of the two layers are interspersed in this compartment, indicating that the layersare formed during a subsequent reorganization phase. thus, there is a significant amount of reorganization that must occur during crystal assembly. overall, the term "aggregation" may be misleading insofar as it suggests a phenomenon based on "stickiness", as for example for misfolded proteins in the endoplasmic reticulum. instead, the interactions that occur between individual proteins in an aggregate may be transient and weak, stimulating formation of aggregates in the tgn due to stabilizing effects provided by multivalent interactions while also allowing for reorganization of the proteins during crystallization, as in figure . some of the nonspecific, low-affinity interactions that occur in aggregates are likely to be mediated by the effects of calcium and ph in charge neutralization, leading to intermolecular interactions of acidic proteins by coordinate association with calcium ions (fig. a) . it is noteworthy that the chromogranins/secretogranins contain a preponderance of acidic amino acids, which endow these proteins with the capacity to bind large numbers of calcium ions with low affinity. ! acidic calcium-binding proteins also form the core of some lrotist dcgs, though they show little overall sequence homology with mammalian proreins.f . an attractive explanation for the similarities is that they reflect a common aggregation-based dcg synthesis mechanism between protozoa and multicellular organisms, and that the amino acid sequences have evolved under similar constraints. following dcg synthesis, the regulated secretion of dcg cargo proteins is dependent on mechanisms that bring the vesicles to the cell surface and control their fusion with the plasma membrane. these activities are dependent on the activity of dcg membrane proteins, for example those that interact with cytoskeleton-based motors for intracellular transpon and those that mediate regulated exocytosis.f it follows that the aggregation ofcore proteins during dcg synthesis cannot by itself be sufficient to form functional dcgs, and that there must be specific, though not necessarily direct, interactions between the lumenal proteins and the membrane constituents in order to ensure efficient sorting of these proteins to the same vesicles. these interactions have been difficult to detect, although some possible examples are discussed in a later section. what is clear, however, is that many lumenal proteins can themselves associate with membranes in unconventional ways. however, the nature and the functional significance of those associations are largely unsettled. five to ten percent of cgb adheres tightly, in a calcium and ph sensitive manner, to mernbranes. ! whether this fraction is in dynamic equilibrium with the remaining~ % is not known, but there is no known chemical difference between the two cohorts. the membrane binding ofcgb is associated with an n-terminal domain defined by a disulfide-anchored loop, which is sufficient to confer membrane association when linked to an otherwise soluble protein. importantly, the chimeric protein was sorted to dcgs in spite of the fact that it did not appear to aggregate, suggesting that the n-terminal domain constitutes an independent targeting signal. that same domain may promote homodimerization at neutral~h, implying that it may mediate different interactions in sequential secretory compartments. cgb, as discussedearlier,also showsa strong tendency to aggregatein a controlled fashion. the coexistence in a single protein of domains that facilitate both protein-membrane binding and homo-or heterotypic protein-protein aggregation, offers the potential to generate cooperative networks with physiologically-useful properties (fig. b) . first, the total concentration of dcg proteins needed to reach the aggregation threshold in the tgn may be reduced for any proteins that interact with the membrane, since the local concentration may be increased depending on local membrane geometry. secondly, the avidity of a cgb aggregate for the membrane will be greater than that ofa monomer, since multiple n-terminal domains are availablefor independent membrane binding. validation of this came from an extension of the experiments with cgb chimeras outlined above. while a single n-terminal cgb domain was able to direct sorting to dcgs, efficient sorting only occurred when two such domains were present. this suggests that the membrane affinity of a single domain may be only marginally sufficient, but is more than adequate if two or more such domains are linked, as would be the case in a cgb aggregate. in a nonconventional sense, cgb could be considered as a dcg sorting receptor: a membrane-associated protein that is itself targeted to dcgs, and that can potentially cotranspon any proteins with which it associates. a similar argument has been made for the enzyme carboxypeptidase e (cpe), which is targeted to dcgs by a c-terminal amphipathic alpha helical domain. • in addition to acting as an enzyme to modify dcg cargo, cpe can also bind a subset ofdcg proteins , for example the hormone precursor pro-opiomelanocortin (pomc). the cpe recognition site involved is different from the enzymatic cleft, and binding may be important for efficient sorting ofpomc, a conclusion based on experiments with cpe knockout mice and from cpe -deficient cell lines. . cpe has been called a receptor for pomc and perhaps for other cargo proteins, though use of the term "receptor" has remained contentious since cpe can also aggregate with pome, chromogranins, and other cargo proteins in a conventional ca + and ph-dependent fashion. , o membrane association of cgb and cpe may be a property that has arisen convergently in these proteins, albeit by different mechanisms, reflecting the importance of this activity in dcg cargo sorting. an n-terminal disulfide bonded loop such as that found in cgb is found in several dcg proteins, including pomc and chromogranin a (cga), though the homology does not extend beyond the structural level, and evidence to date suggests that its role in sorting may be protein-specific. as in cgb , n -terminal disulfide loop domain in pomc is both necessary and sufficient for sorting, but sur~risingly,it appears to interact with the membrane indirectly, through interaction with cpe. the disulfide loop in chromogranin a was not necessary for the sorting of this protein in pcl cells, and instead an interior domain is essential for sorting in these cells,via interaction with membrane-associated secretogranin . • these studies find no evidence for a conserved dcg targeting signal, but they do indicate that specific protein-protein interactions can be important for efficient sorting oflumenal cargo. precisely how cgb, cpe, secretogranin iii, and other ostensibly soluble lumenal proteins associate with membranes is not resolved. there is some evidence that they associate preferentially with cholesterol-rich membranes, so-called lipid rafts. . consistent with this, depletion of cholesterol from tissue culture cells decreased the sorting efficiency of both cpe and cgb, though it is difficult to distinguish direct from indirect effects in such experiments. . in addition, because both constitutive and regulated secretion were inhibited by cholesterol withdrawal, the results do not demonstrate a specific role for cholesterol in dcg formation. the experimental limitations notwithstanding, these data suggest that the association of cpe and cgb with specific membrane sub-domains could be an important aspect ofsorting. iflipid rafts are indeed involved in this pathway, it could add another level of complexity to the cooperative mechanisms that may perrain (fig. ) . interestingly, cgb is also differentially sorted between the apical and basolateral pathways in polarized epithelial cells, which do not make figure . selective association ofocg proteinswithlipidraftsin thetgn. implications ofsuchassociation includethe following possibilities: . independentassociation ofproteinswith a singleraftwouldpromote protein-protein aggregation . . protein aggtegates could stabilize rafts with which they associate. large aggregates couldleadto formationofextensive rafis, in principle, thisprocess couldbesufficient to generate ocgs with a highly biased lipid composition, which is indeed observed. l l the thickened, patterned regions of the cisternal membrane representputativelipid subdomains. dcgs, and this also requires signals within the n-terminal domain. this may suggest a similarity in sorting mechanisms used in epithelial and regulated secretory cells. a final complication in dissecting dcg sorting signals is that the requirement for the disulfide loop in cgb depends on cell type. disulfide bond reduction led to the constitutive secretion of newly synthesized cgb in pcl cells. as expected, this treatment did not affect the sorting of secretogranin ii, a protein that undergoes aggregation but does not contain cysteine residues. however, in gh ci cells, the same treatment did not perturb the sorting of cgb. similarly, cga soning also appears to exhibit cell~e specificity: a c-terminal truncation was correctly sorted in pcl but not in gh ci cells, and an n-terminal region, which does not contain a disulfide loop, was important for sorting in pcl cells. thus, the sorting requirements for cga, cgb, and granule proteins more generally, may depend on the cell type, specificallybecause the efficiency of any protein's sorting will depend on the available interacting parmers . in some cases, a protein's interacting parmer could be a membrane raft, whereas in other cases, the same protein may be delivered to dcgs by virtue of its ability to aggregate with other lumenal cargo proteins. our current understanding of signals involved in dcg membrane protein targeting is relatively primitive. in principle, membrane proteins could be targeted by signals in their lumenal, transmembrane and/or cytoplasmic domains; however the characterization ofsuch signals has not been straightforward. a significant obstacle has been the fact that relatively few membrane proteins have been identified that are exclusivelylocalized to granules. phogrin (phosphatase homolog in granules of insulinoma) localizes to dcgs in a range of neuronal and endocrine tissues. it is a transmembrane protein with an n-terminallumenal domain and c-terminal cytoplasmic domain, and is synthesized with a large n-terminal proregion that is later cleaved within isgs. either the pro-domain or the lumenal domain of the processed protein can be independently stored in dcgs, indicating that each contain signals sufficient for rargeting. one possibility is that these, and by implication the full length phogrin as well, can be sorted by associating with the condensing core of granule cargo in the tgn. this may also be true for two dcg membrane proteins of the anterior pituitary and adrenal medulla, peptidylglycine a-amidating monooxygenase (pam) and dopaminẽ -hydroxylase. in these cases, there is physiological evidence that the lumenal domains can sort independently of the transmembrane or cytosolic domains, since both the soluble forms and the transmembrane forms occur naturally in dcgs. loo nonetheless, efficient storage of the transmembrane form of pam also requires signals within the cytoplasmic tail the idea that sorting of transmembrane proteins in dcg involves cytosolic signals is also supported by analyses ofvamp , a widely distributed dcg v-snare, and p-selectin, a protein of platelets and endothelial cells. the sorting ofvamp to insulin-containing dcgs is impaired by a point mutation in the cytosolic portion of the protein, and the expression of this incorrectly sorted mutant protein is unable to support regulated exocytosis in the absence of wildrype vamp . analysis ofp-selectin targeting is complicated by the fact that it can be found in more than one intracellular compartment, suggesting that it contains hierarchical targeting signals. in addition, the dcgs of platelets and endothelial cells share some properties with lysosomes, and mechanisms involved in their biogenesis may differ from those in neuronal and endocrine cells. . · nonetheless, p-selectin expressed heterologously in the neuroendocrine cell line att- was targeted to dcgs, and this depended on a tyrosine-containing motif in the cytoplasmic domain.l . the same motif is important in the endogenous endothelial cell context, indicating that the rargeting mechanisms may be similar. the tyrosine-based motif suggests that this protein can interact with a coat-associated adaptor, and indeed a functional role for ap- in the sorting ofp-selectin to dcgs has been suggested,llo but no ident ified coats are involved in the formation ofisgs in the tgn. one possibility is that conventional adaptor/coat-mediated sorting of p-selectin occurs at a step distinct from the known budding and maturation steps in dcg bio~enesis; a second is that adaptors may have noncanonical roles unrelated to coat recruitment.i i the studies ofp-selectin have revealed clear evidence that a cytosolic signal can be important for the sorting of transmembrane proteins to dcgs. further analysis of the targeting mechanism will likely be an important topic in future research, as it represents an activity that is topologically distinct from the relatively well characterized aggregation-based sorting events in the lumen. in an intriguing set of experiments, cutler and colleagues found evidence that the function ofthis cytosolic sorting determinant can be coupled to the expression ofa lumenal dcg protein. when the lumenal dcg protein von willebrand factor (vwf) was coexpressed with p-selectin in neuroendocrine att- cells, the vwf was stored in vesicles that were distinct from dcgs containing endogenously-expressed cgb, l , l a finding that is consistent with previous results.i the novel and intriguing finding was that p-selectin was preferentially targeted to the vwf-containing vesicles, indicating that vwf and pvselectin, which are normally expressed in platelet and endothelial cells, could be cosorred in a cell type in which they are heterologously expressed (fig. ) . there was no indication, however, of a direct interaction between the two proteins, and the sorting ofp-selectin in this context was instead dependent on the same tyrosine-containing cytoplasmic motif that had previously been shown to be necessary for targeting to dcgs. the targeting of one class of membrane proteins, those linked via a gpi-anchor, cannot depend on cytosolic signals, since anchors of this type do not penetrate the cytoplasmic membrane leaflet.i for gp- , the major membrane protein of zymogen granules in pancreatic acinar cells, sorting may occur via a coaggregation mechanism. its lumenal domain has been found to associate with a lectin (zg p), sulphated matrix proteoglycans, and syncollin, the last a lumenal protein that may itself interact with the membraney these proteins have been postulated to form a membrane-associated matrix that could serve as a sorting intermediary between the membrane and the zymogen core contents. l this is a variation on the model described for cgb and cpe as sorting receptors , and suggests by analogy that gp- or syncollin might serve as the membrane anchor for the zymogen core. however, dcg assembly is normal in the absence of either protein, indicating that neither is playing a unique role in that regardys, l in summary, the relatively limited evidence to date suggests that a mechanism similar to that involved in cargo protein condensation is involved in sorting of some, but not all membrane proteins with lumenal dcg contents. in principle, indirect interactions between membrane and core proteins may be equally important in the cosorting of membrane and lumenal cargo. iflumenal proteins like cpe preferentially insert into membrane sub-domains based on their lipid composition, then any membrane proteins that independently partition into the same sub-domains would be cosorted. in support of this hypothesis, recent studies have suggested that prohormone convertases and , which are responsible for proteolytic cleavageof lumenal proteins during granule maturation, are sorted to isgs by virtue of c-terminal membrane raft-associated tails, which are by themselves necessary and sufficient for targeting to dcgs,uo additionally, cytoplasmic signals on some transmembrane proteins appear to play important roles in sorting, but the mechanisms are unknown. the canonical mechanism of vesicle budding, as for example that involved in the emergence oflysosome-bound carriers from the tgn, involves transmembrane receptors, adaptors , and coat proteins . since there is no evidence that transmembrane proteins or coat proteins are relevant in isg budding, other mechanisms are likely to apply. there has been some progress in reconstituting this process using cell-free systems, though the field has generally suffered from a lack of in vivo models, for example a well developed genetic system with mutations that affect this step in the pathway. the general approach has been to start with labeled dcg protein in the tgn of permeabilized cells or in golgi-enr iched fractions, then measure the transfer of the label from the relatively large and pelletable golgi membranes to nonpelletable vesicles,using medium speed centrifugation to separate the two pools. the appearance oflabel in smaller vesiclesis taken as an indication of cargo transfer to isgs via vesicle budding. since little is known about dcg biogenesis, it is important to note that "budding" as defined by this assay may include a large number of steps, including the establishment of golgi/tgn microdomains, and the release of previously budded, weakly associated vesicles. thus, the results of these experiments could depend upon on the nature of the starting material. in addition, it has not yet been rigorously demonstrated in any system that the released vesicles are bona fide isgs, for example by testing whether they are competent to fuse with their appropriate target membrane. the reconst ituted buddin~reactions utilize atp, as expected , and most but not all require a cytosol extract . , - the small gtpase arf is required, although the targets for this regulatory protein are not yet clear. one potentially relevant arf target is phosfholipase d (pld) , the binding of which to the membrane can enhance isg buddingp pld converts phospharidyl choline to phosphatidic acid, perhaps thereby effecting a change in membrane curvature. this idea is appealing because, in the absence of coat proteins, the membrane curvature required for isg budding must be induced by other mechanisms. in addition, the indirect products of pld activity may recruit additional effectors to the budding site, including the unconventional gtpase dynamin_ . dynamin mediates membrane scission events, such as pinching off vesicle buds . however, pld does not stimulate budding in all reconstituted systems; the differences may reflect the variery of ways in which donor fractions are prepared. there is evidence that kinases and phosphatases, heterotrimeric g-proteins, and a phosphatidyl inositol transfer protein (pitp) are involved in isg budding, but the enzymatic substrates have not been established. [ ] [ ] [ ] [ ] [ ] an important unanswered question is whether any of these activities, the majoriry of which are as yet unidentified at the molecular level, is specifically required for the formation ofdcgs and not other membrane carriers. pld, for example, has been implicated in tgn tubularion, but the downstream effectors, as for dcg budding, the reaction depends upon ap-l, which can be recruited by the cytoplasmic tails of furin , the mannose-o-phospharereceptor,and other membrane proteins. the mannose- -phosphatereceptorcan in turn bind any soluble lysosomal enzymes in the isg lumen, so thesewillalsobewithdrawn in the budding vesicles. other soluble proteins may also be included based on random partitioning, but the aggregated deg proteins will be excluded.at the end of this process, the mature secretory granule is no longer a budding donor compartment, perhaps becauseit no longer contains membrane proteins that can recruit ap-l (representedby stars). are unknown. the noncanonical gtpase dynamin has been implicated in dcg budding as well as in constitutive secretion. . one possibility is that these activities are only indirectly involved in dcg synthesis. according to the "sort ing by exclusion" model ( fig. a ), isgs are created by a passive process, as aggregation prevents dcg cargo from entering into outbound vesicles and tubules that bear lysosomal or constitutively secreted proteins. instead of being actively budded from the tgn, aggregated proteins would be enriched in a separate subset of relatively large membrane carriers, while non-dcg proteins are removed from the compartment by active coat-dependent processes . thus, the cyeosolic components identified as isg budding factors by in vitro reconstitution assays may really be parts of the mechanisms for other secretory pathways. according to this model, the so-called sorting receptors need only act as membrane tethers in associating the lumenal aggregates with membrane rafts. as there is no need to transport this material to a new compartment, the receptors do not recruit cytosolic coat proteins for vesicle budding, as the traditional membrane receptor proteins do in sorting proteins to other pathways. an alternative to the "sorting by exclusion" model proposes that isg budding is indeed an active process, and that the same mechanism is also involved in driving the budding and tubulation of constitutive secretory carriers from the tgn. although the two pathways give rise to vesicles of vastly different sizes, it is possible that the difference is caused by the cargo proteins (large aggregates versus soluble material) and is not a reflection of different cytosolic budding machinery. the formation ofconstitutive secretory carriers, like isg budding, differs from clathrin-dependent transport at the tgn in that is not associated with the appearance of vesicle coats. there are similarities between the budding of constitutive and regulated vesicles at the molecular level as well: in addition to rab proteins, constitutive traffic has been shown to rely on the activity of dynamin- , protein kinase d, and heterotrimeric g-proteins , factors which may also be associated with isg budding (above). cholesterol depletion has been shown to inhibit both pathways, however it is difficult to know whether the treatment has a direct effect on both pathways, or whether the inhibition of one pathway could inhibit the second via some indirect mechanism. thorough testing ofthis model requires experiments that avoid this problem. the only concrete indication that there are dcg-specific budding factors is that, at least in one reconstituted system, the cytosol re~uirement cannot be substituted for by an extract from hela cells, which do not make dcgs. one possibility that is compatible with both sorting models is that specific cytosolic proteins are involved in establishing or facilitating golgi subdomains in which dcg proteins condense. the structural and functional analysis of the tgn is at a very early stage, but the existence of sub-domains is consistent with the observed nonuniform protein distribution within a single cisterna, as well as with live imaging of heterogenous budding structures. , however, the cisternal dilations involved in isg budding do not necessarily reflect the active maintenance of sub-domains. a simpler view is that the cisterna are passively stretched around the forming granule protein cores, like the bulges in a pancake around blueberries. future work with in vitro systems may provide molecular identification ofactivities that are required for isg budding, but the question ofwhether cells have machinery that is specifically used for this purpose will need to be addressed by other types of analyses. if the budding mechanism is specific to isgs, and not indirectly required for isg production, as in the "sorting by exclusion" model, the prediction is that knocking out individual components would inhibit isg formation without also inhibiting the exit of lysosomal or constitutive proteins from the tgn. depending on cell type, the importance of isgs as a locus of protein sorting may be as important as that of the tgn. sorting at this level involves the budding of vesicles from isg membranes, resulting in the remodeling of membrane and lumenal contents by selective withdrawal (fig. b ). this targeted removal occurs via clathrin-coat recruitment to isgs occurs via the ap- adaptor, wh ich is recruited by membrane proteins in an arf-dependent, bfa-inhibitable step. proteins known to be withdrawn from isgs include the cargo protease furin and the mannose- -phosphate receptor, both of which can interact directly with ap_l. . the mannose-s-phosphate receptor can bind any lysosomal enzymes that may have been incorrectly sorted upon exit from the tgn, and this step therefore leads to selective withdrawal of some lumenal proteins by classical receptor-based sorting. mature dcgs do not support ccv formation, the simplest explanation for which is that isgs become progressivelydepleted ofproteins that act in the recruitment ofap- . consistent with this, myristoylated arf binds to isgs but not mature granules in vitro. recent evidence suggests that the full cohort of arfs and adaptors present on isgs includes arf , and , and ap- and _ . these may all be present on a uniform population of vesicles, or may reflect heterogeneity within isgs. coated vesicles budding from the isg will also withdraw any soluble proteins that randomly partition by diffusion into the vesicle lumen during budding. however, large aggregates of proteins that are condensing in the isg are too large to fit into the buds , and are therefore selectively retained. the efficiency of this separation is increased by the tendency of trafficking imide cells: pathways, mechanisms and regulation nonaggregating proteins to be concentrated at the periphery of the vesicle lumen , as they are excluded from the dense core forming in the center ofthe isg. as a result, the soluble proteins accumulate in a place where they can readily enter the vesicles that are budding from the membrane. these may include proteins that randomly partition at the tgn into budding isgs, but will also include some soluble products of dcg proprotein processing. the best characterized of these is derived from proinsulin, which is processed into and a, b, and c peptides. the first two are disulfide linked, and crystallize to form the granule core. the c peptide is soluble and is largely excluded from the core, and is selectivelywithdrawn. . a collateral consequence of isg maturation is the generation of a set of coated vesicles bearing newly synthesized proteins, some ofwhich have undergone processing by isg-specific enzymes. at least in some cell types, these can deliver their cargo to the plasma membrane, probably via an endosomal intermediate. this has been called "constitutive-like" secretion: constitutive-like in that it is independent ofextracellular stimulation, but with kinetics that are slower than those of true constitutive secretion. in pancreatic~cells, the c peptide that is withdrawn from isgs is secreted via this route . the model that describes the progressive enrichment of granule cargo during isg maturation has been given the name "sorting by retention" and essentially posits that sorting in isgs can be based on a protein's ability to aggregate, rather than depending on specific targeting signals. the concepts are like those of the "sorting by exclusion" model that may apply at the tgn, and the similarity in models may be a reflection of similar molecular mechanisms in vivo. thus, isgs may simply be a functional extension of the tgn, which becomes progressivelyenriched in dcg contents as nonaggregating proteins are actively removed during maturation. thus, there may not be any mechanistic differences between coat mediated sorting at the tgn versus the isgs, though the material that is included in the budding vesicles could change as the compartment matures. alternatively, modification of the coat mediated sorting machinery may be required in order to facilitate sorting from a compartment that is progressively changing. for example, such modifications may be necessary for the trafficking of proteins that are allowed to enter isgs but are not stored in mature dcgs, such as proteases (see "structural maturation of isgs" section) , or for adapting to differences in membrane composition between the tgn and isgs. indirect evidence in support of this possibility comes from the study of the membrane lipid component phospharidyl inosirol-i-phosphare (pi- -p) and its derivatives. in the tgn, these molecules play important modulating roles, including the recruitment of ap-l/clathrin coat proteins for vesicle budding. the levels on pi- -p in the tgn are affected by the activity of pi- kinase, which is stimulated by myristoylated arf -gti~a part of the coat formation machinery. , interestin~r: isgs have been found to contain a pi- -k activity that is not stimulated by arfi-gtp' the tgn has two different pi- kinases (ii and iii) , and it is possible that isgs only recruit one of these. . coat recruitment at the tgn vs. isgs may also be differentially regulated by modification of the vesicle cargo, since the binding of ap-l to the cytoplasmic tails of both furln and the mannose- ~hosphate receptor is stimulated following their phosphorylation by casein kinase ii. , in this regard, a very interesting observation is that newly-budded isgs are rapidly transported to the cell periphery, at least in some cell types, and therefore primarily inhabit a different cellular microenvironment from the tgn. this may be relevant for differential regulation of similar activities at the tgn vs. isgs, for example if receptors in isgs are selectively modified. although the data is not yet conclusive, the emerging view of sorting from isgs is that it is directed by the core elements of a "flexible" ap-l/clathrin dependent sorting mechanism that is differentially controlled at the isgs versus the tgn. the model holds that the sorting events ofisg maturation are not mediated by a unique vesicle trafficking mechanism, but are instead accomplished by pathway-specific modifications ofmachinery that is common to all cell rypes. a similar phenomenon may occur at an earlier stage ofthe pathway, where the coat-independent machinery that drives the formation ofconstitutive carriers from the tgn may be adapted for the budding of isgs, as discussed in the "mechanisms of immature secretory granule (isg) budding" section. this apparent mechanistic conservation may explain the abiliry offibroblast cells, which do not normally make dcgs, to make dense-cored vesicles when expressing heterologous chromogranin genes or vonwillebrand factor. . . however, these observations do not preclude the possibility that specialized dcg-producing cells express proteins that specifically modify parts of the conserved cellular trafficking machinery to enhance dcg synthesis. the cores of newly-budded isgs apftear less electron-opaque than those in mature dcgs, and are also lower in buoyant density, indicating that granule cargo becomes increasingly condensed during granule maturation. this is one reflection of the larger remodeling of protein and lipid constituents during the maturation process, which includes the selective withdrawal of components that are present in im matu re, but not mature, granules . this overall process serves important structural functions. the tighter packing offers increasingly efficient storage, and not simply because more material can be contained in a fixed vesicle volume. protein condensation overcomes an energetic barrier that is posed by a vesicle filled with concentrated soluble macromolecules, which is hyperosmolar when compared to cytosol. maintaining such a vesiclewould require constant pumping ofosmolytes to counter vesicle swelling, an expensive cellular proposition. within dcgs, aggregated proteins are no longer solvated, and are therefore osmotically inert. the progressive condensation durin~maturation parallels, and is likely to be controlled by, changes in the lumenal environment. in neuroendocrine cells, the tgn is acidified to ph . by vacuolar atpases. these are also present in the isg membrane, with the result that the isg continues to acidify - at the same time there is an increase in calcium that , along with other cations, is important for charge neutralization of the largely acidic core proteins. this calcium may be cotransported from the endoplasmic reticulum with calcium-binding dcg cargo proteins, or imported via isg membrane ion exchangers. the ionic changes can trigger changes in dcg protein conformations or interactions . for example, cgb forms horno-oligomers under the conditions found in isgs. . the functional significance is as yet unknown, but these are presumably based on contacts different from those involved in aggregative sorting. one well-established consequence ofisg acidification, in combination with increased ca +, is the activation of proteases that are specifically localized to dcgs. the contents of neuronal and endocrine dcgs are largely synthesized as proteins that are proteolytically processed to generate bioactive peptides, the species that are eventually released during exocytosis. l proteolytic processing involves a variety of enzymes including amino-and carboxypeptidases, and a family ofaspartyl proteases called prohormone convertases. [ ] [ ] [ ] [ ] members of this family are differentially active over a range of proton and calcium concentrations, and may thus act sequentially on their substrates during isg maturation, in a cell type-dependent fashion [davidson, # ;laslop, # ;goodge, # ;. though isgs are considered to be the major compartment of proprotein processing, in some cell types processing may begin in the tgn, and moore and colleagues have begun to resolve the requirements for isg budding from those required for the onset of processing. . . in their cell-free system, the onset of processing precedes budding. both require hydrolyzable gtp, but at two distinct concentrations . this difference suggested a model in which the former requires arf, while the latter depends upon a heterotrimeric g-protein. in addition to generating mature peptides, proprotein processing may drive the physical reorganization of the core, in cases where mature peptides can pack more tightly than the precursors . the best example of this is found in~-cell granules, in which mature insulin but not proinsulin can assemble into hexagonal crysrals, simply because processing relieves a packing constraint . , (fig. ) . the control of assembly via proteolytic processing is strongly reminiscent of mechanisms involved in viral capsid formation. the process ofdcg maturation, which includes the generation of active peptides by proteolytic processing and the condensation of cargo into a densely packed, osmotically inert form, serves to increase the efficiency of the regulated secretory pathway in several ways. first, the condensation ofmaterial allows great quantities of protein to be stored in the vesicles,with the consequence that a small number ofexocytic events can generate a relativelylarge secretory response. second, proteolytic processing in isgs allows the cell to combine multiple dcg peptides into a single proprotein, thereby linking the sorting of these proteins at earlier stages of the pathway. in neuroendocrine isgs, for example, the chromogranin proteins are cleaved into multiple biologically active peptides with different postexocytic functions. , furthermore, limiting the site of proteolytic processing to isgs may provide a failsafe mechanism, ensuring that the active forms of the proteins are only found in a compartment that is under direct control of the regulated secretory pathway, therefore leaving any incorrectly sorted proteins as uncleaved precursors. the remodeling of the membrane attending the budding of clathrin coated vesicles does not simply serve to remove proteins that may have been incorrectly targeted at the tgn. rather, it also underlies differences in the activity ofisgs and mature granules. this was suggested by the observation that isgs and mature granules differ dramatically with regard to exocytosis: whereas mature dcgs undergo efficient exocytic fusion with the plasma membrane in a stimulus-dependent fashion , isgs exhibit an increased tendency to fuse with the plasma membrane in the absence of stimulation. in att cells, unregulated release ofdcgs from isgs proceeds for - hours after isg budding from the tgn. these isgs contain two snares, vamp and synaptota~min iv (syt iv) , which are withdrawn during maturation in a brefeldin a-inhibitable step.i , , during the same period , the maturing granules become responsive to exocytic stimuli, a process also blocked by bfa. that the two phenomena may be linked is suggested by the observation that overexpression ofsyt iv itself decreased the responsiveness of maturing granules to secretory stimuli. l syt iv is thought to act as a negative regulator of calcium-induced exocytosis, and the withdrawal of this inhibitory factor from isgs may foster maturation. a recent study showed that the removal ofvamp from isgs depends upon interactions with ap-l and the coat protein pacs-l, thereby providing genetic confirmation and molecular detail to this model. however, a complication ofthis model is that syt iv is thought to inhibit membrane fusion by forming inactive heterodimers with synaptotagmin i, and the mechanism by which the heterodimers are separated and syt n is selectively removed from the isgs is unknown. another functional characteristic that may, in some cell types, distinguish isgs from dcgs, is that isgs can undergo homotypic fusion, a reaction that has been more extensively characterized in vitro than in vivo. , , the specific function ofthis reaction is not clear. in some systems homotypic-like fusion might allow for the synthesis ofspecialized dcg cores in which the contents are not randomly distributed. in pseudomicrotborax dubius, two kinds of isgs, containing morpholo §ically-distinguishable cargo, fuse during the process ofassembling a complex core structure. more generally, consolidation could potentially define the size of the granules, which in many systems appear to be controlled. disruption of the gene encoding rab d, an exocrine granule-associated small gtpase, resulted in a doubling ofmature granule volume, and one possibility is that rab d acts as a negative regulator of homotypic fusion. at some level, membrane remodeling must account for the difference in the fusogenic behavior of isgs vs. mature granules, and attention has focused on the snares, due to their importance in regulating membrane fusion. isgs from pcl cells contain syntaxin , which must be present on both donor and acceptor membranes for efficient homotypic fusion in vitro.in syntaxin is also present in clathrin-coated vesicles which bud from the isg rnernbrane , consistent with the idea that it is selectively removed during maturation via several likely ap-i binding sites in its cytoplasmic domain. as isg maturation appears to involve the removal of specific factors via the budding of clathrin coated vesicles, it is possible that more thorough analyses of the target proteins and their interacting partners will help to uncover isg-specific machinery that regulates clathrin-dependenr sorting in this compartment. more broadly, the identification of the molecules that define the functional maturity ofdcgs by their presence or absence in the vesicle will provide insights into the nature of organelle identity, a topic that is central to an understanding the general principles of vesicular traffic. finally, recent evidence hints at aspects of granule maturation that have not previously been recognized. functional maturation of secretory granules may extend beyond the period of morphological change, based on the observation that the distribution and fusogenic activity of granules may change with vesicle age. the majority of the work on dcg synthesis has focussed on the sorting of the lumenal content proteins in the tgn and isgs. these studies have, for the most part, supported the nonspecific aggregation-based model for sorting that was proposed by chanat and huttner in . not surprisingly, studies of many granule cargo proteins in multiple systems have revealedsome casesthat are possible exceptions to this general rule, where specificprotein-protein interactions are required for the sorting of a particular protein, as discussed in the "protein sorting into isgs" section of this chapter. overall, the precise requirements for the sorting of any particular protein is likely to be both context (which other granule cargo proteins are being expressed, and in what quantities) and cell type dependent (protein aggregation is sensitive to physiological properties of the lumen, such as calcium concentration and ph, which may vary between cell types), though it is likely that the general principles of aggregation-based sorting apply in all cells that produce dcgs. further analysis of the specific sorting requirements for individual proteins may lead to a greater knowledge of the details ofaggregation-based sorting, but the next leap forward in our understanding of they system will more likely come from experimental approaches that expand beyond the level of individual proteins and consider the dcg synthesis pathway more broadly. for example, cargo protein aggregation is known to be sensitive to lumenal calcium concentration and ph levels, but the mechanisms that control these physiologic parameters have not been elucidated. secondly, how are granule cargo proteins sorted to the same destination as other proteins that are essential for dcg function, such as membrane fusion machinery? the answers to these questions may be learned from studies in genetic systems, such as c. elegans, drosophila, and ciliated protozoans, which offer promising avenues for further experimentation. these orf.anisms have recently been used to identify elements of the regulated exocytosis machinery.' , , and similar studies could uncover genes that are involved in vesicle synthesis. another major gap in our understanding of the granule synthesis pathway is the extent of its functional relationship with other branches of the secretory pathway. two decades ago, dgc formation was considered to be one of a small number of distinct, post-tgn secretory pathways. this carried the assumption that vesicles bound for constitutive or regulated exocytosis, or toward lysosomes, would rely on distinct mechanisms for their biogenesis. that view now seems, paradoxically, to have been both too simple and too complex. it was too simple because post tgn traffic cannot be neatly divided into three branches: for example, what was called the constitutive pathway may in fact consist of multiple branches. . this was initially established for apical vs. basolateral targeting in polarized epithelia, but there is evidence in other cell types as well. furthermore, the mechanisms for dcg formation are not easily separated from those that are directly involved in other pathways, implying that the secretory pathway cannot be divided into distinct, independently functioning branches. for example, ap- dependent sorting of proteins to the lysosomal pathway is associated with isg maturation, and may also be part of the driving force for the "sorting by exclusion" of dcg contents in the tgn (see "protein sorting in isgs" section). at the same time, the fact that the dcg synthesis pathway and lysosomal pathway use some of the same machinery argues that the historical view of distinct mechanisms was too complex. similarly, the historical view that constitutive and regulated secretory carriers are fundamentally different may also be incorrect. the idea that constitutive traffic is based on small vesiclesis being modified by the recognition that tgn tubularion may be as, if not more, important in this pathway, at least in some cell types (referencesin ref ) . thus coat-mediated vesicle formation may be the exception rather than the rule for anterograde traffic to the plasma membrane, and the formation of constitutive and regulated secretory carriers may share common mechanisms . in the extreme, the mechanisms may be mostly conserved, and the end 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secretory granules in vitro and in vivo procathepsin l self-association as a mechanism for selective secretion drosophila caps is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion ca( +)-dependenr activator protein for secretion is critical for the fusion of dense-core vesicles with the membrane in calf adrenal chromaffin cells a novel kd brain cyrosolic protein reconstitutes ca + -regulated secretion in permeable neuroendocrine cells the synaptic vesicle cycle: exocytosis and endocytosis in drosophila and c e egans isolation and ultrastructural characterization of secretory mutants of tetrahymena thermophila protein secretion in tetrahymena thermophila: characterization of the secretory mutant strain sb genetic characterization of tetrahymena thermophila mutants unable to secrete capsules maturation of dense core granules in wild rype and mutant tetrahymena thermophila mutational analysis of regulated exocytosis in tetrahymena mutations affecting the trichocysts in paramecium aurelia. i morphology and description of the mutants evidence for defects in membrane traffic in paramecium secretory mutants unable to produce functional storage granules overall lack of regulated secretion in a pci variant cell clone a pci variant lacking regulated secretory organelles: aberrant protein targeting and evidence for a factor inhibiting neuroendocrine gene expression analysis of a mutant exhibiting conditional sorting ro dense core secretory granules in tetrahymena thermophila biogenesis of von willebrand factor-containing organelles in heterologous rransfected cv-i cells induction of specific storage organelles by von willebrand facror propolypeptide chromogranin a, an «on/off. switch controlling dense-core secretory granule biogenesis chromogranin b-induced secretory granule biogenesis: comparison with the similar role of chromogranin a molecular sorting in the secretory pathway protein secretion: puzzling receptors proteins synthesized and secreted during rat pancreatic development phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas ph-and ca +-dependent aggregation properry of secretory vesicle matrix proteins and the potential role of chromogranins a and b in secretory vesicle biogenesis milieu-induced, selective aggregation of regulated secretory proteins in the trans-golgi network signal-mediated sorting to the regulated pathway of protein secretion +)-induced conformational change and aggregation of chromogranin b. comparison with chromogranin a and implication in secretory vesicle biogenesis secretory granule content proteins and the luminal domains of granule membrane prot eins aggregate in vitro at mildly acidic ph the granin (chromagranin/secretogranin) family the chromogranins: th eir roles in secretion from neuroendocrine cells and as markers for neuroendocrine neoplasia chro mogranin b (secretogranin i) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor gorr suo aggregation chaperones enhance aggregation and storage of secretory proteins in endocrine cells in vitro aggregation of the regulated secretory protein chromogranin a reconstitution in vitro of the ph-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of gp- exocrine granule specific packaging signals are present in the polypeptide moiery of the pancreatic granule membrane protein gp and in amylase: implications for protein targeting to secretory granules identification of a chromogranin a domain that mediates binding to secretogranin iii and targeting to secretory granules in pituitary cells and pancreatic beta-cells identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin a sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary multiple neuropept ides derived from a common precursor are differentially packaged and transported structure et ultrastructure de lacrymaria olor (o.f.m. ) cocrystallization of proinsulin and insulin vayssie let ai. a large multigenic family codes for the polypeptides of the crystalline trichocyst matrix in paramecium protein secretion in tetrahymena thermophila. characterization of the major proteinaceous secretory proteins growth and form of secretory granules involves stepwise assembly but not differential sorting of a family of secretory proteins in paramecium quality control in the endoplasmic reticulum proteolytic processing and ca +-binding activity of dense-core vesicle polypeptides in tetrahymena identification and characterization of a novel secretory granule calcium-binding protein from the early branching eukaryote giardia lamblia myosin va facilitates the distribution of secretory granules in the f-actin rich cortex of pc cells chromogranin b (secretogranin i) a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in pc cells the disulfide-bonded loop of chromogranin b mediates membrane binding and directs sorting from the trans-golgi network to secretory granules the disulfide-bonded loop of chrornogranins, which is essential for sorting to secretory granules, mediates hornodimerizarion identification of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway carboxypeptidase e, a prohormone sorting receptor, is anchored to secretory granules via a c-terminal transmembrane insertion carboxypeptidase e is a re~lated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in cpe at mice identification of a novel prohormone sorting signal-binding site on carboxypeptidase e, a regulated secretory pathway-sorting receptor depletion of carboxypeptidase e, a regulated secretory pathway sorting receptor, causes misrouting and constitut ive secretion of proinsulin and proenkephalin, but not chromogranin a proinsulin targeting to the regulated pathway is not impaired in carboxypeptidase e-deficient cpefat/cpefat mice carboxypeptidase e, a peripheral membrane protein implicated in the targeting of hormones to secretory granules, coaggregates with granule content proteins at acidic ph secretogranin iii binds to cholesterol in the secretory granule membrane as an adapter for chromogranin a sorting of carboxypeptidase e to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts cholesterol is required for the formation of regulated and constitutive secretory vesicles from the trans-golgi network selective delivery of secretory cargo in golgi-derived carriers of nonepithelial cells reduction of the disulfide bond of chromogranin b (secretogranin i) in the trans-golgi network causes its missorting to the constitutive secretory pathways disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins n-and c-terminal domains direct cell type-specific sorting of chromogranin a to secretory granules molecular cloning of phogrin, a protein-ryrosine phosphatase homologue localized to insulin secretory granule membranes the lumenal domain of the integral membrane protein phogrin mediates targeting to secretory granules differential trafficking of soluble and integral membrane secretory granule-associated proteins identification of routing determinants in the cytosolic domain of a secretory granule-associated integral membrane protein biogenesis of weibel-palade bodies mutational analysis of vamp domains implicated in ca +-induced insulin exocytosis targeting of p-selectin to two regulated secretory organelles in pc cells p-selectin targeting to secretory iysosomes of rbl- h cells regulated secretion of conventionallysosomes angelica ec the molecular machinery for the biogenesis of lysosome-related organelles: lessons from hermansky-pudlak syndrome p-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in att- cells a complex web of signal-dependent trafficking underlies the triorganellar distribution of p-selectin in neuroendocrine pc cells ap- adaptor functions in targeting p-selectin to secretory granules in endothelial cells coat proteins: shaping membrane transport selective and signal-dependent recruitment of membrane proteins to secretory granules formed by heterologously expressed von willebrand factor weibel-palade bodies recruit rab by a content-driven , maturation-dependent mechanism that is independent of cell type assembly of multimeric von willebrand factor directs sorting of p-selectin how glycosylphosphatidylinositol-anchored membrane proteins are made interaction of syncollin with gp- , the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains a submembranous matrix of proteoglycans on zymogen granule membranes is involved in granule formation in rat pancreatic acinar cells the major zymogen granule membrane protein gp- in the rat pancreas is not involved in granule formation loss of the zymogen granule protein syncollin affects pancreatic protein synthesis and transport but not secretion recycling of raft-associated prohormone sorting receptor carboxypeptidase e requires interaction with arf requirement for gtp hydrolysis in the formation of secretory vesicles the use of permeabilized cells to investigate secretory granule biogenesis exocytic transport vesicles generated in vitro from the trans-golgi network carry secretory and plasma membrane proteins reconstitution of constitutive secretion using semi-intact cells: regulation by gtp but not calcium phospholipase d stimulates release of nascent secretory vesicles from the trans-golgi network secretory vesicle budding from the trans-golgi network is mediated by phosphatidic acid levels implications of lipid microdomains for membrane curvature, budding and fission role of dynamin in the formation of transport vesicles from the trans-golgi network a role for phosphatidylinositol transfer protein in secretory vesicle formation multiple trimeric g-proteins on the trans-golgi network exert stimulatory and inhibitory effects on secretory vesicle formation t rimeric g-proteins of the trans-golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules formation of nascent secretory vesicles from the trans-golgi network of endocrine cells is inhibited by tytosine kinase and phosphatase inhibitors cooperativity of phosphatidylinositol transfer protein and phospholipase d in secretory vesicle formation from the tgn-phosphoinositides as a common denominator? role of diacylglycerol in pkd recruitment to the tgn and protein transport to the plasma membrane functional diversity in the dynamin family protein kinase d regulates the fission of cell surface destined transport carriers from the trans-golgi network biogenesis of processing-competent secretory organelles in vitro two independent targeting signals in the cytoplasmic domain determine trans-golgi network localization and endosomal trafficking of the proprorein convertase furin direct and gtp-dependent interaction of adp-ribosylation factor with clathrin adaptor protein ap-l on immature secretory granules the ap-l adaptor complex binds to immature secretory granules from pc cells, arid is regulated by adp-ribosylation factor interaction of furin in immature secretory granules from neuroendocrine cells with the ap- adaptor complex is modulated by casein kinase ii phosphorylation mannose -phosphate receptors are sorted from immature secretory granules via adaptor protein ap- , clathrin, and syntaxin -positive vesicles differential distribution of mannose-e-phosphare receptors and furin in immature secretory granules differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta cells site-specific cross-linking reveals a differential direct interaction of class , , and adp-ribosylation factors with adaptor protein complexes and proinsulin endoproteolysis confers enhanced targeting of processed insulin to the regulated secretory pathway the role of assembly in insulin's biosynthesis protein discharge from immature secretory granules displays both regulated and constitutive characteristics protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment protein traffic from the secretory pathway to the endosomal system in pancreatic beta-cells phospharidyiinosirol phosphate regulates targeting of clathrin adaptor ap-l complexes to the golgi arf mediates recruitment of ptdlns- - h kinase-beta and stimulates synthesis of ptdlns( , )p on the golgi complex type i phosphatidylinositol -phosphate -kinase directly interacts with adp-ribosylation factor and is responsible for phosphatidylinositol , -bisphosphate synthesis in the golgi compartment regulation and recruitment of phosphatidylinositol -kinase on immature secretory granules is independent of adp-ribosylation factor dynamics of immature secretory granules: role of eytoskeletal elements during transport, cortical restriction, and f-actin-dependent tethering mechanisms of ph regulation in the regulated secretory pathway endoproteolytic cleavage is mediated by a vacuolar atpase that generates an acidic ph in the trans-golgi network ph-independent and -dependent cleavage of proinsulin in the same secretory vesicle biosynthesis and secretion of pituitary hormones: dynamics and regulation inhibition of the vacuolar h+-atpase perturbs the transport , sorting, processing and release of regulated secretory proteins low-molecular-weight constituents of isolated insulin-secretory granules . bivalent cations , adenine nucleotides and inorganic phosphate endoplasmic reticulum ca + is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic beta-cell effects of ph and ca + on heterodimer and heteroretramer formation by chromogranin a and chromogranin b prohormone and proneuropeptide processing calcium-and ph-dependent aggregation and membrane association of the precursor of the prohormone convertase pc ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in att- cells molecular and cellular regulation of prohormone processing the proprotein convertases furin and prohormone convertase / are major convertases in the processing of mouse pro-growth hormone-releasing hormone an antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in att cells before its cleavage lau et ai. biogenesis of regulated exocytotic carriers in neuroendocrine cells comparison of secondary structures of insulin and proinsulin by ftir nmr and photo-cidnp studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition the role of proteolytic processing in the morphogenesis of virus particles peptides derived from the granins (chromograninslsecretogranins) distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin a homotypic fusion of immature secretory granules during maturation requires syntaxin synaptic function modulated by changes in the ratio of synaptoragmin i and iv ap-l recruitment to vamp is modulated by phosphorylationdependent binding of pacs- cytoplasmic granule formation in mouse pancreatic acinar cells. evidence for formation of immature granules (condensing vacuoles) by aggregation and fusion of progranules of unit size, and for reductions in membrane surface area and immature granule volume during granule maturation homotypic fusion of immature secretory granules during maturation in a cell-free assay immunological characterization of trichocyst proteins in the ciliate pseudomicrothorax dubius lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland rab d is not required for exocrine exocytosis but for maintenance of normally sized secretory granules functional and spatial segregation of secretory vesicle pools according to vesicle age genetic approach to regulated exocyrosis using functional complementation in paramecium: identification of the nd gene required for membrane fusion novel secretory vesicle proteins essential for membrane fusion display extracellular-marrix domains post-golgi biosynthetic trafficking multicolour imaging of post-golgi sorting and trafficking in live cells gaip participates in budding of membrane carriers at the rrans-golgi network macro-and micro-domains in the endocrine pancreas key: cord- -pjjvfdnq authors: fontana, juan; lópez‐montero, noelia; elliott, richard m.; fernández, josé jesús; risco, cristina title: the unique architecture of bunyamwera virus factories around the golgi complex date: - - journal: cell microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: pjjvfdnq viral factories are novel structures built by viruses in infected cells. during their construction organelles are recruited and build a large scaffold for viral replication and morphogenesis. we have studied how a bunyavirus uses the golgi to build the factory. with the help of confocal and d ultrastructural imaging together with molecular mapping in situ and in vitro we have characterized a tubular structure that harbours the viral replication complexes in a globular domain. numerous ribonucleoproteins were released from purified tubes disrupted in vitro. actin and myosin i were identified by peptide mass fingerprinting in isolated tubes while actin and the viral nsm non‐structural protein were detected in the tubes' internal proteinaceous scaffold by immunogold labelling. studies with nsm deletion mutants and drugs affecting actin showed that both nsm and actin are key factors for tube and virus assembly in golgi. three‐dimensional reconstructions based on oriented serial sections of infected cells showed that tubes anchor cell organelles to golgi stacks and make contacts with intracellular viruses. we propose that this new structure, unique among enveloped viruses, assembles in association with the most stable component of golgi stacks, the actin‐containing matrix scaffold, connecting viral replication and morphogenesis inside viral factories. rna viruses replicate their genome in intracellular membranes of infected cells (mackenzie, ; salonen et al., ) . modified membranes harbouring viral replication complexes (rcs) frequently integrate into a complex structure known as the 'viral factory' where the cytoskeleton participates, cell organelles are recruited and the different steps of the virus life cycle are sequentially connected. characterizing how this happens would help to understand how viral factors take control of cells and modify their architecture. there is no description of the mechanisms involved in the formation of most viral factories, although there is evidence that some viruses induce aggresomes and autophagosomes to generate sites of replication (novoa et al., a; wileman, ) . expression of early proteins such as viral polymerases is probably sufficient to program the cell for organelle recruitment and factory formation, as recently observed in cells transfected with rubella virus replicons (fontana et al., ) . the viral replication machinery is usually inserted in single-or double-membrane vesicles that can be associated with a variety of organelles, such as the rough endoplasmic reticulum (rer), mitochondria, the endolysosomal system and chloroplasts or vacuolar membranes in plants (hagiwara et al., ; novoa et al., a; salonen et al., ; kopek et al., ) . some viruses, e.g. polioviruses, induce proliferation of specific membranes creating a 'novel compartment' (cherry et al., ) where the viral rna polymerase molecules can assemble bidimensional arrays (hobson et al., ; lyle et al., ) . although the purpose of this targeted localization has not been elucidated, one possibility is that association of rcs with membranes provides a structural framework for efficient replication and transfer of replicated rna to assembly sites in contiguous membranes. bunyaviruses comprise a large family of rna enveloped viruses that includes serious emergent pathogens for humans, animals and plants, and are responsible for severe episodes of encephalitis and haemorrhagic fevers in humans (elliott, ) . they assemble a large factory involving the golgi complex where virus particles bud and mature (salanueva et al., ; novoa et al., b) . bunyamwera virus (bunv) serves as a model for the many pathogens within this family. it contains three rna segments of negative-sense polarity. the large segment (l) codes for an rna-dependent rna polymerase (l protein), the medium segment (m) codes for a precursor polyprotein (nh -gn-nsm-gc-cooh), which is cotranslationally cleaved to yield the two virion glycoproteins (gn and gc) and a non-structural protein called nsm, and the smallest segment (s) codes for the nucleoprotein n and a second non-structural protein nss in overlapping reading frames (elliott, ) . the nh -terminal domain of nsm is essential for bunv morphogenesis (shi et al., ) while nss is a non-essential protein that contributes to viral pathogenesis (bridgen et al., ) . there is no ultrastructural description of the bunyavirus replication site that has been defined as 'cytoplasmic' (nichol et al., ) . morphogenesis of coronaviruses, arteriviruses, rubiviruses and bunyaviruses is associated to the golgi complex. the choice of the golgi for viral replication or assembly is somehow surprising. the golgi is a highly dynamic organelle whose function requires continual membrane and protein flow (james morre and mollenhauer, ) . on the other hand, the golgi may be an autonomous organelle with a stable framework (seemann et al., ) . its unique architecture is thought to depend on cytoplasmic matrix proteins and the cytoskeleton. actin seems to participate in the preservation of the flattened shape of golgi cisternae (lazaro-dieguez et al., ) and several actin-binding proteins are known to play some role in golgi function. these include myosins and spectrins (beck, ) . we have studied how a bunyavirus modifies cell structure and builds factories around the golgi complex from early to late steps of its life cycle. in a previous study we observed that golgi stacks contain peculiar virus-induced tubular structures in bunv-infected cells (salanueva et al., ) . with the help of three-dimensional reconstructions and molecular mapping in situ and in vitro we have characterized these tubular elements and discovered that they represent a new structure of viral and cellular origin. tubes assemble in golgi stacks where they seem to be involved in multiple functions including viral genome replication, transfer of viral ribonucleoprotein complexes to assembly sites and viral morphogenesis. we propose that this new multifunctional structure associates with the actin-containing matrix of the golgi stacks providing a stable scaffold for viral replication and early morphogenesis. changes in cell organization during assembly of the viral factory were studied by confocal microscopy (fig. ). control monolayers (fig. a) showed round-shaped nuclei (blue), perinuclear golgi elements (red) and welldefined straight actin stress fibres (green). infected cells (fig. b) have a completely different aspect: nuclei are elongated, the golgi is rounded and concentrates on one side of the nucleus, and stress fibres have moved to the cell periphery. no changes in microtubules or vimentin filaments were observed (not shown). in addition to the wga trans-golgi marker shown in fig. a and b, two additional golgi markers (giantin and galactosyltransferase or gal-t) exhibited a similar transition from a perinuclear distribution in non-infected cells ( fig. c and d) to a more compact pattern on one side of the nucleus in infected cells ( fig. e and f). when observing just nuclei-associated staining a depression was frequently detected, corresponding to the location of the viral factory ( fig. g and h). electron microscopy (em) showed factories as groups of organelles near the nucleus (fig. i) . higher magnifications showed round-shaped and tubular structures in golgi stacks ( fig. j and k). we have detected these structures in several mammalian cultured cell types that support bunyavirus replication such as bhk- (this study), vero (salanueva et al., ) and cho cells (not shown). their number was higher early in infection and their viral origin was demonstrated in previous studies (salanueva et al., ) . in the present work we have studied a large number of serial sections and discovered that viral tubes actually contain a tubular domain and a bigger globular domain on one of the extremities (fig. l ). the tubes are open to the cytoplasm (arrows in fig. k -m). bunv generates round-shaped and tubular structures in infected cells whose representative views in thin sections of epoxy resin are summarized in fig. a -h. the golgiassociated morphogenetic pathway of bunv and the structural and biochemical characterization of the three viral forms have been previously described in detail (salanueva et al., ; novoa et al., a,b) . global understanding of all these structures has been possible through a careful analysis of consecutive serial sections (see below). condensation of material in individual golgi sacculi originates arcs opened to the cytosol ( fig. a) , elongated globular structures of low internal electrondensity (fig. b) and complete viral tubes with both globular and cylindrical domains (fig. c) . the two types of cross-sections originated by a viral tube are shown in fig. d and e. cross-sectioned globular heads are always > nm in diameter and most of these structures have a diameter of - nm. the cylindrical domain of viral tubes has a cross-section of - nm in diameter. virus particles have smaller diameters (fig. f-h) . budding profiles in golgi membranes generate immature viruses or vi that are released into the lumen of a golgi sacculus (fig. f ). intermediate intracellular virus or vii exits the golgi in secretory vesicles (fig. g ) and mature extracellular virions or ve are seen in the extracellular environment (fig. h ). all three viral forms have a diameter of around nm in thin sections of epoxy resin for ultrastructural studies. in cryosections for immunogold detection of specific components structural details of viral tubes and viruses change ( cell fractionation and centrifugation in optiprep gradients allowed isolation of viral tubular structures (fig. a) . negative staining confirmed their general morphology: they contain a cylindrical and a globular domain ( fig. b and c) similar to tubes in situ (fig. l ) and a filamentous internal texture (fig. d ). dsrna was detected in isolated tubes by dot-blot (fig. e ). viral proteins involved in genome replication such as the rna pol and the nuc protein were detected by western blot and coomasie blue staining, respectively (fig. f ). controlled disruption of isolated tubes by saponin treatment showed a proteina-ceous internal scaffold (fig. g ) that was labelled with anti-pol and anti-nuc antibodies ( fig. h and i). complete disruption of tubes provoked the release of viral ribonucleoprotein complexes or rnps (fig. j ). similar release of rnps was observed when disrupting isolated viruses in vitro (novoa et al., b) . nsm is a transmembrane non-structural protein involved in bunv morphogenesis (shi et al., ) that accumulates in the golgi region of infected cells (lappin et al., ; shi et al., ) . immunogold and em showed that nsm localizes both in the cylindrical ( fig. a and b) and globular (fig. c ) domains of golgi-associated viral tubes. nsm was detected in golgi membranes where new virus particles are assembling (fig. d ). western blot analysis confirmed the presence of nsm in isolated viral tubes and intracellular viral intermediates i and ii while extracellular mature virions lacked the protein (fig. e ). the same result was obtained by immunogold detection of nsm and em of isolated viruses disrupted in vitro (not shown). this distribution is typical for the behaviour of scaffolding proteins that assist in the assembly of immature viral precursors and are later degraded by proteolysis during maturation (steven et al., ) . to obtain functional information about the role of nsm in viral tubes and viral particles three nsm mutants were characterized at the ultrastructural level ( fig. f -h). these viruses obtained by a reverse genetics approach have deletions in nsm domains iii and iv (shi et al., ) . they grow slowly generating maximum virus yields -to -fold lower than wild-type bunv. when studying ultrathin sections of cells infected with nsmd mutant virus, viral tubes were rarely seen. more than cells infected with wild-type bunv and an equivalent amount of cells infected with nsmd mutant virus at , and h post infection (p.i.) were studied by oriented serial sectioning. while more than viral tubes per cell were detected when infecting with the wild-type virus, a total of complete tubes (longitudinal sections of the cylindrical domain) were seen in~ cells infected with the mutant virus. tubes assembled by the nsm mutant were apparently less rigid than normal, had small heads and exhibited a diameter of - nm (fig. f ). intracellular immature viral intermediates (type i viruses) accumulated in golgi membranes which suggests a blockade in viral particle maturation (fig. g) . a significant amount of viral glycoproteins was visualized at the plasma membrane by confocal microscopy (not shown) and budding profiles were also frequently seen at the cell surface (fig. h ). similar results were obtained with nsmd and nsmd deletion mutants, with small differences in the amount of intracellular virus particles and budding profiles in plasma membrane compared with nsmd virus (not shown). these data confirmed an active participation of nsm protein in tube structure and virus assembly in golgi, and pointed to viral tubes as elements involved in viral morphogenesis. matrix-assisted laser desorption ionization (maldi) peptide mass fingerprinting and database searching (navarro-lerida et al., ) was performed to detect cellular proteins in purified tubes. the highest scores obtained from this study corresponded to cytoskeletonassociated proteins (actin, myosin i and tubulin), several ribosomal proteins, the eukaryotic translation elongation factor and a retinoblastoma-like protein (table ) . considering the filamentous internal structure of the tubes ( fig. d ), we focused our attention on the cytoskeletal proteins. confocal microscopy showed that although actin is mostly removed from the viral factory some spots of actin remained in the modified golgi region (fig. a ). omitting the actin and golgi-associated signals showed a weak to-pro blue staining that most probably corresponds to rna accumulation in the factory (fig. b ). when the golgi signal is omitted, thin filamentous elements of actin are distinguished (fig. c ). the dimensions of these filamentous structures are compatible with those of viral tubes as seen by em. actin was detected by western blot in isolated tubes and intracellular viruses while it was absent in extracellular mature virions (fig. d ). actin was also detected by immunogold labelling in tubes and viruses as shown in cryosections of infected cells ( fig. e and f) and on isolated tubes ( fig. g -i). intact tubes exhibited a very weak or no immunogold signal (fig. g ), while tubes opened with saponin 'on grid' showed an enhanced signal on the released protein aggregates ( fig. h and i). an actinassociated signal was also detected on protein aggregates released from intracellular immature viruses disrupted in vitro (not shown). these results confirmed the presence of actin as a component of the internal fibres of viral tubes and its incorporation into immature viruses. assembly of viral tubes in golgi stacks could involve golgi membrane components or more stable golgi matrix proteins. to test these possibilities we used brefeldin a (bfa), a drug that induces the rapid fusion of golgi membrane with pre-golgi membranes and rer leaving a separated scaffold of matrix components (seemann et al., ) . from previous studies we knew that drugs disrupting cis-and medial-golgi subcompartments block bunv replication when added at - h p.i. (salanueva et al., ; novoa et al., a) . thus, short bfa treatments were applied to already infected bhk- cells. drug was added to the cultures at h p.i., followed by immunofluorescence staining for galt or giantin ( fig. a-f) . at or min after adding bfa the immunofluorescence signal for galt, a golgi membrane enzyme, was rapidly fragmented and dispersed while giantin, a golgi matrix protein, stayed in the perinuclear location for longer times. similar effects were seen after just min of treatment (data not shown). viral tubes deprived of surrounding golgi membranes had normal globular and cylindrical domains ( fig. g -i) and maintained their internal proteinaceous scaffolds and contacts with mitochondria ( fig. g ). tubes with apparent connections with cytoplasmic fibres that remind cytoskeletal intermediate filaments were observed (fig. h ). viral tubes with short bfa treatments then exhibited the same behaviour than giantin and remained as distinguishable structures when golgi stacks have already disappeared. several drugs that affect actin such as latrunculin a (lta), cytochalasin d (cyd) and jasplakinolide (jpk) were also used to target actin associated to golgi membranes. among them, the actin-stabilizing drug jpk provided the clearest results. lta and cyd had very little or no effect, respectively, on viral replication and assembly as determined by measurement of infectious viral particles released to the culture supernatants at and h p.i. complete tubes, virus budding profiles and viral particles assembled in golgi stacks of cells treated with lta, as observed in thin sections of treated cells studied by em (not shown). however, treatment with jpk led to a decrease in infectious virus release by - % in cell cultures that simultaneously lose the actin stress fibres as observed by fluorescence microscopy (fig. j and k) . em showed that in cells treated with jpk cell organelles were displaced to the cell periphery against the plasma membrane (not shown) and golgi fragments were occasionally distinguished (fig. l) . intact viral tubes were not detected although their globular domains were seen in golgi remnants (fig. l ) and attached to mitochondria (not shown). viral assembly was massively displaced to the plasma membrane where budding profiles were frequently seen (fig. m) . intracellular budding and viral particles in golgi remnants were very scarce and observed exclusively in cells less affected by the drug according to characteristic jpk effects in ultrastructure. budding profiles were not distinguished at the plasma membrane of normally infected cells where budding events were exclusively located in golgi stacks, as confirmed by analysis of oriented serial sections covering all planes from each infected cell. at least cells per condition were studied. our results strongly support a functional participation of golgi-associated actin in assembly and function of viral tubes as well as in anchoring virus assembly sites in golgi membranes. as viral tubes could play multiple functions in bunyavirus factories (replication, morphogenesis and organelle recruitment) a three-dimensional view of how they integrate in the factory and the contacts they establish with factory components can help us to understand how they work. we obtained serial sections of factories for a detailed study of the whole, complex structure. analysis of all planes provided a much better characterization of factories compared with random planes with a more accurate quantification of numbers and dimensions of viral structures. most tubes have a total length of . - . mm although tubes longer than mm were occasionally seen. d reconstructions from serial sections were done for early ( fig. a -f) and late (fig. g-i) factories. early factories with few viral particles are round shaped structures near the nucleus and surrounded by mitochondria (fig. a) . a close-up of the central areas of this factory showed viral tubes that connect golgi stacks with rer cisternae (fig. b) . the consecutive sections corresponding to these tubes are shown in fig. c . this frequent contact was never detected after random analysis of factories in d which points to the importance of covering all the planes of very complex structures in an oriented manner. an important contact previously detected in d analysis occurred between tubes and mitochondria ( fig. d and e) . careful study of d maps revealed that early factories are indeed composed of repetitive units of a basic set integrated by a golgi stack with mitochondria, rer and one or more viral tubes (fig. f ). by contrast, late factories are less compact structures that have abandoned the juxtanuclear location and contain many viral particles (fig. g ). mitochondria and rer do not completely surround the factory and virus particles are moving towards the cell periphery. physical contacts between viral tubes and new viral particles were detected as shown in the encircled area of fig. g and in fig. h and i. the existence of these contacts supports a role for the viral tubes as the physical connections between viral replication and morphogenesis inside the golgi stacks of the virus factory as proposed in the model of fig. . viral factories are probably the most extreme examples of how viruses manipulate cell organization. it is remarkable that just a few different classes of viral macromolecular complexes can transform a whole eukaryotic cell in minutes. we are interested in understanding how this happens. at the same time, by studying viruses, we have a very valuable tool to study cell architecture. in the present work we have characterized the bunyavirus factory built around the golgi complex. although the whole factory is large and complex it is in fact composed of repetitive units constituted by one or more golgi stacks, mitochondria, rer elements and a tubular structure that acts as a link between these organelles. further, the tubes could play multiple functions such as transfer of replicated viral genomes to assembly sites. actin is needed both for assembly of these tubes and for virus morphogenesis in golgi membranes. the presence of tubular structures in thin sections of mouse brain infected with bunv was reported in old literature where they were interpreted as elongated virus particles or nucleocapsids (murphy et al., ) . we also detected these structures in a previous study (salanueva et al., ) and estimated a discreet number of tubes, or , per golgi stack. analysis of serial sections has provided us with a much more accurate appreciation of their real numbers, as all planes of the cell are analysed. as a consequence several new data have been obtained: tubes are in fact more abundant (more than in many cells) and frequently connect with mitochondria and rer cisternae. our structural characterization of viral tubes in golgi membranes was completed with a molecular mapping of the structures both in situ and in vitro after their purification from infected cells. identification of viral factors involved in genome replication and morphogenesis as well as cell proteins such as the translation elongation factor or ribosomal proteins suggested that tubes may harbour fig. . d maps of early and late factories obtained by serial sectioning, tem, d-reconstruction and image processing. a. large round-shaped early factory near the nucleus. mitochondria (red) surround a network of membranes (yellow for the rer and beige for golgi stacks) where viral tubes (grey) are embedded. b. d reconstruction of the encircled central region of the factory in (a) elaborated with the central sections of the series to show contacts between viral tubes and cell membranes in more detail. the large tube is on the centre, a smaller one is encircled. c. three original serial thin sections included in the d map shown in (b). the central tube is marked with arrows. d and e. whole volume (d) and one single plane (e) of the same area of a factory showing a clear contact between a viral tube and a mitochondrion (arrows). f. basic factory unit constituted by a golgi stack, rer elements, mitochondria and a viral tube. a viral particle is coloured in purple. g. d map of a late factory. a significant displacement of components (mitochondria in red, rer in yellow) from the nuclear cavity (arrow) to peripheral areas of the cell is observed. golgi-associated density has been omitted in this volume for better visualization of viral particles (purple). a contact between viral tubes (grey) and viral particles (purple) is encircled. h and i. original thin section and d reconstruction, respectively, of a viral tube touching a viral particle (arrow). n, nucleus; ne, nuclear envelope; pm, plasma membrane; v, extracellular virions; c, centrioles. bar: nm in c; nm in h. post-replication events. a functional analysis of how altered nsm sequences or actin integrity affect factory architecture and viral assembly demonstrated that viral nsm protein and golgi actin are essential for the structure and function of the tubes. a sequence database comparison with bunv nsm showed homology with nsm proteins from other bunyaviruses and a low ( %) similarity with human atp p x receptors. immunoprecipitation assays showed that nsm interacts with n protein and the viral glycoproteins in infected cells (shi et al., ) . interestingly, nsm from tospoviruses, the plant-infecting bunyaviruses, is a movement protein associated with transport of rnps through plamodesmata (storms et al., ) . in mammalian cells tube-associated nsm could facilitate the transport of rnps from the rcs to the assembly sites. our data suggest that nsm could act also as a 'matrix' protein, facilitating binding of rnps to the cytosolic domains of viral glycoproteins gc and gn in golgi membranes. in fact its behaviour as a scaffolding protein that assists in viral assembly is supported by its presence in immature viral intermediates and its absence in mature extracellular virions. during tube assembly nsm aggregates seem to grow from the cytosolic side towards the interior of golgi sacculi creating a new space (fig. k) . as in situ labelling experiments showed that actin and giantin are inside viral tubes when they are normally facing the cytosolic side of golgi membranes, interactions of viral proteins with golgi actin and matrix proteins could be essential for tube assembly. despite its highly organized structure the golgi complex is a very dynamic organelle (james morre and mollenhauer, ) . nevertheless, the golgi manages to maintain its high degree of structural organization thanks to a large group of golgi-resident proteins that form a matrix. various cytoskeletal networks together with coiled-coil proteins of the golgin family, such as the cis-golgins p , gm and giantin, are components of this golgi matrix (short et al., ) that has been visualized by em of detergent-extracted golgi stacks (fath and burgess, ) . microtubules and associated motor proteins and the actin cytoskeleton are of particular importance in golgi organization. accordingly, several myosin motors localize to the organelle, where they are thought to contribute to the formation and transport of golgi vesicles. complex molecular machineries regulate actin dynamics involved in transport events in golgi membranes (fath et al., ; beck, ) . viruses manipulate actin in many different ways. they use actin for entry, intracellular transport, or cell-to-cell transmission (pelkmans et al., ; fackler and krausslich, ; radtke et al., ) . actin is involved in replication and transcription of both nuclear and cytoplasmic viruses and the infectious particles of retroviruses, herp-esviruses, and picornaviruses contain actin (grunewald et al., ; radtke et al., ) . when the actin cytoskeleton is destroyed with specific drugs a compaction of the golgi complex is observed (lazaro-dieguez et al., ) . curiously, this altered golgi is very similar to the round-shaped organelle we observed in bunv factory whose assembly occurs simultaneously with relocation of stress fibres to the cell periphery (fig. ) . in an attempt to test the potential function of actin in the structure and function of viral tubes we have used three actin-disrupting drugs. two of them depolymerize filamentous actin (cyd and lta) while the third one stabilizes filamentous actin (jpk). jpk stabilizes actin filaments in vitro, but in vivo it induces polymerization of monomeric actin into amorphous masses enhancing the rate of actin filament nucleation and disordered polymeric actin (bubb et al., ) . we used three drugs with different actin binding sites and different mechanisms of action because we wanted to target molecules associated to a very particular structure, the tubes inserted in golgi stacks. although we knew that viral tubes are open to the cytosol, we were not sure about efficiency and accessibility of drugs to actin molecules integrated in the tubular structure. in fact, while actindepolymerizing drugs had little effect on viral tube structure and virus production, the f-actin-stabilizing drug jpk altered the structure of viral tubes, the location of virus assembly sites and the release of infectious virus particles to the culture supernatants. golgi-associated actin seems to play a structural role in maintaining the structural integrity of the viral tubes in golgi membranes. brefeldin a is a drug that induces redistribution of most golgi-localized proteins to the er. studies on the nature of the golgi matrix scaffold showed that giantin, gm , grasp and grasp end up in membranes called bfa remnants that are distinct from the er (seemann et al., ) . these results supported the idea of a preexisting template for golgi stacks composed by these and other proteins. by using bfa we wanted to study if viral tubes disappear immediately when golgi membranes fuse with the er or if they stay for longer times following a common behaviour with the golgi matrix scaffold. like giantin viral tubes resisted short bfa treatments, which suggests they could be bound to the stable scaffold of golgi stacks. interestingly, integrity of golgi actin and viral nsm protein is also important for viral tube structure and normal production of infectious virions. our working hypothesis is that assembly of these viruses happens in golgi membranes because replication machinery anchors in golgi matrix components. a detailed study with silencing experiments and transfections with mutated components will be necessary to determine if particular golgi matrix components are specifically involved in the organization and activities of viral tubes. also to explore if myosin i molecules detected in isolated tubes are partici-pating in transport of rnps from the rcs to the assembly sites in nearby golgi membranes. according to our data, in particular the information coming from d maps and molecular detection, multiple interactions and movement of molecules must take place in viral tubes, as proposed in the working model of fig. . tubes represent a new structure that, in communication with the cytosol, would host viral rna replication and assembly of rnps in a protective environment, facilitating the posterior transfer of rnps to the assembly sites. the unusual cylindrical shape of this viral rc-containing structure might be related with the organization of golgi sacculi where viral tubes are anchored. actin and other matrix proteins can form the cellular protein scaffold for nsm interactions and tube growth while the actomyosin-based motors might mediate macromolecular movements. contacts with rer and mitochondria most probably provide necessary factors for tube functions. future work aims to locate viral and cellular proteins in tubes after d reconstruction by electron tomography (cyrklaff et al., ; mcintosh et al., ) . although factories are very large and complex structures for electron tomography we plan to use d maps from serial section reconstructions to assist in segmentation and interpretation of tomograms. in fact, our results also demonstrate that methods of d reconstruction from serial sections can benefit from the same principles of segmentation and noise reduction common to electron tomography. these procedures provide three-dimensional maps with enough resolution and details to analyse contacts between small elements such as virus assemblies inside very large and complex structures such as whole eukaryotic cells. this will hopefully help us to understand how viral macromolecular complexes interact with cell components to create the unique architecture of virus factories. bhk- (c ) cells supplied by the american type culture collection (atcc) were grown in dulbecco's modified eagle's medium supplemented with % foetal calf serum from reactiva s.a. (barcelona, spain). bunv (atcc vr- ) was propagated and subjected to titre determination in bhk- cells by a lysisplaque-forming assay, as previously described (salanueva et al., ) . bunvs containing deletions in the gene for nsm protein and designated rbunm-nsmd (aminoacids - were deleted from the m polyprotein), rbunm-nsmd (deleted aminoacids, - ) and rbunm-nsmd (deleted aminoacids, - ) were generated by reverse genetics as described (shi et al., ) . the mab monoclonal antibody against bunyamwera gc glycoprotein, and an anti-nsm rabbit antiserum against the peptide tdqkytldeiadvlqa (residues - of the m segment precursor) were described previously (watret et al., ; nakitare and elliott, ; lappin et al., ) . rabbit antisera against the amino-and the carboxyl-terminal domains of viral l protein were previously characterized (jin and elliott, ) . rabbit anti-n antiserum was obtained by immunization with a synthetic peptide corresponding to the aminoterminal peptide of the protein (mielefhdvaantsst) following standard procedures. the anti-giantin rabbit antiserum was a kind gift of m. renz (institute of immunology and molecular genetics, karlsruhe, germany). anti-b actin (clone ac- ) and a rabbit anti-actin antiserum were from sigma. anti-gal-t and anti-dsrna k mabs were kindly provided by t. suganuma (department anatomy, miyazaki medical college, japan) and n. lukacs (biological research center, institute of plant biology, szeged, hungary), respectively. anti-dsrna j mab was purchased from english and scientific consulting (hungary). anti-dsrna antibodies have been validated as markers of viral replication for different rna viruses (schonborn et al., ; westaway et al., ) . fluorescent secondary antibodies, alexa , phalloidin (green), wga (wheat germ agglutinin, red) and topro (marker for nuclei) were purchased from molecular probes/invitrogen. monolayers of bhk- cells were infected with bunv or nsm deletion mutant viruses at a multiplicity of infection (moi) of or plaque forming units (pfu) per cell. at , , or h p.i. culture supernatants were removed and cell monolayers processed for immunofluorescence or em. monolayers at h p.i. were incubated , or min with culture medium containing mg ml - bfa. for treatment with drugs for actin culture supernatants were removed at h p.i. and substituted by medium with mm cyd, mm lta or . mm jpk. at h p.i. cells were fixed and processed for microscopy. cell monolayers grown on glass coverslips were fixed min at °c with % paraformaldehyde in pbs before processing for immunolabelling of viral and cellular components as previously described (novoa et al., b; fontana et al., ) . a zeiss axiophot fluorescence microscope equipped with a micromax digital camera and a bio-rad radiance confocal laser microscope were used for image collection. cell monolayers were fixed h at room temperature with a mixture of % glutaraldehyde and . % tannic acid in hepes buffer (ph . ) and processed by conventional embedding in the epoxy-resin eml- (taab laboratories) or in lowicryl k m (taab) after cryo-processing by freeze-substitution as described (fontana et al., ) . ultrathin sections ( - nm) were collected on cupper grids, stained with uranyl acetate and lead citrate or processed for immunogold labelling, and studied in a jeol -ex ii electron microscope operating at kv. cryosections were obtained in an ultracryomicrotome (leica em fcs) operating at - °c by the standard tokuyasu method as described (salanueva et al., ) . for immunogold labelling primary antibodies were diluted in saturation buffer (pbs containing % bsa) as follows: : for anti-giantin, anti-l, anti-nsm and rabbit anti-actin, : for anti-n and : for anti-dsrna. secondary antibodies conjugated with nm colloidal gold particles from bbc were diluted : in the same buffer. negative staining of isolated tubes and viruses was performed with uranyl acetate using standard procedures. for immunogold labelling samples adsorbed to em grids were submitted to short incubations with primary and secondary antibodies before negative staining as described (novoa et al., b) . the newly synthesized viral rna in bunv-infected bhk- cells was labelled from to h p.i. with mm brutp (sigma). at h before labelling, mg of actinomycin d (sigma) per ml was added to the medium to block cellular rna synthesis. brutp was introduced into cells using dotap liposomal transfection reagent (roche) as described (westaway et al., ) . incubation with the mixture brutp-dotap-actinomycin d was maintained h at °c. cells were then washed with pbs, fixed with % paraformaldehyde + . % glutaraldehyde in pbs and processed for cryosectioning and immunogold labelling using a monoclonal anti-bromodeoxyuridine antibody (sigma) diluted : in saturation buffer followed by a secondary antibody-colloidal gold conjugate as described above. the established protocol for the isolation of intracellular viruses (novoa et al., b) was slightly modified for the purification of more labile structures such as the viral tubes. bhk- cells were infected at an moi of pfu/cell and maintained h at °c. cells were washed and collected in ten buffer containing protease inhibitors (roche), and frozen at - °c. after two consecutive cycles of freezing and thawing to release the viral tubes, cell lysate was clarified by centrifugation at g, min at °c. supernatant was centrifuged . h at g through a % (w/v) sucrose cushion. pellet was re-suspended in ml of ten with protease inhibitors and applied to a - % (v/v) optiprep iodixanol density gradient (sigma). centrifugation of samples was performed for . h at g. fractions of ml were collected from the top of the gradient and processed for structural and biochemical characterization. complete intact tubes localized in fractions - from the top, well separated from intracellular viral particles (fractions - ). controlled disruption of isolated tubes was done with saponin after adsorbing them on hydrophilic em grids. tubes were incubated with saponin ( . % in pbs) during s, s or min. after washing with pbs grids were processed for negative staining or immunogold labelling. purified viral tubes were processed by polyacrylamide gel electrophoresis (page) in the presence of sodium dodecyl sulfate (sds) with %, % or % acrylamide gels. coomasie blue staining was used to visualize protein bands (novoa et al., b) . for western blot analysis proteins were transferred from gels to nitrocellulose membranes by standard blotting procedures. membranes were saturated overnight at °c with pbs containing non-fat dry milk and . % tween , and incubated h at room temperature with primary antibodies diluted in saturation buffer ( : for anti-l, : for anti-nsm and : for anti-b actin). after washing with this buffer, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody diluted : . immunoreactive bands were detected by chemiluminescence (ecl kit, amersham). for rna dot-blotting samples were processed under rnasefree conditions as described (brown et al., ) . samples were incubated overnight at °c in a mixture of mg ml - proteinase k and % sds in depc water. a positive control (dsrna birnavirus ibdv, e strain) kindly provided by dr. d. luque (cnb, madrid), a negative control (yeast rna from sigma), purified extracellular bunv virions and fractions enriched in intracellular viral tubes were applied to nitrocellulose membranes. membranes were allowed to dry at room temperature during min and processed for immunodetection of total rna. after incubation with saturation buffer ( % dry milk in pbs) membranes were incubated h with a mixture of j and k anti-dsrna antibodies (diluted : each in saturation buffer), washed three times with the same buffer and incubated h with a secondary antibody conjugated with horse radish peroxidase diluted : in saturation buffer. after several washes in saturation buffer signal in membranes was visualized by chemiluminescence (ecl kit, amersham). fractions containing tubes as detected by negative staining and em and fractions without tubes were submitted to sds-page using % and % acrylamide gels to cover a wide molecular weight range. exclusive bands in fractions containing viral tubes were processed by maldi peptide mass fingerprinting and database searching as described (navarro-lerida et al., ) . ultrathin consecutive sections of approximately nm were collected on formvar-coated parallel-bar copper grids and stained with uranyl acetate and lead citrate and studied by em. after selecting an interesting region in the series, photographs were taken at ¥ magnification. using an epson perfection photo scanner the photographs were digitized at dpi as -bit images with a final pixel size of . nm. the images were then normalized using the bsoft software (heymann and belnap, ; http://lsbr.niams.nih.gov/bsoft/). sections were aligned by selected traces between two consecutive sections using the free editor for serial section microscopy 'reconstruct' (fiala ; http://www.synapse-web.org/tools/index.stm/) taking into account the 'tips for aligning sections' of the user's manual. as sections had an average thickness of nm the voxel in the serial section d reconstruction had an anisotropic size of . nm in xy axis and nm in z axis. segmentation and d visualization were done with amira (http://amira.zib.de). for noise-reduction, images were subjected to three iterations of a median filter (van der heide et al., ) . isosurface was used for d visualization, and the optimal threshold for the different materials was determined using a previously implemented algorithm (cyrklaff et al., ) . only elements with unequivocal identity were included in the d representations. a total of factories were studied by d reconstruction of serial sections and - sections per series were included. as individual sections have a thickness of - nm, the total thickness of the d maps in the z axis was around mm. spectrins and the golgi bunyamwera bunyavirus nonstructural protein nss is a nonessential gene product that contributes to viral pathogenesis analysis of rna by northern and slot blot hybridization effects of jasplakinolide on the kinetics of actin polymerization copi activity coupled with fatty acid biosynthesis is required for viral replication cryo-electron tomography of vaccinia virus molecular biology of the bunyaviridae emerging viruses: the bunyaviridae interactions of human retroviruses with the host cell cytoskeleton golgi-derived vesicles from developing epithelial cells bind actin filaments and possess myosin-i as a cytoplasmically oriented peripheral membrane protein molecular motors and a spectrin matrix associate with golgi membranes in vitro reconstruct: a free editor for serial section microscopy novel replication complex architecture in rubella replicon-transfected cells three-dimensional structure of herpes simplex virus from cryo-electron tomography subcellular localization of host and viral proteins associated with tobamovirus rna replication efficient automatic noise reduction of electron tomographic reconstructions based on iterative median filtering bsoft: image processing and molecular modeling for electron microscopy oligomeric structures of poliovirus polymerase are important for function microscopic morphology and the origins of the membrane maturation model of golgi apparatus function mutagenesis of the l protein encoded by bunyamwera virus and production of monospecific antibodies three-dimensional analysis of a viral rna replication complex reveals a virus-induced miniorganelle localization of bunyamwera bunyavirus g glycoprotein to the golgi requires association with g but not with nsm actin filaments are involved in the maintenance of golgi cisternae morphology and intra-golgi ph visualization and functional analysis of rna-dependent rna polymerase lattices wrapping things up about virus rna replication new views of cells in d: an introduction to electron tomography electron microscopic observations of mouse brain infected with bunyamwera group arboviruses expression of the bunyamwera virus m genome segment and intracellular localization of nsm proteomic identification of brain proteins that interact with dynein light chain lc virus taxonomy. fauquet virus factories: associations of cell organelles for viral replication and morphogenesis key golgi factors for structural and functional maturation of bunyamwera virus local actin polymerization and dynamin recruitment in sv -induced internalization of caveolae viral interactions with the cytoskeleton: a hitchhiker's guide to the cell polymorphism and structural maturation of bunyamwera virus in golgi and post-golgi compartments viral rna replication in association with cellular membranes monoclonal antibodies to double-stranded rna as probes of rna structure in crude nucleic acid extracts matrix proteins can generate the higher order architecture of the golgi apparatus requirement of the n-terminal region of orthobunyavirus nonstructural protein nsm for virus assembly and morphogenesis golgins and gtpases, giving identity and structure to the golgi apparatus virus maturation: dynamics and mechanism of a stabilizing structural transition that leads to infectivity the nonstructural nsm protein of tomato spotted wilt virus induces tubular structures in plant and insect cells synthesis of bunyavirus-specific proteins in a continuous cell line (xtc- ) derived from xenopus laevis nascent flavivirus rna colocalized in situ with double-stranded rna in stable replication complexes aggresomes and autophagy generate sites for virus replication this work was supported by grants bmc - and bfu - /bmc from the ministerio de educación y ciencia of spain (to c. risco), ja-p -tic and mec-tin - (to j.j. fernández) and wellcome trust programme grants and (to r.m. elliott). juan fontana and noelia lópez-montero are recipients of contracts from the comunidad de madrid. we thank sylvia gutiérrez erlandsson for expert support with confocal microscopy and silvia juárez for her support with peptide mass fingerprinting studies, reyes novoa and gloria calderita for technical assistance, sonia ruiz and carmen lópez-iglesias for support with cryosections, xiaohong shi for manuscript reading and alberto fraile-ramos for helpful discussions and critically reading the manuscript. key: cord- - j gq authors: elbein, alan d.; molyneux, russell j. title: alkaloid glycosidase inhibitors date: - - journal: comprehensive natural products chemistry doi: . /b - - - - . - sha: doc_id: cord_uid: j gq nan polyhydroxy alkaloids with glycosidase inhibitory properties have been isolated and identi_ed in the s and s\ with few exceptions[ discovery of the indolizidine alkaloids swainsonine and castanospermine\ with their potent and speci_c inhibitory activities towards a!mannosidase and a! and b!glucosidase\ respectively\ created a recognition that additional nitrogen!containing analogues of simple sugars might have similar properties and stimulated the search for new members of the class[ as a result\ more than naturally occurring members of the group have been discovered\ almost doubling the number discussed in a previous review[ another review has discussed these alkaloids\ with particular reference to their ecological signi_cance[ numerous synthetic analogues have been prepared\ but the scope of this chapter will be restricted to the chemistry and bioactivity of those alkaloids isolated from natural sources\ their glycosidase!inhibitory properties and consequent e}ects on glycoprotein processing[ the alkaloid glucosidase inhibitors discovered up until do not conform to a single structural class but do have several features in common\ including two or more hydroxyl groups and a nitrogen atom\ generally heterocyclic in character[ a small group of glycosidase inhibitors isolated from microorganisms also exists\ which are structurally more closely related to amino sugars[ however\ it is possible to integrate the major class of heterocyclic compounds into structural groups based upon _ve! and six!membered rings\ which may also be fused into bicyclic ring systems[ five di}erent subclasses can be de_ned\ from the simple monocyclic examples to the more complex bicyclic rings\ as follows[ "commonly used alternative or abbreviated names for individual alkaloids are shown in parentheses[# alkaloids of the pyrrolidine class\ with _ve!membered rings\ are exempli_ed by \ !dihydroxy! methyl! \ !dihydroxypyrrolidine "dmdp#\ " \ r oh#\ which is fully "tetra!#substituted at all carbon atom ring positions[ the trisubstituted representatives are !deoxy!dmdp " \ r h#\ \ !dideoxy! \ !imino!d!arabinitol "d!ab # " \ r b!oh#\ \ !dideoxy! \ !imino!d!ribitol " \ r a!oh#\ \ !dihydroxy! !hydroxymethyl! !pyrroline "nectrisine# " \ r b!oh^ \ !double bond#\ and n!hydroxyethyl! !hydroxymethyl! !hydroxypyrrolidine " #\ the only alkaloid in this group bearing a substituent on the nitrogen atom[ only a single disubstituted member of the group is known\ namely !hydroxymethyl! !hydroxypyrrolidine "cyb # " \ r h#[ a pentahydroxy alkaloid\ \ !dideoxy! \ !imino!dl!`lycero!d!manno!heptitol "homodmdp# " \ r ch oh# and its !apioside " \ r ch o!apiose# have been isolated and structurally identi_ed[ homodmdp is thus the most highly hydroxylated representative of the pyrrolidine class[ ( ) ( ) ( ) alkaloids with six!membered rings of the piperidine class encompass nine members\ one of which\ !deoxyfagomine " \ r h\ r b!oh#\ is disubstituted\ while two\ namely fagomine " \ r oh\ r b!oh# and !epi!fagomine " \ r oh\ r a!oh#\ are trisubstituted[ an additional three alkaloids\ !deoxynojirimycin "dnj# " \ r a!oh# and its n!methyl derivative\ and !deoxymannojirimycin "dmj# " \ r b!oh#\ are tetrasubstituted[ the latter has also been found to occur as a series of glycosides\ namely] !o\ !o\ and !o!a!d!glucopyranosides^ !o\ ! o\ !o\ and !o!b!d!glucopyranosides^and\ !o and !o!a!d!galactopyranosides[ the remaining four alkaloids are characterized by complete substitution at all carbon atoms\ and include the glucose analogue\ nojirimycin " \ r a!oh\ r a!oh#\ the mannose analogue\ nojirimycin b "mannojirimycin# " \ r b!oh\ r a!oh#\ and the galactose analogue\ galactostatin " \ r a!oh\ r b!oh#[ a!homonojirimycin "hnj\ " ## has a hydroxymethyl group at the !position\ in place of the hydroxy group found at that position in nojirimycin\ and the alkaloid has also been isolated as its the pyrrolizidine alkaloids that are inhibitors of glycosidases may be regarded in a formal structural sense as the result of fusion of two pyrrolidine ring systems\ with the common nitrogen atom at the bridgehead[ the tetrasubstituted pyrrolizidines\ australine " # and alexine " # di}er only in the stereochemistry at the bridgehead carbon atom "c! a#\ all other substituents having identical con_gurations[ a certain amount of confusion has arisen in the naming of epimers of these compounds because those having a bridgehead con_guration identical to that of australine have been classi_ed as a!epi!alexines[ in fact\ alexine itself is the only member of this group isolated to date which has an a bridgehead proton[ harris et al[ have proposed that all alkaloids having the r stereochemistry be named as australines and those with the s stereochemistry as alexines[ adopting this convention\ the three known naturally occurring epimers would therefore be named as follows\ "with the alternate name in parentheses#] !epi!australine " \ a!di!epi!alexine# " \ !oh\ a# !epi!australine " \ a!di!epi! alexine# " \ !ch oh\ a#\ and !epi!australine " \ a!di!epi!alexine# " \ !oh\ b#[ a unique tetrasubstituted pyrrolizidine alkaloid is a!epi!alexa~orine\ which also has a a!"r# bridgehead con_guration\ consistent with all the other alkaloids except alexine\ and may be regarded as an oxidized form of australine[ on the basis of its physical properties\ including resistance to melting and insolubility in all solvents except for water\ together with evidence of a carboxylate ion in its infrared spectrum\ this alkaloid was shown to exist in the zwitterionic form " #[ h an interesting addition to the class has been casuarine " \ r h#\ a highly oxygenated penta!substituted pyrrolizidine[ this alkaloid has also been found as the !glucoside " \ r a!d! glucosyl#[ the occurrence of several australine:alexine epimers suggests that epimeric forms of casuarine will ultimately be discovered[ in an analogous manner to the pyrrolizidine alkaloids\ the indolizidine group may be visualized as a pyrrolidine ring fused with a piperidine ring\ yielding a bicyclic : ring system[ seven naturally occurring members have been discovered\ the simplest of which are the dihydroxylated alkaloids\ lentiginosine " \ r b!oh# and !epi!lentiginosine " \ r a!oh#[ the familiar trihydroxylated alkaloid swainsonine " # \ Ð is unique within the indolizidine class as the only member with an a!"r# bridgehead con_guration[ a second trihydroxyindolizidine\ !deoxy! !epi!castanospermine " \ r b!oh\ r h# has the a!"s# con_guration charac! teristic of the tetrahydroxy alkaloid\ castanospermine " #\ and its epimers !epi!castanospermine " \ r b!oh\ r b!oh# theoretically\ pentahydroxylated indolizidines\ corresponding to casuarine\ could occur but none have yet been isolated from natural sources[ the polyhydroxy pyrrolidine\ piperidine\ pyrrolizidine\ and indolizidine groups have been estab! lished for some time but the nortropane group is a relatively new addition to the catalog of alkaloid classes with glycosidase!inhibitory properties[ whereas tropane alkaloids are well!known in nature\ nortropanes "i[e[\ compounds in which the nitrogen atom is not methylated# are relatively rare[ the nortropane ring system can be conceptualized as a result of fusion of a _ve!membered pyrrolidine ring with a six!membered piperidine ring\ but in contrast to the indolizidines the fusion points are a to the nitrogen atom of each monocyclic system[ the polyhydroxy nortropane group now consists of more individual alkaloids than any of the other classes\ and the chemistry of these compounds has been the subject of a review[ the alkaloids have been named calystegines after the source of the _rst member to be isolated\ the bindweed calyste`ia sepium[ \ a consistent feature of all calystegines\ in addition to the absence of n! methylation\ is the presence of an a!oh group at the bridgehead junction "c! # of the bicyclic ring system "i[e[\ an aminoketal functionality#[ three subclasses have been de_ned\ namely calystegines a\ b\ and c\ each of which corresponds to tri!\ tetra! and pentahydroxylation\ respectively[ four trihydroxylated alkaloids\ calystegines a " \ r a!oh\ r h#\ a " \ r h\ r a!oh#\ a " #\ and a " # are known[ although the majority of calystegines bear an equatorial hydroxyl group at the c! position\ the latter two alkaloids lack this substituent\ while calystegine a is unique within the a subgroup in possessing a secondary hydroxyl group on the _ve!membered ring moiety[ the calystegine b alkaloids consist of _ve tetrahydroxylated compounds\ namely b " #\ b " \ r a!oh\ r a!oh#\ b " \ r b!oh\ r a!oh#\ b " \ r a!oh\ r b!oh#\ and b " #[ calystegine b is the only alkaloid within this subgroup that does not have a !oh substituent[ although both calystegines b and b have secondary hydroxy groups on the _ve!membered ring moiety\ these occur at di}erent positions\ namely c! and c! \ respectively[ the remaining three members\ calystegines b \ b and b \ di}er only in the stereochemistry of the hydroxy groups located at c! and c! on the six!membered ring^the c! hydroxy substituent is b in all three alkaloids[ an alkaloid named calystegine n \ corresponding to calystegine b but with an amino group\ rather than a hydroxy group\ at the bridgehead c! position has also been obtained[ however\ reactions of nojirimycin derivatives with ammonia!saturated methanol\ resulting in replacement of the !oh group by an nh substituent\ suggest that calystegine n is an artifact of the isolation procedure\ which involves elution from an ion!exchange column with dilute ammonium hydroxide[ two pentahydroxylated calystegine c alkaloids are known\ having identical substitution patterns\ including a hydroxy group at c! analogous to calystegine b [ these alkaloids\ calystegines c " \ r a!oh# and c " \ r b!oh#\ di}er only in the stereochemistry of the c! hydroxy substituent[ oh oh two additional alkaloids\ bearing axially oriented methyl groups on the nitrogen atom\ have been isolated and structurally characterized[ these compounds\ n!methylcalystegine b " \ r h# and n!methylcalystegine c " \ r oh# should strictly be classi_ed as tropane alkaloids but the preponderance of polyhydroxy nortropanes isolated to date suggests that these new alkaloids are the result of n!methylation of the latter rather than products of the normal biosynthetic route to tropane alkaloids[ for the purposes of this chapter they are therefore classi_ed within the nortropane group[ the polyhydroxy alkaloid glycosidase inhibitors have been isolated primarily from plant sources\ but also occur in microorganisms and have occasionally been found in insects[ the sources of the individual alkaloids are listed in table [ many of the earliest polyhydroxy alkaloids to be discovered\ particularly the bicyclic pyrrolizidines and indolizidines\ were found in the plant family legumin! osae[ this apparent taxonomic relationship has now become far less secure with the isolation of casuarine " # from the casuarinaceae and myrtaceae[ moreover\ swainsonine " # has been identi_ed as a constituent of several ipomoea species "convolvulaceae#\ co!occurring with calys! tegines[ similarly\ the initial isolation of calystegines "" #Ð" ## from the convolvulaceae \ \ has now been overshadowed by a much more widespread occurrence in the solanaceae\ \ Ð \ \ \ and a limited presence in morus species "moraceae#[ certain individual alkaloids\ predominantly dmdp and swainsonine\ have a particularly wide! spread pattern of occurrence[ thus\ dmdp " \ r oh# has been isolated from plants in the families araceae\ campanulaceae\ euphorbiaceae\ hyacinthaceae\ and leguminosae\ \ \ as well as from the body of a lepidopteran "urania ful`ens#\ and from a streptomyces species[ similarly\ swainsonine " # has also been discovered in two unrelated microorganisms\ rhizoctonia le`uminicola and metarhizium anisopliae\ \ in addition to its quite widespread occurrence in plants[ it has been shown that the biosynthetic pathways to swainsonine in the diablo locoweed\ astra`alus oxyphysus\ and r[ le`uminicola are identical\ implying either a direct or indirect relation! ship between plant and microorganism[ thus\ the genetic ability to produce this alkaloid could have been transferred from one to the other in the course of evolution[ alternatively\ microorganisms capable of producing the alkaloid may have an endophytic association with the plants[ the presence of a calystegine!catabolizing rhizobium meliloti strain in roots of calyste`ia sepium but not within plants that do not produce calystegines emphasizes the complexity of such interactions[ in contrast to the previous examples\ castanospermine " # and its epimers " # \ \ and the australine:alexine "" #Ð" ## Ð alkaloids have so far been restricted to the monotypic castanospermum australe and species of alexa\ which are closely related genera in the leguminosae[ it is apparent from these examples that no consistent conclusions can be drawn regarding the distribution of polyhydroxy alkaloids at the present time[ it may be that these natural products are quite widely distributed and many new sources will be discovered in the future[ the comparative newness of their discovery relative to many other classes of alkaloids is probably a consequence of their cryptic nature\ due to exceptional water solubility and relative insolubility in non!hydroxylic organic solvents[ the increasing number\ regio! and stereochemical potential for structural vari! ation and signi_cant biological properties of these glycosidase inhibitors will no doubt result in discovery of new members of the known classes[ the identi_cation of the nortropane group is also an indicator that new structural groups may yet remain to be discovered[ the hydrophilicity of the polyhydroxy alkaloids renders them incapable of being isolated by conventional extraction and puri_cation methods which involve extraction into nonpolar organic kifunensine " # kitasatosporia kifunense a!mannosidase nagstatin " # streptomyces amakusaensis b!n!acetyl! glucosaminidase * ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ solvents and partitioning between aqueous acid and base[ ion!exchange chromatography is therefore generally employed for puri_cation\ following extraction from the natural source by water\ methanol or ethanol\ either alone or in various mixtures[ subsequent separation can be achieved by paper\ column\ or thin!layer chromatography[ the alkaloids are particularly amenable to detection by thin!layer chromatography in association with speci_c spray reagents\ gas chromatography with ame ionization or mass spectrometric detection\ and by their glycosidase inhibitory properties[ all of these techniques have been reviewed in detail[ structural determination places a particular reliance on nuclear magnetic resonance spectroscopy which generally permits establishment of the speci_c ring system present\ the substitution pattern\ and relative stereochemistry of the hydroxy groups[ mass spectrometry provides similar information\ with the exception of stereochemistry[ the isolation of increasing numbers of these alkaloids has furnished a spectroscopic database which renders the determination of structures increasingly facile[ determination of the absolute stereochemistry is dependent upon x!ray crystallography\ which can be used whenever well!re_ned crystal data can be obtained\ either from the alkaloid itself or a crystalline derivative such as the hydrochloride salt [ the inhibitory activity of individual alkaloids may be remarkably speci_c\ as with swainsonine\ which inhibits only a!mannosidase and golgi mannosidase ii\ or can be more general\ showing a spectrum of activity against a series of glycosidases[ additionally\ the potency may vary with the source of a particular enzyme\ its purity\ and the conditions\ such as ph\ under which the assay is performed[ for these reasons the inhibitory properties of individual alkaloids are presented here only in a summary form " table # [ the inhibition of n!linked glycoprotein processing by the most potent and speci_c of the alkaloids is discussed in detail in section [ [ ^particulars regarding other alkaloids should be obtained from the publications referenced in table [ early approaches to correlation of structure of the polyhydroxy alkaloids with their glycosidase inhibitory properties appeared to indicate a rather straightforward relationship[ swainsonine " # was perceived as an aza!analogue of d!mannopyranose\ lacking the hydroxymethine group at c! \ but otherwise having the same relative disposition of the remaining hydroxyl groups\ which therefore accounted for its ability to inhibit a!mannosidase[ the structures of !deoxynojirimycin " \ r a!oh# and castanospermine " # correlated even more closely\ as monocyclic and bicyclic {{aza sugars||\ with that of glucose\ and they inhibited glucosidases as expected[ this naive approach had to be reconsidered with the isolation of !epi!castanospermine " \ r b!oh\ r b!oh# which\ in spite of its stereochemical similarity to mannose\ failed to inhibit either a! or b!mannosidase but instead proved to be an e}ective inhibitor of a!glucosidase\ with a level of activity only slightly less than that of castanospermine[ numerous additional examples of inhibitory speci_cities due to both naturally occurring alkaloids and synthetic analogues have further undermined this empirical approach and it is obvious that structureÐactivity correlations can only be developed with the aid of sophisticated molecular modeling techniques[ molecular orbital calculations and molecular modeling have been applied to a series of known mannosidase inhibitors and others which were expected to inhibit but failed to do so[ the results showed that good inhibitors _t closely with a single low!energy conformer of the mannosyl cation and demonstrated that !epi!castanospermine did not comply with the structural requirements[ \ the electronegative binding groups present in the inhibitor necessary for speci_city and activity were established\ as were those which were of little signi_cance[ additional studies of this type should provide valuable information regarding the receptor sites on the various enzymes but the inhibition data available is compromised by the variability in enzymes and the conditions under which measurements have been made[ a comprehensive screening program using standardized conditions would provide much more useful information for structureÐactivity correlations and consequently the design of speci_c and potent inhibitors[ the crystal structures of glucoamylase and its complex with the inhibitor !deoxynojirimycin " \ r a!oh# have recently been reported[ this structural data has now been used in a molecular modeling study\ using !deoxynojirimycin and other deoxynojirimycin derivatives\ dmdp " \ r oh#\ australine " #\ and castanospermine " #\ to probe the active site of the enzyme[ pre! liminary results indicated that binding to speci_c residues within the active site were essential for inhibitory activity and that the inhibitory potency was dependent upon the number of hydrogen bonds involved in such binding[ however\ although castanospermine is an excellent inhibitor of the enzyme it lacked these requirements and therefore did not conform to the model[ nevertheless\ this approach illustrates the potential value of such methods for understanding enzymeÐinhibitor interactions\ which should prove useful with increasing re_nements in the models and available structural data[ in the absence of more comprehensive molecular modeling studies\ the inhibition results obtained have been rationalized on the basis of generally accepted models for glycosidase inhibition[ this approach has been developed most e}ectively for the calystegines\ which provide a comprehensive series of structurally related natural polyhydroxy alkaloids[ for b!glucosidase inhibition\ the model involves the presence of two carboxylic acid groups at the active site of the enzyme\ one responsible for generation and the other for stabilization of the glycosyl cation intermediate[ it has been speculated that for calystegines b " # and c " \ r a!oh#\ the exo hydroxy group at the !position is protonated by the acidic group responsible for catalytic activity within the active site\ in an analogous manner to the inhibitor conduritol b epoxide[ in contrast\ calystegine b " \ r a!oh\ r a!oh#\ which shows a similar level of inhibitory activity towards b!glucosidase\ is supposed to be bound to the glucosyl cation binding site through the hydroxyl group at the !position[ the essential requirement of equatorial hydroxyl groups at the ! and !positions is in accord with earlier studies of interaction of other inhibitors with b!glucosidase[ thus\ the interaction of inhibitory calystegines with glycosidases can be envisioned as binding to the sites determining speci_city and to the catalytic center\ through speci_c hydroxyl groups and through the imino group[ the mechanism of galactosidase inhibitory activity is less apparent[ calystegines b \ b and c are potent inhibitors of either a or b!galactosidase\ yet calystegine b " \ r b!oh\ r a!oh#\ with a much closer con_gurational similarity to d!galactose than any of the former\ has no inhibitory activity against these enzymes\ an observation which is reminiscent of the situation with !epi! castanospermine in the indolizidine alkaloid series[ obviously\ a much larger set of natural or synthetic epimers\ enantiomers and structural analogues is needed before a complete understanding of structureÐactivity relationships can be applied to prediction of inhibitory activity[ some progress in this direction has been made through a comparison of glycosidase inhibition by synthetic analogues and derivatives of "¦#!calystegine b [ the nonnatural "−#!enantiomer showed no gly! cosidase inhibitory properties\ whereas n!methylation of natural b suppressed inhibition of b!glucosidase while activity towards a!galactosidase was retained[ in addition to the synthesis of known naturally occurring alkaloids for the purpose of structural con_rmation\ many epimers\ enantiomers\ and structural analogues have been prepared[ the num! ber of these synthetic alkaloids\ particularly those related to swainsonine\ castanospermine\ and australine\ now approaches or perhaps exceeds those isolated from natural sources[ the natural product focus of this review does not permit a comprehensive survey of these compounds[ various aspects of the synthetic approaches\ either a priori syntheses or those routes commencing from carbohydrate!based templates\ have been summarized in a number of publications[ Ð nonnatural epimers have been prepared by modi_cation of natural alkaloids which are available in large quantities\ such as castanospermine\ and ring!expanded analogues of pyrrolizidine and indolizidine alkaloids have also been synthesized[ \ it is probable that at least some of the synthetic compounds\ especially epimers of known naturally occurring alkaloids\ will subsequently be found to occur in nature[ in addition\ new structural classes have already been generated which might reasonably be expected to be biosynthesized by plants[ predominant among these are polyhydroxy quinolizidine alkaloids\ consisting of two six! membered rings fused into a bicyclic system\ which are ring!expanded homologues of the indolizidine alkaloids[ \ although quinolizidine alkaloids are a well!established class of natural products\ none have yet been isolated that bear more than two hydroxyl groups[ this is probably a consequence of the high water solubility of polyhydroxylated alkaloids which renders them unextractable into the nonhydroxylic solvents normally used for alkaloid puri_cation[ the combination of novel natural polyhydroxy alkaloids\ together with synthetic analogues tailored to have speci_c structural features\ will ultimately lead to a full comprehension of the interaction of these alkaloids with receptor sites on the enzyme which results in their glycosidase inhibitory properties[ as might be expected from a class of compounds that inhibits glycosidases and consequently the fundamental cellular function of glycoprotein processing\ the polyhydroxy alkaloids exhibit an exceptional diversity of biological activities[ discovery and isolation of many of the alkaloids has been a result of observations of the ultimate clinical e}ects which result from the consumption by animals of plants containing these bioactive compounds[ predominant among such examples is the occurrence of swainsonine " # in swainsona species "poison peas# of australia and astra`alus and oxytropis species "locoweeds# of north america[ the potent a!mannosidase inhibitory activity of swainsonine disrupts glycoprotein processing by mannosidase ii in the golgi\ resulting in neuronal vacuolation due to abnormal storage of mannose!rich oligosaccharides\ leading to the neurological damage so characteristic of the locoism syndrome[ however\ the clinical e}ects are not limited to the nervous system since emaciation\ reproductive failure in both males and females\ and congestive right!heart failure are also observed[ since the discovery of swainsonine as the causative agent\ locoweed poisoning has now been established as a widespread phenomenon\ with additional occur! rences being reported from south america and many parts of china and tibet[ swainsonine has been reported to co!occur with calystegines b " \ r a!oh\ r a!oh# and c " \ r oh# in ipomoea species of australia which cause poisoning of sheep and cattle\ in contrast to the above examples which exhibit a complexity of e}ects\ the alkaloids concentrated in the chestnut!like seeds of castanospermum australe "black bean#\ primarily castanospermine " # and australine " #\ together with several less potent epimers of both\ produce gastrointestinal disturbances in livestock and humans but no discernable neurological damage[ this is consistent with the ability of the alkaloids to inhibit a! and b!glucosidase\ resulting in a syndrome phenotypic of the genetic defect\ pompe|s disease[ although this relationship has not been directly established in _eld cases of poisoning\ rodent feeding experiments with castanospermine resulted in vacuolation of hepatocytes and skeletal myocytes\ and glycogen accumulation\ consistent with pompe|s disease or type ii glycogenesis[ gastrointestinal problems and lethargy have also been observed in livestock grazing bluebells "hyacinthoides non!scripta# in the uk\ and the demonstration of the presence of dmdp and homodmdp in this plant may account for the syndrome[ all of the above poisoning syndromes are relatively obvious once signs develop\ although this may take several weeks of consumption of the plant because the alkaloids implicated often are present at very low levels[ nevertheless\ they are potent inhibitors and it has been estimated that a swainsonine content of [ ) of the dry weight of the plant may be su.cient to induce locoism[ for those alkaloids which are less active or which are present at extremely low levels\ it seems probable that the signs of poisoning would be subclinical\ with no overt changes being apparent[ in such cases\ toxicity may only be manifested as minor digestive disturbances\ failure to gain weight and other deviations from optimal health which could be attributed to stress or infectious diseases[ the occurrence of various calystegines in human food plants from the family solanaceae\ such as potatoes\ eggplant and peppers\ could account for a variety of complaints\ primarily gastrointestinal\ reported in certain individuals consuming these vegetables[ it should be anticipated that compounds capable of inhibiting glycosidases would have an inhibitory e}ect on digestive enzymes\ and defense against herbivorous insects may be one of the roles played by the polyhydroxy alkaloids in plants which contain them[ conversely\ it is well established that insects co!evolve with their host plants to circumvent such defenses and utilize the active constituents for their own defense[ such strategies involving speci_c alkaloids have been demonstrated for several plantÐinsect relationships[ castanospermine " # added to an arti_cial diet is highly inhibitory to feeding by the pea aphid\ acyrthosiphon pisum\ with a ) deterrency level of ppm\ and a consequent very low survival rate[ although the alkaloid does not inhibit aphid trehalase\ it has been shown di}erentially to inhibit a number of disaccharidases from a wide taxonomic distribution of insects[ cas! tanospermine has also been shown to be an antifeedant compound to the egyptian cotton leafworm\ spodoptera littoralis\ as are d!ab " \ r b!oh#\ dmdp " \ r oh#\ and swainsonine " #[ dmdp also appears to be a particularly e}ective feeding deterrent to nymphs of the locusts schistocerca`re`aria and locusta mi`ratoria\ at levels as low as [ ) of the body weight[ since these alkaloids inhibit di}erent enzymes\ it is di.cult to correlate antifeedant activity with inhibition of digestive enzymes alone[ it is possible that deterrency may also be a consequence of blocking of the sensory response to glucose[ insect resistance to the e}ects of the alkaloids has been observed[ thus\ the bruchid beetle callosobruchus maculatus\ a feeder on legumes that do not produce dmdp\ has a gut a!glucosidase which is times more sensitive to the alkaloid than that of ctenocolum tuberculatum\ which has adapted to feed exclusively on dmdp!containing species of the legume subtribe lonchocarpinae[ among the lepidoptera\ the aposematically!colored moth\ urania ful`ens\ accumulates dmdp and a!homonojirimycin " # from its food plant\ the vine omphalea diandra\ but does not sequester the other alkaloid present\ !deoxymannojirimycin " \ r b!oh#\ while the death|s!head hawkmoth procures calystegines from its solanaceous hosts[ the mechanism of resistance to the e}ects of the alkaloids is not understood but it appears likely that those which are accumulated serve a protective role in the insect[ in contrast\ alkaloids which may be harmful can be speci_cally excreted[ for example\ pea aphids feeding upon the spotted locoweed\ astra`alus lenti`inosus\ excrete in their honeydew swainsonine " # acquired from the phloem of the plant\ while showing no feeding deterrency[ since this plant was colonized opportunistically in the laboratory and is not a normal host for the pea aphid\ the implication is that certain insects my have a general ability to com! partmentalize and eliminate polyhydroxy alkaloids that might otherwise be harmful[ polyhydroxy alkaloids from several of the structural classes have been shown to be inhibitory to the growth of plants[ particularly noteworthy in this respect is castanospermine " # which has been demonstrated to be a potent root elongation inhibitor of lettuce\ lactuca sativa\ alfalfa\ medica`o sativa\ barnyard grass\ echinochloa crus`alli\ and red millet\ panicum miliaceum[ the alkaloid was much more e}ective against the dicots\ showing ) inhibition of root length growth at ppb\ while the monocots were times less sensitive[ the structurally related indolizidine alkaloid\ swainsonine " #\ failed to exhibit any phytotoxic activity against these species\ indicating that the bioactivity is a consequence of a! or b!glucosidase inhibition but not of a!mannosidase inhibition[ nojirimycin " \ r a!oh\ r a!oh# is inhibitory to cell extension of pisum sativum stem segments and of coleoptiles of avena and triticum\ induced by auxins[ there is considerable evidence that elongation is a consequence of cell!wall loosening due to degradation or depolymerization of xyloglucans by exo!b!glucanases and inhibition of these enzymes by the alkaloid could therefore account for the failure of the cells to elongate[ the phytotoxic e}ects of the polyhydroxy alkaloids may confer a major competitive advantage upon plants which biosynthesize them through the phenomenon of allelopathy[ the alkaloids are highly water soluble so that excretion into the surrounding soil or leaching from various parts of the plant can suppress the growth of encroaching species through creation of a zone of inhibition[ at the same time\ movement of water would transport the compounds through the soil so that concentrations in the vicinity of the secreting plant itself do not attain levels high enough to induce self!inhibition[ however\ there is some evidence that castanospermum australe seeds may be inhibited from germination by the presence of castanospermine " #[ considerable irrigation is required before the seeds commence to sprout and this may be a valuable strategy in the native environment where rainfall is highly seasonal\ enabling germination and rooting to take place only when the rainy season is well!established[ natural calystegine b \ that is the "¦#!enantiomer " \ r a!oh\ r a!oh#\ showed sig! ni_cant inhibition of alfalfa seed germination\ and growth and lateral production of roots trans! formed by a`robacterium rhizo`enes\ but corresponding e}ects were not observed with the unnatural "−#!enantiomer[ root length was reduced by ) after treatment for h with mm "¦#!calystegine b \ while under the same conditions the unnatural "synthetic# alkaloid caused an ) increase in root length[ such results demonstrate the dependency of bioactivity upon speci_c structural conformations and stereochemistry[ there has been little information reported in regard to the e}ect of polyhydroxy alkaloid inhibitors on growth or function of microorganisms\ although nojirimycin " \ r a!oh\ r a!oh# was discovered as a result of the antimicrobial activity of streptomyces nojiriencis\ s[ roseochromo`enes\ and s[ lavandulae against a drug!resistant strain of shi`ella~exneri[ the antibiotic activity of the same alkaloid towards xanthomonas oryzae renders it capable of preventing the bacterial leaf blight of rice[ the calystegines were _rst isolated from roots of the bindweed\ calyste`ia sepium[ although these alkaloids have now been detected in other plant parts\ there appears to be a relatively high abundance in subterranean organs of the convolvulaceae and solanaceae and they are therefore believed to be nutritional mediators between such plants and associated rhizosphere bacteria[ over ) of the bacteria isolated from the rhizospheres of calystegine!producing plants were capable of catabolizing the alkaloids\ whereas no bacteria with this ability were obtained from plants which did not elaborate calystegines[ in addition\ wild!type rhizobium meliloti was capable of using natural "¦#!calystegine b " \ r a!oh\ r a!oh# as an exclusive source of carbon and nitrogen\ whereas a catabolism!de_cient strain of r[ meliloti was not[ furthermore\ neither organism could utilize the unnatural\ synthetic enantiomer\ "−#!calystegine b [ the ability to catabolize such compounds\ which at the same time may have antibiotic properties towards other microorganisms\ has an obvious competitive advantage for those speci_c bacteria capable of utilizing them[ the capability of polyhydroxy alkaloids to disrupt the general cellular function of glycoprotein processing leads to the expectation that these compounds should have therapeutic potential for the treatment of various disease states[ the signi_cant mammalian toxicity of certain of the alkaloids is an obvious hindrance to their utility[ however\ this is frequently true of many drug candidates and it is not unreasonable to assume that an appropriate doseÐresponse relationship could be achieved[ moreover\ adverse e}ects\ such as the neurological damage caused by swainsonine\ often develop quite slowly and appear to be reversible if ingestion of the alkaloid is terminated\ as would be the situation with most drug regimens[ investigation of the alkaloids for therapeutic potential has so far concentrated on three major disease states\ namely for treatment of cancer and inhibition of metastasis\ as antidiabetic drugs\ and for antiviral activity[ swainsonine " # has received particular attention as an antimetastatic agent[ in vivo experiments with mice have shown that pulmonary colonization is reduced by over ) if the animals are provided with drinking water containing mg ml − of swainsonine for h prior to injection with b !f murine melanoma cells[ this e}ect has been shown to be due to enhancement of natural killer t!cells and increased susceptibility of cancerous cells to their e}ect[ the pharmacokinetics of swainsonine in such experiments indicate that the levels of alkaloid and period of administration would not be su.cient to produce neurological damage[ it has been suggested that post!operative metastasis of tumor cells in humans could be suppressed by intravenous administration of the alkaloid prior to and following the surgery[ clinical trials in humans with very advanced malig! nancies showed that lysosomal a!mannosidases and golgi mannosidase ii were inhibited and some improvement in clinical status occurred[ castanospermine has also been reported to suppress metastasis in mice but experiments with this alkaloid have not been as extensive as those with swainsonine[ castanospermine " # and !deoxynojirimycin " \ r a!oh# have been shown to be capable of suppressing the infectivity of a number of retro viruses\ including the human immunode_ciency virus "hiv# responsible for aids[ Ð this e}ect is a consequence of inhibition of glycoprotein processing which results in changes in the structure of the glycoprotein coat of the virus[ cellular recognition of the host is thus prevented and syncytium formation is suppressed[ in spite of this signi_cant e}ect\ both of these alkaloids su}er from the disadvantage that they are highly water! soluble and therefore excreted very rapidly[ this defect has been overcome by derivatization to give !o!butyryl!castanospermine and n!butyl!deoxynojirimycin\ and both of these compounds have undergone clinical trials against aids in humans\ either alone or in combination with azt[ as might be expected\ gastrointestinal disturbances have been reported as a signi_cant side e}ect[ another structural modi_cation of !deoxynojirimycin\ the n!hydroxyethyl derivative\ miglitol\ an inhibitor of a!glucosidase\ has been clinically evaluated and released as an antidiabetic drug in insulin! and noninsulin!dependent diabetes[ the alkaloid was shown potently to inhibit glucose! induced insulin release and also suppressed islet a!glucoside hydrolase activity\ thus controlling postprandial glycemia[ the structurally related alkaloids\ !o!a!d!galactopyranosyl!dnj and fagomine\ have also been shown to have antihypoglycemic activity in streptozocin!induced diabetic mice but have not been tested in humans[ the ability of polyhydroxy alkaloid glycosidase inhibitors to prevent cellular recognition has resulted in their evaluation for clinical situations where suppression of an immune response would be desirable\ or for use against parasitic diseases[ thus\ in vivo experiments have shown that castanospermine can be used as an immunosuppressive drug\ promoting heart and renal allograft survival in rats[ parasitic diseases may also be controlled by altering cellular recognition processes[ castanospermine provides protection against cerebral malaria by preventing adhesion of plasmo! dium falciparum to infected erythrocytes\ while swainsonine inhibits the association of trypano! soma cruzi\ the causative agent of chagas| disease\ with host cells by formation of defective mannose! rich oligosaccharides on the cell surface [ there is no doubt that the polyhydroxy alkaloids have considerable potential for treatment of a variety of disease states in humans and animals[ the primary challenge in introducing them as commercial drugs is to minimize their toxicity and enhance the speci_city of their bene_cial e}ects[ improvement of their pharmacokinetic properties should result in much lower dose rates being necessary so that undesirable side!e}ects are limited[ increased speci_city of action can be achieved by preparation of synthetic derivatives and a comprehensive understanding of structureÐactivity relationships[ glycoproteins are widespread in nature\ being found in all eucaryotic cells[ they have also been shown to be present in various archaebacteria as well as in some lower bacteria[  in addition\ it has become eminently clear that carbohydrate sequences on glycoproteins\ glycolipids\ and proteoglycans are critically important as ligands in molecular recognition[ at least with regard to the n!linked glycoproteins\ on which this review focuses\ these molecules have been implicated in a number of important physiological functions\ especially cellÐcell recognition reac! tions involving such critical phenomena as in~ammation\ pathogenesis\ parasitism\ devel! opment\ cell adhesion\ and symbiosis\ to mention only a few[ n!linked oligosaccharides are also involved in lysosomal enzyme targeting\ in the uptake or removal of glycoproteins from the blood\ in protein folding in the endoplasmic reticulum\ and in many other physiological phenomena of potential signi_cance[  although the carbohydrate portion of the glycoprotein has not been shown to participate in every case of recognition\ speci_c oligosaccharide structures are clearly central to many of these cases[ thus\ inhibitors that block speci_c steps in the assembly of the various n!linked oligosaccharides and cause the formation of altered or immature oligosaccharide structures should be valuable tools for probing the role of carbohydrates in glycoprotein function[ figure shows three representative structures of the n!linked oligosaccharides[ all of these oligosaccharides have the same core structure shown within the box\ and are composed of a branched trimannose structure linked to a disaccharide of glcnac "i[e[\ n\n?!diacetylchitobiose#[ the imma! ture or initially synthesized oligosaccharide is a high mannose structure shown in "a#\ and this oligosaccharide is the biosynthetic precursor that gives rise to all of the other n!linked oligo! saccharides[ high!mannose "or oligomannose!type# oligosaccharides are most commonly found in glycoproteins from lower eucaryotes such as fungi and yeast\ although a small percentage of the n! linked oligosaccharides of animal cell surface proteins are of the high!mannose type[ the lower structure "c# of figure is a hybrid type of oligosaccharide that is produced by partial processing down to the glcnac transferase i step\ and then addition of various sugars to the ! linked mannose branch[ however\ hybrid structures are apparently the result of an absence of mannosidase ii action or activity[ it is not clear whether hybrid structures are formed normally\ but they are found in glycoproteins produced in individuals with hempas disease\ a condition where individuals lack mannosidase ii activity[ hybrid structures can also be induced by treating cultured cells with swainsonine " #[ the middle structure "b# in figure is an example of one type of complex oligosaccharide that is frequently found in cell surface glycoproteins of higher eucaryotes\ such as the low density lipoprotein receptor and many other membrane receptors[ this particular structure is referred to as a biantennary complex chain\ but other complex oligosaccharides may have three of the sialic acid!galactose!glcnac chains "triantennary chains#\ or four of these trisaccharide sequences "tetraantennary chains#[ the biosynthesis of the n!linked oligosaccharide chains involves two rather distinct series of reactions[ the _rst of these pathways gives rise to the precursor\ or immature oligosaccharide\ which is then transferred cotranslationally to the protein chain while it is being synthesized on membrane! bound polysomes[ in contrast\ the second series of reactions involves the modi_cation of this precursor oligosaccharide by the removal of some sugars and the addition of others\ to give a large number of di}erent oligosaccharide structures[ this _rst pathway requires the participation of a lipid carrier and the involvement of lipid!linked saccharide intermediates[ the reactions leading to the production of the _nal lipid!linked oligosaccharide precursor are presented in figure [ figure biosynthetic assembly of the core n!linked oligosaccharides[ as shown in figure \ the assembly of the n!linked oligosaccharide chain is initiated in the endoplasmic reticulum "er# by the transfer of a glcnac! !p from udp!glcnac to dolichyl!p to form glcnac!pp!dolichol[ a second glcnac is then added\ also from udp!glcnac\ to produce glcnacb \ glcnac!pp!dolichol[ then\ _ve mannose residues are added\ the _rst in a b \ linkage to the terminal glcnac\ and the next four in a linkages to form the important intermediate\ man glcnac !pp!dolichol[ these _rst seven reactions are believed to occur on the cytosolic side of the er membrane\ since they involve nucleoside diphosphate sugars as the sugar donors\ and these activated sugar donors are biosynthesized in the cytoplasm by soluble sugar nucleotide pyrophosphorylases[ it seems likely\ therefore\ that the sugar acceptor\ dolichyl!p\ is initially ori! ented in the er membrane in such a way that the phosphate group is exposed to the cytoplasm\ and is therefore able to accept sugars from the cytosol[ after the addition of the _rst seven sugars to give man glcnac !pp!dolichol\ this lipid!linked oligosaccharide is believed to undergo a {{~ip! op|| in the membrane so that the oligosaccharide chain now becomes oriented towards the lumen of the er[ the assembly of the oligosaccharide is completed by the addition of four more mannose residues and then three glucose units to give a glc man glcnac !pp!dolichol[ these last seven sugars "i[e[\ four mannose and three glucose units# are all added in the lumen of the er\ and are donated by the activated lipid precursors\ mannosyl!p!dolichol and glucosyl!p!dolichol[  these two sugar donors are synthesized using the sugar nucleotides\ gdp!mannose and udp!glucose\ by transfer of the respective sugar to dolichyl!p[ the reactions for the synthesis of the activated lipid! linked monosaccharides are proposed to occur on the cytosolic side of the er membrane and are catalyzed by the enzymes\ dol!p!man synthase and dol!p!glc synthase[  the _nal step in this pathway is the transfer of the glc man glcnac from its lipid carrier to speci_c asparagine residues on the polysome!bound protein\ catalyzed by the enzyme oligo! saccharyltransferase[  the asparagine residue that acts as the acceptor of this oligosaccharide chain must be in the tripeptide consensus sequence\ asn!x!ser"thr#\ where x can be any amino acid except proline\ but certain amino acids are favored over others[ in addition\ the tripeptide sequence must be in a speci_c conformation or orientation\ such as a b!turn of the protein\ in order to be glycosylated[ in spite of the fact that all of the reactions in this pathway are well known\ it is still not clear how the pathway is regulated\ nor where the control points are located[ after the oligosaccharide is transferred to protein and while the protein chain is still being synthesized in the er\ the oligosaccharide begins to undergo a number of processing or trimming reactions[ the initial reactions in this second pathway encompass the removal of three glucose residues and up to six mannose residues\ but later processing reactions involve the addition of a number of other sugars\ principally glcnac\ galactose\ neuraminic acid\ l!fucose\ and possibly galnac[ the processing pathway is outlined in figure [ the _rst processing step involves a membrane!bound glucosidase\ called glucosidase i\ which removes the outermost a \ !linked glucose[ this enzyme is quite distinct from the common glycosidases\ such as the lysosomal enzymes that are involved in the degradation of polysaccharides\ glycolipids and other complex carbohydrates\ since those enzymes usually have a ph optimum of around \ whereas glucosidase i has a ph optimum of about [ to [ [ in addition\ the common glycosidases are only speci_c for the sugar at the nonreducing terminus and the anomeric con! _guration of the glycosidic bond\ but do not have strong speci_city for the group to which this sugar is attached\ nor the speci_c glycosidic linkage if that group is another sugar[ glucosidase i\ on the other hand\ will only cleave a terminal glucose that is attached in a \ !linkage to another glucose[ thus\ glucosidase i will not work with p!nitrophenyl!a!d!glucopyranoside[ finally\ these kinds of enzyme can be distinguished by their location^the processing glucosidases are in the er\ while the other hydrolytic a!glucosidases are usually in the lysosomes[ glucosidase i is the enzyme that initiates the trimming or maturation of the n!linked oligo! saccharide chains and therefore may play a key role in controlling the rate of transport or exit of newly formed glycoproteins from the er to the golgi apparatus[ this enzyme has been puri_ed from a number of sources\ including calf and porcine liver\ and bovine mammary glands\ as well as plants "mung bean seedlings# and yeast "saccharomyces cerevisiae#[ the pig liver glu! cosidase i was cloned from a human hippocampus cdna library and expressed in cos cells[ the expressed enzyme had a molecular mass of kda and was degraded by endoglucosaminidase h "endo h# to a kda form\ indicating that the enzyme has a high!mannose oligosaccharide at the asparagine glycosylation site[ the hydrophobicity pro_le of the enzyme and the fact that trypsin treatment of microsomes released a kda fragment\ support the view that the glucosidase i is a transmembrane glycoprotein containing a short cytoplasmic domain of about amino acids\ followed by a transmembrane domain and a large c!terminal catalytic domain on the luminal side of the er membrane[ a yeast mutant gls \ has been isolated that is lacking glucosidase i and produces glycoproteins with glc man glcnac structures[ this alteration in the normal structure of the oligosaccharides on these yeast proteins has no e}ect on their secretion[ a cho mutant that is missing glucosidase i was also isolated by virtue of its resistance to the toxic e}ects of the lectin l!pha[ the mutation in these cells\ called lec \ has profound e}ects on the secretion or targeting of glycoproteins[ a second glucosidase\ located in the lumen of the er and called glucosidase ii\ removes the other two a \ !linked glucoses to give a man glcnac !protein[ interestingly\ this enzyme removes the outermost a \ !linked glucose quite rapidly "t : min#\ whereas removal of the innermost a \ !linked glucose is considerably slower "t : Ð min#[ those earlier observations on the activity of this enzyme correlate well with the more recently described role of this enzyme in protein folding[ that is\ a single a \ !linked glucose on the high!mannose chain functions as a recognition site to bind a chaperone to those proteins that are improperly folded or denatured\ and that chaperone expedites or assists their proper folding [ thus\ it has been shown that the er contains a protein called calnexin that functions to help newly synthesized membrane proteins fold into their proper conformation\ a step that is apparently necessary for many of these proteins to be transported to the golgi apparatus at the proper rate[ calnexin is a lectin that recognizes a single a \ !linked glucose on the high mannose chains of unfolded or denatured proteins[ since glucosidase ii acts fairly slowly on the _nal a \ !linked glucose\ there must be a time period when the glycoprotein has only a single glucose on its oligosaccharide[ this glucose on the high!mannose chains of unfolded proteins is the recognition site for calnexin to bind to those proteins that have not yet assumed their proper conformation[ Ð the er also contains a safety mechanism to assure that unfolded or improperly folded glyco! proteins can interact with this chaperone to obtain the conformation that is required for exit from the er into the golgi apparatus[ thus\ an unusual glucosyltransferase that is localized in the er functions to transfer a glucose from udp!glucose to high mannose chains on denatured\ but not on native\ glycoproteins[ once this glucose has been added\ calnexin can recognize and assist this protein in its proper folding and transfer to the golgi[ as a result\ a glycoprotein that has had all of its three glucose residues removed by glucosidase i and ii but has failed to fold into the proper conformation can be reglucosylated by this novel enzyme\ and this signal then allows the protein another opportunity to interact with calnexin and fold properly[ this mechanism\ involving the removal of glucoses by the glucosidases and reglucosylation by the glucosyltransferase\ is postulated to be part of a unique {{glycoprotein!speci_c folding and quality control mechanism|| in the er that allows this organelle to control and pass properly folded glycoproteins on to the next step in transport and processing[ glucosidase ii has a fairly high ph optimum of about [ to [ \ but also hydrolyzes p!nitrophenyl! a!d!glucoside[ on the other hand\ the enzyme does appear to be fairly speci_c for the a \ !linked glucose since hydrolysis of glc man glcnac is inhibited by nigerose\ an a \ !linked disaccharide of glucose\ but not by the corresponding a \ !\ a \ !\ or a \ !linked disaccharides of glucose[ the enzyme from pig kidney was shown to have a subunit molecular mass of kda and to contain a high!mannose oligosaccharide\ while the enzyme from mung bean seedlings had two kda subunits as well as high!mannose oligosaccharides\ although in some other animal systems\ glucosidase ii subunits were reported to have molecular masses of kda[ / this enzyme has been reported to be located in the rough and smooth er of pig hepatocytes but has also been located in post!golgi structures in tubular cells of pig kidney[ the cdna for glucosidase ii was cloned using degenerate oligonucleotides based on the amino acid sequences derived from a puri_ed pig liver glucosidase ii[ a [ kb cdna was isolated with an open reading frame of [ kb[ the amino acid sequence did not contain any known er retention signals or any hydrophobic regions that might represent transmembrane domains\ but it did contain a single n!linked oligosaccharide consensus site near the amino terminus[ the processing glucosidases can best be assayed\ in vitro\ using the radiolabeled oligosaccharide substrates\ ð hŁglc man glcnac and ð hŁglc man glcnac[ these substrates are readily prepared in cultured animal cells infected with an enveloped virus\ such as in~uenza virus\ that has an n! linked glycoprotein coat[ thus\ mdck cells are infected with in~uenza virus\ and progeny virus are produced in these cells in the presence of a glucosidase or mannosidase processing inhibitor to prevent the removal of those speci_c sugars[ for example\ if the virus is grown in the presence of castanospermine " #\ the oligosaccharide chains on its envelope glycoproteins will be mostly of the glc man glcnac structure\ whereas if the virus is grown in the presence of deoxymannojirimycin " \ r b!oh# or kifunensine " #\ it would have mostly man glcnac structures[ the oligo! saccharides are radiolabeled by growing the virus in the presence of either ð hŁgalactose to label the three glucose residues of the oligosaccharides\ or in ð ! hŁmannose to label the nine mannose units[ the virus!infected mdck cells are incubated for h to allow the virus to replicate and lyse the cells and the virus particles are isolated from the culture medium by ultracentrifugation[ the viral pellet is then treated exhaustively with pronase to digest the proteins and the resulting glycopeptides are isolated by gel _ltration[ these glycopeptides are then incubated with endo h "i[e[\ endo! glucosaminidase h# to cleave the high!mannose and glucose!containing high!mannose glyco! peptides\ and the resulting oligosaccharides\ having a single glcnac at the reducing end\ are isolated by gel _ltration on columns of biogel p! [ once the two glucosidases have removed all three glucoses from the n!linked oligosaccharide as shown in figure \ a number of a!mannosidases can remove one or more of the four a \ !linked mannose residues to ultimately give a man glcnac !protein "i[e[\ mana \ "mana \ #mana \ ðmana \ Łmanb \ glcnacb \ glcnac!protein#[ there are believed to be at least three di}erent a \ !mannosidases involved in the conversion of man glcnac to man glcnac ^a n er a!mannosidase\ a golgi man !mannosidase\ and a golgi mannosidase i[ these enzymes di}er in a number of properties including their substrate speci_city\ their sensitivity to various mannosidase inhibitors\ and their intracellular location[ the er mannosidase presumably removes only a single mannose to generate a unique and speci_c man glcnac structure[ this enzyme is reported to cleave the a \ !mannosidic linkage in man glcnac that is normally resistant to hydrolysis by the golgi man !mannosidase[ however\ a soluble form of the er a!mannosidase has been shown to exhibit rather low speci_city\ in that it can release several di}erent a \ !linked mannose residues from the man glcnac substrate[ these mannoses are removed in a random fashion so that three di}erent man glcnac structures are produced\ as well as a number of man glcnac isomers[ the discrepancy in speci_city between the er mannosidase and the soluble mannosidase reported in these two studies may be due to the e}ects of the protein itself on substrate speci_city "i[e[\ the er a!mannosidase may act di}erently in its speci_city on the free oligosaccharide# compared with the protein!bound oligosaccharide[ the man !mannosidase\ at least the enzyme from pig liver\ cleaves both free and peptide!bound man glcnac to give a speci_c man glcnac isomer[ thus\ the er mannosidase and the man ! mannosidase may be complementary to each other[ another a \ !mannosidase\ isolated from rat liver golgi and requiring ca ¦ \ apparently cleaves each of the four a \ !mannoses in the man glcnac at a comparable rate\ indicating that it alone could produce the man "glcnac# that is involved in the formation of complex types of oligosaccharides[ the exact function of these di}erent a!mannosidases is not currently known[ the fact that each of these enzymes removes a \ !linkages\ and that there is considerable redundancy in their action\ indicates that each has a speci_c role in the processing\ and perhaps the targeting pathway\ and that they may function to produce oligosaccharides with speci_c signals for particular roles in the cell[ in addition to these exo!a \ !mannosidases\ some animal cells and tissues contain an endo!a \ ! mannosidase that cleaves the glucose branch of the glc ! man glcnac between the two terminal mannoses to release a glc man\ glc man or glc man from the oligosaccharide and leave a man glcnac !protein[ this enzyme presumably prefers oligosaccharides with a single glucose on the high!mannose chain and may represent an alternate route to that utilizing glucosidase i and glucosidase ii[ nevertheless\ the speci_c role of this interesting enzyme in the processing pathway is still not clear^it may represent a new targeting route in some cells [ the cdna encoding an endoplasmic reticulum a!mannosidase was isolated from a rat liver gt library[ two degenerate oligonucleotides were prepared based on the amino acid sequences obtained from the puri_ed enzyme[ these oligonucleotides were used as primers in pcr with liver cdna as the template to generate an unambiguous cdna probe[ the base!pair cdna fragment was then used to isolate cdna clones by hybridization[ two overlapping clones were used to construct a full length cdna of bases which encoded an open reading frame of amino acids and a kda protein that contained six of the known peptide sequences[ no signal sequence or membrane spanning domains were found in the amino acid sequence[ northern blots of various animal tissues using the cdna as a probe revealed that a [ kb mrna was present in all tissues examined\ but was enriched in adrenal glands and testis and was less abundant in spleen\ intestine\ and muscle[ the rat liver er a!mannosidase bears striking homology to the vacuolar a!mannosidase from saccharomyces cerevisiae[ the man !mannosidase was also cloned in gt \ using a mixed pig liver cdna library[ three isolated clones allowed the construction of a base!pair full length cdna[ this cdna construct contained an open reading frame of bp and encoded a kda protein of amino acids[ the kda active enzyme expressed in cos cells had the same substrate speci_city\ sensitivity to inhibitors and metal ion requirements as a previously isolated kda active fragment[ structural and hydrophobicity analysis of the coding region as well as other studies indicated that this enzyme is a nonglycosylated\ type ii transmembrane protein with a residue cytosolic tail\ followed by a amino acid membrane anchor\ a luminal residue stem and a kda c!terminal catalytic domain[ immuno~uorescence studies indicted that the pig liver enzyme expressed in cos cells resides in the er[ on the other hand\ the human kidney enzyme expressed in cos cells was localized in the golgi apparatus[ the authors speculate that localization is likely to be sequence dependent[ after removal of the four a \ !linked mannose units\ the man glcnac !protein is a substrate for glcnac transferase i\ a glycosyltransferase in the medial golgi stacks\ that transfers a glcnac from udp!glcnac to the mannose on the a \ !branch to give glcnac!man glcnac !protein[ > this enzyme was puri_ed to homogeneity from various sources and shown to be a type ii integral membrane protein[ the enzyme is speci_c for the mana \ \manb \ glcnac arm of the n!glycan core\ and transfers a glcnac in b ! !linkage to the terminal \ !linked mannose[  this reaction is necessary before mannosidase ii can remove the a \ and a \ mannoses from the mana \ arm to give the trimannose structure[ the gene for this enzyme was disrupted by homologous recombination in embryonic stem cells and transmitted to the germ line[ mice lacking glcnac transferase i activity did not survive to term\ and biochemical and morphological analysis of embryos showed that they were developmentally retarded especially in regard to neural tissue[ once the glcnac has been added to the !linked mannose\ mannosidase ii can remove the two mannoses that are linked to the a \ !linked mannose branch[ the result of this reaction is a glcnacb \ mana \ "mana \ #manb \ glcnacb \ glcnac!protein[ mannosidase ii has been puri_ed to homogeneity from rat liver and mung bean seedlings[ the animal enzyme and the plant enzyme had apparent molecular masses of about kda on sds gels\ and both enzymes appeared to be glycoproteins[ however\ the primary sequence of the murine mannosidase ii derived from cloning studies predicted a molecular mass of kda for the deglycosylated enzyme[ this discrepancy may be explained by anomalous migration on sds!page "sodium dodecyl sulfate!polyacrylamide gel electrophoresis# by the deglycosylated or glycosylated protein\ since the glycosylated enzyme migrates as a kda protein[ the full length mannosidase ii cdna has been isolated from a t cdna library[ the murine enzyme is a type ii transmembrane glycoprotein with a cytoplasmic tail of _ve amino acids\ a single transmembrane domain\ and a luminally oriented catalytic domain[ the cdna was overexpressed in cos cells\ resulting in the appearance of immunoreactive material in a perinuclear membrane array indicating golgi localization[ the human a!mannosidase cdna has also been isolated and this gene was mapped to chromosome [ although the enzyme has been located in the golgi apparatus\ its {{subgolgi|| location depends on the cell type[ thus\ in exocrine pancreatic cells\ hepatocytes\ and intestinal goblet cells\ the enzyme is found in the medial to trans golgi[ but in cho cells\ it was restricted to the medial golgi[ thus\ in some cells\ mannosidase i and mannosidase ii appear to colocalize in the same region of the golgi[ a!mannosidase ii activity has been demonstrated in all mammalian tissues that have been examined[ however\ the level of the enzyme is very low in brain[ interestingly enough\ this tissue has been found to have an alternate hydrolytic enzyme that has a \ \ a \ \ and a \ !mannosidase activity and can cleave man glcnac down to man glcnac [ this enzyme is clearly distinct from mannosidase ii in terms of its substrate speci_city and its reaction to various mannosidase inhibitors "see section [ [ [ #[ its speci_c role in glycoprotein processing is still to be determined[ a lack of mannosidase ii has also been observed in hempas disease\ a hereditary a/iction that is characterized by altered expression of one or several of the glycoprotein processing enzymes[ one form of the disease results from a de_ciency in mrna expression of a!mannosidase ii[ lymphocytes derived from patients having this defect contain less than ) of control mannosidase ii levels\ and their glycoproteins contain mostly hybrid types of oligosaccharides[ the catalytic domain of the murine mannosidase ii cdna shows a considerable amount of similarity in sequence to the lysomal a!mannosidase cloned from the slime mold\ dictyostelium discoideum[ nevertheless\ these two enzymes have considerable di}erences in ph optimum\ sub! strate speci_city\ and localization within the cell[ based on the sequence similarity\ it has been proposed that the two enzymes were derived from the duplication and divergence of a primordial a!mannosidase gene with later acquisition of localization information and substrate speci_city[ a lesser degree of sequence similarity was observed between murine a!mannosidase ii and the endoplasmic reticulum a!mannosidase or its cytoplasmic homologue\ or the yeast vacuolar a!mannosidase [ following the action of the various glycosidases in the trimming part of the pathway\ a number of glycosyltransferases act on the glcnacman glcnac !protein to produce the complex types of n!linked oligosaccharides[ thus\ in the trans!golgi apparatus\ there are a number of glcnac transferases\ galactosyltransferases\ fucosyltransferases\ and sialyltransferases\ that can add these sugars to the n!linked chains to give a great diversity of complex chains\ having biantennary\ triantennary\ or tetraantennary structures[ many of these enzymes have been well characterized and a number of the genes for these important proteins have now been cloned[ although there are not any good inhibitors of these enzymes currently available\ the search for\ or the chemical synthesis of\ such compounds should be a rewarding future goal[ a number of low molecular mass compounds have been isolated from natural sources\ or synthesized chemically\ that speci_cally inhibit the glycosidases in the trimming pathway[ these inhibitors have become valuable tools to use in biological systems to determine the role of n!linked oligosaccharide processing on the function of various membrane or secretory glycoproteins[ the inhibitors are of special interest since they are small molecules which are able to permeate most cells and therefore can be used with intact cells and tissues to study {{in vivo|| situations[ in addition\ these inhibitors have been very useful in distinguishing the various processing enzymes from each other[ the best example is shown in table where it is clear that the many di}erent a!mannosidases have very di}erent sensitivities to the various mannosidase inhibitors[ the remaining sections of this chapter describe the chemistry and biological activities of the various classes of alkaloidal and alkaloidal!like compounds that function as inhibitors of n!linked oligosaccharide processing[ a number of naturally occurring\ sugar!like compounds\ in which the ring oxygen is replaced by a nitrogen\ have been isolated and are described in section [ [ [ many of these alkaloids have been shown to be potent inhibitors of various glycosidases[ the nitrogen in the ring apparently mimics the catalytic intermediate in the reaction "i[e[\ an oxycarbanion intermediate# but these compounds are still speci_cally recognized and bound to the active site of a particular glycosidase because of the resemblance in chirality to speci_c sugars like d!glucose and d!mannose[ thus\ they function as valuable inhibitors of glycosidases\ such as those that are involved in glycoprotein processing[ castanospermine " #\ as indicated earlier\ is an indolizidine alkaloid that was _rst isolated from the seeds of the australian tree\ castanospermum australe[ the initial studies on the e}ect of this compound in biological systems demonstrated that it was a reasonably potent inhibitor of b!glucosidase[ later studies also showed that castanospermine inhibited a number of isolated a!glucosidases\ including the glycoprotein processing enzymes\ glucosidase i and glucosidase ii\ sucrase\ maltase and lysosomal a!glucosidase[ since this compound is such a potent inhibitor of intestinal maltase and sucrose\ it prevents the degradation of the disaccharides sucrose and maltose\ and therefore blocks the normal digestion of starch and sucrose[ as a result\ the seeds of cas! tanospermum australe are toxic to animals and cause severe diarrhea and other gastrointestinal upsets[ in addition\ when castanospermine is fed to mice over a four or _ve day period\ it inhibits the lysosomal a!glucosidase and causes the accumulation of partially degraded glycogen particles within the lysosomes "i[e[\ a situation similar to that which occurs in pompe|s disease\ a genetic disease where a/icted individuals are lacking the lysosomal a!glucosidase# [ when various cultured animal cells are grown in the presence of castanospermine\ the processing of the n!linked oligosaccharides is blocked at the _rst step "i[e[\ glucosidase i#\ and the asparagine! linked glycoproteins have mostly oligosaccharides with glc man ! glcnac structures[ however\ in some cells there is an endomannosidase in the golgi that can release a glc Ð a \ man from glucose!containing n!linked oligosaccharides[ although this enzyme prefers to act on the mono! glucosylated oligosaccharide and release the disaccharide glca \ man\ it can apparently also cleave the oligosaccharide containing three glucose residues[ thus\ cells that contain this enzyme may be able to get around a castanospermine block[ as mentioned above\ the role of the endomannosidase in glycoprotein processing is not yet understood[ there are other glucosidase inhibitors that act at the level of glucosidase i and have similar e}ects to that of castanospermine but may have somewhat di}erent levels of activity\ or di}erent speci_cities[ these include !deoxynojirimycin " \ r a!oh#\ which is a polyhydroxylated pip! eridine analogue that corresponds to d!glucopyranose\ but has a nitrogen in the ring[ this compound also inhibits a! and b!glucosidases[ another inhibitor is the pyrrolidine alkaloid\ \ !dihydroxy! methyl! \ !dihydroxypyrrolidine "dmdp# " \ r oh#[ the latter compound is much less e}ec! tive than the above two inhibitors\ which suggests that a six!membered ring structure is preferred for inhibitory activity[ nevertheless\ dmdp does inhibit a! and b!glucosidase [ the e}ect of preventing the removal of the glucose residues from the n!linked oligosaccharides on the targeting of the glycoproteins can be quite dramatic[ thus\ when the hepatocyte cell line\ hep!g \ was incubated for various times in the presence of !deoxynojirimycin\ the rate of secretion of the serum protein\ a !antitrypsin\ was greatly diminished\ while the rate of secretion of other serum n!linked glycoproteins\ such as ceruloplasmin and the c! component of the complement\ were only marginally a}ected[ cell fractionation studies indicated that the antitrypsin had accumu! lated or was held up in the erÐgolgi compartment\ suggesting that the presence of glucose on the oligosaccharides might retard the movement of those proteins from the er to\ or through\ the golgi apparatus[ similar results were obtained when the biosynthesis and targeting of the low density lipoprotein receptor of _broblasts and smooth muscle cells were examined\ in the absence and presence of castanospermine[ in these studies\ it could be shown that cells grown in the presence of the inhibitor had only about one!half the number of receptor molecules at their cell surface\ and therefore bound much less i!ldl[ however\ these inhibited cells still had the same total number of ldl receptor molecules in the cells[ the missing receptor molecules were found to be located in the er or golgi\ based on cell fractionation studies[ an interesting study was done in im! lymphocytes where castanospermine was used to examine the role of oligosaccharide processing in the biosynthesis and targeting of the insulin receptor[ cells treated with castanospermine had a ) decrease in the number of insulin receptors at the cell surface\ as demonstrated by the binding of i!insulin[ the studies showed that removal of glucose residues from the n!linked glycoprotein was not necessary for the cleavage of the insulin proreceptor\ that is for the maturation of the receptor[ however\ as shown in other systems\ the presence of glucose apparently slowed the transport of this glycoprotein out of the er to the golgi\ resulting in a decrease in the number of receptor molecules at the cell surface[ in the case of the e glycoprotein of coronavirus\ both castanospermine and deoxynojirimycin caused a signi_cant drop by log in the formation of virus\ and also a dramatic inhibition in the appearance of e glycoprotein at the cell surface[ signi_cantly\ the e that was formed in the presence of the glucosidase inhibitors was still acylated with fatty acids as was the control viral e [ however\ the drug!induced e accumulated in an intracellular compartment that was not de_nitively identi_ed\ but was probably the er[ another study dealing with the sodium channel of rat brain neurons also showed that addition of palmitic acid to this protein was not prevented by the processing inhibitors[ the sodium channel is composed of a! and b!subunits that form a complex during maturation of the channel[ the a!subunit undergoes post!translational modi_cation by the addition of a palmitate\ and the incorporation of this fatty acid into the glycoproteins was prevented by tunicamycin\ a glycosylation inhibitor that completely prevents formation of n!linked oligosaccharides[ on the other hand\ castanospermine prevented processing of the oligosaccharide chains and the addition of sialic acids\ but had no e}ect on the addition of palmitic acid[ this alkaloid also did not a}ect the covalent assembly of the a! and b!subunits or the biological function of the channel[ thus\ the oligo! saccharide is apparently necessary for palmitate addition\ but the speci_c structure of the oligo! saccharide "i[e[\ high!mannose or complex# is presumably not critical for the addition of palmitate groups[ gp is the envelope protein of hiv\ the aids associated virus\ and this protein is a glycoprotein with many oligosaccharide chains[ these oligosaccharides are involved in the recognition and mechanism of attachment of hiv to the cd receptor on t lymphocytes and other susceptible cells[ gp interacts with target molecules on the susceptible cells to cause the fusion of the cells with the formation of syncytia\ which are necessary for viral formation and infectivity[ the glucosidase inhibitors\ !deoxynojirimycin "dnj# and castanospermine\ caused a signi_cant decrease in the formation of new virus and in syncytium formation[ \ as a result of these interesting results\ these inhibitors have been tested in human clinical trials as potential antiaids drugs[ although the results have not been published\ one reported side e}ect in humans was the occurrence of diarrhea and other gastrointestinal problems in individuals taking these compounds[ as shown in table \ there are a number of other compounds in addition to castanospermine and "dnj# that are also inhibitors of glucosidases and glycoprotein processing[ one such compound is the pyrrolidine alkaloid dmdp " \ r oh#\ which occurs in several di}erent plant families[ when placed in a medium of cultured animal cells\ dmdp inhibits the same step and gives the same oligosaccharide structure "i[e[\ glc man ! glcnac # as do castanospermine and dnj[ however\ dmdp is much less e}ective than these other inhibitors and therefore considerably higher concentrations are necessary in the medium[ the fact that a _ve!membered ring structure can show glycosidase activity against enzymes that act on hexopyranosides is signi_cant and would certainly warrant modeling studies of this structure in comparison to the indolizidine and piperidine alkaloids[ several other unusual structures that show increased selectivity towards the two processing glucosidases "i[e[\ glucosidase i and glucosidase ii# are discussed below[ australine " # is a tetrahydroxypyrrolizidine alkaloid that was found in the same seeds that contain castanospermine\ namely castanospermum australe[ however\ australine is present in the seeds in much lower amounts than is castanospermine[ this compound is a good inhibitor of fungal amyloglucosidase\ but it also inhibits the processing glucosidase i[ however\ in contrast to the other glucosidase i inhibitors discussed above which are also fairly e}ective against glucosidase ii\ australine is a very poor inhibitor of glucosidase ii[ thus\ australine is the _rst glucosidase inhibitor to distinguish between these two processing enzymes[ nevertheless\ the key a}ect of australine in cell culture is to block glucosidase i and cause the accumulation of glycoproteins having glc man glcnac structures[ additional compounds such as australine\ and especially ones with more potent activity\ will be useful tools to help understand the di}erences between glucosidase i and glucosidase ii inhibitors[ another interesting glucosidase inhibitor is \ !diamino! \ !imino! !o!"b!d!glucopyranosyl#!d! lycero!l!guloheptitol "mdl #[ this compound\ referred to in the following discussion as mdl\ was synthesized chemically to resemble a disaccharide that would function as a transition state analogue of the intestinal enzyme\ sucrase[ as anticipated\ mdl did inhibit rat intestinal maltase\ sucrase\ isomaltase\ glucoamylase\ and trehalase when present in micromolar amounts[ most interesting was the observation that mdl also showed speci_city for the glucosidases but in the opposite manner to that of australine[ thus\ mdl was much more e}ective against glucosidase ii than it was against glucosidase i[ in cell culture\ mdl was quite di}erent from the other glucosidase inhibitors in that it caused the accumulation of glycoproteins having glc man "glcnac# structures[ however\ the overall e}ects of mdl on glycoprotein function in cell culture are likely to be similar to those observed with castanospermine and other inhibitors of glucosidase i[ a compound named trehazolin " # was isolated as a trehalase inhibitor and has also been tested as an inhibitor of the processing glucosidases[ this compound inhibited glucosidase i quite well\ but was a very poor inhibitor of glucosidase ii[ the isolation and demonstration that structures like australine\ mdl or trehazolin do exist\ and that these compounds have selective actions against the processing glucosidases should stimulate the search for more and better inhibitors[ such inhibitors will be useful tools for additional studies on the role of carbohydrate and especially of the glucose residues in the function and localization of n!linked glycoproteins[ in the last few years\ it has become clear why and how inhibitors of glucosidase i cause many n!linked glycoproteins to accumulate in the er[ helenius as well as other investigators have elegantly shown that the er has a {{protein correction and folding system|| that helps newly synthesized er proteins fold into the proper conformation that is necessary for transport to the golgi apparatus[ this system involves the action of a chaperone "i[e[\ a protein that helps other proteins fold#[ the chaperone\ named calnexin\ is also a lectin that recognizes a monoglucosylated high!mannose oligosaccharide on the unfolded glycoprotein[ in the presence of castanospermine or other glucosidase i inhibitors\ the _rst glucose cannot be removed\ and therefore the unfolded protein cannot be recognized by calnexin and cannot be helped to fold[ most proteins will fold on their own given enough time\ but the folding of some may be very slow and interaction with calnexin can help speed up this process[ thus\ proteins like the ldl "low density lipoprotein# receptor\ or the insulin receptor\ or a !antitrypsin\ are transported to the golgi at a much slower rate in the presence of glucosidase inhibitors because of the inability of calnexin to bind to the protein[ a number of a!mannosidase inhibitors have been identi_ed from natural sources or synthesized chemically[ in addition to their use as tools to examine the role of mannose oligosaccharides in the function of n!linked glycoproteins\ they have also been valuable in distinguishing the various a!mannosidase activities from each other[ the _rst glycoprotein processing inhibitor to be reported was the indolizidine alkaloid\ swain! sonine " #\ an inhibitor of mannosidase ii[ this compound was initially shown to be an inhibitor of the lysosomal a!mannosidase and to cause symptoms of the lysosomal storage disease a!mannosidosis when administered to animals[ thus\ swainsonine was essentially the prototype which chemists could use to design other glycosidase inhibitors[ that is\ based on the structures of swainsonine\ castanospermine and !deoxynojirimycin\ it appeared evident that a useful glycosidase inhibitor should have the following characteristics] "i# a ring structure\ probably of the pyranose type\ with nitrogen replacing the heterocyclic oxygen" ii# a number "unknown at the time and still not certain# of hydroxyl groups^and "iii# stereochemistry of the hydroxyl groups matching that of the sugar for which the glycosidase to be inhibited is speci_c[ in this section on mannosidase inhibitors\ they will be discussed in the order in which they act in the glycoprotein processing pathway " figure #\ rather than in order of their historical identi_cation[ based on the fact that !deoxynojirimycin "dnj# " \ r a!oh# was a good inhibitor of a!glucosidases\ it was reasonable to assume that a related structure\ but with mannose chirality\ would be an inhibitor of a!mannosidases[ the !epimer of dnj\ namely !deoxymannojirimycin "dmj# " \ r b!oh# was synthesized chemically and was indeed found to be a potent inhibitor of the glycoprotein processing mannosidase i[ \ most interestingly\ dmj did not inhibit jack bean or lysosomal a!mannosidase\ nor did it inhibit mannosidase ii[ those observations on the selective speci_city of dmj demonstrate that it is dangerous to screen for new glycosidase inhibitors by using the commonly occurring aryl!glycosidases "i[e[\ a! and b!glucosidase\ galactosidase\ or mannosidase# to test for the inhibitory activity[ that is\ if the goal is to _nd a new glycoprotein processing inhibitor\ such as an inhibitor of er a!mannosidase\ then one would desire a speci_c inhibitor that does not work on golgi mannosidase i or mannosidase ii\ or jack bean or lysosomal a!mannosidase[ thus\ if one used the enzymes that hydrolyze aryl!mannosides "such as p!nitrophenylÐd!manno! pyranoside# to screen for such a compound\ the screens would obviously be negative and any potential inhibitor would be discarded[ in the period since deoxymannojirimycin was synthesized and shown to be a speci_c inhibitor of golgi mannosidase i\ a number of other neutral a!mannosidase activities have been reported in animal cells[ these enzymes have all been discussed in section [ [ [ on glycoprotein processing\ although it is still not clear what role\ if any\ some of them play in the trimming of n!linked oligosaccharides[ as also indicated earlier\ these enzymes have di}erent substrate speci_cities from mannosidase i\ and thus many of them are resistant to inhibition by dmj[ as these new man! nosidases are puri_ed and separated from each other\ and from other competing activities\ and as rapid assays for measuring their activities become available\ it will be easier to identify or synthesize speci_c new inhibitors for each of these enzymes[ nevertheless\ at this time\ a number of a!mannosidase inhibitors have been identi_ed and the activities of these various compounds on di}erent a!mannosidases are presented in table [ in animal cells\ dmj inhibited the golgi mannosidase ia:b and caused the accumulation of glycoproteins having a high mannose oligosaccharide\ mostly of the man glcnac structure[ in contrast to the e}ect of the glucose analogue dnj\ which prevented the secretion of igd and igm by cells in culture\ dmj had no e}ect[ as suggested above\ this e}ect of dnj is due to the function of calnexin on protein folding and its interaction with glucose[ however\ once the protein has folded and the glucoses are removed\ the protein is treated normally with respect to targeting\ regardless of whether it has a high mannose or modi_ed chain[ in one interesting study\ dmj was used as a tool to determine whether glycoproteins were recycled through the golgi during the endocytic process[ in this experiment\ membrane glycoproteins were synthesized in cho cells in the presence of dmj to inhibit mannose trimming\ together with ð ! hŁmannose to label the n!linked glycoproteins[ after an appropriate incubation\ the medium was changed to remove inhibitor and label and the cells were incubated for additional times[ during this second period\ the oligosaccharide structure of the transferrin receptor was determined under conditions where it would undergo endocytosis[ before the chase\ the oligosaccharide structure of the transferrin receptor was of the high mannose type\ but during the chase period\ a small percentage of the recycled receptor molecules underwent processing and gave complex types of structure[ these studies indicated that some endocytosed glycoproteins do recycle through the golgi compartments and may undergo oligosaccharide processing[ however\ the amount of glycoprotein molecules that were actually modi_ed in this experiment was small\ indicating that recycling through the golgi is probably not a major route[ ut! cells were used to examine the role of the er a!mannosidase in glycoprotein targeting and function[ ut! cells are cells that overexpress hmg coa reductase\ a glycoprotein enzyme that resides in the er of the cell[ the oligosaccharide chains of this protein are of the high mannose type and mostly man glcnac and man glcnac structures[ since previous studies had shown that the er mannosidase is not inhibited by dmj\ this inhibitor was used to determine whether the initial trimming of mannoses involved the er mannosidase[ in these studies\ the hmg coa reductase produced in the presence of dmj had mostly man glcnac structures and the smaller oligosaccharides were not found\ indicating that the er enzyme was involved in the removal of the _rst mannose\ but other mannoses were trimmed by dmj!sensitive mannosidase"s#[ dim " \ !dideoxy! \ !imino!d!mannitol# is another inhibitor that was synthesized from benzyl! a!d!mannopyranose and shown to be a good inhibitor of jack bean a!mannosidase[ it also inhibited glycoprotein processing in cultured mdck cells\ and gave rise to glycoproteins having mostly man glcnac structures suggesting that it inhibited the golgi a!mannosidase i[ in keeping with these observations\ in vitro studies with a partially puri_ed preparation of mannosidase i showed that dim did inhibit release of ð hŁmannose from ð hŁman glcnac[ however\ dim is not nearly as e}ective an inhibitor of a!mannosidases as is either swainsonine or kifunensine "see below#[ on the other hand\ dim is of considerable interest as an inhibitor since] "i# it has a furanose rather than a pyranose ring structure\ and "ii# it is synthesized chemically and therefore can be produced in large amounts and readily modi_ed to produce various structural analogues[ it is not clear whether this compound also inhibits the er mannosidase since this activity may not be present in mdck cells[ kifunensine " # is an alkaloid produced by the actinomycete\ kitasatosporia kifunense\ and it corresponds in structure to the cyclic oxamide derivative of !amino!dmj[ this alkaloid is a very weak inhibitor of jack bean a!mannosidase\ as is dmj\ but is a strong inhibitor of the golgi mannosidase i "ic to × − m#[ this inhibition is almost times higher than the inhibition of mannosidase i by dmj[ interestingly\ kifunensine had no e}ect on either the er mannosidase or on mannosidase ii[ in~uenza virus!infected mdck cells incubated in the presence of kifunensine produced in~uenza virus particles in which the envelope glycoproteins had n!linked oligosaccharides mostly having man glcnac structures[ this is the same e}ect as that seen in the presence of dmj[ however\ kifunensine was much more e}ective in causing this change in structure and only : as much of this inhibitor was needed compared with dmj[ a compound that mimics the mannopyranosyl cation\ the intermediate proposed as being involved in the enzymatic hydrolysis of a!mannopyranosides\ was synthesized chemically and named mannonolactam amidrazone[ this compound not only inhibited golgi mannosidase i with an ic of mm\ and mannosidase ii with an ic of nm\ but was also a potent inhibitor of er a!mannosidase "ic of mm#[ furthermore\ the compound also inhibited the aryl!a!mannosidase "ic of nm# and the aryl!b!mannosidase "ic of mm#\ although it clearly preferred a!linkages[ in cell culture studies\ mannonolactam amidrazone gave rise to glycoproteins with the same type of high mannose oligosaccharide as seen with dmj and kifunensine[ thus inhibition of golgi mannosidase i "and:or er mannosidase# appears to prevent trimming of most if not all mannose residues[ the designers of this compound hypothesize that the reason that it is so e}ective as a general mannosidase inhibitor is that it is the _rst analogue of mannose that mimics the true half!chair conformation of the cationic intermediate that is believed to be involved in catalysis of the a!mannosides[ mannonolactam should serve as a model for the synthesis of more speci_c mannosidase inhibitors[ as mentioned earlier\ the _rst processing inhibitor to be described was the indolizidine alkaloid\ swainsonine " #[ in early studies\ swainsonine was added to the culture media of mdck cells infected with in~uenza virus\ and these cultures were labeled by the addition of ð ! hŁmannose[ this inhibitor caused a signi_cant inhibition in the amount of mannose!labeled\ endo h!resistant oligosaccharides "i[e[\ complex oligosaccharides# and a great increase in the amount of mannose! labeled endo h!sensitive structures[ these latter oligosaccharides were shown to be hybrid types of oligosaccharides[ \ however\ the change in the structure of the viral oligosaccharides from complex to hybrid types did not a}ect the production\ maturation or release of the in~uenza virus particles[ these early studies did not identify the speci_c site of swainsonine inhibition\ but later in vitro studies with the puri_ed a!mannosidases demonstrated that swainsonine speci_cally inhibited mannosidase ii\ and was inactive towards mannosidase i[ in keeping with this site of action\ swainsonine caused the formation of hybrid structures when it was added to the medium of cultured animal cells producing vsv glycoproteins "i[e[\ g protein#\ _bronectin\ and bhk cell surface glycoproteins[ in most studies where swainsonine was used to determine the e}ect of changes in oligosaccharide structure on glycoprotein function\ this inhibitor had little e}ect on functional aspects of the proteins in question\ although it did cause alterations in structure to hybrid chains[ the inhibitor did prevent the receptor!mediated uptake of mannose!terminated glycoproteins by macrophages[ this inhibition was probably due to the formation of hybrid structures on the macrophage surface which could then react with and bind the mannose receptors[ swainsonine proved to be a valuable tool in determining the sequence of addition of certain sugars during the assembly of the n!linked oligosaccharides[ thus\ the addition of l!fucose or sulfate to the in~uenza viral protein was studied in the presence of various processing inhibitors[ when the glycoproteins were produced in the presence of castanospermine or dmj\ there was no ð hŁfucose or ð sŁsulfate associated with the glycoproteins\ suggesting that fucose and sulfate were added after the mannosidase i step in processing[ however\ in the presence of swainsonine\ the glycoproteins contained both l!fucose and sulfate indicating that the transferases that added these groups worked after the glcnac transferase i processing step[ these results agree with the reported acceptor oligosaccharide speci_city "i[e[\ glcnac!man glcnac of the fucosyltransferase and the sulfotransferase[ in some studies\ swainsonine did cause a loss in the function of speci_c proteins[ thus\ gluco! corticoid stimulation of resorptive cells\ involving the attachment of osteoblasts to bone\ is inhibited by swainsonine[ treatment of either the parasite\ trypanosoma cruzi\ or the macrophages with swainsonine inhibits the interaction of these cells with each other[ this alkaloid also caused a dramatic decline in the ability of b melanoma cells to colonize the lungs of experimental animals[ as a result of these and similar studies\ swainsonine has been undergoing tests and consideration as a drug to treat certain types of cancers[ these are only a few of the many studies that have been done with this interesting compound[ many of these other studies are summarized in a review [ another inhibitor of mannosidase ii\ named mannostatin " #\ was isolated from the fungus\ streptoverticillium verticillus[ this compound is of special interest because it has a very unusual structure with an exocyclic nitrogen\ a _ve!membered ring\ and a thiomethyl group\ but is still a glycosidase inhibitor[ mannostatin was found to be a potent inhibitor of jack bean a!mannosidase as well as mannosidase ii "ic nm#[ in cell culture studies\ mannostatin caused the formation of the same types of hybrid oligosaccharides as are formed in the presence of swainsonine[ interestingly\ acetylation of the amino group of mannostatin resulted in loss of mannosidase activity[ while this compound does not have any functional advantage over swainsonine as an inhibitor\ it is of considerable interest\ since it adds a great deal of additional structural information to our understanding of the requirements necessary for a compound to be a glycosidase inhibitor[ waterman\ phytochemistry\ \ \ [ [ b[ dra Ãger\ a[ van almsick\ and g[ mrachatz\ planta med[\ \ \ [ [ n[ asano\ e[ tomioka\ h[ kizu\ and k[ matsui\ carbohydr[ res[\ \ \ leach\ royal society of chemistry\ london\ "see also] http]::www[ch[ic[ac[uk:ectoc:echet :#[ [ g[ w[ j[ fleet\ in {{swainsonine and related glycosidase inhibitors\|| eds their composition\ structure and function\|| elsevier\ new york\ [ \ \ \ [ [ b[ kalz!fuller\ e[ bieberich\ and e[ bause\ eur[ j[ biochem[\ \ \ [ [ b[ esmon\ p[ c[ esmon\ and r[ scheckman\ j[ biol[ chem[\ \ \ biophys[ biochem[\ \ \ [ [ b[ ganem and g[ papandreou\ j[ am[ chem[ soc[\ \ \ key: cord- -j ehiwn authors: verheije, monique h.; raaben, matthijs; mari, muriel; te lintelo, eddie g.; reggiori, fulvio; van kuppeveld, frank j. m.; rottier, peter j. m.; de haan, cornelis a. m. title: mouse hepatitis coronavirus rna replication depends on gbf -mediated arf activation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: j ehiwn coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of rna replication. not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (mhv) replication complexes (rcs). we now show that mhv replication is sensitive to brefeldin a (bfa). consistently, expression of a dominant-negative mutant of arf , known to mimic the action of the drug, inhibited mhv infection profoundly. immunofluorescence analysis and quantitative electron microscopy demonstrated that bfa did not block the formation of rcs per se, but rather reduced their number. mhv rna replication was not sensitive to bfa in mdck cells, which are known to express the bfa-resistant guanine nucleotide exchange factor gbf . accordingly, individual knockdown of the golgi-resident targets of bfa by transfection of small interfering rnas (sirnas) showed that gbf , but not big or big , was critically involved in mhv rna replication. arf , the cellular effector of gbf , also appeared to be involved in mhv replication, as sirnas targeting this small gtpase inhibited mhv infection significantly. collectively, our results demonstrate that gbf -mediated arf activation is required for efficient mhv rna replication and reveal that the early secretory pathway and mhv replication complex formation are closely connected. viruses rely on cellular host factors for virtually all steps of their infection cycle. however, the cellular proteins required and the cellular pathways hijacked by viruses have hardly been elucidated. all positive-strand rna viruses assemble in infected cells their replication complexes (rcs) in association with intracellular membranes [ , , , , ] . the induction of such local microenvironments is likely advantageous for the virus, as membrane association may facilitate the recruitment of both the viral and cellular components involved in rna replication. alternatively, membrane association may provide a shielded environment that prevents the activation of, or protects against, antiviral host cell responses like those mediated by interferon. coronaviruses belong to a family of enveloped positive-strand rna viruses in the order nidovirales. upon translation of the viral genomic rna, two very large polyproteins (approximately , and , amino acids) are synthesized, the autoproteolytic cleavage products of which collectively form the rcs. these rcs are associated with double membrane vesicles (dmvs [ , , ] ), which appear as cytoplasmic foci when analyzed by fluorescence light microscopy and increase in number during the course of the infection [ , , , ] . it is plausible that the nonstructural viral proteins (nsps) mediate the formation of dmvs by modifying intracellular membranes and by recruiting cellular components to their need. recent studies suggest the endoplasmic reticulum (er) to be the lipid donor compartment of the membrane-bound coronavirus rcs [ , , , ] , although colocalization of nsps with markers for endosomes, golgi and autophagosomes has also been described [ , , , , ] . brefeldin a (bfa) is a well known fungal metabolite that induces the redistribution of golgi proteins into the er [ , ] , effectively resulting in the block of transport though the secretory pathway [ , ] . this drug inhibits the activation of adpribosylation factor (arf) small gtpases by targeting the large guanine nucleotide exchange factors (gefs) gbf (golgi-specific resistance factor ), and big (bfa-inhibited gef) and [ , , ] . more specifically, bfa locks arf*gdp when bound to gef, thereby blocking the gef activity at an early stage of the reaction, prior to guanine nucleotide release [ , ] . the large gefs function in the er to golgi transport pathway [ ] and localize to the cis-(gbf ) and trans-sides (big and big ) of the golgi complex [ ] . the cellular effectors of these gefs, arfs, are divided into three classes: class i (arf - ), class ii (arf and ), and class iii (arf ) [ ] . class i arfs regulate the assembly of coat complexes onto vesicles budding from compartments along the secretory pathway and activate lipid-modifying enzymes (reviewed in [ , ] ). while the function of class ii arfs remains largely unclear, the class iii arf is thought to regulate endosomal membrane traffic [ , ] . gbf and the bigs are likely to activate distinct subclasses of arfs at specific locations in order to regulate different types of transport routes [ ] . in the field of virology, bfa has been used, besides for studying viral protein transport and virus assembly [ , , , , , ] , to investigate the formation of rcs and rna replication of several positive-strand rna viruses [ , , , ] . for example, poliovirus rna replication was shown to be sensitive to bfa. in the presence of this drug, poliovirus replication sites were not formed and rna replication was completely blocked [ , ] . remarkably, other members of the picornavirus family appeared to differ in their sensitivity to bfa. whereas echovirus rna replication was strongly inhibited by bfa, rna replication of encephalomyocarditis virus was not affected at all, while parechovirus exhibited an intermediate sensitivity to it [ ] . relatively little is known about the host pathways involved in coronavirus rna replication and in rc formation. recently, we demonstrated the important role of the er in the generation of the rcs. while mhv nsp was localized to this organelle when expressed alone, it was recruited to the replication complexes in infected cells [ ] . furthermore, coronaviral replication was inhibited when the er export machinery was blocked by use of the kinase inhibitor h or by expression of a dominant active mutant of sar [ ] . other cellular proteins and pathways are likely to contribute to the formation of the coronavirus rcs as well. here, we studied the involvement of bfa-sensitive pathways in mhv replication and rc formation. our results demonstrate that gbf -mediated arf activation is required for efficient mhv rna replication. moreover, together with our recent observation about the relevance of the er in the same process, our data reveal that the early secretory pathway and mhv replication are intimately connected. bfa is known to disturb membrane traffic in most cell types, resulting in a redistribution of golgi proteins into the er [ , ] . we first confirmed the sensitivity of murine lr cells to bfa by immunofluorescence using antibodies directed against the golgi protein marker gm [ ] . indeed, after treatment of the cells with mg/ml bfa for h, the typical golgi staining pattern of gm was lost, concomitant with a reticular redistribution of the protein marker (data not shown). next, we tested whether mhv infection was sensitive to bfa. therefore, lr cells were inoculated with a luciferase-expressing recombinant of mhv-a (mhv-eflm) in the presence or absence of mg/ml bfa. after h, the inoculum was removed and the cells were further incubated either in the presence or in the absence of bfa. at h p.i., the intracellular luciferase expression level was determined relative to untreated cells. luciferase expression was inhibited more than % when bfa was present from - h p.i., whereas bfa treatment during virus inoculation had only a minor effect on reporter gene expression (fig. a) . although this latter decrease might have resulted in part from a reduced entry, the negative effect of bfa on mhv replication and transcription is evident from the profoundly impaired mhv reporter gene expression when bfa was added post inoculation ( - h p.i.). in a control experiment, the effect of bfa on sindbis virus replication in lr cells was assayed by using sindbis pseudovirus particles containing luciferase-expressing replicons. as described previously [ ] , sindbis virus replication was not affected by the bfa treatment (fig. a) . this result indicates that the observed effect of bfa on mhv-driven luciferase expression was not due to non-specific drug-induced toxicity. although we have demonstrated in previous studies that reporter gene expression by mhv is a reliable measure for coronavirus replication [ ] , we wanted to confirm that the reduction in luciferase expression resulted from a corresponding decrease in viral rna synthesis rather than from inhibition of viral protein translation. to this end, a similar experiment as shown in fig. a was performed, in which the amount of intracellular genomic viral rna was determined by real-time taqman pcr. as for the luciferase expression levels, the amount of genomic rna was found to be severely reduced when bfa was added directly after the virus inoculation (fig. b) , whereas a less profound effect was observed when cells were treated during virus inoculation. very similar results were obtained when targeting the taqman pcr to a different region of the viral genome (data not shown). to more directly check for an effect of bfa on the translation of viral mrnas, we performed an additional experiment. lr cells were infected at high multiplicity with the recombinant virus mhv- afls, which expresses the firefly luciferase, and subsequently transfected with a synthetic mrna encoding renilla luciferase. this synthetic mrna mimics viral mrnas as it contains ' and ' untranslated regions identical to those found in the viral genome. the cells were incubated in the presence or absence of bfa ( - h p.i.) after which the intracellular renilla and firefly luciferase expression levels were determined. the results show that bfa treatment did not inhibit the synthesis of renilla luciferase from the synthetic mrna, while firefly luciferase expression driven by the recombinant virus was severely affected (fig. c) . renilla luciferase expression was also not affected in the absence of a viral infection (data not shown). all together, these results indicate that bfa inhibits mhv rna replication while translation of viral mrnas is not affected. next, we determined the post inoculation period during which mhv replication was most sensitive to bfa, by analyzing the luciferase expression levels as they are a reliable measure for rna replication. thus lr cells infected with mhv-eflm were treated with bfa for overlapping h periods. at the end of each incubation period the intracellular luciferase expression levels were coronaviruses are the causative agents of many respiratory and enteric infections in humans and animals. as with all viruses, virtually all of the steps of their infection cycle depend on host cellular factors. as the first and most crucial step after their entry into cells, coronaviruses assemble their replication complexes (rcs) in association with characteristic, newly induced membranous structures. the cellular pathways hijacked by these plus-strand rna viruses to create these ''factories'' have not been elucidated. here, we study the involvement of the secretory pathway in mouse hepatitis coronavirus (mhv) replication by using the drug brefeldin a (bfa), which is known to interfere with er-golgi membrane traffic by inhibiting the activation of adp-ribosylation factor (arf) small gtpases. our observations show that mhv rna replication is sensitive to bfa. in agreement herewith we demonstrate, by using various techniques, that the bfasensitive guanidine nucleotide exchange factor gbf and its downstream effector arf are of critical importance for coronavirus replication. from our results we conclude that mhv rna replication depends on gbf -mediated arf activation. our study provides new insights into the close connection between mhv replication and the early secretory pathway. determined and compared to those in mock-treated cells. the results showed that replication was affected throughout the course of the infection (fig. d) ; however, the effects were most pronounced during the early phases of infection. to confirm our observation that bfa inhibits mhv replication but also to prove that the effects of this drug are due to the inhibition of gef activities, we next analyzed to what extent the expression of a dominant-negative mutant of arf (t n) would affect mhv infection. this arf mutant has a decreased affinity for gtp and, following gdp displacement, it remains 'nucleotidefree' for a longer period than wt arf [ ] . as a consequence, expression of arf -t n mirrors the effects of bfa [ ] . in addition to this protein, we included a constitutive-active arf mutant (arf -q l), which persists in the gtp-bound state longer than wild-type arf, resulting in a prolonged arf activation. expression of this latter mutant is known to inhibit transport at later steps in the secretory pathway, e.g. from vesicular tubular clusters (vtc) to the golgi complex and between golgi stacks [ ] . lr cells were transfected with plasmids expressing yfp fusions of either wild type arf , arf -t n or arf -q l. after transfection, the cells were inoculated with an rfpexpressing mhv-a recombinant (mhv-rfp) that allows flow cytometric analysis of mhv replication [ ] . the percentage of rfp-positive cells in the yfp-expressing population was determined relative to that of the wild type arf expressing cells (fig. e) . overexpression of the wt arf fusion protein itself did not significantly affect mhv infection when compared to nontransfected cells (data not shown). the results indicate that overexpression of the dominant-negative arf mutant inhibited mhv infection profoundly, thereby confirming the results obtained with bfa. in contrast, expression of the constitutiveactive mutant of arf did not influence mhv replication. as bfa is known to affect intracellular vesicle formation and transport, and because mhv replicates its genome in association with dmvs, we next investigated the effect of bfa on the assembly of the mhv rcs. first, we checked whether the morphological integrity of the rcs was affected in the presence of bfa. therefore, lr cells infected with mhv-a were treated with bfa for minutes starting . h p.i. they were subsequently fixed and processed for immunofluorescence using antibodies both against nsp , which served as a protein marker for the mhv replication sites [ , ] , and against the viral structural protein m, known to reside in the golgi [ ] . the nsp antibody revealed the typical perinuclear staining pattern in both treated and non treated infected cells ( fig. a) . in contrast, a dispersed distribution of m protein was observed in bfa-treated cells reflecting the collapse of the golgi, whereas in non-treated cells the m protein showed a clear golgi-like staining ( fig. a) . these results indicate that, once formed, the replication sites are not disrupted by bfa. subsequently, we investigated whether bfa inhibited rc formation early in the infection. bfa was therefore added to lr cells directly after inoculation with mhv-a and staining was performed at h p.i using the nsp antibody. although some perinuclear staining of nsp could be detected in bfa-treated cells, the number and intensity of the nsp containing foci were clearly reduced when compared to non-treated cells (fig. b ). we next investigated whether these nsp puncta represented mhv replication sites. therefore, we studied the ability of the nsp foci to recruit the nucleocapsid protein n, a protein previously shown to localize to the rcs [ , ] . three parallel cultures of lr cells were transfected with a plasmid coding for a mhv n-gfp fusion protein and h post transfection two of them were infected with mhv-a . bfa ( mg/ml) was added to one of these latter cultures directly after inoculation (t = h p.i.). at h p.i., the cells were fixed and subsequently processed for immunofluorescence using the anti-nsp antibody (fig. c ). as expected, n-gfp was diffusely localized to the cytosol in non-infected cells (indicated by an arrowhead in fig. c ). in contrast, when cells were infected with mhv, this fusion protein also appeared in foci that co-localized with nsp (indicated by arrows in fig. c ). this co-localization was observed both in mock-and in bfa-treated cells, indicating that the nsp foci that had been formed in the presence of bfa, though decreased in number and intensity, correspond with the replication sites. in complete agreement with the luciferase expression data shown above, this result demonstrates that bfa inhibits, but does not completely block, the formation of rcs. to study the effects of bfa on the dmvs at an ultrastructural level, mhv-infected lr cells were fixed at h p.i. and embedded in epon resin in order to be analyzed by electron microscopy. dmvs (indicated by the asterisks in fig. a ) were always seen organized in clusters often located in the perinuclear area. the morphology and dimensions of these vesicles were similar to those previously described for the dmvs harboring the rcs [ , , , , , ] . importantly, these vesicles were not observed in mock-infected cells (data not shown). fig. b shows a close view of these dmvs, in which the translucent interior is surrounded by a double membrane. the presence of an inner web-like structure is most likely artificial [ ] . treatment of cells with bfa ( - h) led to the expected disappearance of an apparent golgi complex with the concomitant expansion of the er volume (not shown). in these cells, vesicles with a morphology almost identical to those present in non bfatreated cells were observed (fig. a) . however, the number of these dmvs was significantly decreased (p, . ) in bfa-treated cells as compared to non-treated cells ( . vs. . on average per section, fig. c ). the reduction in the number of dmvs is likely to be an underestimation as only em sections were included in the analyses in which at least one replication vesicle could be detected. strikingly, the double membrane of the replication vesicles was visually more pronounced in bfa-treated cells than in untreated cells (fig. b) , which might relate to the swelling of the er observed after bfa addition. the dmvs were slightly bigger in the bfa-treated cells ( . nm +/ . compared to . nm +/ . in non-treated cells; p, . ; fig. d ), although the significance of this latter observation is not clear at present. overall, our ultrastructural analysis of mhv-infected cells confirms that treatment of cells with bfa decreased the number of replication vesicles, consistent with the reduced viral rna replication in the presence of bfa. to address which arf gefs contribute to mhv replication, we next focused on the bfa-sensitive gefs localized in the secretory pathway, i.e. gbf , big and big . first, we studied whether coronavirus replication was affected by bfa in mdck cells. these cells have a bfa-resistant golgi-apparatus due to a point mutation in gbf (m l; f. van kuppeveld, unpublished results). however, the trans-golgi network (tgn) and the endocytic organelles in mdck cells are still sensitive to bfa [ , , ] . mdck cells stably expressing the ceacam a receptor (mdck(mhvr); [ ] ) were inoculated with mhv-eflm and bfa was added either during ( - h p.i.) or after ( - h p.i.) the inoculation. the results show that mhv replication was not affected by bfa treatment of the cells during either time period (fig. a) , pointing toward a possible involvement of the bfa-sensitive gbf protein in mhv replication. to confirm that gbf , rather than big or big , is required for mhv replication, each one of these gefs was specifically and singularly depleted by rna interference before assaying mhv replication. for each target gene, three sirna oligos were transfected into hela-ceacam a cells. at h post transfection, the cells were infected with the luciferase-expressing mhv- afls. six h later, the number of viable cells and the luciferase expression levels were determined ( fig. s a and s b) as described in the materials and methods. in fig. b the results are presented as relative luciferase expression (rii) levels, i.e. the luciferase activity expressed relative to mock-treated cells after correction for the number of viable cells. transfection of control sirnas targeting the housekeeping protein glyceraldehyde -phosphate dehydrogenase (gapdh) did not change the rii, whereas sirnas targeting firefly luciferase reduced the rii up to % (p, . ) demonstrating the efficiency of the sirna transfection. importantly, down-regulation of gbf resulted in a drastic inhibition of rii (p, . ) whereas sirnas targeting big and big did not have a significant effect (fig. b) . almost identical results were obtained when the three sirna oligos for each gene were singly transfected (data not shown). in a parallel experiment, we demonstrated that the down-regulation of the major target of gbf , arf , had a similar phenotypic effect on mhv replication as seen for gbf (fig. b) . to prove the specificity of our results, we performed a series of controls. first, the specific knockdown of the respective mrnas after sirna transfection was confirmed by quantitative rt-pcr analysis. at h after transfection of the sirnas, the corresponding mrna levels for big , big , gbf and arf were found to be reduced by %, %, %, and %, respectively. the mrna levels were not affected after transfection of non-corresponding sirnas, demonstrating the specificity of the mrna depletion (data not shown). second, the functional knock-down of gbf and arf at the protein level was demonstrated by cotransfection of plasmids encoding gbf -yfp and arf -yfp together with either the gbf -or arf -specific sirnas, respectively. this approach was chosen because of the unavailability of specific anti-antibodies. twenty-four h after transfection, the cells were fixed and yfp-positive cells were counted. fig. c demonstrates that gbf and arf expression are prohibited in the presence of their specific sirnas. next, we analyzed whether inhibition of mhv replication after depletion of arf coincided with a collapse of the golgi complex as observed after bfa treatment. again, hela-ceacam a cells were transfected with sirnas targeting arf and subsequently processed for immunofluorescence at h post transfection using the gm antibody. in the arf sirna-transfected cells, the gm staining was indistinguishable from that in mock-treated cells (fig. d) indicating that loss of arf did not lead to the collapse of the golgi into the er. this is in complete accordance with the results of volpicelli-daley et al. [ ] , who demonstrated that arf depletion alone is not sufficient to mimic the bfa effect on the golgi complex, but rather requires a simultaneous depletion of arf and arf [ ] . having established that depletion of gbf or arf affects mhv replication profoundly, we studied whether the formation of the mhv rcs was similarly affected. to this end, we performed a similar knock down experiment in which we transfected sirnas targeting either arf or gbf and subsequently infected the cells with a recombinant mhv, which expressed an additional copy of nsp , now fused to gfp. the nsp -gfp fusion protein co-localizes with nsp and provides an additional marker for the rcs (data not shown). six hours after infection the cells were fixed and processed for immunofluorescence with the nsp antibody. in mock transfected cells, many gfp and nsp positive foci were observed, which largely co-localized (fig. e ). in agreement with the relative luciferase expression values shown in fig. b , both in arf -and gbf depleted cells, the number and intensity of the nsp positive foci was reduced, similar to what had been observed in bfa-treated cells (fig b) . apparently, the number of mhv rcs is reduced in these cells. strikingly, however, it appeared that the nsp -gfp expression was much more affected than that of nsp by the depletion of either arf or gbf , as hardly any gfp fluorescence could be detected. while nsp is expressed directly from the viral genome, the nsp -gfp fusion protein is expressed from a subgenomic mrna and hence replication and transcription is required for its expression. these results therefore indicate that not only fewer rcs are formed in the absence of either gbf or arf , but that these rcs are also impaired in their rna synthesis. in conclusion, our results demonstrate that depletion of gbf and arf reduces mhv replication as well as the number of rcs. furthermore, our results indicate that the rcs formed in the absence of either gbf or arf are less active. in addition, inhibition of mhv replication is not caused by the collapse of the golgi apparatus per se, as in arf -depleted cells virus replication is severely affected whereas the overall morphology of the golgi complex is unaltered. we next addressed the question whether arf is recruited to the replication sites. to this end, lr cells expressing wild type arf fused to yfp were infected with mhv-a and either fixed at an early ( h) or a late ( h) time point p.i. before identifying the replication sites by immunostaining the cells with nsp antibodies. figure a shows that arf -yfp was predominantly localized to the golgi apparatus (indicated by the arrowhead on the left panel of fig. a ) both at h p.i. and h p.i. at h p.i., only in a minority of the cells co-localization between arf and nsp was observed (indicated by the arrows in fig. a ). no co-localization could be observed in infected cells at h p.i. similar results were obtained for gbf (data not shown). many downstream effectors of arf have been described, and the list is still growing. one of the best known functions of arf involves the regulation of copi-mediated vesicular transport. for the bfa-sensitive poliovirus, copi has been found to localize at the replication vesicles [ ] . to study whether a similar recruitment of copi to the replication vesicles occurs during mhv replication, we determined its localization in mhv-infected cells. thus, hela-ceacam a cells were infected with mhv-nsp gfp. this recombinant virus allowed us to directly visualize the replication vesicles without having to perform an immunostaining with the anti-nsp antibodies. this was desirable as both the antibody against accop (two subunits of the copi coat) and the nsp antibody had been raised in rabbits. at h p.i. the cells were fixed and processed for immunofluorescence analysis using the accop antibody. the results show that, in addition to a diffuse staining throughout the cell, copi was primarily localized in a golgi-like pattern (fig. b) . copi did not co-localize with the nsp -gfp positive sites, indicating that copi was not recruited to the replication sites of mhv. another well known effector of arf is phospholipase d (pld), a lipid-metabolizing enzyme involved in membrane dynamics and vesicular transport [ , ] . to analyze whether rcs recruit pld, lr cells were transfected with a construct expressing pld b fused to gfp and subsequently infected with mhv-a . the cells were fixed at h p.i. before identifying the replication sites by immunostaining the cells with nsp antibodies. no co-localization between the rcs and pld b could be observed (fig. s a) . furthermore, specific inhibition of pld by -butanol [ ] did not affect mhv luciferase expression compared to controls (fig. s b) . further studies will be required to examine the role of other arf effectors. finally, we studied whether normal vesicular trafficking is affected in mhv-infected cells. to investigate this, we made use of a gaussia reporter gene, the protein product of which is secreted upon expression [ , ] . cells were transfected with a plasmid encoding this gene under the control of a cmv promoter and subsequently infected with either mhv-a , mock-infected, or treated with bfa. at . h p.i. the intracellular and extracellular levels of gaussia luciferase were measured. thus, the ratio of the luciferase activity in the cell lysate and in the culture supernatant was determined for each condition. while in mock-infected cells almost % of the total amount of gaussia luciferase was found in the culture supernatant, in mhv-infected cells, the amount of secreted gaussia luciferase was decreased about -fold to % (fig. ) . bfa treatment inhibited, as expected, gaussia protein secretion almost completely. from this we conclude that although mhv rna replication depends on gbf mediated arf activation, mhv infection does not drastically impair the secretory pathway. this result is not unexpected, as coronaviruses require a functional secretory pathway for the release of their progeny virions. rna viruses use and manipulate cellular membranes for the assembly of their replication and transcription structures. we and others have shown that coronaviruses exploit the early secretory pathway, but the way in which they do so is not understood. in this report we have demonstrated using several different approaches that mhv requires a functional gbf -arf pathway for efficient rna replication. first, we showed that mhv, but not sindbis virus replication is sensitive to bfa in murine lr cells. second, we observed that mhv replication is not sensitive to bfa in mdck cells, which contain a bfa-resistant gbf . third, we showed that the specific sirna-based knockdown of the bfasensitive gef gbf , but not big and big , strongly affects mhv infection. fourth, also arf , a downstream effector of gbf , appeared to be required for efficient mhv replication, as shown by the inhibition of mhv-driven reporter gene expression during sirna-mediated down regulation of arf as well as during expression of an inactive arf mutant. the inhibition of coronavirus rna replication in the presence of bfa is either caused by direct inhibition of rc formation, resulting in reduced rna replication, or by inhibition of rna replication via another mechanism, resulting in reduced de novo formation of rcs. though it is difficult to distinguish between these two scenarios, our results indicate the latter option to be most plausible. although bfa reduced the number of rcs, their formation was not completely blocked as demonstrated by immunofluorescence staining of the rcs using the nsp antibody and by quantitative electron microscopy. apparently, bfa did not prevent the formation of rcs after translation of the incoming genomic rna. in addition, mhv replication was inhibited by bfa throughout the infection. early in infection the inhibition was more profound than at later time points, when many transcriptionally active rcs have already been formed. furthermore, while the inhibition of reporter gene expression in the presence of bfa, or after depletion of either gbf or arf , is in complete agreement with the reduced numbers of rcs, our results also indicate that the few rcs that are formed in the absence of gbf or arf are less active. therefore, we hypothesize that bfa inhibits mhv rna replication by affecting rc maturation or functioning rather than rc formation per se (fig. ) . replication of several viruses has now been shown to be sensitive to bfa. these viruses, which include poliovirus [ , , ] , grapevine fanleaf nepovirus [ ] and mhv (this study), all appear to use er-derived membranes for the formation of their rcs ( [ ] , [ ] and [ , , ] , respectively). strikingly, picornaviruses belonging to different genera were found to differ in their sensitivity to bfa, which was suggested to correspond with differences in the assembly of their rcs [ ] . replication of equine arterivirus, a distant relative of coronaviruses, was observed not to be sensitive to bfa [ ] , while other nidoviruses have not been studied to date. unlike for poliovirus [ ] , arf is hardly recruited to coronavirus rcs. we therefore hypothesize that downstream effectors of gbf -arf are involved in mhv replication. to date, more than downstream effectors of arf have been identified [ , , , ] , and each one of these might thus be somehow implicated in the functioning of the mhv rcs. the most well known effector of arf is copi. for picornaviruses, bfa sensitivity was suggested to correlate with the recruitment of copi to these sites [ ] . however, no co-localization between copi and the mhv rcs could be observed. this is in agreement with the almost complete absence of arf at these sites. in addition, coronavirus rcs did not co-localize with pld nor was coronavirus replication affected by inhibition of phospholipase d, a lipid-metabolizing enzyme involved in membrane dynamics and vesicular transport [ , ] . it might be that the gbf -arf pathway simply functions to deliver lipids to the rcs. in agreement herewith, cerulenin, an inhibitor of phospholipid biosynthesis, severely inhibits mhv replication (c.a.m. de haan, unpublished results). nonetheless, the observed inhibition of mhv infection after bfa treatment is probably not an indirect consequence of the collapse of the golgi complex as, unlike bfa treatment, arf depletion did not affect the morphology of the golgi complex (fig. d) . consistent herewith, another recent study showed that arf depletion did not affect the golgi morphology or protein transport [ ] . several studies have indicated that coronavirus replication and the er are closely connected. electron microscopical analyses of infected cells showed the partial co-localization of coronavirus replicase proteins with the soluble er resident protein disulfide isomerise [ ] , while the dmvs were often found in close proximity to the er and occasionally in continuous association with it [ , ] . furthermore, when expressed in the absence of a coronavirus infection, the nsp and nsp proteins were inserted into the er and became modified by the addition of n-linked sugars [ , , ] , whereas expression of tagged mhv nsp in mhv-infected cells resulted in the recruitment of the protein to the replication complexes [ ] . in addition, coronavirus replication was inhibited when the er export machinery was blocked by the use of the kinase inhibitor h or by expression of dominantactive mutant of the small gtpase sar [ ] . we now show by using several approaches that mhv rna replication also depends on gbf -mediated arf activation. apparently, an intimate association exists between the early secretory pathway and mhv replication. interestingly, whereas h blocked rc formation completely [ ] , this was not the case when the gbf -mediated activation of arf was impaired by bfa. rather it appears that the rcs formed in the absence of gbf or arf are less active, suggesting a role for these proteins in rc maturation or functioning (fig. ) . clearly, further investigations are needed to unravel the precise mechanism by which the secretory pathway contributes to the biogenesis of functional coronavirus rcs and to rna replication. hela-ceacam a cells were generated by transfecting hela cells (obtained from the mpi-cbg high-throughput technology development studio [ ] ) with the expression plasmid pmhvr [ ] as described before [ ] . murine lr [ ] , hela-ceacam a, and madin-darby canine kidney-ceacam a [mdck(mhvr); [ ] cells, which all stably express the mhv receptor mceacam a, were maintained as monolayer cultures in dulbecco modified eagle medium (dmem; cambrex) containing % fetal calf serum (fcs), iu of penicillin/ml, mg of streptomycin/ml (all from life technologies), and . mg/ml g (life technologies, paisley, uk). split cells, i.e. bhk- cells stably expressing sindbis virus structural proteins [ ] , were maintained in glasgow mem (invitrogen) containing % fcs, iu of penicillin/ml, mg of streptomycin/ml, mg/ml g and mg/ml hygromycine b (boehringer gmbh) and used to generate sindbis pseudovirus particles containing a replicon expressing firefly luciferase. to this end, the firefly luciferase gene was cloned into the psinrep vector (invitrogen) using conventional cloning procedures. the resulting vector was subsequently processed further according to polo et al. [ ] to produce the pseudovirus particles. lr cells were used to propagate the wild type and recombinant mhvs (based on strain a ). the recombinant viruses expressing the firefly luciferase gene (mhv-eflm and mhv- afls) or the red fluorescent protein (rfp) gene have been described before [ , ] . the recombinant virus mhv-nsp gfp, which expresses a nsp -green fluorescent protein (gfp) fusion protein, was generated in a similar way as described previously for mhv-nsp gfp [ ] . briefly, an nsp -gfp fusion construct was cloned behind an additional transcription regulation sequence into a derivative of the rna transcription vector pmh [ ] . targeted recombination to obtain the recombinant mhv-nsp gfp was performed as described before [ ] . antibodies directed against the mhv nsp (anti-p , kindly provided by m. denison, vanderbilt university medical center, nashville, usa [ ] ), the amino terminus of the mhv m protein (j . , kindly provided by j. fleming, university of wisconsin, madison, usa [ ] ), against accopi (anti-accopi, kindly provided by f. wieland, university of heidelberg, germany), against gbf (anti-gbf ) and against the golgi marker gm (anti-gm ) (the latter two from bd transduction laboratories, figure . model of mhv rcs and their links to the early secretory pathway. two major steps in the anterograde protein secretion route (reviewed in [ ] ) are linked to mhv rc formation and/or rna replication. first, transport of proteins out of the er requires er exit site formation controlled by sar p [ , , ] . blocking this early step by using the drug h [ ] or by expressing of a dominant mutant of sar p [ ] blocks mhv replication profoundly [ ] . next, er exit sites develop into, or form de novo, vesicular-tubular clusters (vtcs) (also called ergic), for which gbf and arf are required. this step, which can be blocked by bfa, by expressing a dominant-negative mutant of arf or by down-regulating arf using sirnas [ ] , is also involved in mhv rc formation (this manuscript). however, a fully functional secretory pathway is not essential, as a dominantactive mutant of arf , which blocks transport between vtcs and cis-golgi [ ] , does not impair mhv replication. doi: . /journal.ppat. .g san jose, usa) were used. the conjugated secondary antibodies were purchased from jackson immunoresearch laboratories. plasmids containing the different arf and gbf genes in frame with either a gfp or a yellow fluorescent protein (yfp) tag were obtained from g. romero [ ] and c. jackson [ ] , respectively. pgbf -yfp and parf -yfp encode the wild type proteins fused to yfp. parf t n-yfp and parf q l-gfp encode a dominant-negative and a dominant-active mutant of arf fused to yfp and gfp, respectively [ ] . the pn-egfp plasmid, which encodes the mhv nucleocapsid (n) protein extended at its c-terminus with gfp was constructed by cloning a pcr fragment, specifying the n gene without its stop codon, into pegfp-n (clontech), using conventional cloning procedures. the plasmid encoding the gaussia reporter gene behind a cmv promoter was generated by replacing the egfp gene in pegfp-c (clontech) with the gaussia luciferase gene from pgluc-basic (new england biolabs) using conventional cloning methods. the viral expression plasmid pm f-rl-m was generated by cloning a synthetic dna segment (genscript ß ) corresponding to the extreme ' nt and the extreme ' nt of the mhv-a genome, separated by a nhei restriction site and flanked by a t promoter and a poly(a) sequence, upstream and downstream, respectively, into puc . subsequently, the coding region for renilla luciferase, obtained from prlnull (promega), was cloned into the nhei-digested vector. subconfluent monolayers of lr cells grown on coverslips in cm tissue culture dishes were overlaid with transfection medium consisting of . ml of optimem (invitrogen) that contained ml lipofectamine (invitrogen) and mg of dna. after hours, the medium was replaced with dmem containing % fcs. at h after transfection the cells were processed further as indicated. the plasmid pm f-rl-m was linearized using a paci restriction site directly downstream of the poly(a) sequence, and subsequently rna transcripts were produced using the t messagemachine kit (ambion) according to the manufacturer's instructions. of the transcripts, . pmol of rna was transfected into mock-or mhv- afls-inoculated lr cells at h p.i. using lipofectamine (invitrogen). next, the cells were treated with or without mg/ml bfa from h until h p.i., after which the cells were lysed and intracellular renilla and firefly luciferase activity was measured with the dual-luciferase assay kit (promega) according to the manufacturer's protocol. cells were fixed using a % paraformaldehyde solution in phosphate buffered saline (pbs), and subsequently permeabilized with . % triton-x in pbs. next, the cells were incubated for h with the first antibody diluted in pbs containing % normal goat serum. after several washing steps, the cells were incubated with an appropriate dilution of secondary antibody in the same buffer for h. after three subsequent washing steps, the coverslips were mounted in fluosave (calbiochem). the immunofluorescence staining was analyzed using a confocal laser-scanning microscope (leica). gfp/yfp and fitc were excited at nm and cy at nm. virus replication was quantified by determining either the virusdriven luciferase expression levels or the amount of genomic rna. to this end, lr or mdck(mhvr) cells were inoculated at a multiplicity of infection (moi) of with mhv-eflm, mhv- afls or sindbis pseudovirus particles in the presence or absence of mg/ ml bfa in dmem. after h, the culture medium was replaced by dmem containing % fcs and antibiotics, again in the presence or absence of mg/ml bfa. at the indicated time points, the luciferase expression in the cells was determined using the firefly luciferase assay system (promega) according to manufacturer's instructions and using a single-tube luminometer (turner designs, td- / ). alternatively, rna was isolated from the cells using the qiagen rneasy kit (qiagen) according to the manufacturer's protocol. taqman single-tube reverse transcription-pcr (rt-pcr) assay (pe biosystems, foster city, california, usa) was performed essentially as described by de haan et al. [ ] . the reactions were performed in triplicate according to the manufacturer's instructions by using the taqman rt-pcr kit (pe biosystems) and an abi prism sequence detector. small interfering (si) rna-mediated knockdown experiments sirna duplexes targeting different sites within the coding sequences of gbf , big , big , and arf were designed by and obtained from ambion inc. (three sirnas per gene; nucleotide sequences available on request). sirnas targeting gapdh, luciferase gl +gl , and kif (all from ambion) were taken along as controls in each experiment. one day after seeding the hela-ceacam a cells, they were transfected with a final concentration of nm sirna using oligofectamine (invitrogen). seventy-two h after transfection, the cells were inoculated with mhv- afls at such a moi that approximately % of the mocktreated cells became infected. at h post infection (p.i.), the cell number and viability was measured by wst- assay according to the manufacturer's protocol (roche diagnostics gmbh). subsequently, the medium was replaced by dmem lacking phenol red (cambrex) and steadylite hts firefly luciferase substrate (perkin elmer) was added. luciferase expression was determined using a luminescence plate reader (berthold centro lb ). each sirna experiment was performed in triplicate. for each well, luciferase values were corrected for the cell number and viability as determined by the wst assay relative to the mock-treated cells. to validate the functional knockdown of the targeted genes, mrna levels of each gene were determined after sirna transfection using taqman gene expression assays (applied biosystems, ca, usa), according to the manufacturer's protocol. to determine whether sirnas targeting the arf and gbf genes effectively depleted hela-ceacam a cells from the corresponding proteins, a sirna transfection experiment was performed in which ng of the plasmids encoding either arf -yfp or gbf -yfp were added to the transfection mixture containing the corresponding sirnas. twenty-four h after transfection, the cells were fixed and representative images were taken by an automated cellworxtm microscope (applied precision) with a objective. lr cells transfected with parf -yfp, parf t n-yfp, or parf q l-gfp were inoculated with mhv-rfp (moi of ) at h post transfection. two h p.i. mm hr peptide [ ] was added to inhibit syncytia formation. at h p.i., the cells were collected and fixed using a % paraformaldehyde solution. after two washes with pbs, the samples were analyzed employing a facscalibur tm flow cytometer (becton dickinson) gating for yfp/gfp-positive cells in the forward and side scatter, such that a limited cell population with similar arf expression levels was selected. from the yfp/gfp-positive population, the fraction of cells expressing rfp was determined. lr cells infected with mhv-a and treated from to h p.i. with or without mg/ml bfa were resuspended in % glutaraldehyde in . m cacodylate buffer (ph . ) for at least h at room temperature (rt). this buffer was then replaced with fresh one and the fixation was continued overnight. cells were then centrifuged, washed times with the . m cacodylate buffer before being post-fixed in % oso , . % ferrocyanide at uc for min. next, the cell pellet was washed times with distilled water and left sit in the last wash for min before being centrifuged and resuspended in warm % low melting point agar (roche, basel, switzerland) and immediately spun down. after solidification of the agar on ice, the tip containing the cells was cut into small mm blocks. these blocks were then dehydrated by immerging them into increasing amounts of ethanol ( %, %, %, %, % and times %) by incubation on a rotatory wheel for at least min at rt for each step. these amalgamations were followed by others in , -propylene oxide (merck, haarlem, netherlands)-epon resin ( : ) for min, , propylene oxide -epon resin ( : ) for min, , -propylene oxide-epon ( : ) for min and epon resin overnight. the epon solution was prepared by mixing g of glycid ether , g of dodecenylsuccinic acid anhydride, g of methylnadic anhydride and ml of benzyldimethylamine (all from serva, heidelberg, germany). the epon resin was then replaced the following day with freshly made resin and the incubation continued for h at rt. after centrifugation at rpm for min, the epon resin was polymerized by heating 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inhibitor h three distinct steps in transport of vesicular stomatitis virus glycoprotein from the er to the cell surface in vivo with differential sensitivities to gtp gamma s we would like to thank d. duijsings for helpful discussions, m. denison and j. fleming for providing us with the antibodies directed against the mhv nsp and m protein, respectively, and g. romero and c. jackson for providing the plasmids containing arf and gbf , respectively. key: cord- -sufwsu authors: bär, séverine; rommelaere, jean; nüesch, jürg p. f. title: vesicular transport of progeny parvovirus particles through er and golgi regulates maturation and cytolysis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: sufwsu progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. this process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. by colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny pv particles become engulfed into copii-vesicles in the endoplasmic reticulum (er) and are transported through the golgi to the plasma membrane. besides known factors like sar , sec , rab , the erm family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing copii-vesicles. these proteins also contribute to the transport through er and golgi of the well described analogue of cellular proteins, the secreted gaussia luciferase in absence of virus infection. it is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. finally, parvovirus egress via er and golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). while not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through er and golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway. egress of enveloped viruses is typically associated with the cell secretory pathway guiding the particles and/or their precursors through cellular organelles, in particular the endoplasmic reticulum (er) and the golgi cisternae [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this regard, enveloped viruses usurp the cellular secretory machinery in order to achieve the efficient transport of progeny viruses to the plasma membrane and the maturation of precursor particles into infectious virions [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in contrast, non-enveloped viruses are thought to be released as mature virions through a cytolysis burst at the end of infection [ ] [ ] [ ] . however, there is evidence on non-enveloped virus egress through active transport besides the major lytic pathway. a few non-enveloped lytic viruses were shown to be actively transported to the cell periphery and released prior to cell lysis. for instance sv was detected in intra-cytoplasmic smooth membrane vesicles and described as being released from the cell before cytopathic effects are seen [ ] . similarly, cocksackie b virus was found to be transferred from cell to cell through microvesicles [ ] . we also recently reported that in the case of non-enveloped lytic parvoviruses that progeny virions are actively transported from the nucleus to the plasma membrane (pm) through vesicles in a gelsolin-dependent manner [ ] . the cellular secretory pathway has been characterized in great detail and a number of proteins involved in the specific recognition of cargos, the formation and loading of vesicles, and the guided transport through the cytoplasm have been identified [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . once in the er, transport of cargos is mediated by coat protein complex ii (copii) vesicles. engulfment of cargos into copii vesicles at er exit sites is triggered by the activation of the small gtpase sar , a process which in turn induces the recruitment of two heterodimeric complexes, sec -sec and sec -sec , and their assembly into a protein coat. in this complex, sec -sec is responsible for the selection and binding of the cargo. sec can either bind directly to the transported protein if it features a transmembrane domain, or interact with a transmembrane receptor specific to the cargo in the case of a secreted protein. in both cases, sec -sec bound to the cargo forms a ternary complex which concentrates the cargo and bends membranes. the budding vesicle is then enveloped by the sec -sec cage and released from the er by fission. during the last step of this budding, cellular factors responsible for directing the vesicle to the next compartment and fusing the vesicular membrane with the target membrane, associate with the departing vesicle [ , ] . targeting of cargo-vesicles to distinct organelles/compartments through the cytoplasm is achieved by distinct small gtpases, rab-proteins, which are associated with the surface of moving vesicles. for instance, rab targets vesicles from the er to the golgi apparatus, while rab is responsible for transport within the golgi cisternae and for retrograde transport from the golgi apparatus to er. transport from the transgolginetwork (tgn) to the plasma membrane (pm) can take different routes and is accordingly controlled by a variety of rab proteins. among these, rab is responsible for guiding the vesicles along the direct route from the tgn to the pm, while rab shuttles tgnvesicles through recycling endosomes to the pm [ ] . a number of other rab proteins are also involved in vesicle targeting to the endocytosis and secretion pathways, and new routes are constantly being discovered. when the vesicles reach the periphery of the cell, the release of the cargo is generally achieved by fusion of the vesicular membrane with the pm [ , ] . parvoviruses (pv) are small icosahedral non-enveloped particles with a . -kb linear single-stranded dna genome. during productive infection, pvs induce dramatic morphological and physiological changes in their host cells, culminating in cell death and lysis [ , , ] . this is mainly attributed to the large nonstructural viral protein ns , a multifunctional protein involved in particle production and spread (reviewed in [ ] ). ns was shown to modulate cellular pathways by physical interactions with distinct cell components [ , ] and/or induction of post-translational modifications of cellular target proteins [ , , , ] . these targets might be modified either directly by ns /ckiia, a recently described complex formed by ns with the catalytic domain of cellular ckii [ ] , or indirectly through activation/modulation of the pdk /pkc/pkb signaling cascade [ ] . the latter activation is mediated by the virus-induced association of the erm family protein radixin with pkcg, forming a complex that controls the activity and substrate specificity of the pdk . the cellular proteins targeted by the ns /ckiia complex include the erm-family protein radixin [ ] . erm proteins are known mediators of the interplay between filamentous actin and membrane structures [ ] . in particular, radixin (rdx) has been implicated in the parvovirus mvm infectious cycle, controlling progeny particles production and spreading as measured through the formation of lysis plaques [ ] . gelsolin, an actin-severing protein was found to be another target for the ns /ckiia complex. gelsolin shown to contribute to the egress of pv progeny virions [ ] through its function in the formation and/or the pvloading of cellular vesicles, which are then targeted through the cytoplasm to the pm [ ] . ns /ckiia-mediated phosphorylation seems to play a crucial role in this process. the present investigation aims to characterize vesicular egress of non-enveloped lytic parvoviruses in more detail and to determine the impact of this process on the pv life-cycle. using fluorescence microscopy, biochemical fractionation and selective inhibition of proteins involved in vesicular transport (e.g. rabproteins), we show that pv progeny particles become engulfed into vesicles in the er and are actively transported through the golgi apparatus to the pm. the erm family proteins moesin and radixin play an important role in the formation/loading and targeting of progeny virus-containing vesicles. finally, egress through the golgi apparatus appears to play an essential role in post-assembly modification, infectivity of progeny virions, and the stimulation of virus-induced cytolysis. previous investigations have shown that progeny parvoviral particles become associated to vesicles in a gelsolin-dependent manner and are actively transported from the nuclear periphery to the plasma membrane [ ] . to further characterize this pathway, we analyzed potential association of progeny particles in mvm infected a cells with cellular factors known to be involved in generation/loading of perinuclear vesicles. an obvious candidate for viral cargo shuttling out of the cell is the secretion pathway, which starts in the endoplasmic reticulum (er), with sar , sec / and sec / -dependent formation of copii vesicles. we therefore investigated the role of these factors in the association of progeny pv virions with vesicles and the further transport to the cell periphery. in a first step, we determined whether out-going mvm virions colocalize with sec , sec and b-cop in infected a cells using laser scanning microscopy. previous reports pointed that progeny particles are associated with lamp , a protein present in the membrane of lysosomes and late endosomes/multivesicular bodies [ , ] . we therefore used colocalizations with lamp and mitochondria as positive and negative controls, respectively. the absence of any significant capsid fluorescence at h p.i. was taken as a reference for the selective detection of newly produced virions after onset of virus propagation and not in-coming virus during the infection process. furthermore, cell reinfection with progeny particles was prevented by addition of neutralizing antibodies to the medium after the initial infection. as illustrated in fig. a , progeny pv particles showed clear colocalization with the cellular vesicle forming complex, giving a first hint that mvm virions may associate with cellular vesicles assembled at the er membrane. to functionally challenge this finding, we knocked down vesicle formation through transfection of neutralizing sec antibodies and expression of dominant-negative sar . association of progeny particles with vesicles was determined by cell fractionation and monitoring single-stranded virion dna presence in the corresponding biochemical fractions [ ] . to confirm association of virus with membranes structures and to assess internalization into vesicles, cell extracts were treated, or not, with or triton x- (disruption of membranes, release of membrane associated proteins into cytoplasm) and proteinase k (elimination of all proteins that are unprotected by membrane structures) prior to previously, it was thought that non-enveloped lytic parvoviruses were released through a lytic burst of cells at the end of infection. however, recent work demonstrated that these small non-enveloped single-stranded dna viruses are actively transported through vesicles from the nucleus, the site of replication and assembly, to the cell periphery. the current investigation demonstrates that progeny particles become engulfed into copii-vesicles in the endoplasmic reticulum (er) and are transported through the golgi to the plasma membrane (pm). erm family proteins radixin and moesin appear to play an essential role in this cellular secretion pathway. while passing through er and golgi cisternae, pvs maturate through post-assembly modifications, which significantly increase the infectivity of progeny virions. finally, the vesicular transport of parvoviral particles was shown to regulate virus-induced cytolysis, thereby accelerating the further release and spread of progeny virions. as rodent pvs are currently viewed as oncolytic agents for cancer virotherapy, it is important to further investigate the mechanism of pv egress -not only to improve the spreading of these agents through the tumor mass, but also to optimize the induction of an anti-tumor immune response upon virus -induced cytolysis. mvmp ( pfu/cell), and single rounds of infection were monitored in presence of neutralizing antibody b after initial infections. cells were fixed with paraformaldehyde at h (input) or h (mvm h) p.i., and analyzed by confocal laser scanning microscopy after double-staining with antisera specific for the indicated cell proteins (red) and mvm capsids (green). lack of interference from input virions is revealed by the absence of detectable capsid signal at h p.i. (nomarsky picture). colocalization areas appear yellow in the merge panel and are expressed as percent of total cytosolic capsids after quantification by image j analysis of infected cells from three individual experiments. to better visualize colocalization, zoom-ins were performed on indicated areas. bars indicate scales (in mm). (b-d) a cells were infected with mvmp ( pfu/cell), nch cells with hgh -pv ( pfu/cell) and harvested/fixed at h p.i. when indicated, sar functioning was jeopardized by over-expression of the dominant-negative sar k m variant (dnsar ) through transduction by raav h prior to parvovirus infection. sec was inhibited by transfection of neutralizing antibodies (asec ) h prior to infection, apkb served as control igg. to measure potential signals from incoming capsids cells untreated cells were harvested at h p.i. (b) cellular extracts were fractionated by differential (density) centrifugation to separate organelles. nuc, purified nuclei; hmf, large organelles; cyt, cytosol. the lmf-fraction was fractionated by centrifugation through iodixanol gradients allowing cellular vesicles to be identified according to their density (fractions [ ] [ ] [ ] [ ] . the presence of progeny particles was determined by southern blotting (revealing their singlestranded dna). to determine the nature of pv virion-containing vesicles, cell lysates were treated with triton x- (triton) or proteinase k (prot k) prior to fractionation. to rule out contamination from infecting particles, a fractionation experiment was performed after cell transfection with mvm infectious clone in the presence of neutralizing antibodies (transf+b panel). the migration of lamp (lysosomes/late endosomes) was determined by western blotting. (c) cells were fixed with paraformaldehyde and stained for mvm capsids (green) and counterstained with lamin b (red). fractionation. a h p.i. time-point was chosen to discriminate egress from the infection process. transfection experiments in the presence of neutralizing antibodies were performed to discriminate egress from (re-)infections. as shown in fig. b and in agreement with the above hypothesis, viruses were found in the subcellular vesicle fractions, from which they were removed by exposing cell lysates to triton prior to fractionation. this vesicle association was not detected when target cells were either transfected with sec neutralizing antibodies or if the cells expressed dominant-negative sar mutants. this suggests that mvm progeny virions become engulfed into vesicles by components of the cellular secretory pathway in the perinuclear er. similar results were obtained with the related rodent parvovirus strain hgh -pv after infection of the human glioblastoma cell line nch , suggesting that vesicular egress of progeny virions is a general feature of rodent pvs. we next studied the effect of functional sec / and sar on pv egress. to this end, mvm-infected a cultures were analyzed for the proportion of cells supporting cytoplasmic transport of progeny virions, as determined by immunofluorescence microscopy and the amount of infectious particles shed into the medium supernatant. cells lacking functional sec or sar were unable to transport progeny virions away from the nucleus, resulting in a marked increase in the proportion of cells showing only nuclear virus (fig. c ). while total virus production was not much affected by sec and sar knockdowns, the observed retention of virions in the (peri)nuclear area correlated with a significant reduction of virus release in the culture medium as determined by measuring the amounts of virion dna (dot blots) and/or infectious virions (plaque assays) in the supernatant (fig. d ). we next characterized the pathway followed by vesicles carrying pv progeny particles. many enveloped viruses are transported through one or the other compartment of the cellular secretory pathway before mature virions are released by budding at the plasma membrane [ ] [ ] [ ] [ ] [ ] [ ] . to determine the mode of parvoviral egress, we first examined the presence of mvm particles within cell organelles, and the association/colocalization of the particles with proteins involved in the targeting of intracellular vesicles. the latter proteins include the small gtpases, rab that are located on the outside of vesicular membranes and are guiding the vesicles between subcellular compartments. colocalisation with mitochondria (mitotracker), er (calnexin, sec ) and golgi (gm ) as well as the small gtpases rab (er to golgi), rab (trans golgi network [tgn] to pm), and rab (recycling endosomes) were investigated by confocal laser scanning microscopy. rab which is involved in retrograde transport from golgi to er and is mainly detectable in the golgi complex was used as a second marker for this organelle. it was shown previously that pvs become associated with lamp -containing vesicles (lysosomes, late endosomes/ multivesicular bodies (mvb) [ ] . therefore, this marker served as a positive standard. (re-)infections were inhibited by addition of neutralizing antibodies after the initial infections. as shown in fig. s and summarized in fig. a , strong colocalizations were observed between mvm particles and the er resident protein calnexin, the golgi complex marker gm , and the small gtpases rab and rab , known to control vesicular transfer between these compartments. interestingly, significant co-staining was seen with rab (tgn-re-pm), while only background levels were obtained with mitotracker and rab (tgn to pm). the presence of mvm progeny particles in er and golgi was then confirmed by biochemical fractionation of cellular extracts, separating er membranes from the golgi complex by ultracentrifugation on a nycodenz step gradient (fig. b ). altogether these results strongly argue for the active transport of progeny pv particles through er and golgi. to functionally support these findings, secretion of intracellular proteins was inhibited by selective inactivation of distinct rab proteins through (over-)expression of the corresponding dominantnegative mutants lacking gtpase activity [ , , ] . to this end, a cells were transduced with raav virions expressing dominantnegative (and for controls functionally active) rab proteins prior to mvm infection. to ensure efficient expression, the dnrab mutant genes were placed under the control of the parvoviral p promoter, and cells were co-transduced with raav:p -dnrab and, in absence of the pv ns protein (i.e. in absence of pv infection), with a second recombinant aav virus expressing a transactivator protein specific for this promoter (raav:p -transactivator). this synthetic transactivator protein is comprised of two distinct ns domains, the n-terminal site-specific dnabinding domain (aa - ) and the c-terminal transactivator domain (aa - ) linked by gfp. dimerization of this polypeptide takes place through an n-terminal gst-tag. in contrast to the natural pv ns protein, this polypeptide expressed from the pv p -promoter proved to be non-toxic as assessed by measuring metabolic activity and cell lysis (fig. s a ). under these conditions, selective inhibition of the secretion pathway was first tested independently of mvm infection, using cells transfected with a plasmid expressing secreted gaussia luciferase (gluc). indeed, this enzyme is known to be transported by vesicles from er to golgi and further to the plasma membrane following the regular rab and rab dependent secretion pathway [ ] . as shown in fig. a , secretion of gluc was efficiently blocked as a result of the expression of dnsar (driver for the formation of transport vesicles at er exit sites), dnrab and dnrab but only marginal dnrab , demonstrating the functional impact and specificity of these variant proteins. we next tested the effects of rab , rab and rab inhibition on the vesicular egress of mvm through er (sec ) and golgi (rab ) by colocalization with corresponding marker proteins ( fig. b; fig. s ), and on the release of progeny particles in the medium by dna dot blot analyses ( fig. c ) and plaque assays (fig. d ). as expected, none of the dominant-negative mutants prevented mvm particles from entering the er compartment. in contrast, mvm transport from er to golgi, and virus secretion into the supernatant were strongly impaired upon expression of dnrab . moreover, a very limited reduction of secreted particles was observed with dnrab , and nothing at all upon inhibition of rab , suggesting that pv particles take a different route from the golgi apparatus to the plasma membrane as compared to gluc. due to their impact on mvm plaque morphology in a cells, the erm family proteins moesin (moe) and radixin (rdx) have been implicated in the spreading capacity of this parvovirus [ ] . this observation, together with evidence of moesin being involved in endocytosis [ ] , led us to determine whether erm proteins may play a role in the vesicular egress of progeny pv virions. in agreement with this possibility, we first showed that moe and rdx protein can be detected together with sec and virion dna in vesicular fractions of mvm-infected a cells (fig. a, top panels). to put moe and rdx contribution to the test, we (over)expressed mutants thereof previously shown to interfere with endogenous protein functioning [ ] . impact on viral egress was then evaluated measuring virion dna presence in vesicles (fig. a , bottom), viral capsid intracellular distribution (fig. b ) and colocalization with cellular compartment marker proteins (figs. c and s ), as well as progeny virion release into the medium (fig. d ). the dominant-negative moesin (moet a) and radixin (rdxdl[p]) mutants strongly interfered with the loading of progeny virions into cytoplasmic vesicles ( fig. a and c), resulting in a dramatic inhibition of virion release into the medium (fig. d) . accordingly, viral capsids were hardly detectable in the cytoplasm under these conditions (fig. b ). in the presence of the functionally active rdxy f mutant (consensus phosphorylation site for receptor tyrosine kinases [ ] ), mvm virions became associated with the vesicular fractions ( fig. a ) and were readily found in the cytoplasm of infected cells (fig. s ). virus neutralizing antibodies were added after the initial infection to prevent cell re-infections with progeny viruses. colocalization was quantified by image j analyzing infected cells from three individual experiments with . cells. lys/le, lysosomes/late endosomes (lamp ); mit, mitochondria (mitotracker); er, endoplasmic reticulum (calnexin [clnx]); tgn, trans golgi network; re, recycling endosomes; pm, plasma membrane. (b) cellular extracts were fractionated to separate golgi-versus er-membrane structures by nycodenz gradient centrifugation. progeny virions were detected by southern blotting measuring single-stranded virion dna, the presence of gm (golgi) and calnexin (er), respectively, by western blotting. to preclude interference from input viruses, a control was made by transfecting cells with mvm infectious dna clone. transfected cells were further incubated in the presence of neutralizing b antibodies to ensure that infection is limited to a single round and cell do not get reinfected with progeny viruses (transf+b panel). doi: . /journal.ppat. .g (fig. b) . however, when vesicular egress through er and golgi was monitored, a strong decrease was observed in the colocalisation with the golgi marker (rab , see fig. s ), indicating the use of an alternative route seemingly involving secretory vesicles (increased rab colocalization) and recycling endosomes (preserved rab colocalization). interestingly, in the presence of rdxy f, progeny virions were not released efficiently into the medium (fig. d) , suggesting that virion transport through er and golgi plays an important role in the efficient release and spreading of pvs. erm proteins are known mediators of the interplay between actin cytoskeleton and membrane structures. the involvement of rdx and moe in the formation/loading of pv-containing vesicles raised the question, whether these proteins are selectively recruited by pvs or are genuine components of the cellular secretory machinery. this was investigated by expressing the secreted protein gluc in mouse a and human nch cells, and by determining the impact of over-expressing dominant-negative erm variants on the proportion of enzyme secreted in culture supernatants in the absence of pv infections. as shown in fig. e , inhibition of endogenous rdx by rdxt a strongly reduced the amount of gluc in the medium from both cell cultures. a lesser but significant inhibition of gluc secretion was also caused by dnmoe (moet a). in contrast dnez (ezt a) failed to impair secretion. these data strongly argue for the essential role played by the erm family proteins rdx and moe in the secretion of cellular proteins. the fact that dn mutant forms of these proteins inhibited in similar way pv egress gave further support to the involvement of vesicular transport in this process. a productive pv infection typically culminates in the rupture of the cell plasma membrane, as apparent from the generation of lysis plaques [ ] and the release of intracellular polypeptides into medium supernatants [ ] . this final lytic event clearly contributes to virus release and spreading, questioning the role of the vesicular egress of pv particles taking place prior to cell death. the above-mentioned impairment of progeny particle release upon down-modulation of vesicular transport (fig. d, d , and d) made us wonder whether the latter transport may not only contribute to pre-lytic virion release but also control the lytic process which follows. to this end, time-course experiments were performed with mvm infected a cells under conditions where viral egress and release were modulated as shown in previous experiments. permeabilization of the plasma membrane was then measured by propidium-iodide incorporation using fluorescent microscopy and image j software quantification. mock-treated a cells served as negative controls. in addition, the relation of the lytic activity observed to the presence/absence of pv egress was tested by performing experiments with a recombinant mvm vector (rmvm). this vector produces similar amounts of ns as the wild type virus in a single-round infection, yet it fails to produce capsid proteins, and as a result, does not lead to virus egress [ ] . therefore, infection of a cells with rmvm was used to measure the mere lytic activity induced upon ns expression. the results are summarized in fig. . mvm-induced cytolysis was strongly reduced when (i) the virus lacked the capacity for progeny particle production and in consequence transport to the pm (rmvm), or (ii) vesicular transport was either inhibited (dnsar , rdxdl[p], dnrab ) or by-passing the golgi apparatus (rdxy f). inhibition of particle loading (dngln, moet a) resulted in an intermediate phenotype as apparent from the delayed and significantly reduced pi incorporation. this was also the case of cells expressing dnrab , correlating with a reduction of virus release into the medium (see fig. c and d), presumably due to inhibition of the golgi-tgn-re pathway. in contrast, no or little effects on cell permeabilization were seen with dnrab , which did not impair egress of pvs ( fig. c and d) . altogether these results suggest that vesicular transport of progeny particles through the golgi complex plays an important role in the induction of cytolysis and to ensure the further release of progeny particles at the end of infection. progeny mvm particles become modified during egress through er and golgi, resulting in a gain of infectivity vesicular trafficking through the golgi apparatus raises the intriguing possibility of assembled pv particles undergoing maturation and gaining full infectivity in this compartment. this prompted us to first determine whether the egress process was accompanied with post-assembly phosphorylations of mvm capsids. unlike ns whose phosphorylation is targeted at serine and threonine residues [ ] , mvm (as aav ) progeny particles purified from infected cells were also phosphorylated at tyrosines (fig. a&b) . after metabolic p-labeling, these phosphorylations resolved into a complex pattern of seven distinct tryptic phosphopeptides in two-dimensional electrophoresis/chromatography analysis (fig. c) . to determine the impact of vesicular transport on mvm capsid phosphorylation, effector protein variants known to inhibit viral egress (ckiiae a [ ] , moet a, and rdxy f [see above]) were tested for their effects on the capsid tryptic phosphopeptide profile. as shown in fig. c , the inhibition of vesicular egress or its re-routing away from the golgi compartment, led to remarkable changes in the capsid phosphorylation pattern, in particular the characteristic loss of three distinct phosphopeptides (a, c, and e). these data provide strong evidence of parvoviral capsid modifications during egress through er and golgi. we next determined the impact of vesicular egress on viral infectivity. virus stocks were produced both in parental a cells, and in stable a -transfectants, which have been previously shown to suppress vesicular egress of progeny pv particles due to the expression of variant cellular proteins (glny a, ckiiae a [ ] , rdxy f, moet a [see above]). virus stocks were purified on cscl-gradients to remove empty capsids, matched for their ssdna content (fig. a ) and tested for their infectivity in a cells by counting both ns -positive cells (fig. b ) and lysis plaques (fig. c ) in infected cultures. progeny particles that had transited (at least in part) through the golgi complex in parental a cells proved to be significantly more infectious (lower particle (dna) to infectivity ratio) than their counterparts that had bypassed this compartment (rdxy f, moet a, glny a, ckii-e a). for the same amounts of viral genome, virus produced in the latter four cells gave rise to to times less ns -positive cells and up to fold fewer plaques than virus produced in vesicular egressproficient cells (a ). together with the above finding that egress through er and golgi is accompanied by capsid modifications, these results suggest that the transport pathway to the plasma membrane represents an important step in the maturation of progeny virions. progeny virion shuttling from the nucleus, the site of parvovirus replication and assembly, to the plasma membrane is an important step in virus release and cell to cell spread. the present analysis shows that the release of non-enveloped pvs is not a mere consequence of the cellular lytic burst occurring at the very end of the viral life-cycle but involves a pre-lytic active vesicular transport of virions. this transport makes virions transit through er and golgi, and appears to have two major implications: the maturation of progeny particles into fully infectious virions, and the induction of cytolysis with the ensuing virus spread. for this transport process, pvs usurp cellular components allowing progeny virion engulfment into copii vesicles at the er and moving through the golgi to the plasma membrane under control of small rab gtpases. this vesicular trafficking is a general feature of rodent parvoviruses as it was demonstrated for both, the mouse virus mvm and rat virus h- pv in mouse and human cancer cells, respectively. besides known cellular components of vesicular transport (sar , sec / , sec / ), the erm family proteins radixin and, to a minor extent, moesin were found to be involved in the formation of pv-loaded copii-vesicles. given their known mediator function between actin and membrane structures [ ] , we hypothesize that erm proteins may control the engulfment of viral cargo into membrane structures. in keeping with this possibility, we recently reported that progeny pv particles associate with actin fragments, and that the actin-severing protein gelsolin controls pv loading [ ] . interestingly, the present results indicate that, radixin and (at least in human cells) moesin are essential players not only for pv egress but for the secretion of gluc as well. this speaks for the fact that erm family proteins are not simply involved in pv virion sensing, but play a general role in the copii vesicle-mediated secretion. in contrast, the actin-processing protein gelsolin, whose knock-down had no impact on gluc secretion, seems to be recruited specifically to promote (parvo)virus egress and is apparently not required for cellular secretion in general. during their traffic from the nucleus to the pm, pvs appear to transit through the golgi compartment, as evidenced by biochemical fractionations and co-staining data. this is unexpected, since in contrast with enveloped viruses, pvs do not acquire, during this transit, a lipid envelope harboring proteins mediating cell attachment and/or entry. however, passage through the er-golgi pathway still proved to be important for pv maturation in that capsids gained post-assembly modification such as phosphorylations. these capsid modifications did not occur under conditions inhibiting the vesicular secretory pathway. mvm viruses lacking the golgi-associated capsid modifications were less efficient at infecting naïve cells as compared with viruses produced in cells with an intact secretory pathway. this is not without precedent for non-enveloped lytic viruses. indeed, tyrosine phosphorylations at the capsid surface of the defective parvovirus aav also seem to control the infectivity of this agent [ ] . furthermore, other viruses become processed after assembly. for instance, assembled polioviruses present in autophagosomes, were shown to mature upon acidification of these vesicles prior to their fusion with lysosomes. this acidification induces the cleavage of poliovirus capsid proteins, an essential step for these virions to gain full infectivity [ ] . interestingly, mvm and h- pv virions were shown to colocalize with the late endosomal/lysosomal marker lamp (see also below). this leads us to speculate that the maturation of these viruses may involve the induction of additional post-assembly capsid modifications in these compartments besides the above-mentioned golgi-associated phosphorylations. these observations raise the question how parvoviruses get sorted into the intracellular cisternae system. a number of enveloped viruses (e.g. filo-, retro-rhabdo-, arena-, paramyxo viruses) transit through multi vesicular bodies (mvb) during their transport to the pm. a common feature of these viruses consists in the presence in their structural proteins, of (a) short recognition motif(s) facilitating their interaction with cellular factors that participate in mvb sorting. in the case of hiv- , the ptap-motif of gag interacts with tsg , thereby inducing the recruitment of the escrt complex which constitutes the sorting machinery for incorporation of ubiquitylated proteins into mvbs. thus, this interaction promotes entry of viral particles into mvbs during hiv-infection [ ] [ ] [ ] [ ] . other motifs known to mediate vesicular uptake include ypx(n)l and py (ppxy), which bind to the cellular factors aip /alix and nedd ubiquitin ligase, respectively [ , ] . it is noteworthy that ppxy motifs are present in the parvovirus vp protein. together with the strong and weak colocalization of pv capsids with the late endosome markers lamp [ , ] , and rab (present study), respectively, this feature raises the possibility of late endosomes/mvbs playing a role in the transport of pvs from the golgi to the pm. besides contributing to virus intracellular trafficking, vesicular transport of progeny pv particles through er and golgi apparently serves additional purposes. importantly, this vesicular transit was found to result in post-assembly modification (in particular phosphorylation) of capsids, and in a marked increase in virion infectivity. furthermore, vesicular egress proved to have an impact on the still poorly understood mechanism of pvinduced cytolysis. the present results indicate that progeny virus transport through er and golgi up-regulates this process. this is apparent from the delay in or inhibition of infected cells lysis upon alteration of the virus egress route. the mechanism underlying this dependence of cytolysis on vesicular pv transport remains a matter of speculation. it may be traced back to the co-transport of cellular or viral products involved in cytolysis, which become engulfed into virion-containing vesicles, are brought to the pm and cause its permeabilization. besides cellular or viral phospholipases, the viral ns protein is an intriguing candidate for this process, since it appears to exert lytic activities upon (prolonged) ectopic expression [ ] and is known to be present in cellular vesicles [ , ] . at late stages of infection, the vesicular co-transport of cytolytic factors might not be required anymore since the cytoskeletal collapse [ , ] would allow direct access of cellular and viral lytic factors to the pm. this would explain the delayed cell lysis and small plaque phenotype observed after pv infection of cells deficient in er-golgi vesicular transport. as a whole, the present study revealed not only the subcellular pathway followed by progeny pvs to exit infected cells, but also the unexpected role played by this vesicular egress in the interconnected process of cytolysis, virus maturation and spread. the latter processes are of particular importance in the context of the current development of pvs as oncolytic agents for anti-cancer therapeutic applications [ ] . it is noteworthy in this regard that tumor cell killing by parvoviruses is immunogenic [ ] , resulting in an immune bystander effect that takes over from the initial direct oncolytic effect of the viral treatment to complete eradication [ ] . it will be of great interest to determine whether the hereby reported vesicular egress of parvovirus particles also contributes to the display and/or release of tumor-associated antigens and/or damage/pathogen-associated molecular patterns that stimulate the immune component of pv oncosuppression. primary antibodies. sec , sec , calnexin (abcam ab ), gm , rab a (sta. cruz biotechnologies sc ), rab (sta. cruz biotechnologies sc ), rab a (sta. cruz biotechnologies sc ), rab a (abcam ab ), radixin (sta. cruz biotechnologies sc ), moesin (sta. cruz biotechnologies sc ) antibodies as well as antibodies against flag-(m ) and myc-tag (sigma: f , m ), lamin b (sta. cruz biotechnologies: m- sc ), lamp (abcam ab ), sar (abcam ab ), sec (sta. cruz biotechnologies sc ); sec (sta. cruz biotechnologies sc ) and pkb (sta. cruz biotechnologies sc ). rabbit antiserum recognizing the mvm ns (ans c ) [ ] , antiserum recognizing vp (avp ) [ ] , and monoclonal anti-capsid antibody b [ ] were described previously. phosphotyrosine specific mouse monoclonal antibodies ( ca ) were kindly provided by prof. dr. angel alonso (german cancer research center). secondary iggs. horseradish-peroxidase-conjugated (hrpconjugated) anti-rabbit and anti-mouse iggs (promega), hrpconjugated anti-goat iggs (sta. cruz), alexa fluor fluorescent-dyelabeled igg (dianova and invitrogen). others: protein g sepharose beads (pharmacia amersham), [ p]-labeled a-dctp (perkin elmer), [ p]-orthophosphate (mp biomedicals). protein kinases. flag-tagged ckiiae a (dominant-negative) [ , ] . erm-family proteins. fl-ezt a (dominant-negative), fl-rdxt a (dominant-negative), fl-rdxt e (constitutiveactive), fl-rdxdl[p] (dominant-negative), fl-rdxy f (active), fl-moet a (dominant-negative) [ ] . gelsolin. fl-gln y a and fl-d n [ ] . site-directed mutagenesis was performed by single or chimeric pcr, cloned into pcr . vectors (invitrogen) and verified by sequencing [ ] . effector genes. dnsar was obtained by the substitution of lysine at position by a methionine using following primers: sar k m-f: ggataatgccgggatgacaactttgctacac and sar k m-r: gtgtagcaaagttgtcatcccggcattatcc. the dnrab mutant corresponds to the substitution of serine by an asparagine [ ] using following primers: rab s n-f: gttggaaagaactgccttctcc and rab s n-r: ggagaaggcagttctttccaac. the dnrab mutant corresponds to the substitution of serine by an asparagine [ ] using following primers: rab t n-f: ggggtggggaagaactgtgtgctg and rab t n-r: cagcacacagttctcccccacccc. the dnrab mutant corresponds to the substitution of serine by an asparagine [ ] using following primers: rab s n-f: ggtgttggaaagaataatctcctg and rab s n-r: gagattattctttccaacacc. all four variants were cloned in order to express an nterminal myc tag using an n-terminal primer harboring the respective sequence in frame with the original start codon. (fig. s a) . production of expression constructs for generating stably transfected cell lines. mvm ns -inducible expression vectors were constructed from plasmid paav :pp -gfp, where myc/ flag-tagged protein variants were transferred from pcr . vectors, replacing the gfp reporter gene [ ] . raav :p -x and raav :(pa)p -x constructs. paav:p -gfp contains the gpf-gene under the control of the mvm flanked by multiple cloning sites. this allows easy replacement with candidate gene-sequences [ncoi,pmei,xbai,eco iii]-gfp-[ecorv,hindiii,xhoi,stui,-noti]. paav :(pa)p -gfp contains the same gfp cassette under the control of the ns -inducible (h -pv) p promoter. potential promoter activity through the left-end itr was blocked by insertion of the mvm poly(a) sequence at h -ns position nt . it was constructed as follows: ptrh -gfp [ ] was first cleaved with pmei/bst i, ligated. the pmei/not cleaved gfp cassette was then inserted into the stui/noti cleaved p -less ptrh -gfp. finally the pcr-generated mvm poly(a) sequence was inserted into the nhei-cleaved construct and the correct orientation was checked by a bamhi-digest due to the presence of a new bamhi-site at the left-end of the poly(a) sequence. effector genes, cut blunt-end (n-terminus) and noti (c-terminus) were then inserted into the eco iii-and noti-cleaved paav :p -x or paav :p -x vectors, respectively, to generate the corresponding paav-p /p -x constructs. reporter construct pcmv-gluc. the gaussia luciferase expressing construct under the control of the cmv promoter was generated by transferring the gluc gene from the pgluc-basic vector (new england biolabs) as a pcr-derived ecori-fragment into similarly cleaved pcr . (invitrogen). all cell lines were maintained as monolayers in dmem containing % fcs. stable transfectants were generated with pp -x and the selection plasmid psv neo or ptk-hyg at the molar ratio of : . colonies were pooled after growth under selection and frozen stocks prepared. experiments were performed in the absence of drugs [ ] . mvm was propagated in a cells. human-glioma propagation-competent hgh -pv strain was obtained after serial passaging in nch cells and propagated in nb k cells [ ] . to produce virus stocks under conditions lacking vesicular egress through er and golgi, a derivative cell lines a :p -fl-glnd n [ ] , a :p -ckii-e a [ , ] , a :p -moet a, and a :p -rdxy f [ ] , respectively were infected with , pfu/cell mvmp and harvested upon detection of severe cpe. all virus stocks were purified after freezing and thawing over cscl density gradient [ ] and quantified by their amount of single-stranded dna by southern blotting and standard plaque assays. pv-infection was limited to a single round by addition of either . u/ml neuraminidase (sigma) to remove sialic acid from the cell receptor / dilution of neutralizing antibody b as from h after the initial infection (supplement s )o recombinant adeno-associated viruses (raavs) were generated in t cells in absence of ad by co-transfection with paav-p -x and pdg, purified by iodixanol step gradients and the genomic titers determined by dot blot hybridization [ ] . transduction efficiencies were determined by immunofluorescence microscopy measuring the proportion of trans-gene expressing cells. toxicity was assessed by measuring metabolic activity through mitotracker incorporation, and cell lysis through pi-staining (supplement fig. s a, b) . determination of infectious titers by standard plaque assays [ , ] to determine the presence of infectious particles monolayer cultures (a for mvmp, nb k for hgh -pv) were seeded at a concentration of cells per mm dish, infected h later and covered with a bacto-agar overlay. after incubation for days, cultures were stained for h by addition of neutral-red containing bacto-agar. stained cells were fixed on the plates with formaldehyde after removing the agar overlay. detection and quantification of viral parvoviral dna by southern blot or dot blot hybridization accumulation of parvoviral dna species (monomer/dimer replication intermediates and single-stranded virion dna) were determined by southern blotting [ ] , total viral dnas by dot blot hybridization. at the indicated times p.i., medium was removed and kept separately. adherent cells were washed, harvested in dmem without serum by scraping from the dish, and collected by centrifugation. medium-and cell-associated virions were quantified, after repeated freezing and thawing, in standard plaque assays (see above) or after proteinase k digest by southern and/or dot blot hybridization: cell associated viral dnas were harvested in vte, digested with proteinase k and total dna was shared through syringe. to determine the amount of virion dna in purified virus stocks serial dilutions were performed, medium associated viral dnas were harvested and adjusted to ''vteconditions'' and digested by proteinase k. to detect different viral dna species, samples were analyzed by agarose gel electrophoresis and transferred onto nitrocellulose membranes. total viral dna was transferred to nitrocellulose using a dot-blot apparatus. viral dna was detected by hybridization with a p-labeled probe corresponding to nts - of the ns -encoding region of mvm dna and when indicated quantified after autoradiography with gelquant.net software. western blot analyses [ ] cellular extracts were produced in co-ip buffer and cleared by centrifugation. protein extracts were then fractionated by discontinuous sds-page and blotted onto nitrocellulose membranes. blocking was performed in % dry milk/pbs or for phosphospecific antibodies in % casein, mm tris ph . , mm nacl, mm edta, . % triton x- for h. staining with horseradish-peroxidase-conjugated secondary antibodies for h followed by chemiluminescence detection (amersham). immunofluorescence microscopy [ ] cells were grown on spot slides (roth). cultures were fixed with % paraformaldehyde and permeabilized with . % triton x- . specimens were pre-adsorbed with % fcs, incubated with primary antibodies, and stained with specific alexa fluor or/ and conjugated anti-species antibodies. dapi ( mg/ml) was added to the secondary antibody solutions. analyses were performed with a leica dmirbe confocal microscope ( lens, laser: red nm, green nm) and powerscan software or with an olympus fluoview fv confocal microscope ( lens, laser: green , red nm, far red: nm) presenting a single slice of a stack. quantitative analyses and mean colocalizations were calculated with imagej software. for pi staining, cells were incubated with propidium iodide ( mg/ml) for min at uc. pictures of living cells were taken on an inverted leicadfc fx microscope using transmitted and fluorescent light, pi positive and the total number of cells counted using imagej and the proportion of pi positive cells calculated. separation of nuclear, mitochondrial, and vesicular fractions from the soluble cytosol [ ] . nuclear components were obtained by pelleting at g and further purification through m sucrose. the -g supernatant was centrifuged at g to pellet large organelles like mitochondria in a ''heavy mitochondrial fraction'' (hmf), and the supernatant was centrifuged at g to pellet smaller organelles, including vesicles, in a ''light mitochondrial fraction'' (lmf). the final supernatant represented the soluble cytosolic fraction. the lmf-fraction was used to determine association of newly synthesized virions with vesicles. the lmf pellet suspended in hypotonic buffer was added to % iodixanol/ mm sucrose ( : v/v), and the components were separated according to their density by centrifugation for h at uc in a self-forming gradient in a vertical rotor at , g. fractions were collected from the top, volume-matched with the nuclear, hmf, and cytosolic fractions, and analyzed individually by southern and western blotting. separation of endoplasmic reticulum (er) and golgi membranes. separation of er and golgi proteins was essentially achieved as previously published [ ] . a cells were washed, scraped into ice-cold pbs and centrifuged at g for min at uc. the cell pellet was suspended in ml homogenization buffer ( mm tris ph . , mm nacl, mm kcl, mm edta) and passed multiple times consecutively through a syringe ( g and g ). nuclei were then separated by centrifugation at g for min and discarded. the remaining supernatant was adjusted to ml and centrifuged through a nycodenz step-gradient ( ml %, . ml %, . ml %, . ml %, . ml . %, . ml %, . ml . % (w/v nycodenz in homogenization buffer) at , g for min using a sw rotor (beckman instruments). nine fractions of . ml were collected from the top and analyzed by southern and western blotting respectively. metabolic labeling, purification, phosphopeptide analyses and phosphor amino acid analyses [ , ] a cell cultures were subjected to labeling medium for h ( . nci/cell of [ p]. labeled cells were harvested and the respective proteins isolated by immunoprecipitations and purified by sds-page. p-labeled proteins blotted on pdf-membranes were revealed by autoradiography, excised and digested trypsin. tryptic peptides were analyzed on thin-layer cellulose plates (merck) in two dimensions, first by electrophoresis in a ph . buffer, and then by chromatography in phosphochromatography buffer. phospho amino acid analyses were performed with excised p-labeled protein bands. individual labeled amino acids were obtained by hydrolization in m hcl at uc for h and phosphoserine, phosphothreonine, and phosphotyrosines were separated by thin-layer electrophoreses in two-dimension using ph . and ph . buffers, respectively. figure s a cells grown on spot slides were infected (mvm h) or not (a ) with mvmp ( pfu/cell) and further incubated under conditions neutralizing progeny particles released in the medium. cells were then fixed with paraformaldehyde h p.i., and analyzed by confocal laser scanning microscopy after double-staining using specific antisera for the indicated cell proteins (red) and mvm capsids (green). colocalization areas appear yellow in the merge and are quantified by image j analyzing infected cells from three individual experiments. scale bar mm. infection. a cells grown on spot-slides were transfected with control serum (apkb) or neutralizing anti-sec antibodies (asec ) at mg/ cells. twenty-four hours post-transfection, cells were infected with mvm, washed extensively after h to remove the inoculum, further incubated for h and fixed with paraformaldehyde. transfected antibodies were detected with alexa-conjugated secondary igg and counterstained with dapi to determine the proportion of transfected cells. the amount of infected cells was determined by means of ns expression. scale bar mm. (pptx) assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively molecular determinants and dynamics of hepatitis c virus secretion herpesvirus assembly and egress characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step viral and host control of cytomegalovirus maturation hepatitis b virus maturation is sensitive to functional inhibition of escrt-iii, vps , and gamma -adaptin roles for the recycling endosome, rab , and rab in hantavirus release from epithelial cells structural maturation of the transmissible gastroenteritis coronavirus polymorphism and structural maturation of bunyamwera virus in golgi and post-golgi compartments silencing the morphogenesis of rotavirus herpesviruses remodel host membranes for virus egress structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation proproteinprocessing endoproteases pc and furin both activate hemagglutinin of virulent avian influenza viruses intracellular processing of the gp hiv- envelope precursor. endoproteolytic cleavage occurs in a cis or medial compartment of the golgi complex replication of the parvovirus mvm. i. dependence of virus multiplication and plaque formation on cell growth the autonomously replicating parvoviruses of vertebrates modulation of minute virus of mice cytotoxic activities through site-directed mutagenesis within the ns coding region release of simian virus virions from epithelial cells is polarized and occurs without cell lysis coxsackievirus b transmission and possible new roles for extracellular vesicles vesicular egress of nonenveloped lytic parvoviruses depends on gelsolin functioning targeting rab gtpases to distinct membrane compartments role of rab gtpases in membrane traffic and cell physiology role of sec isoforms in selective export of membrane proteins from the endoplasmic reticulum the mechanisms of vesicle budding and fusion copii-cargo interactions direct protein sorting into er-derived transport vesicles protein sorting receptors in the early secretory pathway dynamin ii regulates hormone secretion in neuroendocrine cells bridging membrane and cytoskeleton dynamics in the secretory and endocytic pathways coordination of copii vesicle trafficking by sec the copii cage: unifying principles of vesicle coat assembly imaging constitutive exocytosis with total internal reflection fluorescence microscopy fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy selective alterations of the host cell architecture upon infection with parvovirus minute virus of mice molecular pathways: rodent parvoviruses-mechanisms of oncolysis and prospects for clinical cancer treatment regulation of non-structural protein functions by differential synthesis, modifications and trafficking ns interaction with ckii alpha: novel protein complex mediating parvovirus-induced cytotoxicity a viral adaptor protein modulating casein kinase ii activity induces cytopathic effects in permissive cells ezrin-radixin-moesin family proteins are involved in parvovirus replication and spreading parvovirus interference with intracellular signalling: mechanism of pkceta activation in mvm-infected a fibroblasts erm proteins: head-to-tail regulation of actinplasma membrane interaction cytosolic activation of cathepsins mediates parvovirus h- -induced killing of cisplatin and trail-resistant glioma cells a gdpbound of rab inhibits protein export from the endoplasmic reticulum and transport between golgi compartments mutant rab impairs docking and fusion of rhodopsin-bearing post-golgi membranes and causes cell death of transgenic xenopus rods rab is required for trans-golgi network-to-plasma membrane transport and a preferential target for gdp dissociation inhibitor a highly sensitive assay for monitoring the secretory pathway and er stress moesin regulates the trafficking of nascent clathrin-coated vesicles identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin cis requirements for the efficient production of recombinant dna vectors based on autonomous parvoviruses phosphorylation of the viral nonstructural protein ns during mvmp infection of a cells tyrosine-phosphorylation of aav vectors and its consequences on viral intracellular trafficking and transgene expression intracellular vesicle acidification promotes maturation of infectious poliovirus particles structure and functional interactions of the tsg uev domain hiv gag mimics the tsg -recruiting activity of the human hrs protein tsg and the vacuolar protein sorting pathway are essential for hiv- budding ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex retrovirus budding retrovirus budding biosynthetic transport of a major lysosome-associated membrane glycoprotein , lamp- : a significant fraction of newly synthesized lamp- is delivered to lysosomes by way of early endosomes through its nonstructural protein ns , parvovirus h- induces apoptosis via accumulation of reactive oxygen species phase i/iia study of intratumoral/intracerebral or intravenous/intracerebral administration of parvovirus h- (parvoryx) in patients with progressive primary or recurrent glioblastoma multiforme: parvoryx protocol parvoviruses-tools to fine-tune anticancer immune responses immune cells participate in the oncosuppressive activity of parvovirus h- pv and are activated as a result of their abortive infection with this agent the ns proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells novel pkceta is required to activate replicative functions of the major nonstructural protein ns of minute virus of mice augmented transgene expression in transformed cells using a parvoviral hybrid vector intranasal vaccination with recombinant adeno-associated virus type against human papillomavirus type l cabp , a calcium binding protein of the thioredoxin family, is a resident kdel protein of the er and not of the intermediate compartment biochemical activities of minute virus of mice nonstructural protein ns are modulated in vitro by the phosphorylation state of the polypeptide special thanks are due to claudia plotzky for excellent technical assistance. we are also grateful to michèle vogel and drs nathalie salome, rainer pepperkok, britta brügger and angel alonso for providing us with antisera as well as helpful comments. confocal microscopy on the olympus microscope was performed at the light microscopy facility of the dkfz (inf , d- heidelberg). key: cord- -a vcapl authors: gottardi, cara j.; caplan, michael j. title: signals and mechanisms of sorting in epithelial polarity date: - - journal: nan doi: . /s - ( ) -x sha: doc_id: cord_uid: a vcapl this chapter discusses epithelial-membrane polarity, sorting pathways in polarized cells, and the sorting-signal paradigm. polarized epithelial cells have long captured the attention of cell biologists and cell physiologists. at the electron-microscopic level, one of the most apparent and fundamental features of this cell type is its polarized organization of intracellular organelles and its structurally and compositionally distinct lumenal (apical) and serosal (basolateral) plasma-membrane domains. the polarized epithelial phenotype is an absolute necessity for organ-system function. in the most general sense, these cells organize to form a continuous, single layer of cells, or epithelium, which serves as a semi-permeable barrier between apposing and biologically distinct compartments. within the tubules of the nephron, these cells orchestrate complex ion-transporting processes that ultimately control the overall fluid balance of the organism. at the surface of the gastrointestinal tract, specialized versions of this cell type control the digestion, absorption, and immuno-protection of the organism. polarized epithelial cells have long captured the attention of cell biologists and cell physiologists. this is largely because the architecture of these cells so tellingly bespeaks their function. at the electron microscopic level, one of the most apparent and fundamental features of this cell type is its polarized organization of intracellular organelles and its structually and compositionally distinct lumenal (apical) and serosal (basolateral) plasma membrane domains (figures a, b) . through the eyes of the physiologist, the polarized epithelial phenotype is an absolute necessity for organ system function. in the most general sense, these cells organize to form a continuous, single layer of cells, or epithelium, which serves as a semi-permeable barrier between apposing and biologically distinct compartments. within the tubules of the nephron, these cells orchestrate complex ion-transporting processes that ultimately control the overall fluid balance of the organism. at the surface of the gastrointestinal tract, specialized versions of this cell type control the digestion, absorption and immuno-protection of the organism. thus while polarized epithelial cells can carry out myriad functions, they share one defining feature: a structural polarity which serves their underlying functional polarity. the differential distribution of membrane proteins between the plasmalemmal surfaces of polarized epithelial cells enables these cells to both respond to and effect changes upon their environment in a directed fashion. the gastric parietal cell of the stomach, for example, contains a population of h,k-atpase-rich vesicles. upon stimulation, these vesicles fuse selectively with the lumenal membrane, resulting in the massive apical secretion of hcl which initiates digestion. without two important elements of the polarized phenotype, that is, junctional integrity and the precision of this membrane insertion, proton pumps might be delivered to a compartment which would be adversely affected by the secretion of acid. another illustration of the utility of the polarized phenotype is provided by the principal cells of the kidney, which carry out net sodium absorption through a mechanism which is entirely dependent upon the polarized distribution of two membrane proteins. sodium absorption is stimulated by the hormone aldosterone, which increases the amount or activity of na,k-atpase at the basolateral surface, while increasing the number or activity of apical sodium channels and thus the sodium conductance of the lumenal membrane (doucet and barlet-bas, ) . because the na,k-atpase generates low intracellular { na+}, sodium is these morphologically distinct apical and basolateral membrane domains are separated by a unique ultrastructure known as the tight junction (tj). this structure is just visible as an area of close, uniform membrane apposition located at the apices between adjacent epithelial cells. (photo courtesyof dr. marian neutra, children's hospital, boston, ma). able to pass from the lumen of the kidney tubule through apical sodium channels and into the cytoplasm down its electrochemical gradient. the na+ is then pumped across the basolateral membrane and into the interstitum by the sodium pump and is ultimately prevented from leaking back into the lumen by impermeable tight junctions. therefore, it is the differential assignment of na' channels to the apical surface and na,k-atpase molecules to the basolateral domain that ensures the vectoriality of this transport process. how the polarized cell assigns these two proteins (and apical and basolateral membrane proteins in general) to their respective surface domains has been the subject of much investigation and is the general focus of this review. it is perhaps important to point out that the fundamental questions of plasma membrane protein aniosotropy are not unique to surface membrane proteins or even to the study of epithelial polarity. the golgi apparatus, for example, is a polarized organelle whose cis-and trans-most cisternae are structurally and biochemically distinct. this organization is thought to enable the ordered addition and trimming of glycoprotein sugar residues as they traverse the stacked cisternae. as is clearly represented in the breadth of topics covered in this book, numerous cell types adopt a polarized state for some functional purpose. the propagation of a nervous impulse from dendrite to axon requires compositionally different membrane proteins in each of these domains, while the localization of determinants to specific parts of an egg's cytoplasm gives rise to cells with different growth potentials and the necessary assymetries required for embryo development . what we hope will become clear in this chapter and related chapters in this book is that we are beginning to appreciate the universality of polarity. the mechanisms involved in establishing and maintaining the polarized state appear to be so fundamental that some of the schemes through which a cell is able to localize a particular protein to a given cellular domain are turning out to be conserved between epithelia and neurons, and even between epithelia and yeast. while the need for protein asymmetries in development, or membrane polarity in epithelial transport is clear, the means through which it is achieved are only beginning to be elucidated. before we embark upon our review of the field, we first introduce the conceptual framework onto which the results in this field are organized and interpreted. first, a protein destined to accumulate with a polarized distribution needs to be recognized as different from other proteins. we presume that what is recognized is some structural aspect of the protein itself. we refer to that part of the protein that is recognized for polarized localization as a sorting signal or localization determinant. these two terms are often used interchangeably, but in fact there is a subtle difference between the two. "sorting signal" is often taken to imply a signal that is recognized and acted upon before the protein is delivered to its ultimate residence. sorting signals are thought to be those signals that enable a cohort of similar proteins with similar destinations to be sorted and sifted away from all of the other molecules traversing the biosynthetic pathway at the same time. a "localization determinant" is perhaps a more general term that carries fewer mechanistic implications. it is defined here as the determinant that specifies a protein's polarized distribution, but it does not make a distinction between recognition that takes place before the protein has reached its final destination or after (e.g., through a selective retention mechanism). the proteins which serve to recognize a particular signal and act upon it are generally referred to as sorting machinery. often, a distinction is made in the literature between "sorting" and "targeting machinery." in these cases, the sorting machinery is exclusively those elements which recognize the sorting signal. any downstream effectors of this sorter that orchestrate the vectorial directing of a vesicle to its final destination are referred to as targetting machinery. a simple schematic of these elements is presented in figure . as is discussed in the second half of this review, we know much more about general targeting machinery than the sorters themselves. one of many possible ways to think about how a secretory or membrane protein could be sorted into a vesicle. it is presumed that the "sorters" will recognize a sorting signal (" embedded within the protein structure. it seems likely that this recognition event would need to take place in the lumen of the golgi for a secretory protein, but this might not be necessary for a membrane protein, which could interact with a sorter from either a lumenal-or cytoplasmic-facing signal domain. ultimately, the sorted protein(s) could be contained within a "domain-specific vesicle," which would then be targetted (with the help of protein targetting machinery x, y, and z) to the appropriate apical or basolateral surface domain. it is thought that proteins destined for either the apical or basolateral domain of a polarized cell occupy the same golgi cisternae during their biosynthesis ( m a t h and simons, ; misek et al., ; rindler et al., ; fuller et al., ; pfeffer et al., ) . immunoelectron microscopic studies performed on nonpolarized endocrine cells which manifest two biochemically and kinetically different secretory pathways suggested that the process of sorting components away from one another takes place at the tgn (orci et al., ; tooze et al., ) . however, recent studies have demonstrated that sorting may not take place exclusively at the tgn. sorting mechanisms have been suggested to take effect as early along the biosynthetic pathway as the er (balch et al., ) as well as at the recycling endosome (matter et al., ; . in hepatocytes, sorting appears to occur after all newly synthesized membrane proteins are delivered to the basolateral plasmamembrane (bartles et al., ) . similar delivery routes have been detected in polarized intestinal epithelial cell lines (matter et al., ) . finally, in at least one subclone of the canine renal mdck cell line, sorting may take place both at the golgi as well as at the level of the plasma membrane. while most proteins in this cell line are sorted in the tgn, the na,k-atpase can be preferentially localized to the basolateral membrane through domain-specific stabilization mechanisms after random insertion into both plasmamembrane domains (hammerton et al., ; siemers et al., ) . apically and basolaterally sorted proteins have been shown to be packaged into distinct classes of golgi-derived vesicles which are ultimately targeted to their appropriate domains. recently it has been shown that membrane and secretory proteins are segregated into distinct vesicular carriers upon transport from the golgi to the basolateral surface of hepatocytes (saucan and palade, ) the extent to whch distinct basolateral (or apical) proteins are cosorted and incorporated within the same vesicle either due to common localization signals or the ability to co-aggregate has not yet been determined. after proteins are sorted, the targeting of a vesicle to a particular surface domain can occur directly (vectorially) from the tgn to the apical domain (matlin and simons, ; rindler et al., ; fuller et al., ) , basolateral domain (caplan et al., ) or indirectly as has been shown for the poly-immunoglobulin receptor (pigr) (mostov and deitcher, ) . in the latter case, the protein is first targeted to the basolateral surface where the receptor can bind its ligand and is then transported to the apical surface via a process known as transcytosis (reviewed in mostov and simister, ) . as noted above, in hepatocytes all apical proteins studied to date make use of this indirect pathway for apical delivery (bartles et al., ) , while cell lines derived from intestine and kidney can employ both routes for surface delivery (matter et al., ; casanovaet al., ; low et al., ) while the details of the routes have been determined for a number of sorting pathways, the molecular signals and recognition components which control each of them are not well understood. the search for these molecular signals and recognition components has been the focus of much study over the last years. during this period, the subjects of protein sorting and epithelial polarity have been extensively reviewed. several of these reviews are listed here for those seeking more background on specific aspects of this field: for general reviews on protein sorting pathways (burgess and kelly, ) ; general concepts of sorting and targeting (caplan and matlin, ); a discussion of the mechanisms required for the establishment and maintenance of epithelial polarity (rodriguez-boulan and nelson, ) ; polarized transport of surface porteins and lipids in epithelial cells ; comparative epithelial and neuronal polarity (rodriguez-boulan and powell, ) ; the generality of the polarized phenotype (nelson, ) ; cytoskeleton as a component of the protein sorting machinery (mays et al., ) ; summary of the few known sorting signals in polarized epithelial cells ; common signals involved in sorting from the tgn and endosomes . perhaps now more than ever before, it is becoming a rather daunting task to provide a synthesis of the observations relevant to the study of epithelial polarity. this is in part due to the fact that important insights into the mechanisms of sorting are being contributed by fields that are not exclusively focussed on epithelial biology. as we discussed in this review, some important contributions are emerging from studies of endocytosis, secretion in yeast and neurons, and the sorting of yeast lyso-soma enzymes (see chapter i of this volume), in addition to more "classical" approaches to epithelial polarity. in this review, we explore the current paradigm that the generation and maintainance of distinct membraneous compartments requires "sorting signals," the recognition domains embedded within the amino acid sequence or polypeptide structure of the protein, and "sorting machinery," the proteins which interpret and act upon these signals. in the first half, we review and categorize the signals that have begun to be elucidated, as well as discuss the approaches and difficulties associated with finding and interpreting sorting signals. while the polarity field itself has not yet succeeded in characterizing the definitive sorting machinery, numerous components of the membrane budding and fusion apparatus are rapidly being elucidated. we have chosen to review some of the important findings in the field of membrane transport, and in particular examine the potential roles that gtp-binding proteins of the rab, arf and heterotrimeric classes may play. we also discuss a class of proteins referred to as adaptins as well as the implications that the snare hypothesis may have for epithelial polarity. although these components have not been shown to be directly involved in sorting per se, it is becoming increasingly clear that in a general sense, the composition of the membrane vesicle budding and fusion machinery may be part of the overall apparatus which "acts upon" the sorted species and contributes to domain specific surface targeting. as stated above, the paradigm for conceptualizing the mechanisms responsible for biosynthetic sorting requires that each protein contains signal information embedded within its polypeptide sequence/structure (sorting signal) which is interpreted and acted upon by components referred to as sorting machinery. this scheme takes its cue from the process through which ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum to initiate cotranslational protein translocation (blobel, ) . prior to the elucidation of this process, it was suggested that protein targeting might require cellular sorting machinery to recognize certain signals which would be shared by proteins with common destinations (blobel, ) . shortly after this suggestion, it became clear that targeting to the rer, mitochondria and chloroplasts required short, contiguous, n-terminal signal peptides (reviewed in burgess and kelly, ) . in the case of the former, the signal was recognized by a receptor, srp (lingappa et al., ; von heijne, ; kurzchalia et al., ; walter and lingappa, ). subsequently, a number of short, contiguous amino acid domains have been shown to play a role in later stages of post-synthetic targeting. these include: ( ) the kdel and adenovirus e l signals which ensure the retention or recapture of resident er proteins (munro and pelham, ; nilsson et al., ); ( ) a transmembrane domain signal responsible for golgi retention (swift & machamer, ; machamer, ) ; ( ) the cluster of positively charged lysine residues (sv -nls) sufficient for nuclear targeting (richardson et al., ) ; ( ) the critical tyrosine/ "tight-turn'' structural motif which can mediate localization to clathrin coated-pits (goldstein et al., ; pearse and robinson, ; collawn et al., ) ; and ( ) the discovery that lysosomal hydrolases were targeted to lysosomes through the recognition of a phosphorylated sugar residue (mannose- -phosphate; reviewed by kornfeld and mellman, ) . in several of these cases receptors for these signals have been well-characterized: the signal recognition particle (srp) for secretory and membrane proteins (walter and lingappa, ) , the mannose- -phosphate receptor (m pr) for the targeting of lysosomal hydrolases to the lysosome (sly and fischer, ; vonfiguraandhasilik, ) , the kdelreceptor (tanget al., ) and the adaptins which couple coated pit localization sequences to clathrin cages (pearse and robinson, ; robinson, ) . the search for definitive signals which mediate the delivery of proteins to a particular epithelial surface domain has proven to be quite difficult. this is due in part to general limitations imposed by certain molecular biological approaches, as well as to some inherent difficulties specific to the investigation of epithelial polarity. our goal in this section is to outline reasonable criteria for the identification of a sorting signal. the observation that the influenza and vesicular stomatitis viruses bud from opposite surface domains of polarized mdck cells (madin darby canine kidney) (rodriguez-boulan and sabatini, ) spawned an extensive search in which chimeric and deletion analyses were applied to the problem of identifying the underlying apical and basolateral sorting signals (reviewed in caplan and matlin, ) . these efforts to characterize sorting signals have generally involved the generation of chimeric or truncated contructs prepared from portions of apical and basolateral membrane proteins. through analysis of the subcellular distributions of the resulting proteins, sorting information can, at least in theory, be assigned to particular portions of the parent molecules. while a large number of chimeric and truncated viral glycoproteins have been generated and analyzed, it has been difficult to interpret many of the resultant observations. with the benefit of hindsight, we now know that these difficulties can be attributed to a number issues that we discuss in more detail below (including the tertiary stuctures of the experimental constructs, the confounding possibilities introduced by uncharacterized default pathways, and the potential for multiple and hierarchical signals to be embodied within the structures of the studied proteins). until recently (thomas and roth, ) , the analysis of viral spike glycoproteins did not produce a definitive sorting signal. much of the uncertainty associated with this work is likely attributable to the fact that these studies engineered chimeras from portions of structurally dissimilar molecules. the tertiary structures of the resultant chimeras may thus differ substantially from those of either parent molecule, which may in turn exert unpredictable effects upon sorting behavior. clearly, if sorting signals are formed from domains arising from noncontiguous regions of a polypeptide, for example, in much the same manner that heterotrimeric g proteins are thought to "see" their effectors (berlot and bourne, ) , or in the way that the human growth hormone receptor (hghbp) is thought to interact with its ligand (cunningham and wells, ) , it is easy to imagine how the structural integrity of the putative sorting signal could become compromised in a chimeric construct. while producing a rough map of the signal-bearing domain of a protein can be relatively straight forward, determining the exact residues which constitute the signal is turning out to require a collaboration between many different types of mutagenesis approaches. often, contradicting results can arise from alanine scanning, truncation and point mutation/deletion mutagenesis, since a mutated protein can manifest impaired sorting behavior even though the altered residues are not part of the actual sorting signal (aroeti et al., ) . it is becoming clear that a judicious and thorough comparison of many different types of mutagenesis approaches may be necessary to determine definitively the key residues necessary for sorting. perhaps another difficulty in looking for apical or basolateral sorting signals is that the default pathway for "signal-less" membrane proteins is still not known. a protein that is sorted "by default" is, by definition, unable to interact with and be acted upon by any sorting machinery whatsoever. in theory, at least, such "unsorted'' proteins may be distributed with polarity, depending on the nature and characteristics of the membrane vesicular traffic arising from the golgi complex in a particular cell type. obviously, if the localization of a protein construct under study is identical to that produced by the cell's default pathway, elucidation of a signal will be difficult, since elimination of the signal will not alter the protein's distribution. thus, one can appreciate the difficulty in assigning localization information to a particular domain in the context of an undefined default pathway. this caveat accounts for at least some of the reasons which explain why a definitive basolateral sorting signal in the c-terminal domain of vsv-g protein took so long to discern. in the following example we summarize the ha-vsvg spike glycoprotein chimera literature as a means to illustrate the difficulties in interpretating these types of studies. when acdna encoding the influenza ha was expressed in mdck cells, the encoded protein localized to the apical membrane (roth et al., ) , while a cdna encoding the vsvg polypeptide produced a protein that is localized to the basolateral domain (gottlieb et al., b; stephens and compans, ) . when truncation mutants were expressed in which soluble ectodomain versions of these proteins were synthesized, the vsvg ectodomain was secreted from both apical and basolateral domains gonzalez et al., ) while the ha ectodomain was predominantly secreted from the apical domain (gonzalez, et al., ; roth et al., b) . based on evidence that the default pathway for secreted proteins leads to nonpolarized secretion from both surface domains (kondor-koch et al., ; gottlieb et al., a; caplan et al., ) , it was reasoned that the ectodomain of ha encodes an apical sorting signal while the vsvg ectodomain lacks signal information. this was further confirmed by the observation that a hybrid ha-vsvg protein comprising the ha ectodomain fused to the vsvg transmembrane and cytoplasmic tail region was targeted to the apical membrane (mcqueen et al., ; roth et al., a) . but if the vsvg ectodomain is randomly secreted and the vsvg tail domain fused to ha is apical, which domain of vsvg encodes basolateral sorting information? the complementary hybrid comprised of the ectodomain of vsvg (presumably signal-less) tethered to the ha transmembrane and tail region (perhaps also signal-less) was targeted either to the basolateral membrane or to both surface domains (mcqueen et al., ; puddington et a] ., ; roth et a]., a; compton et al., ) . the interpretation of the behavior of this chimera was clearly complicated; it was suggested that this protein could be pursuing its distribution by default. (as discussed above, the default pathway for membrane proteins is still not defined in polarized cells). an alternative interpretation was that the vsvg ectodomain indeed contains basolateral sorting information, but that perhaps this domain needs to be tethered to the plasmamembrane with a transmembrane anchor in order to interact with its presumptive sorting machinery. this interpretation, however, was proved incorrect by the observation that the anchoring of this ectodomain to the membrane through a lipid-linkage resulted in apical targeting (brown et al., ) . interestingly, when the ectodomain of the normally apical placental alkaline phosphatase (plap) was attached to the vsvg transmembrane and cytosolic tail domains (which were though to lack a dominant signal), the resulting chimeric protein was targeted basolaterally. it is difficult to reconcile the ha-vsvg and plap-vsvg chimeras without invoking hierarchical and competing signals. recently, a basolateral targeting signal has been precisely localized to the cytoplasmic domain of the vsvg protein (thomas and roth, ) . in light of the vicissitudes which attended the interpretation of each round of chimeric constructs discussed above, it was certainly unexpected that definitive sorting information would be localized to the cytoplasmic tail of vsvg. the nature and function of this signal will be discussed in depth below. the preceding discussion was presented simply to reinforce the caveat that the default pathway, protein structural considerations and the possible interactions between "dominant" and "recessive" sorting signals can considerably cloud the interpretation of chimera experiments. recent studies of the polymeric immunoglobulin receptor (pigr), the low density lipoprotein receptor (ldlr) and polytopic hetero-oligomeric proteins (h,k-atpase and na,k-atpase) suggest that individual proteins can interact in multiple and complex fashions with the machinery responsible for surface targeting. it is becoming increasingly clear that there can be an array of signals encoded within an individual protein, and the sorting problem is becoming evermore complicated by the apparent redundancy, multiplicity and hierarchical nature of these signals (matter et al., ; mostov et al., ) . for example, brewer and roth's ( ) demonstration that they could completely overwhelm the apical signal present in the ha ectodomain and redirect it to the basolateral surface by changing a single amino acid in this protein's cytoplasmic tail strongly suggests that multiple signals present in a single protein can interact in a heirarchical fashion. the newly created cytoplasmic signal is dominant over the presumed apical sorting signal present in the ectodomain of ha. as discussed below, the ldl receptor has been shown to encode redundant, basolateral sorting information, since either of two cytoplasmic determinants could independently mediate basolateral delivery (matter et a]., ) . moreover, the protein may also contain acryptic apical sorting signal in its ectodomain, since a cytoplasmic tail-minus construct of this protein (ct ) is sorted with great efficiency to the apical membrane in mdck cells (matter et al., ). an ectodomain apical localization signal has also been found within the pigr, whose initial surface delivery is to the basolateral plasmalemma. why do these proteins need multiple signals? what does the ldlr gain by expressing two basolateral localization signals? recent studies (discussed in greater detail in the following section) have more finely decoded these two signals and are revealing functional differences. for ex-ample, the "membrane proximal determinant" encodes coated-pit internalization information, while the "membrane distal determinant" appears to ensure efficient sorting from a basolateral endosome back to the basolateral surface (matter et al., ) . analysis of the sorting behavior of multisubunit ion pumps provides further insight into the possible utility of multiple signals (reviewed in the gastric h,k-atpase and the na,k-atpase are close cousins in the large family of p-type ion transporting atpases. both are composed of kda a-subunits and heavily glycosylated kda p-subunits. they share similar reaction mechanisms and catalytic properties and, not surprisingly, are highly homologous at the amino acid sequence level. the a-subunits are - % identical, whereas the p-polypeptides manifest roughly % identity. while the na,k-atpase is a basolateral protein in most polarized epithelial cell types (with the exception of neural epithelia such as choroid plexus and retinal pigment epithelium), the h,k-atpase occupies the apical membrane and a pre-apical storage compartment in gastric parietal cells. hormonal stimulation of gastric acid secretion induces fusion of the membrane vesicles which comprise the intracellular reservoir with the plasma membrane, resulting in delivery of the h,k-atpase to the apical cell surface. during the interdigestive period, the h,k-atpase is re-endocytosed and returned to its storage compartment. chimera studies reveal that each subunit of the h,k-atpase possesses a sorting signal which participates in regulating this complex traffic . the a-subunit is endowed with a dominant apical targeting signal, which can drive the apical sorting of chimeric pumps expressed in both mdck and llc-pkl renal epithelial cells. the p-subunit of the h,k-atpase possesses a tyrosine-based endocytosis signal (roush et al., manuscript submitted). this signal causes the protein to be sorted basolaterally when it is expressed in mdck cells and apically when it is expressed in llc-pk cells. the na,k-atpase p-subunit does not possess a similar sequence domain. it seems likely that the two h,k-atpase signals participate in distinct stages of pump sorting in the gastric parietal cells. the apical signal in the a-subunit probably mediates the sorting of the entire complex to the apical membrane or the pre-apical storage compartment, whereas the p-subunit signal is responsible for ensuring the re-internalization of the pump following the cessation of secretagogue stimulation (courtois-coutry et al., ) . it remains to be determined why the p-subunit's tyrosine-based signal is differentially interpreted by mdck and llc-pk cells. investigation of this phenomenon may shed light on the nature and function of the epithelial sorting machinery. this apparent trend towards a multiplicity of signals is not entirely surprising, since many proteins are required to perform highly sophisticated feats of membrane targeting during the course of their transits throughout the endomembranous networks of the cell. for example, the pigr receptor expressed in its native hepatocytes or by transfection in mdck cells travels first to the basolateral membrane to pick up ligand and is then transported to the apical surface domain. it appears that an apical sorting signal in this protein's ectodomain might be required for basolateral to apical transcytosis, while a basolateral signal in the cytoplasmic domain ensures the initial basolateral delivery. unlike proteins that are constitutively expressed at one surface domain, a number of distinct and individually acting signals are necessary to orchestrate the more complicated surface targeting events displayed by pigr receptor, and other molecules like it. obviously, the hierarchical (both temporal and spatial) regulation of each signal will be of utmost importance in ensuring that a protein follows a physiologically relevent trafficking pathway. recent evidence, for example, demonstrates that the pig receptor undergoes phosphorylation on a cytosolic serine residue around the time that it is delivered to the basolateral surface (larkin et al., ) . this phosphorylation event appears to inactivate the protein's basolateral signal and thus permit its transcytosis to the apical membrane (casanova et al., ). perhaps not surprisingly, the greatest advances in the elucidation of sorting signals have been made with single membrane-spanning monomeric or homooligomeric proteins (e.g., pigr, ldl-r, tfr). with these molecules the requirements for surface expression are easily met and the effects of mutagenesis on tertiary structure can be assessed through well-characterized functional assays, such as receptor-ligand or antibody binding. through deletion analysis and heterologous expression in mdck cells, it was determined that the pigr (casanova et al., ) and the ldlr (hunziker et al., ) each contained basolateral targeting determinants which mapped to short, contiguous regions of their cytoplasmic domains (table ). both signals could be grafted onto heterologous proteins and cause them to be targeted to the basolateral surface, supporting the idea that each determinant was truly an autonomous basolateral sorting signal. exhaustive mutagenesis studies have more finely mapped each of these determinants. the ldlr possesses two distinct basolateral targeting determinants, one that is "coated-pit related" (proximal determinant) and another which is tyrosine-dependent but not capable of mediating localization into coated-pits (distal determinant) (matter et al., ; . interestingly, the polymeric immunoglobulin receptor (pigr) signal may constitute yet another class of basolateral targetting determinant, since it shares little in the way of sequence homology with either determinant of the ldlr and shows weak tyrosine dependence (aroeti et al., ) . the general characteristics of these three determinants and the degree to which they are related are only beginning to be eluciated thomas and roth, ). an attempt to categorize these basolateral sorting determinants has been made by and is summarized in table . before discussing the nature of the "coated-pit related" basolateral targeting determinant, it is necessary to be familiar with the signals that are known to mediate the accumulation of plasma membrane receptors into clathrin-coated pits (goldstein et al., ) . it is now generally accepted that tyrosine-and dileucine- containing sequence motifs present in the cytoplasmic tails of a number of coated-pit clustering proteins serve as the critical recognition elements for the adaptor components of clathrin coats (pearse and robinson, ; trowbridge, ) . more recently, numerous studies have demonstrated a strong relationship between the signals which mediate localization into coated pits and a subset of those involved in basolateral targeting (brewer and roth, ; hunziker et al., ; lebivic et al., ) . for example, brewer and roth ( ) found that the apically targeted ha molecule could be completely rerouted to the basolateral membrane by replacing a strategically localized cysteine residue (cys ) with a tyrosine in the cytoplasmic domain. this tyrosine was also sufficient to localize this protein into coated-pits and direct the protein's incorporation into endosomes. this observation that an endocytosis signal might also double as a basolateral targeting signal led to the suggestion that the recognition determinants for endocytosis and for tgn-to-basolateral targeting might be similar or identical to one another. thorough mutagenesis studies on the coated-pit localization and basolateral sorting determinants of ha-y (thomas and roth, ; lin et al., ) , vsvg protein (thomas et al., ) , and the ldlr , however, have led to a revision of this initial interpretation. it turns out that the "endocytosis signal" of both the ha-ys and the ldlr (proximal signal) can be resolved into two overlapping but distinct signal components. in other words, there is information recognized for endocytosis that is distinct from that recognized for basolateral sorting, even though the sequences are in part superimposed and share marked similarity. table shows the systematic mutagenesis that ultimately unraveled this relationship. brewer and roth ( ) found that ha-y is capable of both basolateral sorting and endocytosis. the second generation mutant ha-y ,rs , however, behaved as aprotein that was capable of endocytosis, but whose basolateral localization was inhibited (lin et al., ) . similar results were found with the ldlr proximal determinant. matter and colleagues ( ) showed that the truncation mutant ct was basolaterally targetted and rapidly endocytosed, while the removal of terminal acidic residues in ct pro- duced a protein that was not capable of basolateral targetting, but could nonetheless be endocytosed. thus, the initial correlation between endocytosis signals and basolateral targeting has now resolved into two distinct but overlapping signals that can share common residues for their respective activities. the implications of this result are very exciting for the field of epithelial polarity. first, they suggest that the signals for basolateral sortingkargetting may be structurally similar to signals for clathrin-coated pit localization and endocytosis. the involvement of similar signals suggests that the sortinghecognition molecules themselves may be related. at least for endocytosis signals, there is evidence in favor of clathrin "adaptins" (of the ap plasma membrane class) playing a role in recognizing these sequences (pearse, ; glickman et al., ; beltzer and spiess, ; sorkin and carpenter, ; sosa et al., ) . in light of the recent characterization of adaptin related molecules (cops, discussed in section , below), it has been suggested that a family of structurally and functionally similar sorting adaptors may serve as the sorting machinery which interacts with these basolateral sorting signals . the findings support the more general contention that sorting at the level of the tgn may be mechanistically similar to that at the level of the endosome (matter et al., . taken altogether, there now appear to be two general classes of basolateral targeting determinants. one of these is biochemically related to the signals that mediate sorting into coated pits. this type of signal can be colinear with an endocytosis determinant and may share the critical tyrosine residue required for the activity of both, but it is nonetheless distinct and dissociable from an endocytosis signal. the second class of basolateral targeting determinants appears to be unrelated to clathrin-coated pit localization signals, although it may also strongly depend on a tyrosine for activity. this second type of determinant appears to be unique to the ldlr, pigr (casanovaet al., ) and the tw (dargement et al., ) , although these signals share no primary sequence homology with one another. it is possible however, that this second determinant present in these three proteins may be mutually similar in three-dimensional structure but not in primary sequence. in this context it is important to note that adaptor proteins are thought to recognize tyrosine residues in the context of a tight turn, which can be achieved by many different primary sequences (glickman et al., ; collawn et al., collawn et al., , bansal and gierasch, ) . more detailed analyses are revealing that while the dependency on tyrosine is crucial, other residues which are acidic and c-terminal to the tyrosine are also important. demonstrated that the clusters of two or more acidic amino acids downstream from a tyrosine, phenylalanine or di-leucine are important for signal function (see table ). while the authors of this study have argued that it is premature to propose a common motif characteristic of all basolateral targeting determinants, they have found that this critical aromatic amino acid followed by acidic residues can be discerned in the cytoplasmic domains of many known proteins which are targeted to the basolateral membrane of mdck cells, including ecadherin, transferrin receptor, cation-independent and dependent mannose- -phosphate receptors, lap, pigr and fcriib . (see discussion of . as these authors have suggested, it will be exciting to define mutations that will prevent the recognition of these sequences so that the identification and characterization of the molecules which serve to interact with and interpret these signals can be facilitated. an ever growing list of proteins are anchored to membranes through a covalent attachment to glycosylphosphatidylinositol or gpi. proteins of this class are initially synthesized on bound polysomes as transmembrane polypeptides and, while still resident within the er, are cleaved from their transmembrane portions and transferred covalently to lumenally facing glycosyl-phosphatidylinositol molecules (cross, ) . gpi-anchored proteins are widely distributed with respect to both cell type and function. members of this class of proteins include protozoal surface coat proteins (e.g, the variant surface glycoproteins of trypanosomes), differentiation antigens (e.g., thy-i ) , adhesion molecules (e.g., the gpi-linked isoform of n-cam), hydrolases (e.g., alkaline phosphatase and snucleotidase), and receptors (folate receptor). the functional advantages that this membrane linkage confers upon a particular protein is presently unclear, and has been the focus of a great deal of attention (reviewed in brown, ) . in general, the gpi-linkage has been suggested to be important for enabling proteins to "c uster"at a surface density much higher than is possible for single-pass transmembrane proteins (hooper, ) . studies have also shown that these clusters of gpi-anchored proteins may be important for certain cell surface signal transducing events (reviewed in anderson, ) . gpi-linked proteins captured the attention of epithelial biologists because of their polarized distribution in mdck cells (lisanti et al., ) and other cultured epithelial cell lines (lisanti et al., ) . the nearly exclusive correlation of membrane anchoring via gpi with apical localization raised the question as to whether or not the gpi membrane anchor was itself a signal for apical targeting. chimeric analyses showed clearly that the gpi-linkage is sufficient for apical targeting in mdck cells (brown et al., ; lisanti et al., a,b) . of course, in the absence of a known default pathway for membrane proteins, it remains formally possible that the gpi-anchor prevents a protein from gaining entry into the basolateral sorting pathway. moreover, the fact that the cytoplasmic tail-minus versions of the ldl and pig receptors are directly targetted to the apical membrane is consistent with the possibility that apical sorting occurs by default (discussed in . nonetheless, the gpi-linkage is the field's best accepted apical localization signal characterized to date. interestingly, glycosphingolipids (gsls) share the apical preference of gpi-linked proteins and are generally found exclusively in the outer leaflets of the apical membranes of mdck cells. the means through which gpi-anchored proteins and glycosphingolipids (gsls) are sorted and subsequently targetted to the apical membrane are poorly un-derstood. it has been shown that gsls manifest biophysical properties which enable them to self-associate or form clusters in the plane of the membrane (thompson and tillack, ) . these properties have been invoked to support the proposal that gsl clustering occurs at the level of the tgn, and that newly synthesized gpi-linked proteins might co-cluster with these lipids (simons and van meer, ) . it has been further suggested that apically-destined transmembrane proteins could similarly be sorted through an ability to co-cluster with gsls and gpi-linked proteins . according to this model, apical sorting could take place through selective inclusion within these gsl microdomains, while certain basolateral membrane protein components would be sorted by selective exclusion. however, it should be pointed out that there is still no experimental evidence showing that the gsl clusters are important for apical sorting. one cell line in particular suggests that the role of gsls in sorting of gpi-anchored proteins may be more complex. a rat thyroid epithelial cell line (frt) distributes its gsls and gpi-anchored proteins to the basolateral surface while the polarized distribution of a number of transmembrane proteins is identical to that of mdckcells (zurzolo et al., ) . this suggests that at least some of the apical proteins analyzed (e.g., ha) do not partition with basolaterally directed gsls. the frt cell line will serve as an excellent tool for furthering our understanding about the role of glycolipid clustering in the sorting of proteins and lipids in polarized epithelial cells. most of the early studies in epithelial polarity used the kidney-derived mdck cell line as their workbench. however, the last six years has seen the introduction of a number of new cell culture models into the field: caco (pinto et al., ; matter et al., ; costa de beauregard et al., ) ; ht- and t- (human intestinal epithelial), (madara et al., ; polak-charcon et al., ; mikogami et al., ) ; llc-pk (pig kidney proximal tubule) (hull et al., ; gstrauthaler et al., ; gottardi et al., ) ; mdbk (madin-darby bovine kidney) (furuse et al., ) , frt (fischer rat thyroid) (zurzolo et al., ) , as well as primary cultures of choroid plexus and retinal pigmented epithelium (marrs et al., ) . as we have discussed in the first half of this review, we arejust beginning to elucidate the nature of certain "apical" and "basolateral" sorting signals. however, the "nonstandard" sorting of gpi-link proteins in frt cells mentioned above, and the fact that a number of proteins display tissue and cell-type specific membrane localizations (see table ), calls into question the ways in which we think about polarized sorting signals and the mechanisms of sorting. as shown in table , there are notable differences in the localization of certain membrane proteins expressed in different tissue cell-types. the na,k-atpase, nearly ubiquitously expressed at the basolateral domain of most polarized cell types, is localized to the lumenal (apical) domain of both retinal pigmented epithelial and chorid plexus cells (wright, ; steinberg and miller, ; spector and shiel and caplan, a,b; (m) schwartz et al., ; (n) brown et al., . johanson, gundersen et al., ) . when the cdna encoding the ldl receptor was placed under the control of a metallothionein promoter and employed in the generation of a transgenic mouse, the receptor was expressed at the basolateral domains of liver and intestinal epithelial cells, but unexpectedly localized to the apical domains of proximal kidney tubule cells (pathak et al., ) . the polarized budding of certain viruses and the localization of their respective spike glycoproteins was shown to vary considerably between kidney derived mdck and thyroidderived frt cells (zurzolo et al., a) . in some instances, ashift in the type of targeting pathway used by a protein can depend on the differentiated state of the cell culture (zurzolo et al., b) . furthermore, the polarized localization of a particular gpi-linked protein was found to be developmentally regulated in drosophila embryos (shiel and caplan, a) . finally, a remarkable flexibility and "plastic-ity" of protein sorting has been suggested to be present in kidney intercalated cells, which appear to direct the vacuolar proton pump to either surface domain, depending on particular environmental cues (schwartz et al., ; brown et al., a) at the present time, we have little understanding of the signals or sorting mechanisms that mediate the differential sorting of the same protein in distinct cellular types. are different signals recognized by the different epithelial cells or is the same signal interpreted differently? is the sorting machinery itself different between polarized cells, or is the sorting machinery basically conserved between different cell-types while its regulation, adaptation, or wiring to the targeting machinery is different? evidence discussed in the second half of this review on the rab family of proteins suggests that elements of the targeting machinery are in fact highly conserved between different cell types, and it is the cell-type specific adaptation of this machinery which accounts for differences. nonetheless, it is becoming clear that the sorting of a particular protein can be a highly idiosyncratic feature of each polarized cell model. the observation that different epithelial cell lines may handle the same protein (or the same signal) differently has to reflect more than a mere capriciousness of epithelial cells in culture. each of the cultured cell models employed in polarity studies derive from and reflect some of the differentiated features of a tissue or organ system. accordingly, the sorting behavior observed in a particular cell type needs to be evaluated in the context of this cell's functional history. for example, is this cell derived from a tissue specialized for apical secretion or apical endocytosis? studies of the sorting of ion-transporting atpase molecules expressed in distal tubule-derived mdck and proximal tubule derived-llc-pk kidney cells suggest that the distinct cell surface distributions which an atpase subunit achieve in these two lines are consistent with established physiologic differences between the distal and proximal tubule epithelial cells (roush et al., manuscript submitted). these observations have led to the suggestion that sorting mediates delivery to functionally defined rather than topographically defined domains (gottardi and caplan, a) . it is becoming quite clear that the findings in the field of intracellular protein transport (reviewed by rothman, and by mellman, ) will prove to be extremely valuable to the discipline of epithelial polarity. in this field, the convergence of studies on synaptic vesicle (regulated) secretion in neurons, constitutive secretion in yeast, and intra-golgi transport have led to the rapid identifcation and characterization of the basic components necessary for vesicle formation, docking and fusion. clearly, the general components of the bud-ding and docking machinery lie at the heart of any transport process, whether we are considering the transport of a membrane protein from er to golgi, or a secretory protein from the tgn to a particular cell surface. in the following sections we touch upon some of the key discoveries in the field of intracellular transport and focus on the relevant molecules that may contribute to polarized sorting and delivery processes. one of the recent paradigms in intracellularprotein transport is based on the concept that vesicle shuttling between different organellar compartments is regulated through the coordinated efforts of different gtp-binding proteins. there are two broad classes of gtp-binding proteins which have been shown to regulate membrane trafficking events; the small g proteins (rabs and arf) reviewed by (donaldson and klausner, ; pfeffer, pfeffer, , and the trimeric g proteins (reviewed by bomsel and mostov, ) . the role of a gtp-binding protein in regulating vesicular transport was first realized with the analysis of one of the temperature sensitive sec (secretory) mutants in yeast (salminen and novick, ) . sec mutants display a rather striking accumulation of secretory vesicles when cultured at the restrictive temperature. the cloning, sequencing, and characterization of the sec gene revealed that it encoded a ras-like or 'small' gtp-binding protein which was present on the surfaces of the vesicles and could bind and hydrolyze gtp (salminen and novick, ; goud et al., ; kabcenell et al., ) . since the phenotype of cells bearing mutant sec is the accumulation of transport vesicles, it was apparent that sec is necessary for the targeting and/or fusion of secretory vesicles with the plasma membrane. similar results were found with another yeast protein yptl( % identical to sec ), which in its mutant form inhibited vesicular transport between the er and golgi complex (gallwitz et al., ; segev et al., ) . the suggestion that two small gtpases were important in the regulation of two different vesicular transport events in yeast led to the hypothesis that each step in vesicular traffic was regulated by a specific gtpase (bourne et al., ) . these ras-like gtpases are known to zdopt either of two distinct conformations, depending upon whether or not they are complexed with gtp or gdp. consequently, these gtpases have been postulated to serve as key regulators or "molecular switches" for membrane fission and fusion events. the apparent generality of ras-like gtpase in yeast, as revealed by sec and yptl, inspired asearch for these proteins' mammalian counterparts. to date, yptl/seccrelated proteins have been identified and are often referred to as rab proteins ("ras-like" proteins from rat brain) reviewed in (balch, ; hall, ; goud and mccaffrey, ; zerial and stenmark, ) . a number of rabs have been localized to specific organelles within the cell and through the combined efforts of in vitro and in vivo approaches have been shown to regulate membrane traffic between these organelles (reviewed in zerial and stenmark, ) . how this class of molecules contributes to the overall fidelity of membrane trafficking events is still unclear (rothman, ) . the idea that specific rab proteins regulate distinct steps along the transport pathway (e.g., rabl always regulates er to golgi traffic, whether in a kidney cell or neuron) led to the hypothesis that cells which contain unique, cell-type specific transport processes might be regulated by disinct rabs. indeed, the best example of this is the family of rab isofoms which have been found to be localized within cells which are well-adapted for regulated secretory events. rab a has been suggested to be important in the regulation of caz+ dependent secretion in neuronal (fischer von mollard et al., ) , neuroendocrine (darchen et al., ) and endocrine cell types (mizoguchi et al., ) . interestingly, an isoform of rab a, rab d, has been localized to the glucose transporter-containing vesicles of adipocytes, which are known to undergo regulated exocytosis after insulin stimulation (baldini et al., ) . thus, despite cell-specific differences, or vesicle-content differences, these regulated pathways rely on similar rabs (rab ). thus, distinct regulated exocytic events in a variety of cell types make use of similar molecular machinery (lutcke et al., ) . in this context, it has been speculated that polarized epithelial cells, with their distinct apical and basolateral targeting pathways, may employ epithelia-specific rab molecules. recent data suggest that this may be true. there are four rabs which have been implicated in polarized epithelial-specific functions: rab , rab b, rabl , and rab . of the four, only rabl is truly specific to polarized epitheiial cells. in the developing kidney, rabl mrna is detected only after mesenchyme is induced to differentiate into polarized epithelial structures (lutcke et al., ) . interestingly, rabl induction was shown to occur just prior to the appearance of apical markers and has therefore been suggested to be involved in the generation of apicalhasolateral polarity in these cells. rab localizes to the basolateral membrane and to electron dense tubules near the apical membrane. since rab proteins have been shown to regulate transport between the subcellular compartments with which they associate, it has been suggested that rabl regulates epithelial transcytosis. as we stated previously, two isoforms of rab ( a and d) have been implicated in the regulated exocytosis events shared by neuronal, endocrine and adipocyte cell types. interestingly, another isoform of rab , b, has been shown to be specific for polarized epithelial cells and is exclusively localized to the apical pole of cells, near the tight junctions (weber et al., ) . rabl , like rab b, also accumulates at the apical poles of polarized cells and co-localizes with the tight junction associated protein, zo- (zahraoui et al., ) . it has been suggested that these two rabs could regulate events necessary for the establishment of polarity. for example, since the localization of both rabs are completely dependent on the presence the of cell-cell contacts, it is possible that these mole-cules control the recruitment of membrane protein-containing vesicles required for establishing the tight junction "fence," a structure thought to maintain the distinct protein and lipid compositions of apical and basolateral membranes (dragsten et al., ) . it has also been proposed that these rabs control general vesicle targetting to the apical membrane (zahraoui et al., ) . this hypothesis was based on two independent observations. it has been shown that an apical membrane protein (aminopeptidase) inserts preferentially into the apical membrane at regions of cell-cell contact in mdck cells (louvard, ) . furthermore, under conditions in which mdck cells are denied intercellular contacts, apical proteins appear to be sorted and retained within a large subapical vacuolar compartment (vacuolar apical compartment, or vac) which, after initiation of cell-cell contact, is inserted preferentially at regions of cell-cell contact (vega-salas et al., ) . taken together, the localization of r a b l and rab b at this region of cell contact places these monomeric gtpases in a position to regulate the delivery of apical proteins to the cell surface (zahraoui et al., ) . moreover, the localization of a regulated, exocytic compartmentspecific rab (rab ) to a subdomain of the apical membrane of polarized cells is intriguing and suggests possible functional relationships between these subcellular compartments. the last rab worth exploring in the context of epithelial polarity is rab . while rab is not solely expressed in polarized cells, it is the only rab that has been functionally implicated in vectorial targeting. a peptide derived from the c-terminal region of rab can inhibit basolateral but not apical transport of membrane proteins in a permeabilized-mdck cell assay (huber et al., a) . interestingly, rab can also regulate membrane transport to the dendritic plasma membranes of neurons in culture; antisense rab oligonucleotides decrease the level of viral glycoprotein transported to this domain (huber et al., b) . this observation is consistent with the model which suggests that the mechanisms which produce axoddendrite polarity in neurons may be similar to those involved in apicallbasolateral polarity in epithelia (simons et al., ) . taken together, the identification of a polarized epithelia-specific rab (rab ), and the localization of other rabs to specific polarized epithelial domains (rab and rab b, apical; rab , basolateral) suggests that rabs may regulate specific pathways in polarized epithelial cells. for the epithelial cell biologist, the obvious question is, "what brings about the pathway-specific localizations of rab proteins in polarized epithelial cells?" it has been demonstrated that the carboxy-terminal regions of rab proteins are responsible for their unique cellular localizations (chavrier et al., ) . it has been suggested that organelle-specific receptors exist which recognize the c-terminal domains of these molecules. at least in terms of polarized cells, it would be tempting to speculate that identification of such receptors for rabl , b and rab will bring us one step closer to an understanding of the overall machinery that orchestrates domain-specific vesicle formation and targeting. recent evidence, however, suggests that rabs may not provide the primary level of specificity in membrane targeting events (brennwald and novick, ; reviewed by rothman and warren, ) . as we discuss below, a new class of proteins, the snares, may provide the necessary specificity for vesicle-membrane targeting events throughout the cell. the snare hypothesis for vesicle targeting arose from research in three related fields: synaptic vesicle release in neurons, transport between cisternae of the golgi, and secretion in yeast. briefly, a number of synaptic proteins were discovered to be important for the regulated fusion of synaptic vesicles with their targets on the pre-synaptic plasma membranes (reviewed by pevsner and scheller, ) . homologues of these proteins were found in yeast and shown to be required for constitutive vesicle transport (aalto et al., ) . at the same time, key elements of the general machinery for intracellular membrane fusion were being elucidated. in all three .cases, membrane fusion requires an nem-sensitive factor (nsf), adaptors that link nsf to membrane proteins (snaps: soluble nsf attachment proteins) and the membrane receptors for the nsf-snap complexes (snares: snap receptors) (reviewed in rothman and warren, ) . distinct snare proteins are present in the membranes of the vesicle and the target. the snare hypothesis stipulates that each transport vesicle is endowed with its own vesicle-(v-) snare (or vamp-like molecule) that can specifically interact with its cognate target-( t -) snare (or syntaxin/snap -like protein). this 'pairing' could ensure vesicleharget membrane specificity, while a general fusion apparatus consisting of nsf and snaps could be used throught the cell (sollner et al., ) . in the context of epithelial polarity, this hypothesis suggests that vectorial targeting of apical and basolateral proteins will require distinct v-snares. interesting recent data suggest that the situation in at least one epithelial cell type may be somewhat more complicated. when the surface delivery of membrane proteins is examined in mdck cells permeabilized at their apical or basolateral surfaces with streptolysin , it appears that basolateral transport involves all of the machinery discussed above. toxins which cleave snares inhibit basolateral delivery, as do antibodies directed against snaps. in contrast, apical protein insertion is unaffected by these reagents. isolation of apically-bound vesicles from mdck cells reveals the presence of high concentrations of an adducin homologue in their surface membranes. adducins are calcium-dependent phospholipid binding proteins thought to be involved in a number of membrane fusion events (ilkonen et al., ) . it would appear, therefore, that completely distinct classes of vesicular targeting and fusion machinery may operate in the two membrane delivery pathways present in polarized epithelial cells. in the absence of a readily available genetic system with which to identify the genes and gene products necessary for such higher eukarotic functions as transcytosis or polarized targeting, epithelial cell biologists have been resigned to the prospect of "poking" at the epithelial cell with various reagents and watching how it responds. reagents which prevent the polymerization of actin (gottlieb et al., ; jackmon et al., ) and tubulin (achler et al., ; parczyk et al., ) , toxins which modify a particular class of g proteins (stow et al., ; pimplikar and simons, b) , or toxins that inactivate the vamp, syntaxin and snap- molecules described above, second messanger stimulators, analogues of the messangers themselves (apodaca et al., ; cardone et al., ; hansen and casanova, ) and the remarkable fungal metabolite brefeldin a (bfa) are all being incorporated into the repetoire of tools which we hope will enable us to gleen more information from a particular transport pathway. those interested in polarized and nonpolarized cell functions alike have made use of such cell-perturbing reagents. since the focus of this review is epithelial polarity, we have chosen to summarize some of the studies which are providing insights about the mechanisms of polarized sorting and targeting. brefeldin a is a fungal metabolite that endeared itself to cell biologists because of its dramatic effect on the protein secretory pathway (reviewed in . protein secretion is inhibited by bfa: membrane trafficking out of the er is blocked and the golgi appears to breakdown and become redistributed into the er (lippincott-schwartz et a]., ). before golgi redistribution, bfa causes this organelle to form tubular extensions which are devoid of any cytoplasmic (nonclathrin) "coat" material (lippincott- schwartz et al., ) . it has been shown that these morphological changes are not restricted to the golgi but rather are observed in a number of organelles of the endomembranous network such as endosomes, lysosomes and the tgn (hunziker et al., ; lippincott-schwartz et al., ; wood et al., ) , suggesting that the bfa "effector" might play a role in membrane transport events all over the cell. perhaps surprisingly, while membrane transport phenomena are remarkably altered in the presence of bfa, several processes are clearly unaffected, including receptor mediated endocytosis and endocytic recycling . from the standpoint of sorting and polarized delivery, bfa's most interesting property is its ability to differentially affect polarized cell surface targeting events. for example, low and colleagues ( i , ) determined a concentration of bfa where er-golgi trafficking was not inhibited, so that delivery from the tgn to the surface could be assayed for bfa sensitivity. interestingly, bfa inhibited the apical delivery of both endogenous, mdck secretory proteins ( ) and the membrane protein dppiv ( ) while also enhancing their mis-delivery to the basolateral surface. basolateral targeting of the endogenous mdck protein, uvomorulin, was not affected under these conditions. taken together, it would seem that a target molecule for bfa action exists that is exclusively involved in directing apical vesicles or which is simply more sensitive to the effects of bfa than similar molecules participating in the basolateral pathway. either way, these results provide a hint that there are indeed molecular differences between these two targeting pathways. it is important to add that in addition to inhibiting the exocytic apical pathway in mdck cells, basolateral to apical transcytosis is also inhibited by this drug (hunziker et al., ; low et al., ) . these findings have led to the suggestion that sorting mechanisms for apically destined proteins, whether along the exocytic or the transcytotic pathway may be functionally and biochemically similar (hunziker et al., ) . the fact that the loss of the structural integrity of the golgi induced by bfa correlates with a striking absence of its characteristic "coat" (observed at the em level) led to the idea that coat proteins might be rendered non-functional due to bfa action. through a number of studies (reviewed by donaldson et al., ; helms and rothman, ; rothman & orci, ) molecules which make up this "coat" were identified and characterized (e.g., pcop and a m ) . an "order of events" necessary for vesicle budding emerged from these studies and is outlined below. arf is a gtp-binding protein loosely related to ras and distinct from the family of rabs. in its gtp-bound state, it is capable of associating with the membrane by virtue of its myristoyl group, while its gdp-bound form is soluble and not membrane bound. arf binding to membranes appears to be the signal for coatomer binding, that is, the binding of pcop in addition to other as yet uncharacterized coat proteins. coatomer binding is believed to be absolutely necessary for vesicle budding. therefore proper coatomer assembly would be required for any event downstream of budding, such as targeting. recently, it has been determined that bfa inhibits coatomer assembly and vesicle formation through arf, by essentially allowing it to remain in its gdp-bound or inactive form. there exists a class of proteins which are able to catalyze the exchange of gdp for gtp called guanine nucleotide exchange factors (gne). bfa has been proposed to antagonize the action of a gne on arf, thus preventing coatomer assembly and membrane budding helms and rothman, ) . with the recent identification of an ever-growing family of new arf-related genes (kahn et al., ) and the speculation that different cops may exist in the control of membrane budding events from different organelles , there is growing excitement that arfs and cops will turn-out to be essential components for regulating a particular level of specificity inherent to membrane targetting events. in the context of bfas affect on apical sorting and targeting in polarized mdck cells (low et al., (low et al., , , it is likely that distinct arfkoatomer complexes regulate the budding of apical and basolaterallydestined vesicles from the tgn. moreover, the fact that significant missorting into the basolateral pathway was observed in the presence of bfa (low et al., ) suggests that coatomer assembly may be inextricably linked to proper secretory and membrane protein sorting. it has been known for some time that members of the heterotrimeric family of g proteins are associated not only with the plasma membrane but also with intracellular membranes (reviewed by bomsel and mostov, ) . a number of toxins (cholera, pertussis and mastoparan) known to activate or inhibit various classes of g proteins have been applied to studies of polarized sorting and targeting. stow et al. ( ) found that overexpression of gai- in polarized llc-pk cells significantly reduced the level of constitutive basolateral secretion of an extracellular matrix component, heparan sulfate proteoglycan. pertussis toxin, which adp-ribosylates and inactivates the a-subunits of the g a i/o class of heterotrimeric g proteins, relieved this inhibition. similarly, pimplikar and simons ( ) suggested that gi and gs may differentially regulate the trafficking of apical and basolateral vesicles in slo-permeabilized mdck cells, while leyte et al. ( ) found that gi/o and gs associated with the tgn could oppositely regulate constitutive secretory vesicle formation. it should be noted that in no case did the g protein related inhibition or stimulation appear to affect the actual sorting or missorting of apical or basolaterally destined proteins (in contrast to the bfa results discussed above (low et al., ) , but rather may only affect the rate or "efficiency" of sortingkargetting . a possible link between heterotrimeric g proteins and coatomer formatiodvesicle budding was provided by ktistakis et al., ( ) . this group found that activation of a g a protein with mastoparan promoted pcop binding and prevented bfainduced effects. pretreatment of cells with pertussis toxin, which is known to specifically affect g a i subclass of heterotrimerics, prevented mastoparan's antagonizing effects on bfa. stated more simply, these results showed that activation of a pertussis-toxin-sensitive ga promotes the binding of pcop to golgi membranes and thus antagonizes the action of bfa. the authors of this study suggest further that different subclasses or isoforms of ga could be responsible for some of the differences in bfa-sensitivities observed between cell types and organellar membranes. these key observations have led to the idea that heterotrimeric g proteins, by virtue of their membrane topology would be ideal candidates for coordinating the transfer of sorting information to the cytoplasmic surface of the tgn necessary for vesicle budding (bomsel and mostov, ; ktistakis et al., ) . the outer surface of a fruit fly embryo is composed of a monolayer of polarized epithelial cells. the apical membranes of these epithelial cells face the outer shell, or chorion, while their basolateral surfaces face the embryonic interior and yolk space. invaginations of this surface epithelium give rise rise to all of the embryo's internal tissue structures (for review see shiel and caplan, b) . recent investigations have examined the mechanisms through which proteins are sorted in these epithelial cells. human placental alkaline phosphatase (plap) is a gpi-linked protein which has been shown to be sorted to the apical plasma membrane when it is expressed by transfection in mdck cells. a chimeric construct of plap, in which the gpilinkage domain is replaced by the transmembrane and cytoplasmic domains of the vsv g protein (plapg), is sorted to the basolateral surfaces of mdck cells (brown et al., ) . these two proteins have been expressed under the control of heat shock promoters in transgenic flies and their distributions have been examined in embryonic epithelia throughout embryogenesis (shiel and caplan, a) . as would be expected, the plapg protein is restricted to basolateral surfaces throughout ontogeny in the surface epithelial cells as well as in the internal epithelia which derive from invaginations of the surface cells. surprisingly, plap was also restricted to a basolateral distribution in the surface epithelial cells in both early and late stage embryos. biochemical experiments demonstrated that this mis-sorting of the plap protein can not be attributed to problems with the addition of the gpi-linkage, since at all embryonic stages plap is correctly glipiated. internal epithelial cells sorted plap exclusively to their apical surfaces. since in many cases internal epithelia form from surface epithelia without undergoing any mitosis (e.g., salivary gland), essentially the same epithelial cell is capable of differentially sorting plap depending on that cell's physical position within the embryo. examination of epithelia undergoing invagination (e.g., ventral furrow, tracheal placode) demonstrate that the transition in plap sorting occurs in the early stages of the invagination process. while the mechanism responsible for this switch remains unclear, the power of drosophilu genetics will hopefully allow the cellular components responsible for this transition to be readily identified. it is likely that the isolation of the proteins responsible for this phenomenon will shed light on the drosophilu as well as on the mammalian epithelial sorting machinery. a drosophilu mutation whose phenotype includes peturbations of the polarized organization of the surface epithelial cells has recently been identified and characterized at the molecular level wodarz et al., ) . the crumbs gene encodes a transmembrane protein which is normally expressed in the apical membranes of surface and internal epithelial cells. mutation of the crumbs gene results in a loss of crumbs polarity and markedly alters embryonic morphology. genetic studies have demonstrated that the crumbs gene product is necessary not only for its own apical sorting, but for the apical delivery of other proteins as well. furthermore, the product of the stardust gene appears to interact with the crumbs protein and also appears to participate in apical sorting. understanding these proteins' biochemical functions and their intermolecular associations will undoubtedly provide enormous insight into the cellular components responsible for generating and maintaining the polarized phenotype. hopefully, the development of genetic approaches such as these, in concert with the continuing refinement of in vitro and model systems, will allow us to develop a clear and fundamental understanding of how epithelial cells produce their remarkable asymmetry. yeast syntaxins ssolp and sso p belong to a family of related membrane proteins that function in vesicular transport role ofmicrotubules in polarized delivery of apical membrane proteins to the brush border ofthe intestinal epithelium distribution of transport proteins over animal cell membranes podocytosis of small molecules and ions by caveolae the calmodulin antagonist, w- , alters transcytosis, recyclingand morphology ofthe endocyticpathway in madine-darby canine kidney cells mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum cloning of a rab isotype predominately expressed in adipocytes the npxy internalization signal of the ldl receptor adopts a reverse turn conformation biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. in vifro binding oftheasiatoglycoproteinreceptor to the betaadaptin of plasma membrane coated vesicles identificationofeffector-activatingresidues ofgsa lntracellular protein topogenesis. proc. natl. acad. sci. usa role of heterotrimeric g proteins in membrane traffic the gtpase superfamily: aconserved switch sorting of gpi-anchored proteins to glycolipid-enriched membrane an h+-atpase in opposite plasma membrane - . for diverse cell functions . subdomains during transport to the cell surface interactions between gpi-anchored proteins and membrane lipids mechanism of membrane anchoring affects polarized expression oftwo proteins in mdck cells constitutive and regulated secretion of proteins intracellular sorting and polarized cell surface delivery of nqk-atpase, an endogenous component of mdck cell basolateral plasma membranes dependence on ph of polarized sorting of secreted proteins sortingofmembrane and secretoryproteins in polarizedepithelial cells phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in mdck cells phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis an autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor hypervariable c-terminal domain of rab proteins acts as a targeting signal transferrin receptor internalization sequence yxrf implicates a tight turn as the structural recognition motif for endocytosis transplantedldl and mannose- -phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor asortingsignal for the basolateral delivery of the vesicular stomatitis virus (vsv) g protein lies in its luminal domain: analysisofthe targetingofvsv g-influenzahemagglutininchimeras suppression of villin expression by antisense rna impairs brush border assembly in polarized epithelial intestinal cells a tyrosine-based signal targets h,k-atpase to a regulated compartment and is required for the cessation of gastric acid secretion high-resolution epitope mapping of hgh-receptor interactions by alanine-scanning mutagenesis association of the gtp-binding protein rab a with bovine adrenal chromaffin granules the internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information arf: a key regulatory switch in membrane traffic and organelle structure brefeldin a inhibits golgi membrane-catalysed exchange of guanine nucleotide onto arf protein polarized sorting ofglypiated proteins in hippocampal neurons involvement of na,k-atpase in antinatriuretic action of mineralocorticoids in mammalian kidney membrane assymetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane? a small gtp-binding protein dissociates from synaptic vesicles during exocytosis an enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late golgi compartments direct association of occludin with zo- and its possible involvement in the localization of occludin at tightjunctions a yeast gene encoding a protein homologous to the human c-hashas proto-oncogene product specificity of binding of clathrin adaptors to signals on the mannose- -phosphate/insulin-like growth factor receptor receptor-mediated endocytosis: concepts emerging h m the ldl receptor system nonpolarized secretion of truncated forms of the influenza hemagglutinin and the vesicular stomatitis virus g protein from mdck cells an ion transporting atpase encodes multiple localization signals biotinylation and assessment of membrane polarity: caveats and methodological conerns sorting of ion transport proteins in polarized cells secretion of endogenous and exogenous proteins from polarized mdck monolayers sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface ofpolarized epithelial cells small gtp-binding proteins and their role in transport a gtp-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast biochemical characterization ofrenal epithelial cell cultures (llc-pk and mdck) apical polarity ofna,k-atpase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton mechanism for regulatingcell surfacedistributionofna,k-atpase inpolarizedepithelial cells gs alphastimulates transcytosis and apical secretion in mdck cells through camp and protein kinase a inhibition by brefeldin a of a golgi membrane enzyme that catalyzes exchange of guanine nucleotide bound onto arf protein more than just a membrane anchor rab , a small gtpase involved in vesicular traffic between the tgn and the basolateral plasma membrane protein transport to the dentritic plasma membrane of cultured neurons is regulated by rab p the origin and characteristics of a pig cell strain, llc-pk basolateral sorting in mdck cells requires a distinct cytoplasmic domain determinant different requirements for nsf, snap, andrab proteins in apical and basolateral transport inmdck cells inhibition of apical but not basolateral endocytosisofricinandfolateincaco- cells by cytochalasind binding and hydrolysis of guanine nucleotides by sec p, ayeast protein involved in the regulation of vesiculartraffic human adp-ribosylationfactors: a functionally conserved family of gtp-binding proteins crumbs and stardust, two genes of drosophila required for the development of epithelial cell polarity. development suppl exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell mdck the biogenesis of lysosomes action of brefeldin a blocked by activation of a pertussis-toxin-sensitive g protein the signal sequence of nascent preprolactin interacts with the k polypeptide of the signal recognition particle phosphorylationofthe rat hepaticpolymericiga receptor an internal deletion in the cytoplasmic tail reverses the apical localization of human ngf receptor in transfected mdck cells multiple trimeric g-proteins on the trans-golgi network exert stimulatory and inhibitory effects on secretory vesicle formation tyrosine-dependent basolateral sortingsignals are distinct from tyrosine-dependent internalization signals a signal sequence for the insertion of a transmembrane glycoprotein rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er microtubule-dependentretrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway brefeldin a's effects on endosomes, lysosomes, and the tgn suggest a general mechanism for regulating organelle structure and membrane traffic polarized apical distribution of glycosyl-phoshatidylinositol-anchoredproteins in a renal epithelial cell line steady-state distribution and biogenesis of endogenous mdck-glycoproteins: evidence for intracellular sorting and polarized cell surface delivery preferred apical distribution of glycosyl-phosphatidylinositol (gpi) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells selective inhibition ofprotein targeting to the apical domain of mdck cells by brefeldin a inhibition by brefeldina ofprotein secretion from the apical cell surfaceofmadin-darby caninekidney cells rabl , a novel small gtpase, is specific for epithelial cells and is induced during cell polarization targeting and retentioon ofgolgi membrane proteins structural analysis o f a human intestinal epithelial cell line distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different na,k-atpase distributions in polarized epithelia sortingofan apical plasmamembraneglycoproteinoccurs before it reaches the cell surface in cultured epithelial cells sortingofendogenous plasmamembrane proteins occurs from two sites in cultured human intestinal epithelial cells (caco- ) basolateral sorting of ldl receptor in mdck cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants mechanisms ofcell polarity: sorting and transport in epithelial cells structural requirements and sequence motifs for polarized sorting and endocytosis of ldl and fc receptors in mdck cells polarizedexpressionofa chimeric protein in which the transmembrane and cytoplasmic doamins of influenza hemagglutinin have beenreplaced by those of the vesicular stomatitis g protein membranes and sorting. c u r apical-to-basal transepithelial transport biogenesis of epithelial cell polarity: of human lactoferrin in the intestinal cell line ht- c . a intracellular sorting and vectorial exocytosis of an apical plasmamembrane glycoprotein tissue distribution of smg p a, a ras p -like gtp-binding protein, studied by use of a specific monoclonal antibody polymeric immunoglobulin receptor expressed in mdck cells transcytoses iga plasma membrane protein sorting in polarized epithelial short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum the trans-most cisternae of the golgi complex: a compartment for sorting of secretory and plasma membrane proteins microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, madin-darby canine kidney tissue-specific sorting of the human ldl receptor in polarized epithelia of transgenic mice clathrin, adaptors, andsorting receptors compete for adaptors found in plasmamembranecoated pits mechanisms of vesicle docking and fusion: insights from the gtp-bindingproteins inintracellulartransport. trendsincell biology intracellular sorting and basolateral appearance of the g j. , - . nervous system isoformsofthena,k-atpase are presentin both axons and dendrites of hippocampal neurons in culture regulation of apical transport in epithelial cells by a gs class of heterotrimeric g protein role of heterotrimeric g proteins in polarized membrane transport the effect ofmodifying the culture medium on cell polarity in a human colon cell line replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells localization of sodium pumps in the choroid plexus epithelium nuclear localization signals in polyomavirus large-t viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellular transport in doubly infected madin-darby canine kidney cells the distribution of na,k-atpase in the retinal pigmented epithelium from chicken embryo is polarized in vivo but not in primary cell culture the roleofclathrin, adaptors and dynamin inendocytosis morphogenesis of the polarized epithelial cell phenotype polarity of epithelial and neuronal cells asymmetric budding of viruses in epithelial monolayers: a model system for study of epithelial polarity influenza virus hemagglutinin expression is polarized in cells infected with recombinant sv viruses carrying cloned hemagglutinin dna the large extracellular domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells the large external domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells mechanisms of intracellular protein transport molecular dissectionofthesecretory pathway implications ofthe snare hypothesis for intracellularmembrane topology and dynamics a ras-like protein is required for a postgolgi event in yeast secretion membrane and secretory proteins are transported from the golgi complex to the sinusoidal plasmalemmaofhepatocytes by distinct vesicular carriers plasticity of functional epithelial polarity developmental regulation of membrane protein sorting in the generation of epithelial polarity in mammalian and lipid sorting in epithelia polarized sorting in epithelia biogenesis of cell-surface polarity in epithelial cells and neurons the phosphomannosyl recognition system for intracellular transport of lysosomal enzymes snap receptors implicated in vesicle targeting and fusion interaction of activated egf receptor with coated pit adaptins in vitro binding of plasma-coated vesicle adaptors to the cytoplasmic domain of lysosomal acid phosphatase ), c < . drosophila embryos atpase in polarized epithelial cells the mammalian choroid plexus transport and membrane properties of the retinal pigment epithelium nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells a heterotrimeric g protein, gai- , on golgi membranes regulates the secretion o f a heparan sulfate proteoglycan in llc-pki epithelial cells a golgi retention signal in a membrane-spanning domain of coronavirus el protein molecularcloning,characterization, subcellular localization and dynamics of p , the mammalian kdel receptor vesicular stomatitis virus glycoprotein contains a dominant cytoplasmic basolateral sorting signal critically dependent on tyrosine the basolateral targetingsignal inthe cytoplasmicdomainofvsv g protein resembles a variety of intracellular targeting motifs related by primary sequence but having diverse targeting activities organization of glycosphingolipids in bilayers and plasmamembranes of mammalian cells sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans golgi network of att cells endocytosis and signals for internalization exocytosis of vacuolar apical compartment (vac): a c e l k e l l contact controlled mechanism for the establishment ofthe apical plasma membrane domain in epithelial cells analysisofthedistributionofchargedresidues inthen-terminal regionofsignal sequences: implications for protein export in prokaryotic and eukaryotic cells mechanisms of protein translocation across the endoplasmic reticulum distinct transport vesicles mediate the delivery of plasmamembrane proteins to the apical and basolateral domains of mdck cells expression and polarized targeting of a rab isoform in epithelial cells crumbs is involved in the control of apical protein targeting during drosophrlu epithelial development brefeldin a causes amicrotubule-mediatedfusionofthe mechanismsofiontransportacrossthe choroidplexus a small rab gtpase is distributed in cytoplasmic vesicles in non-polarized cells but colocalizes with the tightjunction marker - in polarized epithelial cells opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in fischer rat thyroid epithelial cells key: cord- -ub p ngr authors: mollenhauer, hilton h.; james morré, d.; rowe, loyd d. title: alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity date: - - journal: nan doi: . / - ( ) -z sha: doc_id: cord_uid: ub p ngr monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of principally sodium ions for protons. the outer surface of the ionophore-ion comples is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes in nonpolar solvents. in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. the net effect for monensin is a trans-membrane exchange of sodium ions for protons. however, the interaction of an ionophore with biological membranes, and its ionophoric expression, is highly dependent on the biochemical configuration of the membrane itself. one apparent consequence of this exchange is the neutralization of acidic intracellular compartments such as the trans golgi apparatus cisternae and associated elements, lysosomes, and certain endosomes. this is accompanied by a disruption of trans golgi apparatus cisternae and of lysosome and acidic endosome function. at the same time, golgi apparatus cisternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions. monensin effects on golgi apparatus are observed in cells from a wide range of plant and animal species. the action of monensin is most often exerted on the trans half of the stacked cisternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensin concentrations or short exposure times, near the middle of the stacked cisternae. the effects of monensin are quite rapid in both animal and plant cells; i.e., changes in golgi apparatus may be observed after only – min of exposure. it is implicit in these observations that the uptake of osmotically active cations is accompanied by a concomitant efflux of h(+) and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of cisternae observed in electron micrographs. in the golgi apparatus, late processing events such as terminal glycosylation and proteolytic cleavages are most susceptible to inhibition by monensin. yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the golgi apparatus. in endocytosis, monensin does not prevent internalization. however, intracellular degradation of internalized ligands may be prevented. it is becoming clear that endocytosis involves both acidic and non-acidic compartments and that monensin inhibits those processes that normally occur in acidic compartments. thus, monensin, which is capable of collapsing na(+) and h(+) gradients, has gained wide-spread acceptance as a tool for studying golgi apparatus function and for localizing and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. among its advantages are the low concentrations at which inhibitions are produced ( . – . μm), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or atp levels) and a reversible action. because the affinity of monensin for na(+) is ten times that for k(+), its nearest competitor, monensin mediates primarily a na(+)-h(+) exchange. monensin has little tendency to bind calcium. not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed in cattle. the mechanisms by which monensin interact with coccidia and rumen microflora to achieved these benefits are reasonably well documented. however, the interactions between monensin and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensin poisoning are well known. equine species are particularly susceptible to monensin poisoning, and a common effect of monensin poisoning is vacuolization and/or swelling of mitochondria in striated muscle. other pathological injuries to striated muscle, spleen, lung, liver and kidney also have been noted. a consistent observation is cardiac myocyte degeneration as well as vacuolization. differences in cellular response resulting from exposure to monensin (i.e., golgi apparatus swelling in cultured cells, isolated tissues, and plants vs.mitochondrial swelling in animals fed monensin) suggest that myocardial damage is due either to a monensin metabolite or is a secondary response to some other derivation. however, as pointed out by bergen and bates [ ], the underlying mode of action of ionophores is on transmembrane ion fluxes which dissipate cation and proton gradients. consequently, some or all of the observed monensin effects in vivo in animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes. monensm, a na ÷ ionophore capable of collapsing na ÷ and h + gradients, has gamed wide-spread acceptance as a biochemical and biologacal investigative tool to study golgi apparatus function and to localize and identify the molecular pathways of subcellular vesicular traffic. among its advantages are the low concentrations at which lntubltions are produced ( . - . #m), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or atp levels), and a reversible action [ ] . the purpose of this review is to examine the mechanism of action and specificity of monensln in na+/h ÷ exchange and to attempt to reconcile tlus to the large body of structural and biochemical information on monensin toxicity derived from animal studies in , pressman and co-workers [ ] reported a class of anubiotics that induced alkali ion permeability in mitochondna and other membranous systems. these antibiotics functioned as lonophores (ion-carriers) to carry ions across hpid barriers as complexes soluble in the hpid phase of the membranes. the potential use of tonophores as probes of biological function, or as potential therapeutic agents, was recognized very early [ ] [ ] [ ] [ ] , but major economic importance was not forthcoming until the discovery of monensin in and the recognlnon of tts potential in the poultry mdustrty as a coccxdtostat [ ] subsequently, ~t was discovered that tonophores also could improve feed converston in rumtnants such as cattle [ , ] , thus adding further to their commercial value of the more than lonophores that have been reported [ ] , three, monensln, lasalocld and sahnomysin, have widespread commercial use of those licensed, monensin is probably used most widely. several monensins have been tdentified [ , ] _ monenstn a, and specfftcally the sodium salt of monensin a (hereafter simply designated as monensm) ts derived from streptomvces cmnamonensts [ ] , and a crude mycehal preparation (rumensm') containing about _ % monensin ts used commercially. the on spectfictty of monensm xs ag > na >> k > rb > cs > li > ca [ , ] with approximately a -fold selectlvry for sodtum over potassium [ ] and little tendency to bind calcmm [ ] i! . one of the original interests in ionophores was their percewed potential for directly modifying intracellular iomc gradients, pamcularly ca +, winch would lead, hopefully, to the development of useful pharmacologic agents or, alternatively, provide a tool for studying cellular functtons medmted by changes m ca + [ , ] . of parttcular interest were divalent xonophores such as x- a (normally considered a ca + ionophore -note, however, x- a complexes na ÷ and k + almost as well as ca +) winch have been shown to mduce contractaon of aortic strips and increase the rate of contractility of tsolated perfused rabbit heart [ , ] , and release ca + from energy-loaded vesicles derived from the sarcoplasrmc renculum of muscle [ ] [ ] [ ] x- a may also mcrease blood flow through coronary artenes and mcrease cardiac output [ ] . the emphasis on heart physiology stemmed from this organ's strong dependence on calcmm for proper functioning [ ] . indeed, subsequent studies of x- a used as a feed additive for poultry and cattle has shown that the heart ~s a primary target for lonophore toxicity [ ] . x- a affects many other cellular functions such as release of b~olog~cally active agents and the induction of sperm acrosome reactions of several species [ , ] . it was soon realized, however, that many of the ionotroplc effects of the divalent ionophores could be duplicated with even greater efficiency by monovalent ionophores such as monensm which complexes na + but almost no ca ÷ tins response apparently occurs because the movement of na ÷ into a cellular compartment by monensin fac~htates the entry of ca + by a na+-out/ca +-ln exchange [ , , , ] . thus, a ca ÷ shaft ts stall the pnmary factor medmtlng cellular responses although other factors may also play significant roles m monensm physiology for example, many tono-phores, either directly or as a result ol the lomc imbalance. may transport, promote uptake, or release effector substances such as serotonm, lustamme, prostaglandm and catacholamlne which, m turn. have profound effects on cellular function [ . ] slmdarly. monensm. through alteration of the ph of mtracellular compartments may lnhlbr the release and/or transport of numerous agents and. in so doing, perturbate cellular function in cardiac tissues, both posmve and negative motroplsm has been observed sequentially (the variable factor being time) m tissues following exposure to monensin [ ] concentrahon of monensln may. also. cause similar posttlve/neganve ,notroptc responses [ , . finally. some monensln mgested b}¢ an animal is metabolized to other tonophores, the properties of which are largely unknown thus, ionophores, in spite of many common characteristics, differ indtvtdually in their effects on cell~ moreover. cells may respond to both direct tonophore interaction as well as secondary effects that develop from the initial ionophore reactions. the latter is partlcularly likely in the whole antmal where metabolites with unknown properties are produced from lonophore breakdown and where changes m the products of one organ can affect the function of other organs_ monensin is an open chain molecule that is capable of ton complexation through a cyclic form stabilized by hydrogen bonding between the carboxyl and hydroxyl groups charge transfer bonding wltinn the cavity formed is responsible for on binding (fig ) _ because the affinity of monensln for na + is -ttmes that for k+. tts nearest competitor in biological systems. bergan and bates [ ] ) the aniomc form of the lonophore is stabilized by the polar environment characteristic to the surface of a membrane_ the ionophore is capable of ion pamng with a metal cation either at the terrmnal carboxyhc acid moiety or at other internal sites the binding of a cation initiates the formation of a hpophahc, cyclic catlon-lonophore complex that can diffuse through the interior of the blmolecular membrane structure_ after traversing the membrane, the complex is again subjected to a polar environment where the electrostatic forces that had stablhzed the complex are no longer greater than the unfavorable gibbs free energy change of cyclzzation the ionophore then releases its enclosed cation and reverts to the low energy acyclic conformation monensin, like other carboxyhc lonophores, binds metal ions through liganding s~tes such that the ions become centrally oriented ( fig. ) and masked from the extraceuular environment [ , , ] . the outer surface of the ionophore-lon complex is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes m nonpolar solvents [ , , ] in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse through the membranes from one aqueous membrane interface to the other [ , ] . once the ion traverses the membrane as a monensin-lon complex, the ion is released, and the monensin molecule picks up a proton to form an undissociated molecule which then retraverses the membrane to release the proton to the outside of the cell, vesicle, organelle, or other subcellular compartment [ ] (fig. ) . thus, the net effect for monensin is a trans-membrane exchange of monovalent ions for protons. the on transfer rates may be very high and can approach, or even exceed, normal enzyme diffusion rates [ ] , although the actual rate may be markedly altered depending on factors such as the concentration of k ÷ m the external medium and the type and concentration of permeant ion that accompariles the accumulated k + [ ] . however, effects of lonophores on cation transport and their distribution among different membrane-bounded compartments within the cell, will vary depending upon the physical and chemical properties of the different membranes. membrane fluidity, thickness, curvature, charge and orientation of polar head groups of phosphohpids, cholesterol content, and protein content, all influence solubihty, penetration, and expression of the lonophore [ ] . moreover, in asymmetric membranes (i.e., biological membranes), ionophores generally exhibit asymmetric transport properties [ ] . for example, kovac et al [ ] showed that both vahnomycm and nigericin (an lonophore similar to monensin) crossed the plasma membrane of saccharomyces cereotslae at a rather low rate but then were preferentially located, and active as ~onophores, m the tuner mltochondrlal membrane. thus, the physiological effects of monensln will depend on the membrane composition and functional characteristics of the different compartments involved. although mechanisms for on transfer through a blmolecular leaflet (membrane) have been proposed, questions still remain as to how this action is related to known effects of monensin on cell function and what relationships these may have, m turn, on blochermcal mechanisms leading to ammal toxaclty. while any consideration of ~onophore action must focus on the mechanism of lonophore interaction with biological membranes [ ] , the complexity of the process and the multiplicity of potentml pathways involved suggests that a single causal mechanism cannot explain both the cellular responses and the clinical expressions of toxic-]ty in animals monensin is cost-effective in increasing the yield of meat from both fowl and ruminants [ ] . in fowl, this increase m productivity is derived almost directly through the control of coccid]a that, if present, would adversely affect animal health [ ] in ruminants, increased product]vity appears to result from several factors, the most obvious being an increase in the effectiveness of feed utlhzation [ , , ] . these whole-animal effects (xe, systems effects) are well documented [ , [ ] [ ] [ ] and will be paraphrased only briefly here. the beneficial effects of monensm in cattle accrue, in part, through shifts m rumen rmcroflora population. for example, gram-positive bacteria (that are primarily acetate, butyrate, h , and formate producers) are inhibited by monensin; whereas, gram-negauve bacteria (many of which produce succinate) are less sensmve to monensm [ ] . the outer layer of the multflayered well of the gram-negatxve bacterium may contribute to this resistance by acting as a barrier to the penetration of [able i adapted from ledger and tanzer [ ] in ~e~retlon reduced secretion procollagen [ ] [ ] [ ] [ ] , fibronectm [ ] [ ] [ ] ] , proteoglycans [ , , ] , prolactln [ ] , alburmn [ ] , transfernn [ ] , promsuhn polypepudes [ ] , larmmn [ ] , a-amylase isoenzymes [ , ] , newly synthesized proteins [ ] , secretory proteins [ ] , proteins for fast axonal transport [ , ] , thyroxine-binding globuhn [ ] , acetylchohnesterase [ , ] , chononlc gonadotropin [ ] , phytohemagglutmm [ ] , very-low-density hpoprotem [ ] , maize rootcap polysaccharldes [ ] (see however, sticher and jones [ ] for lack of monensm effect), vesicular stomatltxs virus glycoprotein [ ] , extracellular matrix [ ] , type ii collagen [ , ] , reviews [ , , ] increased secreuon catecholarmne [ ] , catheps'n d [ ] _ defective processing pro-albumin to serum albumin [ ] , receptors for msuhn and somatatomedin c [ ] , pro-oplomelanocortm [ ] incomplete processing of ohgosacchandes (n-hnked and/or o-linked) myeloperoxldase [ ] , prenv glycoprotexn [ ] ; fibronectm [ ] , hcg subumts [ ] , blocked formation of complex ohgo-~acchandes [ ] , herpes simplex glycoprotelns [ ] , hla-dr-as-soclated mvanant chain [ ] , coronavlrus glycoprotem [ ] , review [ ] undersulphauon proteoglycans [ ] , glycosammoglycan chains [ ] , / -d-xyloside glycosammoglycans [ , ] in endocytosts and endosome actdzflcatton intubitlon of mternahzatlon arylsulfatase [ ] , lmmunoglobulln [ ] , a- -macroglobuhn [ ] , semlikl forest varus [ ] , horseradish peroxldase [ ] inhibition of dlssocmtion of mternahzed hgand asmloglycoprotems [ ] , asmlo-orosomucojd [ ] lnhlbxtlon of hgand transfer smb~s virus nuclear capsids to cytoplasm [ ] , epidermal growth factor, / -hexasamlmdase, low-density hpoprotein, lmmunoglobulin, and proteoglycans to lysosomes [ , , ] inhibition of acldfflcauon endocytlc vesicles [ ] [ ] [ ] , lysosomal and prelysosomal compartments [ ] , interference with semllka forest virus genome penetrauon [ ] , expression of diphtheria toxin [ ] ; recycling of ldl receptors [ ] , release of diphtheria toxin from endocyuc vesicles [ , ] lnhlbltton of mtracellular degradation proteoglycans [ ] , insuhn [ ] , lysosomal (methylarmne-sensxtlve) protem degradation [ ] inhibition of contraction of contractile vacuoles paramecmm aureha [ surface formation and growth altered secretion of cell surface molecules proteoglycan [ , , ] , type if collagen and/or procollagen [ , , ] ; fibronectin [ , , ] , lamlmn [ ] , mcorporauon of sulfaudes into myelin [ ] , incorporation of po protein and myehn basic proteins into myelin [ ] inhibition of scale morphogenesis scales of the green alga pyrarmmonas mconstans [ ] inhibition of cell spreading-cultured fibroblasts [ ] ; mesoderm ceils [ ] stimulation of receptor capping mouse t-lymphoma cells [ ] inhtbltlon of growth rye seedhngs [ ] , pelhu ] transport of molecule.~ recognition of independent secretory pathways acetylchohne receptor and acetylchohnesterase [ ] , membrane glycoprotems/ assembly of uukunlerm virus [ ] , ca +-dependent and ca +-mdependent secretion of a-amylase [ ] , proteoglycan and hyaluronate [ ] , prolactln [ ] ; galactosyl receptor [ ] maturation and/or transport of viral coat proteins vesicular stomatltis virus [ , ] , herpes sxmplex varus [ , ] , semhkj forest wrus [ ] , uukumerm wrus [ ] , alphavtrus [ ] , coronawrus glycoprotean [ ] , bovine herpes virus type glycoprotelns [ ] st~mulatlon of sugar and sugar nucleoude transport avian erythrocytes, isolated rat and mouse dmphragm muscle, and red cells [ , , ] ; mouse thymocytes [ ] redirection of secretory product plasmalemma to tonoplast [ ] inhibition of mtracellular transport: protein to rod outer segments [ ] , myeloperoxtdase [ ] , hcg subumts [ ] , accumulation of lammln [ ] ; gp glycoprotein [ ] , procollagen [ ] , fibronectm [ ] interactions wtth other toxm~ enhancement of toxaclty tlamuhn m swine [ ] , dlsulfide-hnked methotrexate-anti-transferrln receptor conjugate [ ] , specific cytotoxlcxty of a breast cancer-associated antigen lmmunotoxan m humans [ ] reduction of toxicity selenmm and vitarmn e [ ] monensm; although, a more direct influence revolving differenes in membrane energetlcs also has been implicated [ ] monensln also may decrease the degradation of dietary protein in the rumen and, thus, increase the amount of protein avaalable for dlgesuon and uptake in the small intestine [ ] . both a reduction in overall cell numbers m the rumen and a direct effect of monensln on bacterial protelnase and dearmnase activity have been suggested as contributing to this effect [ , ] . one of the first subcellular effects observed in relation to the topical apphcatlon of monensin was vacuolatlon of golgi apparatus clsternae [ ] . subsequently, xn vitro studies clearly demonstrated that monensin altered or inhibited numerous membrane-located phenomena (table i) . among these were the transfer of a -macroglobuhn from coated pits to receptosomes [ ] , recycling of low-density hpoprotem receptors [ ] , pinocytosis [ ] , transfer of product from endoplasmac reticulum to golgl apparatus [ ] , maturation and/or transport of viral coat proteins [ , , , , , , , ] , inhibition of transport of membrane proteins to rod outer segments [ ] , xnhibltion of cholesterol transport from the golgl apparatus to the mltochondnal site of steroidogenesis [ ] , blockage of phytohemagglutinln transport out of golgl apparatus and into protein bodies [ ] , inhibition of procollagen and fibronectm secretion from cultured human fibroblasts [ ] , inhibition of carbohydrate processing in cultured human flbroblasts [ ] , and inhibition of processing and cellular secretion [ , , , [ ] [ ] [ ] . additionally, cellular effects of monensm vary markedly depending upon the organism and the route of adrmnlstratlon. cultured cells, cells of tissue shces or explants, and plant organs that have received a topical exposure to monensin sufficient to inhibit growth or some cellular processes, usually show deviations in golgl apparatus structure and function. in contrast, ceils from animals poisoned by ingested monensin often exhibit gross mltochondrlal lesions without the corresponding golgl apparatus modifications. the reasons for these fundamental differences in cellular responses to monensin provide one focus for the present review and illustrate the many complexities surrounding the use of monensln as a probe specific to a single metabolic process. the primary functional unit of the golgi apparatus is a stack of membranous compartments (i e., the clsternae) each of which differs chemically, structurally, and functionally from the others [ ] [ ] [ ] [ ] . the number of cisternae per stack varies widely; although, in most animal cells and higher plant cells, there are about - cisternae per stack. each stack is polarized in the sense that product and membrane maturation appear to occur sequentially from a cls (fornung) face on one side to a trans (matunng) face on the other side [ ] . for simplicity, the stack may be divided into umts (measured from cis to trans faces) each of which represents a known set of functions. currently, there appear to be some - such units that make up each stack [ , ] . in reahty, however, it seems more likely that these changes in function occur gradually across the stack of clsternae rather than in discrete steps. there is, also, one or more membraneous structures (e.g., the trans golgl network [ ] or tgn and the partially coated retlculum [ ] or pcr) that he just off the trans poles of the stacks in plant cells, these structures appear to be derived from sloughed trans clsternae [ ] . tgn and pcr participate in the separation (i.e., sorting) of both secretion and endocytic products [ , [ ] [ ] [ ] [ ] and regulate the release of endocytosed substances through a ph-sensltlve mechamsm [ ] . the functions of these post-golgi apparatus structures are rapidly affected by monensin. several mechanisms for the movement of membrane and product through the golgi apparatus have been postulated. for example, movement may occur by sequential maturation of golgi apparatus elements (i.e., formation, maturation and loss of cxsternae) through the golgl apparatus stack [ ] . this would require the formation of new clsternae on one face of a golgl apparatus sack and commensurate loss of clsternae from the opposite face of the stack the source of these new clsternae is a special region of endoplasmic retlculum which gives rise to transition vesicles that move and condense on the forming (cls) face of the stack where they fuse together to form the new osternae [ ] [ ] [ ] product movement may also occur by shuttle vesicles at the peripheries of the clsternae that move proteins from one cisterna to the next [ ] . however, both direct (i e., nonvesicular) movement of substances into golgi apparatus cisternae and an endoplasmlc reticulum-mediated movement of product through the penpheral tubules of the clsternae must also be considered as viable options for the delivery and transfer of substances in and out of the golgl apparatus [ ] . the post-golgl apparatus structures appear to move membrane and product almost entirely via shuttle vesicles, many of which are coated [ ] [ ] [ ] monensin exerts its most profound effects on the trans cxsternae of the golgi apparatus stacks in those regions of the apparatus primarily associated with the final stages of secretory vesicle maturation and in post-golgl apparatus structures primarily associated with endocytosis and membrane/product sorting. because of its relative specificity, biologists have used monensln extensively as an inhibitor of trans golgl apparatus function. incorporation of radiolabeled [ s]methlonine into secreted lmmunoglobulin m molecules in monensintreated cells was reduced as was slalylatlon of immunoglobulin m and lymphoid cell surface glycoprotelns [ ] . these latter findings showed that the intracellular processing of n-asparagine-hnked oligosacchandes is altered in the presence of monensln with an effect primarily on those sugars (e.g., slalic acid, galactose, fucose) added late in the processing continuum [ ] flbronectin, secreted in human flbroblasts, was incompletely processed in the presence of monensln and exhibited a greater incorporation of mannose than did control protein molecules [ ] inhibition of fibronectln secretion in human melanoma also has been reported [ ] . similarly, when treated with monensln, rat astrocytes in primary culture accumulated lamimn, another matrix glycoprotem involved in cell adhesion [ ] . not only do the monovalent lonophores block transport and surface expression of several secretory glycoproteins in normal cell functioning [ , , ] and the transport of membrane glycoproteins or enveloped viruses [ , , , , , , , ] , they inhibit formation of cell surfaces including assembly of peripheral myelin [ , ] . in mouse thymocytes, monensin leads to ( stimulated incorporation of labeled sialyl-, galactosyl-, and n-acetyl glycosaminyl residues [ ] . this enhanced accumulation was not due to a &rect effect of monensin on glycosyltransferase activities but, rather, as a consequence of a greater entry and accumulation of labeled sugar nucleotides in the swollen clsternae_ galactosyl transferase itself was translocated through the golga apparatus at a slower rate with monensin_ however, the sialylatlon of the o- nked ohgosaccharides of the enzyme was unaffected by monensln treatment [ ] effects of monensin on glycosyltransferases also may be indirect. monensin has been reported to decrease galactosyltransferase activity m golgl apparatus of rat embryo fibroblasts [ ] although it had no effect on this activity in baby hamster kidney cells [ ] monensln is an especially useful inhibitor, since it blocks lntracellular transport of protein at the level of the golgl apparatus without directly affecting protein synthesis [ , ] the effect of monensin is considered to be on transport rather than on processing per se [ , ] one argument is that oligosaccharide processing of those glycoprotelns that reach the appropriate site occurs normally even xn monensin-treated cells [ , , , ] . however, these observations could be explained as well if processing of ollgosacchande chains of different secreted glycoprotelns occurred at different sites, only some of which were sensitive to monensln [ , ] . the ablhty of monensln to effectively 'freeze' processing of molecules at a particular stage had lead to its use in identifying transitory synthetic intermediates. examples include the insulin receptor where several polypeptlde precursors have been described [ ] , the intracellular accumulation of non-cleaved precursors of pituitary hormones that occur in the presence of monensin [ ] , and dissection of the pathway for secretion of gonadotropin by cultured human trophoblastic cells [ ] . in some instances, the effect of monensin may be to redirect, rather than block, the movement of golgl apparatus-derived product. for example, under normal conditions, proteins of developing seeds accumulate in a central vacuole which then partitions into smaller units of storage protein (i.e, the protein bodies). however, when treated with monensin, the golgi apparatus-derived transport of the protein vlclhn in pea cotyledon was redirected from the vacuole to the plasmalemma and the newly synthesized viclhn was released from the cotyledon cells to accumulate between the plasmalemma and cell wall [ ] monensin lnhibmon of golgi apparatus function ~s sufficiently well established [ , , , ] that the phenomenon is used widely as one criterion for verifying the passage of a biochemical entity through the golgi apparatus. thus, based on partial monensm inhibition, hammerschlag and co-workers [ , ] concluded that passage through the golgl apparatus was an obllgator~ step m the lntracellular routing of materials m ta,~t axonal transport_ bartalena and robblns [ ] showed that rnonensin ~mpeded the exit of thyroxin-binding globulin from the golgj apparatus w~thout affecting the terminal glycosylatlon of the protein yanagashito and hascail [ ] reported that monensin reduced and delayed transport of both secretory and membrane-associated forms of proteoglycans, suggesting passage through the golgl apparatus of rat ovarian granulosa cells m culture similarly, an involvement of the golgi apparatus m the transport of sulfatldes to myelin [ ] and phytohemaghitlnln to protein bodies m bean cotyledons [ ] were deduced from monensin mhlbmon fhckinger and co-workers [ ] using [ h]leucine, showed that all, or nearly all, of the protein secretory product of mouse epididymis principal cells pass through the golg~ apparatus in times approximately eqmvalent to those reported m other tissues. this transfer of product from golgi apparatus to the cell surface was largely blocked by monensin swelling of golgi apparatus cisternae observed in the electron microscope following fixation with glutaraldehyde is, perhaps, the most consistent visual in vitro demonstration of a monensin-mduced effect on a membranous cell compartment [ , , , , [ ] [ ] [ ] the swollen clsternae usually appear devoid of contents by electron microscopy (figs. and ) but an electron-dense substance may be precipitated through the osmium tetroxlde-zmc iodide reaction (unpubhshed data) although all clsternae of the golgi apparatus may swell in response to monensin ( fig. and a) , the major effect appears to be associated with the mature, or trans, parts of the golgi apparatus stacks (figs and b) [ , , ]_ gnffiths and co-workers [ ] showed that monensm inhibited the transport of viral membrane proteins from medml to trans golg~ apparatus cisternae, thus indicating a monensln block between medial and trans cisternae monensm also blocked tlamrmng of the high mannose bound to the viral membrane proteins and their conversion to complex ohgosacchartdes similarly, niemann and co-workers [ ] found that monensm blocked glycosylation of e glycoproteln of corona virus in infected mouse cells. srinlvas and co-workers [ ] reported failure to process simple endo-h-sensitive to complex endo-h-reslstant ohgosacchandes and reduced efficiency of cleavage of the prenv glycoprotein precursor to gp for evehne mouse cells infected with friend munne leukemia virus these findings indicate a block prior to entry into the golgl apparatus also, m cultured hepatoma cells, transport of vesicular stomatltis wrus (vsv) g protein was arrested prior to acquisition of endo-h resistance, suggesting a block early in the processing pathway [ ] . strous and co-workers [ ] showed that monensin affects primarily the galactosyltransferase-containlng c~sternae of the golgi apparatus based on studies of the metabohsm, localization, and biosynthesis of n-and o-linked oligosaccharides of galactosyltransferase in helz cells. the accumulation of incompletely processed glycoproteins indicates either an up-stream accumulation of secretory materials behind a golgl apparatus blockage by monensm [ ] or a monensin block near the exit site from endoplasmlc reticulum [ ] . monensin effects on golgi apparatus have been observed in a wide range of plant and animal species and appear to be a universal response to the topxcal applicatton of monensln. as pointed out above, monensln action is exerted on the trans half of the stacked clsternae (fig ) , often near the point of exit of secretory vesicles [ , , , , , , ] or, especially at low monensm concentrations or short exposure times, sometimes m the rmdreglon of the stacked cisternae [ , ] . intracellular transport may be blocked [ , , , , , ] , often wlthxn rmnutes after exposure to monensln [ , , , , ] . swollen units usually accumulate near the golgi apparatus [ , ] a monensm effect is quite rapid in both animal and plant cells; e, changes in golgi apparatus have been observed after only - rmn of treatment [ , , , ] . these early effects have been documented particularly well is suspension cultures of carrot (daucus carota l ) [ ] . when carrot cells were exposed to monensln at - m (which is approxamately the minimum concentration that will elicit a strong monensm response m plants), production of secretory vesicles ceased and, almost immediately, an increased number of clsternae in the dlctyosome stacks was observed. an average of one additional clsterna per stack was formed within the first - mln of monensin treatment and, in some experiments, a second clsterna was formed within about man. these effects occurred without significant swelling of cisternae. thereafter, vacuoles, representing intact swollen clsternae, began to accumulate in the cytoplasm at a rate of about one every - min (fig. ) . the mechanism postulated for this momentary increase of d~ctyosome clsternae was that monensln, acting on the trans pole of the dictyosome, blocked normal formation [ ] for details of procedure) the two samples differed onl~¢ m that the one dlustrated m {a) was adjacent to a 'natural' ( e_ uncut) surface of the hver lobe, whereat (b) was from a cell near the 'cut' surface of the tissue slice in both instances, come golgx apparatus (ga) clsternae were sv~ollen, however, m (a) swelling was progresse~e from cls to trans pole (direction of arrow) whereas m (b) swelling was t.onfined to the trans c~sterna note that m~tochondrm (m) were condensed of secretory vesicles but did not block the formation of new clsternae at the cis face of the apparatus however, as the trans clsternae began to swell, the swollen clsternae were eventually released as intact units that neither fragmented nor integrated (fused) with other cellular constituents (e.g., plasma membrane). with the scale producing green alga pyramimonas lnconstans, exposures of to several hours to monensln resulted m disorientation of the golg~ apparatus and disruption of scale morphogenesls [ ] . these effects were reversible more recent studies indicate that a similar pattern of swelling and accumulation of clsternae in the cytoplasm occurs in cultured animal cells [ ] . when h- hepatoma cells were treated for varying times with ~ to m monensin, one swollen clsterna per stack of clsternae was produced after - rain of treatment during tlus time, approximately one additional clsterna per stack was formed (fig. , inset) . as the clsternae veslculated, vacuoles began to appear m the cytoplasm these large swollen vacuoles were formed at the rate of one sin concentrations, the vacuoles were larger and appeared more rapidly than at low concentrations of monensln but the kinetics of vacuole formation were qualitatively similar. however, by h following treatment with - m momensin, all clsternae of the golgi apparatus appeared as vacuoles. the swollen trans compartments that accumulate in the golgl apparatus region with monensln inhibition may contain regmns that are clathrin coated; e.g., condensing secretory material (prolnsuhn) in pancreatic cells [ ] . the response of golgi apparatus of hver slices to monensin was qualitatively similar to that with hepatoma cells in culture [ ] . with liver shces, a fraction enriched in vacuoles was isolated and demonstrated to contain the trans golgl apparatus markers, galactosyltransferase, and thiarmne pyrophosphatase, in ratios sirmlar to those of golgl apparatus proper [ ] . in barley aleurone layers, the a-amylase and acid phosphatase activities that accumulated within aleurone cells following treatment with monensin, were localized in cellular components with buoyant densities intermediate between endoplasrmc reticulum and mitochondria and cosedimented with latent inoslne diphos-phatase activity, a putative golgi apparatus marker in plants [ ] . heupke and robinson [ ] reported a shift to higher density of golgi apparatus membranes from monensin-treated barley cells, a response no obvious from work with mammalian cells. the golgi apparatus clsternae that accumulated behind a monensin block in semlikl forest virus-infected bhk cells bound viral nucleocapsids, and the resulting increase in density permltted their separation by gradient centnfugatlon from other golgi apparatus elements [ ] . the effects of monensin on golgl apparatus, at least up to several hours of exposure, appear to be fully reversible [ , , , ] in carrot cells, normal secretory activity was resumed within nun after transfer of cells to a monensln-free medium; although, in these cells, the vacuoles formed during the monensln block, remained in the vicinity of the golgi apparatus for several hours or more, even after apparently normal secretory activity had resumed however, with the longer treatment times of several hours [ ] or days [ ] monensin apparently causes swelhng of golgi apparatus cisternae through a na+-in/h+-out exchange across the membranes leading to a net uptake of na ++ ci and entry of water [ , ] evidence in support of this concept was provided by studies with isolated chromaffin granules which lysed readily after brief exposure to monensln in na +-or k+-contalnlng isotomc media for swelling to occur, the membrane must normally be lmpermeant to cations as is known for the chromaffin granule_ the chromaffln granule membrane contains a h+-atpase which is electrogenexc and, in the presence of a permeant anion, acidifies the granule interior to ph _ thus, the operation of this pump in the presence of monensin drives net salt uptake [ ] . to test whether net salt uptake driven by the presence of a proton gradient also would explain the monensininduced swelling of golgi apparatus cisternae, wild-carrot cells in suspension culture were treated with drugs and inhlbltors known to interfere with proton gradients [ ] monensin-induced swelling of golgl apparatus in sjtu could be inhibited by the protonophore, carbonylcyanlde-p-trlfluoromethoxyphenylhydazone (fccp), but was only little affected by the inhibitor of lysosomal acidification, quercetln, or by the lysosomotropic amines, chloroquine, and ammonia. cyanide also dramatically decreased swelling, and arsenate (with prolonged treatments) reduced the number of swollen cisternae organic acids, by providing a readily permeable counterlon, promoted monensln-lnduced swelhng these data imply that the monensin-induced swelling of golgi apparatus cisternae involves a proton gradmnt at or near the mature poles of the golgi apparatus because monensin induces a " na+/h + exchange, and since the van't hoft factors for h + and na + are practically the same [ ] , the osmolanty of the cell content should not increase to cause swelhng without a net proton influx one explanation would be that the ph of golgi apparatus vesicles is highly regulated proton translocatlng atp hydrolyzing enzymes (h +-atpases) are associated with several components of cells that develop acidic intermrs such as endosomes, coated vesicles, lysosomes, and trans golgl apparatus clsternae [ ] . ewdence for the presence of an h +-atpase has been the demonstration of atp-dependent vesicle acxdlflatlon in golgl apparatus isolated from rodent liver [ ] [ ] [ ] , corn coleoptlles [ ] , and sycamore cells [ ] acldificatmn was demonstrated both by [lac]methylamlne uptake and by spectrophotometric assays of amd-quenched dye fluorescence (acridine orange, neutral red, or quinacrme) several lines of evidence confine the golgi apparatus h +-atpase to the trans cisternae. as emphasized in the preceding section, the early in sltu effects of monensm are frequently localized to the trans faces of the golgl apparatus the resultant swelling, whmh is proposed io be due to the accumulation of osmotically active ions in exchange for protons [ . ] , occurs predominantly m trans clsternae additionally, a basic congener o] dmltrophenol ( -( , -dlnitroanlhn o)- '-amlno-nmethyldlpropylamine, damp), which concentrates m acidic compartments as shown in fibroblasts by lmmunocytochemlstry is present onl) clsternae and vesicles associated with the trans faces of the golgl apparatus [ ] moreover, damp rapidly leaves these compartments when cells are incubated with monensm, thus further indicating that accumulation of damp is due to the acid ph some involvement of a low ph compartment is evidenced by the observatmn that some monensln effects on processing, ke, proteolytlc conversion of proalbumln [ ] , are mimicked by amines however, in promyelocytlc leukemia cells, processing of myeloperoxldase, while blocked by both monensln and chloroqulne, was not affected by nh~ cations, thus indicating that processmg is not necessarily influenced by ph-dependent mechanisms [ ] these results were interpreted as indicative of processing in golgl apparatus based on inhibition of transport by monensin and chloroquine rather than processing m lysosomes and other late, acidic compartments involving a ph-dependent mechanism [ confirmatmn of a trans location of the h ~-atpase has come from free-flow electrophoresis separations of golgl apparatus yielding cls, medial, and trans compartments m fractions of diffenng electrophoretic mobility [ , ] _ in these separatmns, proton pumping activity was found exclusively in the most electronegative fractions coming from the trans-most goigl apparatus re-gion_ gnfflng and ray [ ] have offered the suggestion that acldificatmn of clsternal lumlna may be part of an osmotic mechanism to compress and flatten the cisternae the latter, for example, might aid in the transfer of content into secretory vesmles. the inward pumping of protons would tend to favor the exit of na + and k + out of the clsternae furthermore, as the ph falls, those monovalent cations remaining would tend to combine with acidm groups of the golgx apparatus membranes, further reducing the osmolanty of the clsternal content relative to that of the external cytoplasm. thus, water would be driven osmotically out of the clsternae to both compress the clsternae, as seen along a pronounced cls to trans gradient for plant golgl apparatus [ ] , and, perhaps, to account for the condensation of secretory materials m condensing vacuoles and other trans golgi apparatus compartments (e.g, ref ). other cell compartments, including endocytic vesicles [ , ] , lysosomes [ , ] , multivesicular bodies [ ] , and coated vesicles [ ] [ ] [ ] have h+-atpases. all use h+-atpases to acidify their interiors and the enzymes responsible have been solubdized from lyso-somes and reconstituted into liposomes [ ] . vacuole (tonoplast) membranes [ ] , and possibly also plasma membranes of fungi [ ] , contain h÷-atpase. similarly, a gradient of acidification within the endocyuc pathway has been indicated from immuno-electron microscopy with protein a-colloidal gold and monospecific antibodies to the weak base pnmaquine [ ] . however, not all compartments with h+-atpases (e.g., cells, vacuoles, lysosomes, coated vesicles) swell in response to monensin. cell and/or vacuole swelhng may be hmlted due to the very large internal volumes revolved. the contracnon of contractile vacuoles of paramectum was inhibited reversibly by monensin and in a manner dependent upon the presence of na + [ ] but marked swelling was not observed. little is known about the swelhng response, ff any, of lysosomes, coated vesicles and other endocync compartments m response to monensin lntubition. their functions, however, are inhabited by monensin as will be emphasized in the section that follows. carboxyhc lonophores strongly inhibit proton uptake by photosynthenc preparations [ ] . in chloroplasts, swelling of thylakolds (inner membrane compartments) but not of the space between tuner and outer plastld membranes has been observed to result from monensln treatment [ ] . thylakold swelling, in contrast to swellmg of mitochondrial cristae and of golgl apparatus clsternae, was reduced upon mcubanon in darkness, again suggesting a relationship between swelling in the presence of monensln and the hght-driven proton gradient used for photophosphorylatlon [ ] mltochondrla have an outwardly directed energy-hnked proton pump and do not swell with monensln (rather, they tend to condense, see figs. , a and b) while the light-driven proton pump of chloroplasts and chromatophores, and that of the golgi apparatus pump, are directed reward causing the vesicles to swell. evidence for a secretory pathway bypassing the golga apparatus in the monensln-blocked cells is provided by kubo and pigeon [ ] who lnvesngated the effects of monensin on the synthesis and expression of membrane igm of a human lymphoblastold hne. they found altered processing of both /l, k chains and incomplete terminal glycosylations. yet, transport of the altered molecules was observed. that the aberrant processing did not influence markedly the membrane expression of the igm is consistent with a secretory pathway bypassing the golgl apparatus m monensin-blocked cells. slmdarly, a dual secretory pathway, only one part of which was suscepnble to monensin, was deduced from studies of a-amylase secretion m rice seed scutellum [ ] . in zea majze roots, monensln lnhib~ted secretion of aamylase but not polysaccharide slime [ ] . the blocked secretion that results m the intracellular accumulation of secretory products frequently is not absolute. some portion of the material synthesized is released from the monensm-mhabited cells and this material frequently exhibits an abnormal type of posttranslational modification. for example, those proteoglycans from chicken embryo chondrocytes secreted m the presence of monensln are vastly undersulfated [ , , , ] . thus, membrane and secreted molecules leaving the cell following a monensin block appear to have been denied the full range of processing enzymes they would normally encounter during transit through the cell however, whether the incompletely processed molecules bypass one or more parncular lntracelhilar compartments (eg, the golgi apparatus), or whether they pass through functionally incomplete compartments, remains to be determined. during maturation of uukunieml virus m baby hamster kidney cells, monensin appeared to mhibit a terminal step of virus assembly, but not the expression of virus membrane glycoproteins g and g at the cell surface. these findings suggest that both g and g could enter a functional transport pathway in the presence of monensm that bypasses the trans golgi apparatus compartment to become expressed at the cell surface [ ] . evidence for a golgi apparatus bypass has been presented m liver where secretory hpoproteins may move directly from endoplasnuc reticulum to the cell surface without direct golgl apparatus involvement [ , ] . at concentrations of - m or higher, monensin inhibited secretion of albumin, transferrln, and vsv proteins g and x destined for delivery to the cell surface to the same extent m rat hepatoma cells [ ] . this was taken as evidence that the same vesicles were used by all four proteins m their movement from golgl apparatus to the plasma membrane however, the time required to move from er to the golgi apparatus, based on sensitivity to endoglycosidase h, differed for secretory and membrane proteins. an even more striking observation was that following the monensm block, secretory proteins accumulated in an endo-h-sensitive form, whereas, membrane proteins were already endo-h-resistant. this strongly implies that membrane and secretory proteins are not m the same compartment initially and would support the concept of peripheral input of secretory proteins into the secretory vesicles of the golgi apparatus, at least in hver [ , , ] alonso and compans [ ] provided evidence for two distract pathways of glycoprotein transport in madin-darby canine kidney (mdck) cells only one of which was blocked by monensm however, in baby hamster kidney (bhk ) cells, both influenza virus and vsv maturation were sensitive to monensin. the vsv particles were synthesized m both mdck and bhk cells, but transport to the cell surface was blocked only in the mdck cells thus, there appear to be two distinct pathways of transport of glycoproteins to the plasma membrane in mdck cells, only one of which is blocked by monensln there is no information on the nature of the alternative transport vesicle that carries mfhienza virus to the cell surface of mdck cells if, in fact, a vesicle is involved. melroy and jones [ ] reported accumulation of normally secreted a-amylase within barley aleurone layers after monensln treatment. however, only isozyme was secreted normally whereas isozymes , and were not secreted also, in the perfused rat hver, monensin treatment has less of an effect on blliary secretion than on secretion of plasma proteins [ ] . sn-nllarly, in the transport of hla-dr a-and / chains [ ] , processing of n-linked carbohydrate chains to full endo-h resistance occurs however, with the associated i-chain, processing of both o-and n-linked carbohydrate chains ~s inhibited, and carbohydrate chains remain predornlnantly endo-h susceptible. here, the processing of membrane-associated proteins that occurs despite a monensln block may reside in intercalary clsternae that constitute the golgi apparatus region recently termed medial [ a ] there is now considerable evidence for monenslnsusceptible compartments in the endocytotic pathway. transfer of product to secondary lysosomes [ ] as well as virus penetration into cultured cells [ ] are impaired by monensm. stein and co-workers [ ] have shown that monensin blocks transfernn recycling by causing internahzed hgand to accumulate in the perinuclear regmn, primarily in multivesicular bodies, of the k cells used in the study. based on studies of hrp uptake in rat fibroblasts, wilcox and co-workers [ ] suggested that inhibition of endocytic events may be the consequence of an inhibition of membrane recycling within the cell rather than a direct effect of monensin at the cell surface. maxfleld [ ] reported that ~tm monensln resulted m an mcrease in internal ph of endocytic vesicles of cultured mouse fibroblasts from to above to account for its effects on receptor-mediated endocytosis similarly, marsh and co-workers [ ] concluded that the lnhlbmon of semhki forest virus penetration into cultured cells was the result of this increase in ph of endocyuc vacuoles and lysosomes above ph , the threshold for fusion activity of viral membranes monensln has also been shown to inhibit lysosomal degradation of protein by affecting lysosomal ph [ ] and to abolish aslaloglycoprotein degradation in cultures of rat hepatocytes through a ph shift in prelysosomal endocytlc vesicles [ ] . using digttal image analysis, tyco and co-workers [ ] showed that monensin raised the ph of endocytic vesicles in cultured human hepatoma cells and caused a hgand-lndependent loss o! receptors in other studies, monensln did not prevent lnternahzatlon of s-labeled proteoglycans by rat ovarian granulosa cells although their lntracellular degradation was completely inhibited [ ] . yet, degradation pathways involving proteolysls of both dermatin and heparln sulfate and limited endoglycosldlc cleavage of heparln sulfate continued_ these findings, while consistent with an involvement of both acidic and nonacldlc compartments, show that monensm inhibition is primarily on those processes that normally occur in acidic compartments such as endosomes or lysosomes by raising their ph. sirmlarly, with isolated hepatocytes, whittaker et al [ ] found no effect of monensin on insulin internalization but, rather, an impairment of ~ts degradation once lnternahzed. rustan and co-workers [ ] suggested that monensm inhibits both endo-and exo-cytosls by a similar mechanism, namely, disruption of proton gradients their conclusions were based on studies of rat hepatocytes in which monensm inhibited both secretion of very-lowdensity hpoprotelns, and binding and degradation of aslalofetuin both secretion and receptor binding were markedly decreased after only rmn of monensin treatment although no effect on protein synthesis was observed. however, secretion was more sensitive [o monensln than endocytosis, suggesting that monensin independently intubits endocytlc and secretory functions although the mechanisms may be similar. marnell et al [ ] explained the monensin block of the cytotoxic effect of &ptheria toxin on a sirmlar basis following endocytosls of the toxin, the toxin was assumed to penetrate the membrane of the endosome and enter the cytoplasm in response to an acid environment by neutralizing the ability of endosomes to acidify their interiors, monensin, like the lysosomotroplc amines, was able to block the low ph-dependent dissociation of receptor-hgand complexes and subsequent release of ligands either to the cytoplasm (viruses and toxins) or to lysosomes (endocytosed proteins such as ldl). this in turn would prevent recycling of receptors and membrane and eventually bring endocytosls to a halt due, not necessardy to an inhibition of the uptake processes per se, but perhaps, to blockage of an internal step very similar to that believed to be blocked at the goln apparatus. also consistent with sirmlar modes of monensin mhibition in processing both endocytlc and exocytic vesicles are findings that a single mutatmn in chinese hamster ovary cells impaired both golg¢ apparatus and endosomal functions in parallel included were the monensin sensitive steps of virus and toxin penetration from endosomes into the cytoplasm and of golgl apparatus-associated maturation of smbis virus [ ] . the alterations correlated with losses of atp-dependent vacuole acidification as if the atpase of endosome and golg~ apparatus shared a common genetically regulated subunit. ono et al. [ ] studied a monensln-reslstant mutant of mouse balb/ t cells which also proved to be a poor host for either vesicular stomatltis virus or semllkl forest virus multiplication. the mutant cells resistant to monensln, bound virus normally and contained acidic compartments. however, movement of virus from the cell surface to the endosome and lysosome compartments was extremely slow. thus, the ablhty of monensln to block processing of endocytic vesicles by making prelysosomal compartments less acidic, suggests a mechanism for perturbation of endocytosls based on its ionophorlc properties the mechanism could be slmxlar to the monensm-medlated exchange of monovalent alkah tons for protons that induces, by osmotic means, the observed swelhng of golgl apparatus clsternae. clearly, monensm does not interfere with the uptake and binding of particles at the cell surface. monensm is ineffective against activities that occur at the cell surface. hedm and thyberg [ ] showed that uptake of igg prebound to the cell surface was unaffected by monensln similar findings have been made in studies of receptor-medmted endocytosls of various other ligands [ , , ] . however, monensm may secondarily affect mternahzation through depletion of monensln-sens~tive receptor sites at the cell surface. this would occur if the cell surface receptors are recycled back into the cell and then blocked in the post-golgi region by monensin so that they could not return to the cell surface [ , , ] . thus, monensin inhibition of endocytlc events seems to be at the site of transfer from endocytlc vesicles to lysosomes [ , , , ] or, in monensin-sensltive endosomes, inhibition of the dissociation of ligand-receptor complexes [ ] most animal cells show a dose-related response to monensm that falls off rapidly at monensm concentrations less than - m consequently, most studies of monensln effects use monensin concentrations of - m or higher however, a few reports indicate a cellular response at monensm concentrations less than - m for example, receptor capping by lymphocytes is stimulated by low concentrations of monensin in the range - to - m and inhibited by monensm concentrations above - m [ ] cultured adrenal chromaffin [ ] and heart [ ] cells also are stimulated by low concentrations of monensln. though these effects occur at concentrations below the threshold effects for most golgl apparatus responses, they still presumably result from increased levels of cytoplasmic sodium due primarily to the ~onophore insertion at the plasma membrane fig (a) outer cap cells from a control (nontreated) maize root txp preserved by freeze-subsututlon [ ] . the form of dlctyosome (d) was normal and slrmlar to that following glutaraldehyde/osmlum tetroxade fixation (b) same except that root tip was treated for h with -s m monen~m before being preserved by freeze-substitution. the trans clsternae and/or secretory vesicles (arrowheads) were swollen, and mltochondna (m) were condensed (compare with mltochondna of a) w, cell wall, v, central vacuole swelling of golgl apparatus clsternae occurs in a wide range of plant and animal cells (see subsection iii-b ) and may be a universal response to monensln poisoning. however, in plants, and to a lesser extent animals, swelhng is influenced by the fixative used to preserve the cells specifically, morphological evidence of swelhng is less (in animal golgl apparatus) or nonexistent (in plant golgl apparatus) when the tissues are fixed in potassium permanganate as compared to fixation in ghitaraldehyde/osmium tetroxade [ ] . these effects could be due either to fixation artifacts or to differences between plant and animal golgl apparatus (e.g, ref_ ) this problem was evaluated by comparing the images of golgl apparatus preserved by various chemical fixatives as well as preservation by freezing and low temperature substitution in acetone and osmium tetroxade. presumably, the image following freeze sub-stitutlon would reflect the true ultrastructure more closely than the image following chemical fixation. the results of freeze substitution in both animal cells [ ] and maize root (figs a and b) , show swollen golgi apparatus c~sternae following monensln exposure in a pattern similar to that observed after glutaraldehyde/ osmium tetroxide fixation [ ] . however, using videoenhanced light microscopy and cultured bovine mammary epithelial cells, a marked swelling response to monensln was observed only after the addition of glutaraldehyde fixative to the monensln-incubated cells (morr , d j., mollenhauer, h h., spring, h., trendlenberg, m, morr , d m. and kartenbeck, j, unpubhshed data)_ thus, whether monensin-lnduced swelhng occurs m vlvo or is in response to aldehyde fixations remains an important questiion. no swelling was observed in golgl apparatus of protoplasts of carrot cells freshly prepared by digestion of cell walls when exposed to monensin even though such a response was obtained in the same cultures prior to wall dissolution [ ] . similarly, in thin slices of hver incubated in monensin, the golgl apparatus adjacent to cut edges of tissue slices showed a different swelling response than golgl apparatus adjacent to the uncut natural surfaces of the lobe (unpublished data) adjacent to a cut edge, fewer cisternae swelled and those that swelled were only in the most trans positions the basis for such differences is unknown but might, for example, indicate changes in cisternal proton pumping ability, or monensln uptake, in response to changes in the physiological state of the golgi apparatus brought about by the tissue excision the mechanism by which monensin interacts with coccldla and rumen rmcroflora is well documented [ , ] however, the interaction between monensm and the tissue of the host animal is less well understood even though the chnlcal manifestations of monensln poisoning are well known_ most striking are differences between the in wtro monensln effect ohserved in cultured plant and animal cells, and plants, and the in vwo effects observed m animals when used at recommended levels, either as a coccidlostat for poultry [ , ] or for cattle [ , , ] , monensin seldom causes poisoning nonetheless, misuse of the product, usually from improperly mixed or improperly distributed feed, may cause toxlcosis and death [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . horses, ponies and other equine species are particularly sensitive to monensln poisoning [ , . ] . the median lethal dosage (ldso) for the horse js - mg monensin per kg body weight compared to - mg per kg body weight for cattle and mg per kg body weight for poultry [ , , ] in mammals, the physical signs of monensln toxlcosls commonly include anorexia, diarrhea, depression, sweating, ataxm, palpitations of the heart, and sudden death following exercise [ , , , , [ ] [ ] [ ] [ ] stiffness of hindquarters and swollen gluteus muscles [ , ] , elevated pulse rate [ , , ] , and ecg abnormalities [ , , , ] also have been reported in fowl, the outstanding signs of monensln toxlcosls are drowsiness, excessive thirst, anorexia, depression and paralysis [ , , ] marked congestion in a variety of organs also has been noted [ ] severely poisoned birds may die in sternal recombency [ ] . routine clinical tests on serum from horses poisoned by monensm may show abnormally high values for blood urea nitrogen, total billrubin, creatlne kinase, lactate dehydrogenase and aspartate armnotransferase [ , , ] however, chnlcal manifestations are often variable makang interpretation and diagnosis difficult. moreover, serum levels of sodium, potassium, chlorine, calcium, phosphorus, and urea may remain at near-normal levels following monensin treatment [ ] _ in poisoned mammals and fowl, generalized congestion, hemorrhage, and macroscopic injury to striated muscle [ , , , ] , spleen [ , ] , lung [ ] , liver [ ] , and kidney [ , , , , ] have been noted. the most consistent macroscopic observation in ponies, cattle, pigs and fowl, has been cardiac myocyte degeneration and vacuollzatlon [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] in animals poisoned with relatively high doses of monensin, an initial condensation of heart mltochondria is often seen (fig ) however, with longer exposure times, some mitochondria swell and vacuolate with an almost total loss of matnx substance (fig ) _ intracnstae spaces generally remain unchanged with swelling being restricted to the mitochondrial matrix this is followed by loss or dilution of matnx components and a reduction in size of cnstae so that the rmtochondna appear as empty vacuoles with residual cristae typically, only some mitochondrla in a particular fiber become swollen and appear as vacuoles, and fig section of left ventricle from a rat treated wath a single mtraperitoneal mjecaon of mg monensm per kg body weight most rmtochondna (m) were either condensed or swollen in swollen rmtochondna, cnstae (arrowheads) were greatly reduced in extent but, otherwise, were of approxamately normal ttuckness thas dosage level (e_g, mg monensln per kg body wexght) is quite tugh for rats and often resulted m significant generahzed damage to the muscle fiber as dlustrated in the lower part of the rmcrograph however, rmtochondnal swelhng (but not condensauon) may occur as well at lower monensln dosages, even when no fiber damage can be identified ultrastructurally swelling and vacuohzatlon of mltochondna are progresslve with time whereas rmtochondnal condensation was dose-dependent and often occurred rapidly , a few were normal (arrowhead), but most were rmldly condensed swollen tmtochondna always appeared randomly distributed through a fiber although significant differences m rmtochondnal swelling were often noted between fibers (e g, see fig_ ) these are randomly distributed throughout the fiber (fig. ) . we have identified early stages of mitochondrial vacuolation and swelling in both the rat and pony (unpublished data), but these mitochondria were seldom plentiful, suggesting that transition to the vacuolated state, once started, was relatively rapid non-swollen mitochondria may appear condensed, especially after exposure to high levels of monensin ( fig. and ). granulation of mitochondna ( fig. ) was observed only occasionally and those granules that were present appeared similar to the tncalclum phosphate granules often associated with normal mitochondria [ , ] . however, whether monensln-lnduced granules contain ca ~ has not been determined. granules like these have been observed in lschermc and reperfused hearts and are considered indicative of calcium overload [ ] [ ] [ ] [ ] _ ca + overload may be a potential cause of cell death or cellular dysfunction in ischerma [ , [ ] [ ] [ ] [ ] [ ] [ ] although the effects are reversible if the cell is not too severely damaged [ , ] exaggerated matochondrial swelling has also been observed immediately following reperfuslon of hearts rendered lschemlc by occluding blood flow for a few minutes [ , ] whether excess mitochondrial ca + occurs as a result of monensin is not clear under appropriate condiuons, either monensin or na + in excess can block ca + accumulation and promote its release from both mitochondrla and sarcoplasrmc reticulum over a broad range of monensin and na + concentrations [ , , ] it is probable that the release of ca + by monensln can occur as the result of an increase in cytoplasmac na + from a monensin shuttle although monensin (at relatwely high concentrations) has been shown to release ca + directly m a na+-lndependent manner from cardiac sarcoplasmic retlculum [ ] in all probability, however, monensin is not present in myocytes at high concentrations since swelling of golgl apparatus cxsternae (a characteristic response to monensin) ~s never observed in these cells. the picture is further comphcated by the fact that ca + release patterns may vary according to type of cell (e g., white vs red muscle cells [ ] ) as well as the availability of extracellular ca + [ ] . although not related to this report, it may be of some interest to note that both mitochondrlal condensation and granulation are much more intense in x- a-treated animals (and cultured cells as well) than in comparable animals (or cells) treated with monensin [ ] _ the percentage of affected mltochondrla vaned markedly between muscle types and species of animal. analyses of striated muscle m ponies showed that there was a much greater ( -times) hkehhood of finding altered mltochondrla in heart tissues than m diaphragm or appendicular muscle [ ] a similar relationship existed in rats except that most swollen mitochondria were m the &aphragm [ ] . some antibioucs (e g, tiamuhn and avoparcm) may act synergistically with monensln to induce shifts in the distribution of swollen mltochondria [ ] and other cellular damage. differences in distribution patterns of swollen mltochondrla also were observed between red and white muscle fibers of the rat diaphragm [ ] . red and white fibers were dffferentmted structurally by size, mltochondrlal content, and z-band configuration [ , [ ] [ ] [ ] swollen matochondrm were present in all fiber types when the number of affected rmtochondna was small. however, when large numbers of swollen matochondrm were present, the dxsmbutlon pattern was heavily skewed toward the white muscle fibers (fig_ ). a differential effect of monensln was also noted by van vleet and co-workers [ ] who observed in swine severe damage m the diaphragm, vastus lateralls, sem~tendlnosus, triceps and intercostal muscles: moderate damage m longissimus lumborum muscle, and little (or minimal) damage in tongue. damage was greatest m muscles containing a high proportion of type fibers these distribution patterns, coupled with the characteristic form of the degenerating rmtochondrla, are not common observations and, therefore, may be strong indtcatots of monensin poisoning in animals_ swollen rmtochondrla have not been observed in any of the nonmuscle cells of the heart, diaphragm, or appendicular tissues or in hver, adrenal, or kidney cells thus, monensm adrmnistered to mammals in vlvo tends to induce rmtochondrial changes only m selected tissues and/or types of muscle fibers the mechanisms for these rmtochondrlal changes and reasons for the specificity are not known. these problems are compounded by the fact that matochondnal condensation, but not subsequent swelling and degeneration, occurs in cells exposed topically to monensin. whale mitochondnal aberrations are the primary morpholog~c indicators of monensin poisomng in striated muscle, other aspects of muscle ultrastructure may show deterioration [ ] or may remain relatively normal even with gross mltochondnal damage [ ] . generalized fiber and cell degeneration may occur following monensin poisoning [ , , ] . the extent of monensin-induced injuries appears to be time and dosage dependent. if death occurs shortly after monensin exposure, there may be httle or no recognizable evidence of pathologtcal change, at least in liver, kidney, and striated muscle. generalized necrosis appears to occur most often when monensln is administered over long periods of time. even with single doses of monensin, structural aberrations in striated muscle develop progressively over several days and then regress if the animal survives the imtial insult (unpublished data). we have not observed permanent injury in either striated muscle or liver of monensin-treated rats although such effects have been noted in other animals [ ] . many of the effects on striated muscle attributed to monensxn occur also with other lonophores irrespective of their ion specxficlties [ , ] this implies, again, that na + is not the direct cause of muscle perturbation but, rather, that the iomc imbalance resulting from the intracellular influx of na + triggers other cellular responses that lead to the observed perturbations. monensin is known to both inhibit and promote ca + accumulation in myocytes depending on the absence or availability, respectively, of external ca + stores [ , ] ; alter na + gradient-dependent ca + transfer through the basolateral plasma membranes of rat small intestine [ ] ; increase myocardial calcium activity [ ] , inhibit ca + accumulation by cardiac macrosomes or cause release of accumulated ca + stores [ ] ; and release ca + from macrosomes [ , ] . digitalis and other cardiac glycosides (which increase myocardial contractility) appear to act by altering intracellular na + concentration through inhibition of membrane-bound na +-, k+-actlvated adenosine triphosphatase which secondarily results in an increase in mtracellular ca + [see ] . calcium ionophores such as lasalocld and a have been suggested as potential probes for studying the effects of calcium imbalance on myocardial function [ ] . in retrospect, monensin affects the myocardlum in much the same way as do the calcium lonophores and, in some instances, to an even greater extent [ ] . these observations suggest that the xonotropic effects of monensln may be partially indirect; e g., through release of histamine and endogenous amines [ ] , or stimulation of synthesis and/or release of prostaglandln [ , , ] . thus, na + balance plays an indirect but critical role in modulating myocardial function however, a calcium-independent catecholamine depleting action of monensin in cultured rat pheochromocytoma cells [ ] and m bovine adrenal medullary cells and chromaffln granules [ ] suggests that monensln may also play a direct role in altering cellular function. similarly, sutko and co-wor~kers [ ] showed that both monensxn and mgencin produce their effects in guinea pig atria by direct action as well as by releasing catecholamines from tissue stores. differences in subcellular responses to monensin, between the whole ammal and isolated cells or organs, have been noted by us as well as by others [ ] . thus, swelling of golgl apparatus clsternae observed an cultured cells, tissue shces, and plant roots and stems, is not an aberration characteristic of the cells of animals poisoned by monensln. lack of golga apparatus welhng in animals infers that cells from monensin-poisoned ammals are bathed in body fluids containing less than - m monensln which is approximately the minimum effective dose of monensin that will cause sweulng of golgt apparatus clsternae in cultured animal cells. alternatively, lack of vacuolated and/or swollen mitochondna in cultured mammahan cells and plants generally imphes that the vacuolated and/or wollen rnttochondria observed in striated muscle from monensinpoisoned animals are secondary effects of monensin poisoning, perhaps caused by a metabohte of monensin [ , ] . alternatively, monensln could affect the synthesis and/or transport of humoral agents (e.g., catacholanunes) which, in turn, would alter muscle homeostasis and lead to the rmtochondnal aberrations observed in striated muscle. for plants, at concentrations of -s m, monensin was shown to inhibit gernunatlon and growth of ryegrass seedlings [ ] . the effects were primarily associated with poor root development and significant reduction of root mass as compared to controls leaf emergence and leaf mass was only slightly affected. at - m monensln, roots often did not emerge from the seed during germination and root mass of seedlings was often near zero. under these same conditions, shoot mass was reduced about % as compared to controls. monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of prinopally sodium ions for protons. the outer surface of the lonophore-ion complex as composed largely of nonpolar hydrocarbon, which tmparts a high solubility to the complexes in nonpolar solvents. in biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. the net effect for monensin is a trans-membrane exchange of sodium ions for protons. however, the interaction of an ionophore with biologl-cal membranes, and its ionophorlc expression, is highly dependent on the blochemcial configuration of the membrane itself one apparent consequence of this exchange is the neutralization of acidic lntracellular compartments such as the trans golgi apparatus clsternae and associated elements, lysosomes, and certain endosomes. this is accompanied by a disruption of trans golgi apparatus clsternae and of lysosome and acidic endosome function. at the same time, golgl apparatus clsternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions monensin effects on golga apparatus are observed in cells from a wide range of plant and animal species the action of monensin is most often exerted on the trans half of the stacked clsternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensln concentrations or short exposure times, near the rmddle of the stacked cisternae. the effects of monensln are quite rapid in both animal and plant cells; l.e, changes in golgl apparatus may be observed after only - min of exposure it is implicit in these observations that the uptake of osmotically active cations is accompanied by a concornltant efflux of h + and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of clsternae observed in electron rmcrographs. in the golgi apparatus, late processing events such as terrmnal glycosylation and proteolytlc cleavages are most susceptible to inhibition by monensln. yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the golgi apparatus in endocytosls, monensln does not prevent internalization however, intracellular degradation of internalized ligands may be prevented. it is becormng clear that endocytosls involves both acidic and non-acidic compartments and that monensln inhibits those processes that normally occur in acidic compartments. thus, monensln, which is capable of collapsing na + and h + gradients, has gamed wide-spread acceptance as a tool for studying golgi apparatus function and for locahzlng and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. among its advantages are the low concentrations at which inhibitions are produced ( - . /~m), a minimum of troublesome side effects (e g., little or no change of protein synthesis or atp levels) and a reversible action. because the affinity of monensin for na + is ten times that for k +, its nearest competitor, monensxn mediates primarily a na+-h + exchange monensin has little tendency to bind calcium. not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed m cattle the mechanisms by which monensln interact with cocctdla and rumen rmcroflora to achieve these benefits are reasonably well documented. however, the interactions between monensm and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensln poisoning are well known equine species are particularly susceptible to monensm poisoning, and a common effect of monensln poisoning is vacuolizatton and/or swelling of rmtochondna m striated muscle other pathological injuries to striated muscle, spleen, lung, hver and kidney also have been noted a con-sistent observation is cardiac myocyte degeneration as well as vacuohzation differences m cellular response resulting from exposure to monensln (t e, golgi apparatus swelling in cultured cells, isolated tissues, and plants vs. mltochondrial swelling m animals fed monensln) suggest that myocardial damage is due either to a monensln metabohte or is a secondary response to some other derivation however, as pointed out by bergen and bates [ ] , the underlying mode of action of lonophores is on transmembrane ton fluxes which dissipate cation and proton gradients consequently, some or all of the observed monensin effects m vlvo m animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes. the role of membranes in metabohc regulation polyether antibiotics -naturally occurnng acid ionophores polyether antibiotics -naturally occurnng acid ionopbores polyether antlbloucs -naturally occurnng acid ionophores polyether antibiotics -naturally occumng acid ionophores (west-icy polyether antibiotics -naturally occurring acid ionophores the role of membanes m metabohc regulalaon polyether antlbtotlcs -naturally occurnng acid ionophores comp blochem phys- o protoplasma clba found symp ongm and continuity of cell organelles (relnert endocytosls (paston, i and wdhngham proc. natl acad th ann proc equine medacane and surgery current vetennary therapy food ammal practice (howard an atlas of fine structure calcium antagomsts and cardiovascular disease key: cord- -choywmwd authors: nan title: membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: choywmwd the targeting signals of two yeast integral membrane dipeptidyl aminopeptidases (dpaps), dpap b and dpap a, which reside in the vacuole and the golgi apparatus, respectively, were analyzed. no single domain of dpap b is required for delivery to the vacuolar membrane, because removal or replacement of either the cytoplasmic, transmembrane, or lumenal domain did not affect the protein's transport to the vacuole. dpap a was localized by indirect immunofluorescence to non-vacuolar, punctate structures characteristic of the yeast golgi apparatus. the -amino acid cytoplasmic domain of dpap a is sufficient for retention of the protein in these structures, since replacement of the cytoplasmic domain of dpap b with that of dpap a resulted in an immunolocalization pattern indistinguishable from that of wild type dpap a. overproduction of dpap a resulted in its mislocalization to the vacuole, because cells expressing high levels of dpap a exhibited vacuolar as well as golgi staining. deletion of residues of the dpap a cytoplasmic domain resulted in mislocalization of the mutant protein to the vacuole. thus, the cytoplasmic domain of dpap a is both necessary and sufficient for golgi retention, and removal of the retention signal, or saturation of the retention apparatus by overproducing dpap a, resulted in transport to the vacuole. like wild type dpap b, the delivery of mutant membrane proteins to the vacuole was unaffected in the secretory vesicle-blocked sec mutant; thus, transport to the vacuole was not via the plasma membrane followed by endocytosis. these data are consistent with a model in which membrane proteins are delivered to the vacuole along a default pathway. ar~y proteins that reside in the organelles of the secretory pathway of eukaryotic cells have targeting information that directs retention in or sorting to the appropriate compartment (pfeffer and rothman, ) . in the absence of retention or sorting signals, soluble proteins of the secretory pathway are secreted; thus, the default pathway for these proteins is secretion (burgess and kelly, ; pelham, ) . in saccharomyces cerevisiae, mutations in the targeting signal of the soluble vacuolar protein, carboxypeptidase y (vails et al., ) , or the retention signal of the soluble er protein, bip (hardwick et al., ) , result in the secretion of these proteins. likewise, the flow of membrane proteins to the cell surface of nonpolarized mammalian cells is apparently by default, because mutations that disrupt the retention of er or golgi-retained membrane proteins (machamer et al., ; machamer, ; jackson et al., ) or the sorting of a lysosomal membrane protein (williams and fukuda, ) result in localization to the plasma membrane. little is known regarding membrane protein sorting in s. cerevisiae, although a previous study suggested that the cell surface is the default compartment for membrane proteins (fuller et al., b) . in this paper, we characterize the targeting of two membrane proteins of the yeast secretory pathway, dap dipeptidyl aminopeptidases (dpaps) ~ dpap a and dpap b of the golgi apparatus and vacuole, respectively. the biogenesis of two membrane proteins of the yeast vacuole, dpap b (see fig. a), and alkaline phosphatase (alp), has been characterized (klionsky and emr, ; roberts et al., ) . both dpap b and alp are type ii membrane glycoproteins (nomenclature of singer, ) , consisting of nh -terminal cytoplasmic domains of approximately amino acids, single hydrophobic membrane anchors, and cooh-terminal lumenal catalytic domains. these proteins transit the early compartments of the secretory pathway (i.e., er and golgi), but not the later compartments (i.e., secretory vesicles), indicating that the proteins do not transiently reside at the plasma membrane before delivery to the vacuole. the localization signals of these two proteins have not been identified, although the lumenal domain of alp has been shown to be unnecessary for vacuolar targeting (klionsky and emr, ) . the biosynthesis of several membrane proteins that reside this paper is dedicated to the memory of david merrill stevens. in the yeast golgi apparatus has also been examined. in yeast, three membrane-bound proteases, kex p, kexlp, and dpap a (see fig. a) process the mating pheromone a-factor precursor polypeptide as it traverses the secretory pathway (bussey, ; fuller et al., ) . the biosynthetic pathways of kex p and kexlp have been characterized (fuller et al., a,b; cooper and bussey, ) , and kex p has been shown to function in a late golgi compartment (julius et al., ; graham and emr, ) . kex p has been localized by indirect immunofluorescence to three to six punctate structures per cell that exhibit a somewhat random distribution within the cytoplasmic compartment (franzusoff et al., ; redding et al., ) . thus, the golgi apparatus of yeast is not localized to a perinuclear location, as in mammalian cells, but rather is dispersed throughout the cell. both kexlp (a. cooper and h. bussey, manuscript submitted) and dpap a (see below) have been immunolocalized to punctate bodies that are similar to those containing kex p in size, abundance, and distribution. in this paper, a combined gene fusion and mutational analysis was used to show that no single domain of dpap b is required for vacuolar localization. furthermore, both overproduction of dpap a and a mutation in the cytoplasmic domain of dpap a resulted in mislocalization of this protein to the vacuole. finally, the fusion proteins analyzed in this work are shown to be transported directly to the vacuole from the golgi, and not to the plasma membrane. the bacterial strain mc (casadaban and cohen, ) was used for all subcloning steps. oligonucleotide-directed mutagenesis was carded out in strain cj (kunkel et al., ) . the yeast strains used were jhry - aaia (mata, dap a: : h s , stel a : :leu , ura - , leu - , eu , his -a , pep - ; roberts et al., ) , sey zs (match, dap :: leu , emr et al., ) , cjry - b (mata dap a : :leu mnn ura - leu - leu , and sey- ( mata, s e cl-l , dap : : le u , ura - , le u - , leu ) . yeast cultures were grown in yepd or minimal (sd) medium supplemented with the appropriate nutrients as previously described (sherman et al., ) . for the simultaneous induction of the gal/ promoter and the secl secretion defect, ceils were grown to log phase in minimal media plus raltinose and then harvested and resuspended in yep-raitinose. after a -h incubation at ~ galactose was added directly to the cultures and at the same time the cultures were shifted to ~ after h, the cells were fixed immediately and prepared for indirect immunofluorescence as described below. oligonucleotides for mutagenesis were prepared by the university of oregon biotechnology laboratory on an applied biosystems b dna synthesizer (foster city, ca) as described (ito et al., ) . tran s-label and zymolyase t were from icn biomedicals (irvine, ca), endo h was from boehringer marmheim (indianapolis, in), ultra-pure sds was from bdh biochemicals (san francisco, ca), glusulase was from dupont pharmaceuticals (wilmington, de), and all antibodies (except anti-dpap b, anti-dpap a, anti-alp, and anti-vat p antibodies) used for indirect immunofluorescence were from jackson immunoresearch (west grove, pa), cappel products (malvern, pa), or promega biotech (madison, wi). all other reagents were from sigma chemical co. (st. louis, mo). dpap and invertase assays were performed as previously described (gildstein and lampen, ; roberts et al., ) . restriction endonuclease digests and ligations were performed as recommended by the suppliers. plasmid purification, agarose gel electrophoresis, fill-in reactions of sticky-ended dna fragments using t dna polymerase, and dna-mediated transformation of escherichia coli were done according to standard procedures (maniatis et al., ) . lithium acetate transformations of yeast were performed as described (jto et al., ) . a disruption of the chromosomal ste/ locus was constructed by onestep gene disruption (l~thstein, ) , using the plasmid pslk (kindly provided by dr. george sprague). psl consists of pbr containing a . kbp bamhi ste/ fragment, from which a . -kbp bcli fragment within the coding region of the dpap a lumenal domain (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication) was replaced by the . -kbp bglii leu fragment. to create a strain lacking both dpap a and dpap b, a disruption of the chromosomal dap locus with the his gene was made using the plasmid pgp , which contains the . -kbp ecori-bamhi his fragment (sikorski and hieter, ) in place of the l -kbp bsteii-kpni portion of the coding region of dap (roberts et al., ) . the c~-factor signal sequence was fused to the lumenal domain of dpap b as follows: a sail linker was inserted at the hincll site of plasmid p , which contains a portion of the ' region of the mfcd gene (kurjan and herskowitz, ) , including bp of non-coding region (not including the uas) and bp of coding region, including the signal sequence, signal peptidase cleavage site (waters et al., ) , and nh -terminal residues of pro-a-factor, fused to the kre/gene (boone et ai., ) . the acci site at position + of the dap gene was changed to a sali site using oligonucleotide-directed mutagenesis (kunkel et al., ) . mutagenesis of dap was performed using the vector pcjr , which contains the . -kbp bamhi-hindiii da/' fragment in the plasmid ks + (stratagene, san diego, ca). the . -kbp sali-hindui fragment, encoding the lumenal domain of dpap b, was inserted into the sali-hindiii sites of p /sali, fusing the coding regions of mfal and dap in frame, c~fss-b was placed under the control of the ga/_j promoter (johnston and davis, ) by inserting the . -kbp bamhi-hindiii fragment from this plasmid into the bamhi-hindiii sites of pc jr , which contains the -bp ecori-bamhi ga/_,/ promoter fragment inserted into ecori-bamhi sites of the cen plasmid pseyc ca modified version of pseyc ; emr et al., ) . the resulting fusion protein consists of the nh -terminal residues of prepro-afactor and two residues arising from linker sequences fused to residue of the lumenal domain of dpap b (fig. l b ; amino acid sequence nh -mrfpsiftavlfaassala-apvgrphh . . . ). the bb-inv fusion protein expression vectors, pcjr and pc jr ( p and cen plasmids, respectively), were constructed by inserting the . -kbp bamhi-acci fragment (acci blunt ended) into the invertase fusion vectors psey (a derivative of the /z plasmid psey ; emr et al., ) and the cen plasmid pseyc (johnson et al., ) at the bamhi and hin-diii sites (hindlij blunt ended). the resulting fusion protein contains the nh -terminal residues of dpap b fused to residue three of cytoplasmic invertase ( fig. b) . bb-inv was placed under the control of the gal/promoter by cutting pcjr with ecori and hiediii and ligating in the -bp gal/fragment (johnston and davis, ) . this fused the ' end of the gal/fragment at nucleotide - of the dap sequence. vectors encoding a -bb and a -bb were constructed as follows. oligonucleotide-directed mutagenesis (kunkel et al., ) was used to create -and -bp deletions in dap (a -bb and a -bb, respectively) in pcjr . the ~ . kbp bamhi-hindiii mutant dap fragments were inserted into the bamhi-hindiii sites of the cen plasmid pseyc , creating pmnh ( -bb) and pc jr (a -bb). & -bb and a -bb were over-produced by replacing the dap promoter with the gal/promoter as follows. the hindiii site at - of dap was blunt ended and religated, creating an nhei site. the ~ -kbp nhei-hindiii fragments of pc jr and pc jr were inserted at the xbai-hindiii sites of pc jr , creating pc jr and pc jr , respectively. the a and a deletions removed residues - and - of dpap b, changing the nh -terr~nal acid sequence of dpap b from nh -meg-geeeveripdelfdtkkkhlldklirv to nh -mhlldklirv and nh -mirv, respectively ( fig. b) . the pt plasmid encoding dpap a, pcjr , was constructed as follows: the . -kbp xbai-bamhi fragment, containing the stf_j gene (c. a. flanagan, d. a. barnes, m. c. flessel, andj. thorner, manuscript submitted for publication) was isolated from the plasmid p - (julius et al., ) and inserted into puc . the . -kbp sali-bami-ii fragment from this vector was inserted into the xhoi-bglii sites of the # plasmid pckr (c. raymond and t. stevens, unpublished results) . the cen plasmid pcjr was made by inserting the . -kbp eagi-pvuii ste/ fragment into the eaglecorv sites of prs (sikorski and hieter, ) . the plasmid pc jr , encoding the fusion protein aa-b, was constructed by inserting the . -kbp bamhi-mlui (mlui blunt ended) fragment from p - , encoding the stf.j promoter and the cytoplasmic and trans- membrane domains of dpap a, and the . -kbp sall-hindlii fragment of dap (sail blunt ended), encoding the lumenal domain of dpap b, into the bamhi-hindlii sites of pseyc . the . -kbp bamhi-hindlii fragment of the resulting vector (pcjr ) was cloned into the bamhi-hindlii sites of the # plasmid, psey (emr et al., ) . the resulting plasmid encodes aa-b, which consists of the nh -terminal residues of dpap a to residue of dpap b (fig. c) . for the construction of the fusion protein a-bb, xbai sites were created in both the dap and ste/ genes just upstream of the coding regions of the transmembrane domains of dpap b and dpap a, respectively, using oligonucleotide-directed mutagenesis (kunkel et al., ) . nucleotides and of the dap gene were changed from gt to tc (roberts et al., ) , and nucleotides - of ste/ were changed from gcc to aga (with the a of the initiation codon as ) (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). a . kbp saci-xbai fragment (encoding the ste promoter and the cytoplasmic domain of dpap a) and a . -kbp xbal-hindlii dap fragment (encoding the transmembrane and lumenal domains of dpap b) were ligated into the saci-hindlii sites of psey . the resulting plasmid (pcjr ) encodes a protein consisting of residues of the nh -terminal cytoplasmic domain of dpap a fused to amino acid of dpap b (fig. c) . the fusion protein b-a-b was constructed as follows: the . -kbp bamhi-hindui dap fragment, including the xbal site at + , was ligated into the bamhi-hindlii sites of pseyc . a . -kbp xbal-hindlii fragment of ste/ , encoding the transmembrane and lumenal domains of dpap a, was inserted into the xbai-hindlii sites, resulting in the plasmid pc jr . the . -kbp sali-hindiii dap fragment (sali blunt ended), encoding the dpap b lumenal domain, was inserted into the mlui-hindlii sites (mlui blunt ended) of pcjr , resulting in pcjr , a cen plasmid encoding b-a-b. the . -kbp bamhi-hindlii fragment from pcjr was inserted into the same sites of psey , resulting in pcjrt . b-a-b was placed under the control of the gal/promoter by cloning the nhei-hindlii b-a-b fragment into the xbai-hindlii sites of pcjr , creating pc jr . the b-a-b fusion protein consists of residues - of dpap a in place of residues - of dpap b ( fig. c) . oligonucleotide-directed mutagenesis of the portion of ste/ encoding the amino acid cytoplasmic domain of dpap a ( fig. a) was performed in pcjr , which consists of the . -kbp eagi-psti fragment of ste/ inserted into the eagi-psti sites of ks +. a -bp in-frame deletion, removing the amino acids - (z~ ), was created, and the sacl-mlui fragment from this plasmid was inserted into the saci-mlui sites of pcjr , creating the # plasmid psn , which encodes a -aab ( fig. c) . the same saci-mlui fragment was ligated with the -kbp mlui-hindlll (encoding the lumenal domain of dpap a) into the saci-hindiii sites of psey , creating psn , which encodes a -aaa ( fig. c) . a -aa-b was placed under the control of the gal/promoter by fusing a saci-eagi fragment from pcjr (contains the bp hindlii-ecorl gal/fragment in pgem- zf) to an eagl-hindlii fragment from psn , both of which were ligated into the saci-hindlii sites of psey , creating psn . the fusl-laczp expression plasmid pcjrll as follows: a -kb nhei-hindlli fragment, encoding the nh -terminal amino acids of the fus protein fused to ~-galactosidase, under the gall promoter (isolated from pcjrll , a derivative of psb (trueheart and fink, ) ) was cloned into the xbal-hindlll sites of pvt u, a # plasmid (vernet et al., ) , creating pcjr . for the production of dpap a antigen to be used to generate dpap a antiserum, the plasmid pcjr was constructed by inserting the . -kbp mlui-hpal (mlui blunt ended) ste/ fragment into the sinai site of pexp , an e. coli expression vector containing the tac promoter just upstream of the translational start codon of t lysozyme and a multiple cloning site (raymond et al., ) . a amino acid peptide, corresponding to the nh -terminal residues of dpap b followed by a cooh-terminal cysteine residue, was synthesized on an applied biosystems peptide synthesizer. the peptide was coupled to carboxymethylated bsa through the cooh-terminal cysteine with the hifunctional crosslinking agent, mbs, following the manufacturer's recommendations (pierce chemical co., rockford, il), and through lysine residues with gluteraldehyde as previously described (kagen and glick, ) . a : mixture of bsa-peptide conjugates prepared by the two methods was used to immunize rabbits as described previously (vaitukaitis, ) . for affinity purification of the antibody, the poptide was cross-linked to tresyl-activated sepharose b (pharmacia fine chemicals), and affinity purification was carded out as described (raymond et al., ) . dpap a antigen was produced in e. coli cells containing the plasmid pc jr . induction with iptg resulted in the production ofa -kd protein that corresponds to the nhe-terminal portion of the lumenal domain of dpap a (see fig. ) fused to seven residues of t lysosyme and seven residues encoded by the pexpi polylinker. antigen purification was performed as described previously (raymond et al., ) . immunoprecipitations were performed by growing cultures to log phase in supplemented minimal media lacking methionine and cysteine, then pulse labeling in the same media with tran ss-label and chasing by adding /~g/ml methionine and /~g/ml cysteine, followed by the addition of sodium azide to ram. the cells were converted to spheroplasts (stevens et al., ) which were lysed in % sds, m urea plus a protease inhibitor cocktail ( . mm pmsf, /~g/ml leupeptin, and /~g/ml pepstatin) for rain at ~ and adjusted to ml in ip buffer (pbs, . % sds, . % triton x- , mivl edta). for precipitating ~fss-b from the extracellular fractions, the medium was supplemented with m ,,/ml bsa and mm potassium phosphate, ph . . after spberoplasting, the periplasmic and media fractions were pooled and adjusted to ml in ip buffer plus protease inhibitors. after pre-adsorbtion to . % iggsorb, anti-dpap b coohterminal antibody (roberts et al., ) , or anti-dpap a antibody was added, and samples were incubated one hour on ice. iggsorb was added to . %, followed by h on ice, and the immune complexes were precipitated and washed twice with ip buffer. the immune complexes were solubilized, and half of the samples were treated with endoglycosidase h (endo h) overnight at ~ as described (orlean et al., ) . samples were analyzed by sds-page and fluorography as described previously (stevens et ai., ) . the secretion of t~fss-b was quantified using an ambis radioanalytic imaging system (ambis systems, inc., san diego, ca). the fractionation of membranes in the presence of high ph sodium carbonate was performed as described (roberts et al., ) . preparation of fixed, spheroplasted cells for indirect immunofluorescence was carried out essentially as previously described (roberts et al., ) , except that the fixed spheroplasts were treated with % sds for - minutes. antibody adsorption against fixed spheroplasts harboring null mutations in either dap or stej was performed as described elsewhere (raymond et al., ; roberts et ai., ) . the fixed spheroplasts were stained with a : dilution of adsorbed anti-dpap a or anti-dpap b affinity-purified antibody in pbs-bsa (roberts et al., ) . for co-localization with the -kd subunit of the vacuolar h+-atpase (vat p, the product of the vat gene; yamashiro et al., ) , the antibody solution also contained a : dilution of the mab, dll (kane et al., ) . the dpap b or dpap a staining pattern was amplified by subsequent incubations with goat antirabbit antibody conjugated to biotin, followed by a streptavidin-fitc conjugate. the last antibody incubation also contained goat anti-rabbit antibody conjugated to rhodamine. co-detection of alp and fusl-laczp was performed by staining cells with a : dilution of a rabbit polyclonal anti-alp antibody (raymond et al., ) and a : , dilution of a mouse monoclonai anti-/~-gaiactusidase antibody, followed by antibody amplification identical to that used for dpap a and dpap b. the cells were mounted in media containing dapi for staining nuclei, and photomicrographs were made as described previously (roberts et al., ) . the role of the lumenal domain in the sorting of dpap b to the vacuole was addressed in two ways. first, to determine if the lumenal domain was sorted to the vacuole when expressed in the secretory pathway as a soluble protein, a gene fusion was used to create the protein ctfss-b, consisting of the nh -terminal er-targeting signal sequence of prepro-t~ factor fused to the lumenal domain of dpap b at residue ( fig. b) . the signal sequence should direct the translocation of the protein into the er lumen and be cleaved, rendering the lumenal domain a soluble protein in the secretory figure . immunoprecipitations of c~fssb from mnn and mnn strains. jhry - a ( dap a mnn ) or cjry - b ( dap a mnn ) cells were labeled with tran ss-label for rain and chased for min in the presence of izg/ml methionine and /~g/ml cysteine. cultures were separated into two fractions, intracellular (i.e., spheroplasts) and extraceuular (i.e., periplasmic and media), and immunoprecipitated with affinity purified dpap b antibody. half of each immunoprecipitation sample was treated with endo h, and equal amounts were analyzed by sds-page and fluorography. positions of the molecular weight standards are indicated (in kd). pathway. the construct was analyzed in a strain which contains a null allele of dap , the structural gene for dpap b (suarez rendueles and wolf, ) . fig. shows the results of immunoprecipitating o~fss-b from intraceuular and ex-traceuular (i.e., combined periplasmic and medium) fractions of s-labeled cells using an antibody that recognizes the cooh-terminal half of dpap b (roberts et al., ) . analysis of the samples by sds-page and fluorography showed that % of txfss-b was secreted as a heterogeneous population of highly glycosylated species (fig. , lane ) , similar to the secreted protein invertase (esmon et al., ) , whereas the intracellular fraction contained a tightly migrating species (fig. , lane /). treatment of the immunoprecipitates with endo h to remove n-linked carbohydrate demonstrated that the difference in the apparent mobilities was due to glycosylation (fig. , lanes and ) . the glycosylation pattern of the secreted material differs from that of wild type dpap b, which receives only modest glycosyl modifications of the core oligosaccharides in the golgi apparatus (roberts et al., ) . to test whether the alteration in glycosylation caused the secretion of the lumenal domain, otfss-b was expressed in an mnn mutant, which is deficient in the addition of the extensive t~ , outer chain glycosyl groups (kukuruzinska et al., ) . fig. , lanes and , show that t~fss-b was secreted to the same extent ( %) from mnn cells, even though the protein was not aberrantly glycosylated. indirect immunofluorescence microscopy showed that the portion of the lumenal domain that remained intracellular was retained in the er, and no staining of the vacuole was observed (data not shown). unlike the secreted material, the er-retalned material was enzymati- (suc -a ) cells containing a plasmid encoding bb-inv (pcjr ) were labeled with tran s-label for rain and chased for rain in the presence of /~g/ml rrw, thionine and /~g/ml cysteine. the cells were converted to sphemplasts, and extracts immunoprecipitated with either affinity-purified dpap b antibody (lanes - ) or affinity-purified invertase antibody (lanes and ) . half of each immunoprecipitation sample was treated with endo h, and equal amounts were analyzed by sds-page and fluorography. positions of the molecular mass standards for lanes - are indicated (in kd) on the left, whereas standards for lanes and are indicated on the right. cally inactive (data not shown), and thus may be unfolded and incompetent to exit the er (rose et al., ; rothman, ) . the role of the lumenal domain of dpap b in vacuolar targeting was also tested by constructing the fusion protein bb-inv, in which the nh -terminal residues of dpap b were fused to the non-vacuolar protein, invertase (fig. b) . immunoprecipitations of bb-inv using anti-invertase antibody, followed by sds-page and fluorography, showed that the fusion protein was glycosylated, and treatment with endo h showed that the protein was of the expected size (fig. , lanes and ) . bb-inv fractionated with membranes under high ph carbonate conditions, consistent with bb-inv being an integral membrane protein (data not shown). invertase enzyme assays of either permeabilized or non-permeabilized whole cells deleted for the invertase structural gene and expressing bb-inv from a cen plasmid showed that < % of the total invertase activity was extracellular. the subcellular localization of bb-inv was determined by indirect immunofluorescence microscopy using an antibody that recognizes the cytoplasmic domain of dpap b (see materials and methods). fig. shows the staining pattern of wild type dpap b (fig. , a-c) and bb-inv (fig. , g-i) when expressed from the high copy number ( #) plasmids, pgp (roberts et al., ) and pcjr , respectively. for these and the other # plasmid constructs used in this study, the proteins were overproduced - -fold as determined by enzyme activity. both dpap b and bb-inv were localized to the vacuolar membrane as judged by differential interference contrast (nomarski) optics and co-localization with a marker for the vacuolar membrane, the -kd subunit of the vacuolar h+-atpase (vat p; yamashiro et al., ; table i ). dpap b and bb-inv were also localized to the vacuolar membrane when expressed from a single copy (cen) plasmid (data not shown). aside from the differences in signal intensity, no difference in subcellular localization was observed when bb-inv was expressed from cen or # plasmids. the results from the afss-b and bb-inv fusions indi-cate that, similar to alp (klionsky and emr, ) , the lumenal domain of dpap b is neither necessary nor sufficient for vacuolar targeting. the role of the transmembrane domain in the vacuolar targeting of dpap b was tested by constructing the fusion protein b-a-b (fig. b) , in which the membrane anchor of dpap b was replaced by that of the non-vacuolar membrane protein, dpap a. the role of the cytoplasmic domain of dpap b was tested by constructing in-frame deletions in this domain using oligonucleotide-directed mutagenesis of the dap gene. two deletion variants of dpap b were constructed, a -bb and a -bb, in which and amino acids, respectively, were removed from the residue cytoplasmic domain (fig. b; see materials and methods). immunoprecipitations of b-a-b, a -bb, and a -bb from s-labeled cells, followed by sds-page and fluorography, showed that the mutant proteins were glycosylated, and that the deglycosylated proteins were of the predicted size ( fig. , lanes - ) . as with bb-inv, these proteins behaved as integral membrane proteins in high ph carbonate fractionation experiments (data not shown). dpap activity assays of cells expressing b-a-b, a -bb, and a -bb showed that the proteins were fully enzymatically active, and that - % of the total activity was intracellular. indirect immunofluorescence microscopy of ceils expressing b-a-b, a -bb, and a -bb from either # plasmids (fig. , c-h) or cen plasmids, using an antibody that recognizes the cooh-terminal half of dpap b, showed that each of the mutant proteins was predominantly localized to the vacuolar membrane as determined by nomarski optics (fig. ) and co-localization with vat p (data not shown). no staining of the plasma membrane was observed for any of these proteins. thus, the cytoplasmic and transmembrane domains of dpap b are not necessary for targeting to the vacuolar membrane. a significant fraction of cells expressing a -bb and a -bb also showed some er localization, as judged by staining of the perinuclear space and long cisternal compartments (table i; rose et al., ) , whereas b-a-b showed only vacuolar labeling. the z~ -bb and ~ -bb cells in fig. show predominantly vacuolar staining. in many cases, the plane of focus had to be adjusted to observe staining of both vacuolar and er structures within a given cell. the increased residence time of a -bb and a -bb in the er was corroborated by immunoblotting analysis, which showed that a small amount of the er forms of a -bb and a -bb were seen in the steady state, along with the golgi-modified forms, whereas only the golgi-modified form was seen for wild type dpap b (data not shown). the increased er retention of a -bb and a -bb could be due to an impaired ability to achieve a native conformation competent for exiting the er, as has been observed with mutant membrane proteins in mammalian cells (gething et al., ; doms et al., ) . upon exiting the er, however, a -bb and a -bb were transported through the golgi complex to the vacuole, indicating that the cytoplasmic domain of dpap b is not necessary for vacuolar localization. these results, combined with those from the analysis of eefss-b, bb-inv, and b-a-b, demonstrate that, aside from a membrane anchor, no single domain of dpap b is required for transport to the vacuole. * for each construct, the percentages refer to the fraction of stained cells that exhibited staining of a particular organelle. depending on the protein being monitored, some cells showed staining of more than one class of organdie; thus, the percentages for a given protein may add up to more than . w vacuolar localization was determined both by coincidence of staining with the vacuole membrane as determined by nomarski optics, and by co-localization with vat p. i) golgi localization was defined as punctate, non-vacuolar, and non-er staining, characteristic of dpap a, kex p , and kexlp (cooper and bussey, manuscript submitted for publication). ** er localization was determined by staining of perinuclear (as determined by dapi staining of nuclei) and extended cisternal structures. several different models can explain the data presented above (see discussion), including a simple model in which the vacuolar membrane, not the plasma membrane, is the default compartment for membrane proteins of the yeast secretory pathway. to distinguish among these models, we analyzed the retention signal of the golgi membrane protein, dpap a. mutations in the retention signal of this protein should result in dpap a becoming localized to the default compartment for membrane proteins. dpap a is a protein that resides in the golgi apparatus, where it processes the c~-factor precursor polypeptide (julius et al., ) . the structural gene for dpap a (ste ) has been cloned and sequenced (julius et al., ; c. a. flanagun, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication), and the predicted structure of dpap a is similar to that of dpap b in several regards. both are type ii integral membrane proteins, and both have lumenal enzymatic domains of ,x, residues that share a high degree of sequence identity, including % identical residues over the cooh-terminal residues (fig. a) ; however, there is no significant sequence similarity between the cytoplasmic and transmembrane domains of dpap a and dpap b. to assess the subcelhlar distribution of dpap a, an antibody specific for dpap a was generated (see materials and methods). the specificity of the antibody is demonst-rated in fig. . immunoprecipitation of dpap a was carried out with s-labeled cells that varied with regard to the dosage of the ste/ gene. sds-page and fluorography showed that the antibody immunoprecipitated a protein of kd from wild type cells but not from ste a ceils, and that this polypeptide was overproduced ~ -fold in a strain containing the ste/ gene on a t~ plasmid (fig. a) . a protein of * kd was also immunoprecipitated; however, the level of the -kd protein did not vary with the dosage of ste/ . the ste/ dna sequence predicts three possible sites for addition of n-linked carbohydrate (c. a. flanagan, d. a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). treatment of dpap a with endo h resulted in an ~, kd decrease in apparent molecular weight (fig. b) , suggesting that at least two of the three asn-x-ser/thr sites of dpap a were modified, assuming that the core oligosaccharides added were of the typical structure (kukuruzinska et al., ) , and that the carbohydrate moieties were only slightly modified in the golgi apparatus, as is the case for the glycosyl groups of kex p (fuller et al., a,b) and kexlp (cooper and bussey, ) . the apparent molecular weight of deglycosylated dpap a, kd, is consistent with the molecular weight of dpap a predicted from the ste dna sequence, , d (c. a. flanagan, d. a. barnes, m. c. flessel, andj. thorner, manuscript submired for publication). indirect immunofluorescence microscopy of cells containing the $ f,/ gene on a # plasmid showed that dpap a was localized to several non-vacuolar puncture patches dispersed throughout the cell, as judged by nomarski optics and in double staining experiments with vat p antibody (fig. , d-u). this signal was absent in a stei a strain (fig. , a-c). this staining pattern is typical of the golgi apparatus, as determined by the localization of kex p and kexlp (a, cooper and h. bussey, manuscript submitted for publication). attempts to localize dpap a in cells containing only the chromosomal copy of ste/ were unsuccessful due to the low abundance of the protein. because the copy number of yeast /~ plasmids can vary from - copies/cell within a population (rose and broach, ) , the fluorescence signal corresponding to dpap a when expressed from a # plasmid also varied from cell to cell, ranging from a weak signal to a very strong signal, whereas the intensity of the vat p signal was consistent from cell to cell. all of the cells that showed a signal with the dpap a antibody displayed a golgi-staining pattern (fig. , d-u; table i ); however, cells exhibiting a very strong signal, presumably due to a high dosage of the ste/ gene, also showed vacuolar staining ( % of the cells ofapep - strain; table i ). an example of this is shown in fig. , (~ s, and u) . a single cell exhibiting a strong dpap a signal is shown, and the signal stains both vacuolar and punctate non-vacuolar structures, as judged by co-localization with vat p. the percentage of cells showing vacuolar localization of dpap a decreased significantly in an otherwise isogenic pep strain, which contains the full complement of vacuolar proteases (i %; table i) . thus, cells producing high levels of dpap a showed mislocalization of the protein to the vacuolar membrane, with the mislocalized protein degraded in a vacuolar proteasedependent fashion. dpap enzyme assays on permeabilized and non-permeabilized cells expressing dpap a from either cen or g plasmids showed that , % of the total activity was intracellular; thus, it is possible that a small percentage of dpap a is present at the cell surface, even though no plasma membrane staining was detected by indirect immunofluorescence, however, the apparent extraceuular dpap a enzyme activity could in part be due to a small degree of cell lysis during the assay. to determine whether a specific domain of dpap a con-the journal of cell biology, volume , tained the signal for golgi retention, the fusion proteins aa-b and a-bb were constructed ( fig. c) , in which the lumenal domain of dpap a was exchanged for that of dpap b (aa-b), and the cytoplasmic domain of dpap b was exchanged for that of dpap a (a-bb). immunoprecipitation of aa-b and a-bb from s-labeled cells, followed by sds-page and fluorography, showed that both fusion proteins were glycosylated and treatment of the immunoprecipitates with endo h showed that the proteins were of the expected size (fig. b) . the broadly migrating species observed for aa-b and a-bb indicate that the proteins are more extensively modified in the golgi apparatus than dpap a. dpap activity assays on permeabilized or non-permeabilized whole cells demonstrated that the aa-b and a-bb proteins were enzymatically active, and that ~ % of the activity was intracellular. a-bb fractionated with membranes in the presence of high ph sodium carbonate, and thus behaves biochemically as an integral membrane protein (data not shown). indirect immunofluorescence microscopy using anti-dpap b lumenal domain antibody showed that aa-b and a-bb exhibited non-vacuolar punctate staining patterns indistinguishable from wild type dpap a (fig. , a-f) . again, no plasma membrane staining was detected. as with dpap a, all cells that showed a signal displayed a punctate staining pattern, and a subset of those cells that showed a strong signal also exhibited vacuolar localization (table i ). an example is shown in fig. (a-c) for cells expressing aa-b. the cell in the bottom right corner shows a very intense fluorescein signal relative to the other cells in the panel, and clearly exhibits co-localization with vat p. thus, as was seen for wild type dpap a, overproduction of these proteins resulted in some mislocalization to the vacuole. these experiments demonstrate that the cytoplasmic domain of dpap a is sufficient for the retention of an otherwise vacuolar membrane protein in punctate structures typical of the golgi apparatus in s. cerevisiae. the cytoplasmic domain of dpap a also has been shown to be sufficient for golgi retention when fused to the transmembrane and lumenal domains of the vacuolar membrane protein alp (s. nothwehr and ". stevens, unpublished data). to determine if the cytoplasmic domain of dpap a was also necessary for the retention of dpap a in the golgi, a series of in-frame deletion mutations in the cytoplasmic domain were generated using oligonucleotide-directed mutagenesis (see materials and methods). deletion variants that lacked (fig. ) , although both proteins also showed a small amount of er staining (table i). immunoblotting analysis of crude extracts prepared from cells expressing a -aa-b showed a broadly migrating species on sds-page similar to aa-b and a-bb (data not shown; fig. b) , suggesting that the a -aa-b protein transits the golgi apparatus before its transport to the vacuole. similar to the deletions in the cytoplasmic domain of figure . immunolocalization of aa-b and a-bb. jhry' - a (ste a dap a) cells containing plasmids expressing either aa-b (pcjr , a-c) or a-bb (pcjr , d-f) were fixed, spheroplasted, and stained with dpap a antibody and vat p mab. the cells were viewed by nomarski optics (a and c) and by epifluorescence using filter sets specific for fluorescein (b and e) and rhodamine (c and f) fluorescence. dpap b, this deletion delays exit of the fusion proteins from the er; however, upon exit from the er, the enzymatically active a -aaa and a -aa-b fusion proteins were transported to the vacuole. thus, the cytoplasmic domain is both necessary and sufficient for the retention of dpap a in the golgi apparatus, and the signal for the retention of dpap a maps to a -amino acid segment within the ll -residue cytoplasmic domain. we have previously shown that the transport of dpap b to the vacuole does not involve delivery to the plasma membrane followed by endocytic targeting to the vacuole (roberrs et al., ) . this was demonstrated by expressing dpap b in a secl mutant, which at ~ is blocked at a late stage of the secretory pathway and accumulates secretory vesicles salminen et al., ) . to ad-dress the possibility that the mutant constructs analyzed in this study were mislocalized to the plasma membrane followed by rapid endocytic uptake to the vacuole, indirect immunofluorescence experiments were performed on bb-inv, b-a-b, a -bb, and/x -aa-b expressed in a secl strain at ~ as a positive control for accumulation in secretory vesicles, the localization of the fusion protein fusl-laczp was analyzed (trueheart et al., ) . the fus gene product is required for the breakdown of the cell walls after the fusion of a and ct cells during conjugation (mccaffrey et al., ; trueheart et al., ) , and has been shown by protease treatment of whole yeast cells to be a plasma membrane protein (trueheart and fink, ) . the fusion protein fusl-laczp, consisting of the nh -terminal amino acids of fuslp fused to the/acz gene product (/ -galactosidase), has been shown by immunofluorescence microscopy to be localized to the plasma membrane after induction during mating (trueheart et al., ) . for these experiments, the expression of these proteins was under the control of the inducible gal/promoter (see materials and methods; johnston and davis, ) . neither fusl-laczp, bb-inv, b-a-b, or a -bb showed any signal before addition of galactose (data not shown). fig. (a-f) shows that fusl-laczp was localized to the plasma membrane in a sec § strain after h of induction at ~ but accumulated intracellularly, presumably in secretory vesicles, in a secl strain under the same conditions. no colocalization with the vacuolar marker, alp, was detected. figure . immunolocalization in sec* and secl strains. jhry - a (sec*) and sey- (secl) cells containing plasmids encoding fusl-laczp (pcjrll ) or b-a-b (pcjr ) under the control of the gal/ promoter were grown to log phase in media containing raffinose, then shifted to ~ at the same time that galactose was added to the cultures. after h, the cells were fixed, spheroplasted, and prepared for immunofluorescence. cells expressing fusl-laczp were stained with anti-/~-galactosidase mab and anti-alp polyclonal antibody, and were viewed using filter sets specific for fluorescein and rhodamine fluorescence, respectively. cells expressing b-a-b were stained as in fig. . typically, fusl-laczp accumulated in a large patch in the bud and bud neck region of budded cells, and near the cell periphery of unbudded cells. bb-inv, b-a-b, and a -bb were each localized to the vacuole under these conditions (e.g., fig. , g-l; table ii) , as defined by co-localization with vat p. the intraceuular, non-vacuolar signal occasionally observed for b-a-b ( %; table ii ) was different from that of fusl-laczp, in that the signal looked reminiscent of the golgi signal observed for dpap a (data not shown). this was also observed for b-a-b in sec § cells at ~ suggesting that the kinetics of delivery of this protein is slowed at elevated temperatures. the analysis of a -aa-b in the secl strain was complicated by the fact that expression of the protein was leaky; ,~ % of the cells in the population showed a weak vacuolar staining pattern before galactose addition. however, addition of galactose to the culture after shifting the cells to ~ resulted in '~ % of the cells exhibiting an intense vacuolar signal (data not shown). these data show that the bb-inv, b-a-b, a -bb, and a -aa-b proteins are targeted directly from the golgi apparatus to the vacuole without transient appearance at the cell surface; thus, these proteins follow the same route to the vacuole as wild type vacuolar membrane proteins (roberts et al., ) . the search for the vacuolar localization determinant of dpap b led to the surprising conclusion that neither the cytoplasmic, transmembrane, nor lumenal domain of the protein was necessary for vacuolar delivery. analysis of the golgi retention signal of dpap a led to the equally surprising observations that both overproduction of the protein and mutations in the cytoplasmic domain resulted in mislocalization of dpap a to the vacuole, not the plasma membrane. these results were unexpected, given that the targeting of soluble vacuolar proteins in yeast and soluble and membrane proteins of the lysosomes of animal cells require targeting information to prevent delivery to the cell surface (vails et al., ; kornfeld and mellman, ; williams and fukuda, ; machamer, ) . the fusion proteins analyzed in this study are appropriate tools for these experiments for the following reasons. all of the proteins were stable, enzymatically active, membranebound, and glycosylated when expressed in yeast, indicating that they have the correct membrane topology. in addition, all of the fusion proteins containing the dpap b lumenal domain, i.e., a -bb, a -bb, b-a-b, and a -aa-b, were transported to the vacuole after receiving glycosyl modifications in the golgi apparatus. finally, each of these fusion proteins was transported to the vacuole directly from the golgi apparatus, without prior delivery to the plasma membrane followed by endocytic uptake to the vacuole. other golgi membrane proteins have also been shown to be mislocalized to the vacuole rather than the plasma membrane. both mutations in the cytoplasmic domain of kexlp as well as its overproduction result in mislocalization of the protein to the vacuolar membrane (a. cooper and h. bussey, manuscript submitted for publication). similarly, a single amino acid change in the cytoplasmic domain of kex p results in its transport to the vacuole (c. wilcox, k. redding, r. wright, and r. fuller, manuscript submitted for publication). whereas this result might appear to conflict with earlier published data on kex p, more recent analysis indicates that membrane-bound forms of kex p that fail to be retained in the golgi are found in the vacuole rather than the plasma membrane, when these proteins are expressed at wild type or modestly elevated levels (c. wilcox, k. redding, r. wright, and r. fuller, manuscript submitted for publication). given that several golgi membrane proteins all show missorting to the vacuole when either mutant or overproduced, the model we favor to explain these data is the vacuolar default model, which states that vacuolar membrane proteins do not require sorting information, because the default pathway for membrane proteins of the secretory pathway leads to the vacuole. alternatively, one could argue that, given the similarity between dpap b and dpap a, both proteins could contain vacuolar targeting information in their transmembrane domains, even though no significant similarity is apparent from the amino acid sequence data (c. a. flanagan, . a. barnes, m. c. flessel, and j. thorner, manuscript submitted for publication). mutations in the golgi retention signal of dpap a, or saturation of the retention apparatus, would then result in the delivery of dpap a to the vacuole. this model requires that both kex p and kexlp must also have cryptic vacuolar targeting signals. with regard to the cryptic vacuolar targeting signal model, we have found that replacement of the membrane spanning domains of dpap a and a -aaa with a residue hydrophobic sequence, l(lalv)~, creating the proteins axa and a -axa, results in the retention of axa in the golgi and the transport of a -axa to the vacuole (s. nothwehr and t. stevens, unpublished data). these data suggest that the transmembrane domain of dpap a does not contain vacuolar targeting information, and thus support the vacuolar default model. the vacuolar default model is directly testable by analyzing potential localization signals of yeast plasma membrane proteins. in apparent conflict with the vacuolar default model is the observation that the majority of kex p ( %) is missorted to the plasma membrane in cells deficient for clathrin heavy chain (payne and schekman, ) . however, dpap a is missorted to the cell surface to a lesser extent ( %; seeger and payne, a) . it is possible that the majority of dpap a and a significant percentage of kex p are missorted to the vacuole in cells lacking clathrin heavy chain. mutations in clathrin may affect both the retention of golgi membrane proteins and the functional integrity of the sorting pathway (seeger and payne, b) . this could obscure the default pathway for membrane proteins, resulting in the transport of golgi membrane proteins to the plasma membrane as well as the vacuole. an alternative explanation of the data is that one or more of the dpap a and dpap b fusion or mutant proteins analyzed in this study were in a partially unfolded state, and thus recognized as abnormal and transported directly from the er to the vacuole via a "garbage" pathway. there is no clear evidence for the existence of such a pathway in yeast or animal cells. however, several cases have been described in animal cells in which non-lysosomal membrane proteins were transported to lysosomes, either due to mutations (armstrong et al., ) , or in the case of the heptameric t-cell antigen receptor, complexes lacking the ~" subunit are degraded in lysosomes (minami et al., ) . in these cases, the proteins were transported through the golgi apparatus, and it is unclear whether the proteins were transported to lysosomes by a "garbage" pathway, or by the uncovering of a cryptic lysosomal targeting signal. in the great majority of cases, mislocalized membrane proteins of the secretory pathway in animal cells accumulate at the plasma membrane, which is presumed to be the default destination for these proteins (e.g., williams and fukuda, ) . it is well established that proteins that are slow to reach the folded state are retained in the er due to the action of proteins such as bip (pelham, ; rothman, ) , and proteins that are unable to fold or oligomerize properly are degraded in the er (klausner and sitia, ) . several of the proteins analyzed in this study (i.e., a -bb, a -bb, a -aaa, and a -aa-b) showed increased retention in the er. however, all of these proteins eventually exited the er, were enzymatically active, and were transported through the golgi complex to the vacuole. the -amino acid segment of the cytoplasmic domain of dpap a presumably contains the recognition domain for a "retention protein" of the golgi apparatus. this protein could be a permanent resident of the golgi apparatus; alternatively, the "retention protein" could reside in a post-golgi compartment and function as a salvage receptor, such as the proposed receptor for soluble er proteins (pelham, ) . it is interesting to note that the -amino acid stretch identified as the dpap a golgi retention signal contains five phenylalanine residues (fig. ) . in animal cells, one or more aromatic amino acids in the cytoplasmic domains of cell surface receptors have been shown to comprise part of the signal for the clustering into coated pit regions of the plasma membrane (chen et al., ; johnson et al., ; lobel et al., ; mcgraw et al., ) . that the phenylalanine residues in the a region may play a direct role in golgi retention is supported by the observation that mutations in just two of these residues result in a substantial level of missorting of dpap a to the vacuole (s. nothwehr and t. stevens, unpublished data) . the results of this study suggest that the default pathway for membrane proteins of the yeast secretory pathway may be different from that of certain mammalian cell lines that have been examined, where mutant forms of er, golgi, and lysosomal membrane proteins are mislocalized to the plasma membrane with the bulk flow of membrane (machamer and rose, ; jackson et al., ; williams and fukuda, ; wieland et al., ; karrenbauer et al., ; machamer, ) . however, in polarized epithelial ceils, it remains unclear which membrane serves as the default destination for membrane proteins (simons and wandinger-ness, ; mostov et al., ) . even if the default compartments for membrane proteins of the secretory pathways of yeast and certain animal cells are different, the same mechanistic considerations apply, that is, positive sorting information is required for proteins to avoid delivery to the default compartment. according to the vacuolar default model for yeast membrane protein sorting, not only must er and golgi membrane proteins have sorting signals specifying their retention in the appropriate compartment, but plasma membrane proteins are predicted to have sorting information that prevents their localization to the vacuole. we are currently investigating whether yeast plasma membrane proteins have targeting signals that prevent their transport to the vacuole. lysosomal sorting mutants of coronavirus e protein, a golgi membrane protein yeast kre genes provide evidence for a pathway of cell wall/~ glucan assembly constitutive and regulated secretion of proteins proteases and the processing of precursors to secreted proteins in yeast npxy, a sequence often found in cytoplasmic tails, is required for coated pit mediated internalization of the ldl receptor characterization of the yeast kex gene product" a carboxypeptidase involved in processing secreted precursor proteins differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus g protein an mfod-suc (~-factor-invertase) gene fusion for study of protein localization and gene expression in yeast the amino terminus of the yeast ft-atpase ~-subunit precursor functions as a mitochondrial import signal compartmentalized assembly of oligosaccharides on exported glycoprotein in yeast localization of components involved in protein transport and processing through the yeast golgi apparatus enzymes required for yeast prohormone processing yeast prohormone processing enzyme (ke, x gene product) is a ca~+-dependent serine protease intracellular targeting and structural conservation of a prohormone-processing endoprotease expression of wild type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport / -d-fructofuranoside fructohydrolase from yeast compartmental organization of golgispecific protein modification and vacuolar protein sorting events defined in a yeast secl (nsf) mutant erdi, a yeast gene required for the retention of luminal endoplasmic reticulum proteins, affects glycoprotein processing in the golgi apparatus solid phase synthesis of polynucleotides vi. further studies on polystyrene copolymers for the solid support transformation of intact yeast ceils treated with alkali cations identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum cation-dependent mannose -pbosphate receptor contains two internalization signals in its cytoplasmic domain distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar proteinase sequences that regulate the divergent gal -galio promoter in saccharomyces cerevisiae yeast a factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase glycosylation and processing of prepro-a-factor through the yeast secretory pathway assembly and targeting of peripheral and membrane subunits of the yeast vacuolar h+-atpase the rate of bulk flow from the golgi to the plasma membrane protein degradation in the endoplasmic reticulum membrane protein sorting: biosynthesis, transport, and processing of yeast vacuolar alkaline phosphatase a new class of lysosomal/vacuolar protein sorting signals protein glycosylation in yeast rapid and efficient sitespecific mutagenesis without phenotypic selection structure of a yeast pheromone gene (mfa): a putative a-factor precursor contains four tandem copies of mature c~-factor mutations in the cytoplasmic domain of the kd mannose -phosphate receptor differentially alter lysosomal enzyme sorting and endoeytosis golgi retention signals: do membranes hold the key? a specific transmembrane domain of a coronavirus el glycoprotein is required for its retention in the golgi region molecular cloning: a laboratory manual. cold spring harbor laboratory identification and regulation of a gene required for cell fusion during mating of the yeast saccharomyces cerevisiae mutaganesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second-site mutation is capable of reverting an internalization-defective phenotype building a multichain receptor: synthesis, degradation and assembly of the t-cell antigen receptor plasma membrane protein sorting in polarized epithelial cells order of events in the yeast secretory pathway analysis ofglycoproteins from saccharomyces cerevisiae clathrin: a role in the intracellular retention of a golgi membrane protein control of protein exit from the endoplasmic reticulum biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi molecular analysis of the yeast vps gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle immunolocalization of kex protease identifies a putative late golgi compartment in the yeast saccharomyces cerevisiae structure, biosynthesis, and localization of dipeptidyl aminopeptidase b, an integral membrane glycoprotein of the yeast vacuole methods for studying the yeast vacuole cloning genes by complementation in yeast kar , a karyogamy gene, is the yeast homolog of the mammalian bip/grp gene polypeptide chain binding proteins: catalysts of protein folding and related processes in ceils one step gene disruption in yeast a ras-like protein is required for a post-golgi event in yeast secretion selective and immediate effects of clathrin heavy chain mutations on golgi membrane protein retention in saccharomyces cerevisiae a role for clathrin in the sorting of vacuolar proteins in the golgi complex of yeast methods in yeast genetics a system of shuttle vectors and yeast host strains designed for efficient manipulation of dna in saccharomyces cerevisiae polarized sorting in epithelia the structure and insertion of integral proteins in membranes gene dosage-dependent secretion of yeast vacuolar carboxypeptidase y identification of the structural gene for dipeptidyl aminopeptidase yscv (dap ) of saccharomyces cerevisiae the yeast cell fusion protein fusi is o-glycosylated and spans the plasma membrane two genes required for cell fusion in yeast: evidence of a pheromone-induced surface protein yeast carboxypeptidase y vacuolar targeting signal is defined by four propeptide amino acids a family of yeast expression vectors containing the phage fl intergenic region prepro-a-factor has a cleavable signal sequence the rate of bulk flow from the endoplasmic reticulum to the cell surface accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail we acknowledge cathy flanagan, jeremy thorner, antony cooper, howard bussey, celeste wilcox, robert fuller, and greg payne for communication of results prior to publication; scott ernr, charlie boone, and george sprague for plasmids; margaret ho and joe horecka for help in the construction of the a -bb and fusl-laczp constructs, respectively; and jerry gleason and scan poston for the photographic work. we especially thank christopher raymond for numerous insightful contributions to this work, and nick davis, charlie boone, george sprague, antony cooper, carol vater, cynthia bauerle, and margaret ho for comments on the manuscript. received for publication february and in revised form june . key: cord- - r rqqqk authors: nan title: sorting signals in the mhc class ii invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: r rqqqk targeting of mhc class ii molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain (ii). by analysis of ii-transferrin receptor (tr) chimera trafficking, we have identified sorting signals in the ii cytoplasmic tail and transmembrane region that mediate this process. two non-tyrosine-based sorting signals in the ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrin-coated pits and promote rapid endocytosis. leu and ile were required for the activity of the signal most distal to the cell membrane whereas pro met leu were important for the membrane-proximal signal. the same or overlapping non- tyrosine-based sorting signals are essential for delivery of ii-tr chimeras, either by an intracellular route or via the plasma membrane, to an endocytic compartment where they are rapidly degraded. the ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the ii cytoplasmic tail. more than % of the ii-tr chimera containing the ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the ii sorting signals are efficiently recognized by sorting machinery located in the trans-golgi. ii-tr chimera containing the ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the ii sorting signals are efficiently recognized by sorting machinery located in the trans-golgi. m ajor histocompatibility complex (mhc) class i molecules are polymorphic cell surface glycoproteins expressed primarily on specialized antigenpresenting cells such as macrophages, dendritic cells, and b lymphocytes. they bind peptides derived from exogenous proteins and present them to cd + helper t cells as part of the mechanism for recognizing foreign antigens and stimulating an immune response. mhc class ii molecules displayed on the cell surface are ct/ heterodimers, but during their intracellular transport to the cell surface, they are transiently associated with a third nonpolymorphic polypeptide, the invariant chain (ii) . ii is a type ii membrane protein with different isoforms that arise from alternative splicing of one exon and the use of two translation initiation sites (strubin et al., ; o'sullivan et al., ; koch et al., ) . studies of mutant mice lacking ii have established that ii plays a critical role in the surface expression of class ii molecules and in the ability of class ii molecules to present native protein antigens (viville et al., ; bikoff et al., ) .. ii determines the membrane trafficking of mhc class ii a/ dimers (bakke and dobberstein, ; lotteau et al., ) and also blocks their peptide-binding site during transit through the biosynthetic pathway (roche and cresswell, ; teyton et al., ; roche et al., ) . ij is assembled with class ii a and polypepfides in the endoplasmic reticulum, and, after its transport through the golgi region, the a/ ii complex is sorted out of the constitutive biosynthetic pathway to an acidic compartment within the endocytic system (cresswell, ; lamb et al., ; pieters et al., ) . within this prelysosomal (peters et al., ) , early lysosomal (harding and geuze, ) , or endosomal com-pa~i,uent (guagliardi et al., ) , ii dissociates from class ii a/ dimers, permitting binding ofpeptides from exogenous foreign antigens generated in the endocytic pathway. class ii a/ dimers loaded with peptide are then transported to the cell surface, while the free ii is rapidly degraded (blum and cresswell, ; pieters et al., ) . although a/ ii complexes are thought to be delivered to an endocytic compartment predominantly by an intracellular mute, small amounts of ii and aflli complexes have been detected on the cell surface, and rapid internalization of a/ ii complexes has been reported (wraight et al., ; lotteau et al., ; roche et al., ) . these data suggest that at least some newly synthesized li molecules are transported to an cndocytic compartment via the plasma membrane, a route analogous to that taken by some integral lysosomal membrane proteins, e.g., lysosomal acid phosphatase (braun et al., ; peters et al., ) . li contains one or more sorting signals within its aminoterminal cytoplasmic domain required for localization to the cndocytic system (bakke and dobberstcin, ; lotteau et al., ; simonsen ct al., ) . ifli with a truncated cytoplasmic domain is coexpressed with class ii o~/~ dimers, li fails to dissociate, and a~ii complexes are expressed on the cell surface (roche et al., ) . however, the sorting signals within the cytoplasmic domain of li have not been wellcharacterized, and neither the trafficking pathway taken by mhc class ii cd ii complexes nor the cndocytic membrane compartment where o~/~ dimcrs encounter antigen have been clearly defined. in this study, we have constructed ii-human transferrin receptor (tr) chimeras with the aim of characterizing li sorting signals. we show that the cytoplasmic domain of li contains two independent non-tyrosine-based sorting signals, both of which mediate internalization via clathrincoated pits of the plasma membrane. both signals contain two adjacent large hydrophobic nonaromatic residues suggesting that this may be a common feature of non-tyrosinebased sorting signals. trafficking of ii-tr chimeras to an cndocytic compartment where they are degraded is dependent upon these signals but occurs predominantly by a direct intracclhlar route, implying that the non-tyrosine-based sorting signals arc efficiently recognized by sorting machinery located in the trans-golgi. this cndocytic compartment is distinct from the sorting and recycling endosomal compartments traversed by the wild-type tr, and, therefore, is located within the prelysosomal/lysosomal branch of the endocytic pathway. in addition, wc show that efficient delivery of ii-tr chimeras to this endocytic compartment requires an independent signal in the ii transmembrane region. a cla i fragment containing the entire coding region of the wild-type human tr was cloned into the phagemid pbluescript sk (stratagene, la jolla, ca) . oligonucleotide site-directed mutaganesis was used to generate the cytoplasmic taft and transmembrane domain of ii (iic-r and iirm) chimeric constructs from a pbluescript sk phagemid template of the wild-type tr by the method of kunkel ( ) using the mnta-gene mntagenesis kit (bio-rad labs., richmond, ca). the same method was used to generate the iic'r+zm chimeric construct and mutant iict chimeric constructs from pbluescript sk phagemid templates of the cytoplasmic tail of ii (iict) . oligonucleotides were prepared on a model pcr-i~te dna synthesizer using phosphoranddite chemistry and purified by polyacrylamide gel electrophoresis (appl. biosystems, inc., foster city, ca). mutants were selected by restriction mapping or differential hybridization, and cla i fragments encoding the mutant receptors were excised and cloned into the retroviral expression vector bh-rcas (hughes et al., ) . the mutations were verified by dideoxynucleotide sequencing (sanger et al., ; tabor and richardson, ) of the bh-rcas constructs using the sequenase kit (us biochemical corp., cleveland, oh) according to the manufacturer's directions. chicken embryo fibroblasts (cef) were prepared from fertilized eggs (spafas, norwich, ct) and grown in dme supplemented with % (vol/vol) chicken serum, % (vol/vol) defined bovine calf serum (hyclone, logan, lit), and % (vol/vol) tryptose phosphate broth (difeo, detroit, mi) . cef were transfected with /~g retroviral construct dna per -cm tissue culture plate of % confluent cells using the polybrene-dimethyl sulfoxide method (kawai and nishizawa, ) . one to two weeks after transfection with the bh-rcas constructs, the cef stably expressed the wild-type tr and chimeric ii-tr constructs as a result of infection by recombinant virus. surface expression levels of the wild-type tr and chimeric ii-tr constructs was determined by measuring the binding of ilabeled human transferrin (tf) at °c. diferric human "if (miles scientific, naperville, il) was labeled with i to a specific activity of -- #ci/~tg using enzymobeads (bio-rad labs.) according to the manufacturer's directions. cef were plated in triplicate at a density of . × cells/cm in -well tissue culture plates h before the binding assay (costar corp., cambridge, ma). cells were incubated in serum-free dme for h at °c, and then washed once with ice-cold . m nac , . m sodium phosphate buffer (ph . ) containing . % bovine serum albumin (bsa-pbs). t ilabeled tf ( #g/rnl) in . rnl of bsa-pbs was added to triplicate wells and incubated at °c for rain. cells were then washed three times with . ml of ice-cold bsa-pbs, removed from the wells with . ml of m naoh, and the radioactivity was counted in a gamma counter. the apparent internalization efficiencies of the wild-type tr and chimeric ii-tr constructs were estimated from measurements of the steady-state distribution of receptors at °c (tanner and leinhard, ) . cef were plated in triplicate wells as described for the binding studies. the cells were first incubated in serum-free dme, and then incubated with /tg/ml t sllabeled "if in bsa-pbs for i h at °c. the labeling medium was removed, and the cells were washed three times with ml of ice-cold bsa-pbs, and then incubated twice for min with . m] . m acetic acid- . m naci (ph . ) to remove surface-bound - abeled tf (hopkins and trowbridge, ) , and removed from the wells with i m naoh. radioactivity in the acid wash and in the cell lysete was determined. more prolonged incubation with the acid wash did not change the amount of t released. at steady state, the rate of internalization, kint, of cell surface tf.tr complexes, [tr] .... equals the rate of externalization, kext, of the internal pool of apotf.tr complexes, [tr]~; i.e., kint'[tr]sur = kext'~i'r]int, assuming an insignificant rate of degradation of internalized receptors during the time required to achieve steady-state. the values of ['i'r]~ and [tr]mt can be obtained from steady-state binding of tr under saturating conditions at °c. as ke~t of apotf.tr complexes is independent of signals in the tr cytoplasmic domain , k~xt values of mutant and wild-type receptors are identical so that their k~ values are proportional to their steady state distribution, [tr] the internalization efficiencies of wild-type tr and ii-tr chimeras were also determined by measuring their ability to mediate iron uptake. human apo-tf was labeled with fe (feci ; amersham corp., arlington heights, il) to a specific activity of - #ci/ttg using ultrilotriacetate (bates and schlabach, ) . cells were plated in triplicate wells as described for the binding studies. the following day, cells were washed twice in prewarmed ( °c) serum-free dme, and then incubated in dme containing . % bsa and t~g/m fe-tf at °c for , , , , and h. at the indicated times, the medium was removed, and cells were washed three times with ice-cold bsa-pbs. cells from triplicate wells for each time point were removed in . ml m naoh, and the radioactivity was counted in a gamma counter. the relative levels of wild-type tr and chimeric ii-tr constructs expressed on the various cef populations were determined in each experiment by measuring i-labeled tf binding at °c for each weft. after preincubation for h at °c in serum-free dme, triplicate wells of cells were incubated with /~g/rul t i-labeled tf on ice for h, and then washed three times with mi of ice-cold bsa-pbs, and the radioactivity bound to the cells was counted in a gamma counter. cef were plated in triplicate wells as described for the binding studies. cells were preincobated in serum-free dme for vain at °c, and then incubated with i-labeled "iy ( #g/m ) in bsa-pbs for h at °c. the medium was removed and the cells were washed three times with ice-cold bsa-pbs and incubated at °c with prewarmed ( °c) dme containing . % bsa and #g/ml unlabeled "if for , , , , , , or rain. after incubation, the medium was collected, protein was precipitated in % tca and removed by centrifugation, and then the acid-soluble and acid-insoluble radioactivity was counted in a gamma counter. the surfacebound and internalized "if in cef was determined by the acid wash procedure described for the steady-state distribution assay. cef expressing chimeric ii-tr constructs were ~i surface-labeled using lactoperoxidase for min essentially as described , except that iodination was performed in -cm tissue culture dishes in a total volume of . rnl. cef were then washed four times with ice-culd pbs and incubated at °c in complete medium for , , , , or h. at each time point, cells were solub'dized on ice with % np- -pbs, and the chimeric ii-tr constructs were immunoprecipitated using b / monoclonal antibody specific for the extracellular domain of the human tr . immunoprecipitates were analyzed on . % sds-polyacrylamide gels, which were then dried and exposed to preflashed xar film (eastman kodak, rochester, ny). cef were washed twice with methionine-free dme, and then pulse-labeled for rain in ml of dme containing . mci/mi trans-ass-label (icn biomedicals, irvine, ca) and % defined calf serum. pulse-labeled cells were chased for , , , , or h in complete medium. in one experiment, cells were preincubated for h in medium containing #g/ml leupeptin, and then pulse-labeled and chased for , , , , or h in leupeptin-containing medium. at each time point, cells were soluffdized on ice with % np- -pbs. the wild-type tr and chimeric fi-tr constructs were immunoprecipitated from postnuclear supernatants using b / monoclonal antibody and analyzed on . % sds-polyacrylamide gels . dried gels were exposed to prettashed xar film (eastman kodak, rochester, ny). quantitation of radioactivity was performed on a model phosphorimager (molecular dynamics, sunnyvale, ca). the surface-accessible pools of wild-type tr and ii-tr chimeras were calculated from the nsi-labeled "if bound to intracellular and surface receptors at steady-state. the surface-accessible pool of chimera relative to wild ype tr, [ii-tr] s^, equals the cell surface chimem-tf complexes, fii-tr]s~, plus internalized chimera-tf complexes [ii-tr] int, multiplied by , divided by cell surface tr-tf complexes [tr]sur, plus internalized tr-tf complexes, [tr]int; i.e., [ii-tr] receptors. this condition is met as steady-state conditions are achieved in ,~ rain at °c (fling et al., ; see also above). in rain, < % of the total llabeled "if bound to the most rapidly degraded chimera, iicr+xu, is released into the medium as ~ soluble material (see fig. ). the fraction of ii-tr chimeras trafficking via the cell surface is the surface accessible pool, [ii-tr] sa, divided by the rate of biosynthesis of the ii-tr chimera multiplied by the rate of degradation of [ii-tr] sa; i.e., ([ii-trsa x degradation rate of synthesis. as an example of the calculation, the surface expression of iic'r+tm is % relative to wild-type tr, and at stendy-state % of the iicr+~ chimera and % of the wild-type tr are internalized (see results and table i ). thus, [iic'r+t~.dsa ffi ( + [ x / ]) x /( + [ x / ]) = %. quantitation of the data presented in fig. showed that eightfold more iic~+~ chimera was synthesized than wild-type tr, whereas quantitation of the data presented in fig. showed that the [iic'r+tm]sa is degraded eightfold faster than wild-type tr. therefore, the fraction of iic'r+tm chimeras trafficking via the cell surface relative to wildtype tr is x / % or %. cef and cef expressing wild-type or ii-tr chimeras were plated onto glass coverslips and cultured overnight. the cells were fixed with % formaldehyde treatment for rain at room temperature (rt), quenched in . % ni-hc , . % glycine, ph . , and incubated in p/h/s (pbs/ % horse serum/ . % saponin) (for permeabilized samples) or p/h (for nonpermeab'diznd samples) for min at rt. the samples were next incubated with monoclonal antibody b / (undiluted hybridoma supernatants) followed by a second incubation with rat anti-mouse iggi-fric ( : dilution; zymed labs.). each incubation step was followed by three washes in p/h/s or p/h for rain each. in all cases, antibodies were diluted in p/i-us or p/h, and primary and secondary antibodies were incubated for h and rain, respectively. covershps were mounted in mg/ml p-phenylenediamine in a : mixture of pbs:glycerol and sealed with nail polish. slides were analyzed on a nikon optiphot microscope equipped with epifluorescence and a dage mti series sit video camera (dage-mti, michigan city, in) coupled to an image -at (universal ima~ng~ media, pa) image analysis system. micrographs were prepared with a sony color video printer (up- ). for double-label indirect immunofluorescence, ceils were fixed, permeabilized, and incubated with the following combinations of antibodies: rabbit anti-human tr antiserum ( : dilution), goat anti-rabbit ig-fitc ( : dilution; american qualex), mouse monoclonal antibody anti-lep (undiluted tissue culture supernatant, developmental studies hybridoma bank, university of iowa), goat anti-mouse igg-texas red ( : : zymed laboratories). slides were analyzed on a leitz fluorescence microscope equipped with a vario orthomat ii camera system and a dual wavelength (fitc/texas red) epifluorescence filter module. micrographs were prepared with ektachrome pl professional film processed at asa . colloidal gold ( and tun diameter) sols were made as described by slot and ~ ( ) . protein a gold and rabbit anti-mouse ig gold antibody conjugates ( ) were made by standard methods (de mey, ) and cryosections made and immunolabeled essentially as described by tokuyasu ( ) . the sections were incubated rain with a rabbit antiserum a vaingt human tr (trowbridge, i.s., unpublished results) followed by -am gold protein a for rain, and then rinsed for rain with five changes of pbs and incubated with anti-lep mouse monoclooal antibody for rain followed by -nm gold rabbit anti-mouse ig antibody for rain before an additional five rinses in pbs. omitting either first or second specific antibodies indicated nonspecific labeling was less than %. staining and embedding were carried out as described previously (hopkim, ) and the sections were examined at kev in a philips electron microscope. quantitation of the iicr+m chimera in clathrin-coated pits was determined on conventional plastic sections from cells incubated with b / -gold complexes at °c as described earlier (miller et al., ) . to investigate the signal-dependent trafficking of fi, we constructed ii-tr chimeras consisting of the fi cytoplasmic tail, the tr extracellular domain, and the transmembrane region from either tr or from ii (fig. ) . we reasoned that fi-tr chimeras would be expressed in sufficient amounts on the cell surface to identify ii sorting signals using quantitative assays which measure tf internalization and iron uptake . as there is increasing evidence that the same or closely related signals are recognized by the clathrin-based sorting machinery at the plasma membrane and trans-golgi (letourneur and klausner, would also allow identification of ii sorting signals operative along the intracelhlar trafficking pathway. wild-type human tr and ii-tr chimeras containing either the iicr or iicr÷ru were stably expressed in cef using bh-rcas, a replication-competent retroviral vector derived from rous sarcoma virus hughes et al., ) . binding studies at °c using ~ si-labeled human tf indicated that both ii-tr chimeras were expressed on the cell surface of infected cef, but at lower levels than wild-type tr ( + % iicr and ± % iicr÷rm relative to wildtype tr). internalization of the ii-tr chimeras was assayed by measuring the steady-state distribution of internalized receptors and their ability to mediate iron uptake collawn et al., ) . the iicr chimera was efficiently internalized, as judged by either iron uptake or steady-state assays (table i) , indicating that the ii cytoplasmic domain contains internalization signal(s) that can promote rapid endocytosis of the human tr. electron microscopy (see fig. , inset) clearly demonstrates that ii-tr chimeras internalize via clathrin-coated pits, % of the label on the plasma membrane being located within these domains. the apparent internalization efficiency of the iicr+ru chimera was significantly higher than either wild-type tr or the iicr chimera, implying that the transmembrane region of ii also influences trafficking. to locate the ii cytoplasmic tail internalization signal(s), deletion mutants of the iicr chimera were constructed (fig. ) . deletion of residues - or residues - reduced the apparent internalization efficiency of the iicr chimera to ~ %, whereas deletion of residues - completely abolished rapid endocytosis (table ii) . these results suggest that the ii cytoplasmic tail contains two internalization signals, one located between residues - , the other between residues - . since the ii cytoplasmic tail does not contain aromatic residues, the internalization signals identified by analysis of the iicr deletion mutants cannot be tyrosine-based. however, the ii cytoplasmic tail contains three leucine residues (residues , , and ) within the first amino-terminal residues (fig. ) , suggesting that internalization of the ii-tr chimeras may be mediated by signals related to the dileucine-based sorting signals previously identified in the cytoplasmic tails of the t cell receptor cd "y chain and the cation-independent and cation-dependent mannose- -pbosphate receptors (letourneur and klausner, ; johnson and kornfeld, a,b) . although first identified as a lysosomal targeting signal, the cd chain di-leucine signal has been shown to function as an internalization signal (letourneur and klausner, ; white, s., j. f. collawn, and i. s. trowbridge, unpublished results). di-leucine-based sorting signals have not been extensively characterized; however, isoleucine can substitute for the second leucine, but not the first, in the cd ~/di-leucine signal without loss of lysosomal targeting activity, whereas alteration of either leucine to alanine substantially reduces activity (letourneur and klausnet, ) . because of the stricter requirement for leucine in the first position of the cd ~, chain signal, we initially thought that the two internalization signals identified by deletional analysis may involve i.,¢utile s and leu~ pro is, respectively. to investigate this possibility, the two residues of either one or both of these di-peptides were altered to alanine. strikingly, independently altering each di-peptide to two alanine residues reduced the apparent internalization efficiency of the iicr chimera by ~, %, whereas alteration of both dipeptides completely abrogated high efficiency endocytosis (table id . these results supported the idea that the ii cytoplasmic tail contains two non-tyrosine-based signals with similar internalization activity that are related to the cd "y chain di-leucine signal. to further characterize the two sorting signals, each of the two residues that might be important for their activity were independently changed to alanine in mutant receptors in which the second signal had been inactivated by alanine substitutions. the results of this analysis clearly showed that both leu and he s were required for the signal most distal from the transmembrane region (table hi) . however, whereas alteration of pro ~s to alanine abolished the internalization activity of the membrane-proximal signal, alteration of leu to alanine increased its efficiency. this result implied that the membrane-proximal signal was novel and led us to examine whether the two hydrophobic residues, met ~ and leu ~ , on the carboxy-terminal side of pro ~ were required. as shown in table hi , alteration of either met ~ or leu to alanine completely abrogated the activity of the membrane-proximal sorting signal in the ii cytoplasmic tall. we conclude, therefore, that one internalization signal in the ii cytoplasmic taft involves leu and ile s, whereas pro , met , or leu ~ are all important for the second signal. consistent with the distal signal being closely related to the cd , chain di-leucine signal, ile s could be modified to leucine without loss of activity. interestingly, the nonconservative mutation of pro ~ to leucine did not reduce the internalitation activity of the membrane-proximal signal (table hi) . ii is thought to be transported as an ot/~ii complex via the trans-golgi directly to an acidic endocytic compathaent where it dissociates and is rapidly degraded (cresswell, ; lamb et al., ; pieters et al., ) . to determine whether the cytoplasmic tail of ii was sufficient to target tr to this endocytic compartment and whether the non-tyrosine-based sorting signals were involved, metabolic pulse- * the lt--*'a, p"*a, and p~l mutations were introduced into the mutant iicr lp-*aa construct (internalization efficiency of % relative to the iicr chimera; see table ii) , and the l~'-~a, pls~a, p~s-"l, m ~a, and lit-*a mutations were introduced into the mutant iicr li~aa construct (internalization efficiency of % relative to the iicr chimera; see table ii ). internalization efficiencies of mutant iicr chimeras are expressed relative to the iic-r chimeras as described in table ii figure . rapid degradation of ii-tr chimeras in a post-golgi endocytic compartment. equivalent cell numbers of cef expressing either wild-type tr or the ii-tr chimeras, iicr, iier+xm, or iicr li,lp--*aa,aa were pulselabeled for rain with trans- s-label and chased for various periods of time. trs were then immunoprecipitated from post-nuclear supernatants and analyzed on sds-polyacrylamide gels as described in materials and methods. dried gels were exposed to pralashed xar film for two days (eastman kodak, rochester, ny). immunoprecipitates were quantitated on a model phos-phorimager (molecular dynamics). the data shown are from one of two similar experiments. chase experiments were performed. cef expressing either tr, iicr, iicr+r~ or the iicr li,lp---aa,aa mutant were pulse-labeled with trans- ss-label for min and chased for various lengths of time; tr and ii-tr chimeras were then isolated by immunoprecipitation and analyzed by sds-page (fig. ) . notably, both the iicr÷rm and iier chimeras were rapidly degraded with half-lives of < h and '~, h, respectively, similar to the rate of turnover of native ii in antigen-presenting cells. in contrast, the wild-type tr had a half-life of '~ h, consistent with previous estimates of wild-type human tr turnover rates in cef . the iicr÷a~ chimera was consistently degraded > -fold more rapidly than the iier chimera, implying that the ii transmembrane region influences intracellular trafficking. after h (fig. ) , the mr of tr and ii-tr chimeras increased to that of the mature glycoprotein and the molecules became partially resistant to endoglycosidase h digestion (data not shown). this indicates that the ii-tr chimeras traverse the golgi where glycosylation is completed and are degraded in a post-golgi compartment. the ii-tr chimera lacking the non-tyrosine-based signals was degraded more slowly, indicating that these signals are required for delivery to this compartment. further, the rate of synthesis of the iicx and iicr+m chimeras was -and -fold higher, respectively, than that of wild-type tr even though the chimeric receptors were expressed in lower amounts on the cell surface suggesting most of the chimeric molecules were trafficking by a direct intracellular route to the endocytic compartment where they were degraded. degradation of ii is partially inhibited by lysosomotropic agents such as chloroquine, monensin, and leupeptin (ilium and cresswell, ; nguyen and humphreys ; pieters et al., ; zachgo et al., ) . similarly, leupeptin significantly inhibited the degradation of the iicr+rm chimera, as indicated by the slower rate of disappearance of the intact molecule and the appearance of partial proteolytic products derived from the tr external domain (fig. ) , which is consistent with degradation occurring in an endocytic compartment. comparable results were obtained with chloroquine treatment (data not shown). ii-tr chimeras trafficking through the golgi complex were identified using electron microscopy immunocytochemistry. gold complexes bearing antibody specific for the external domain of tr were applied to cryosections and showed ii-tr distributed throughout the flattened cisternae of the golgi stack and within coated vesicles in the trans-golgi area (see fig. ). the intracellular and cell surface distribution of the ii-tr chimeras was determined by immunofluorescence microscopy. the iicr+rm chimera was found predominantly in intracellular vesicles, as indicated by the virtual lack of staining of non-permeabilized cells (fig. a) , compared to the strong punctate staining of permeabilized cells (fig. b) . cef expressing the iicr chimera exhibited more surface staining (fig. e) , but this chimera was also found predominantly in intracellular vesicles (fig. f) . the iicr li,lp ~ aa,aa chimera was expressed mainly on the cell surface ( fig. g) , but some staining of intracellular vesicles was still observed (fig. h) . two-color immunofluorescence studies comparing the intracellular distribution of the iicr+rm chimera with lep , an endogenous chicken lysosomal integral membrane protein fambrough, , ; mathews et al., ) , showed significant colocalization, indicating that the hybrid molecules traffic along the prelysosomal segment of the endocytic pathway (fig. c) . conversely, wild-type tr showed virtually no colocalization with lep (fig. a) . the iicv chimera also partially colocalized with lep (fig. b) , whereas the iicr li,lp-*aa,aa (fig. d) gave a staining pattern similar to that of wild-type tr. to quantitatively determine the altered trafficking of iicr÷m chimera, the steady-state distribution of tr and ii-tr chimeras was determined by gold label immunocytochemistry using antibody specific for the external domain of human tr. the extent to which the tr and iicr+m chimera were colocalized with lep , an antigen located predominantly in the prelysosomal branch of the endocytic pathway fambrough, , ) , was quantified by double labeling. as shown in fig. , the distribution of tr, iicr÷m, and lep is clearly restricted to the perimeter membranes of endocytic elements. quantitative analysis shows that while relatively low amounts ( . %) of tr can be found in lep -containing compartments, % of the labeled compartments in cells transfected with iicr+~m contain both iic~÷~ and lep . to determine whether fi-tr chimeras expressed on the cell surface traffic to the endocytic compartment where degradation occurs, cells were incubated with =i-labeled tf at °c for h to load the endocytic pathway with receptorligand complexes. the cells were then rapidly washed, and the reappearance of intact and degraded tf in the medium time (min) figure . degradation of t bound to ii-tr chimeras. equivalent cell numbers of cef expressing either wild-type tr, iicr, iict+m, or iicr li,lp-*aa,aa were preincubated in serum-free dme for rain at °c, and then incubated with i-labeled tf for h at °c. the cells were then washed and reincubated at °c in dme containing #g/ml unlabeled tf for various times. acid-soluble radioactivity (b) or acid-insoluble i-labeled tf (a) released into the medium, as well as surface-bound i-labeled "it (n) and internalized si-labeled tf ( ) were determined as described in materials and methods and are expressed as a percent of total radioactivity recovered. each point is the average of triplicate determinations from a representative experiment. was monitored by measuring tca insoluble and soluble radioactivity. as expected, the apo-tf released into the medium from cells expressing the wild-type tr was undergraded ( fig. ) , as tr-apo-tf complexes are efficiently recycled back to the cell surface through the sorting and recycling endosomal compartments hopkins and trowbridge, ; dunn et al., ; weissman et al., ; trowbridge et al., ) . in stalking contrast, ,x, % of the l~i-labeled tf released from cells expressing the iicr÷m chimera was degraded, implying that this fraction of the chimeric receptors traffic directly from the cell surface to an endocytic compartment where they are degraded. a significantly smaller fraction (~, %) of tf bound to the iicr chimera was degraded (fig. ) , indicating that the ii transmembrane region influences trafficking from the cell surface to this compartment. the ii-tr chimera lacking nontyrosine-based sorting signals was degraded only to a slightly higher extent than wild-type tr. to confirm the inference from these studies that the ii-tr chimeras traffic from the plasma membrane to an endocytic compartment where they are degraded, cef expressing iict, iicr+m, or iicr li,lp-*aa,aa were surface-iodinated and the rate of degradation of receptors transiently located at the plasma membrane directly determined. as shown in fig. , surface-iodinated iicr chimera (half-life • h) was degraded more rapidly than the chimera lacking non-tyrosine-based signals. surface-iodlnated iicr+~ was degraded at an even faster rate (half-life ~ - h). the iicr+~ chimera was degraded more rapidly than the iicv chimera, whether the chimeras were delivered directly from the trans-golgi to the endocytic pathway or via the cell surface, implying that the ii transmembrane region was influencing trafficking along both pathways. to determine whether the ii transmembrane region was sufficient to promote delivery of tr to the degradative endocytic compartment, a chimera consisting of the tr external domain and cytoplasmic tail and the ii transmembrane region (iim) was constructed. the ii~ chimera was expressed on the cell surface at a level of + % relative to the wild-type tr and was internalized with a relative efficiency of . + figure . degradation of i surface-labeled ii-tr chimeras. equivalent cell numbers of cef expressing either iicr, iict+i"m or iic~ li,lp-*aa,aa chimeras were surface-iodinated, and then incubated for the times indicated. the ii-tr chimeras were immunoprecipitated from post-nuclear snpernatants and analyzed on sds-polyacrylamide gels as described in materials and methods. dried gels were exposed to preflashed xar film (eastman kodak, rochester, ny) for one day (licr l ,lp-~a ,a and licr) or d (licr+rm). figure . li transmembrane region targets the tr to a post-golgi degradative compartment. equivalent cell numbers of cef expressing either wild-type tr or the iitm chimeric receptor were pulse-labeled with trans-ass-label and chased as described in the legend for fig. . trs were then immunoprecipitated from postnuclear supernatants and analyzed on sds-polyacrylamide gels as described in materials and methods. dried gels were exposed to preflashed xar film for two days (eastman kodak, rochester, ny), and immtmoprecipitates were quantitated on a model phosphorlmager (molecular dynamics). . %. importantly, pulse-chase analysis showed that the fully glycosylated iitm chimera was rapidly degraded (fig. ) with a half-life of ,, h, similar to that of the iicr chimera. by constructing -tr chimeras comprised of the cytoplasmic domain of ii, the external domain of the human tr, and the transmembrane region of either ii or tr, we have been able to take advantage of the quantitative assays available for measuring the rate of internalization of trs from the cell surface. this experimental strategy has allowed us to demonstrate that the ii cytoplasmic tall can promote rapid internalization of tr and to identify residues within the cytoplasmic tall of i that are required for this activity. our data indicate that the cytoplasmic tail contains two independent non-tyrosine-based sorting signals that promote rapid internalization. the same or overlapping signals are recognized in the trans-golgi and mediate direct sorting along an intracellular route to the endocytic pathway. one signal involves leu and ile s and appears to be analogous to the di-leucine lysosomal targeting signal identified in the cytoplasmic domain of the cd "y chain (letourneur and klausner, ) . deletion of residues - from the icr chimera cytoplasmic tall reduces the internalization activity to ,~ %, implying that, as for the di-leucine signal of the cd , chain (letourneur and klausner, ) , residues to the amino-terminal side of leuqle s are required for activity. the second sorting signal requires prols, met , and leu ~ , and, therefore, differs significantly from the dileucine and leucine-isoleucine motifs identified previously (letourner and klausner, ; johnson and kornfeld, a,b; ogata and fukuda, ) . nevertheless, all these signals, including both signals, contain two adjacent large nonaromatic hydrophobic residues and may, therefore, represent a family of related structural motifs specified by short linear arrays of amino acids that differ in specific sequence, analogous to tyrosine-based signals (trowbridge et al., ) . additional mutagenesis is required, however, to define these sorting signals more precisely, to identify any common features they may share, and to delineate any differences between signals recognized in the trans-golgi or at the plasma membrane. at present, we believe it most appropriate to refer to them as non-tyrosine-based signals, a neutral term that serves to distinguish them from tyrosine-based signals. our results are consistent with those of bakke and dobberstein ( ), who identified amino acids - in the ii cytoplasmic tail as important for targeting ii to the endocytic pathway. lotteau et al. ( ) also concluded that a sorting signal was located within residues - of the ii cytoplasmic tail. the distal signal involving leuqle s was not identified in either of these studies, however (see also romagnoli et al., ) , illustrating the difficulty of detecting multiple signals without quantitative assays. recently, in agreement with our data, pieters et al. ( ) have independently concluded that the ii cytoplasmic tail contains two sorting signals, one involving leutile , the other localized to residues - . the extent to which newly synthesized molecules, alone or complexed to mhc class ii c~/~ dimers, traffic to the endocytic pathway by an indirect route via the cell surface has been a matter of debate (peters et al., ; pieters et al., ; roche et al., ) . ii-tr chimeras trafficking to the plasma membrane can be quantitated because receptors appearing transiently at the cell surface can be labeled with exogenous tf. thus, the surface-accessible pools of the ii-tr chimeras can be determined by measuring ~ i-labeled tf bound to surface and internalized chimeric receptors under steady-state conditions. the surface-accessible pool of the iicr+rm chimera was only % of the wild-type tr pool despite the fact that the ,,o -fold increase in the degradation rate of the iicr+rm surface-accessible pool is offset by a similar increased rate of biosynthesis relative to wild-type tr (see materials and methods for details of the calculation). assuming that essentially all wild-type trs bind tf under steady-state labeling conditions, it can be calculated that only % of the iicr+rm chimera traffics via the cell surface, and, therefore, that > % of the chimera must be delivered by a direct intraceilular route to the endocytic compartment where it is degraded. similarly, • % of the iicr chimera is degraded without ever being displayed on the cell surface. the lysosomal membrane glycoprotein, lgp-a (lep , lamp- ), also traffics along the endocytic pathway to the lysosome by a direct intracellular route or via the cell surface, although which route is the major pathway is less clear and may depend upon expression level (williarns and fukuda, ; mathews et al., ; halter and mellman, ) . the fate of ii-tr chimeras which traffic via the plasma membrane can also be determined by loading them with exogenous tf and monitoring the reappearance of intact and degraded tf in the medium. the results of such studies indicated that ,~ % of ct+tm chimeras transiently displayed on the plasma membrane were degraded after internalization, whereas the remaining ,, % were recycled back to the cell surface. an even higher fraction (,, %) of the cr chimera recycled back to the cell surface. these results indicate that the ii-tr chimeras displayed on the plasma membrane recycle several times before being sorted to the endocytic compartment where they are degraded. in this respect, -tr chimeras expressed on the cell surface behave similarly to lysosomal acid phosphatase which is transported to the cell surface and undergoes multiple rounds of internalization and recycling before being transferred to the lysosome (braun et al., ) . studies on the trafficking of the cation-independent mannose- -phosphate receptor (m pr) have clearly established that a direct route from the trans-golgi to the endocytic pathway exists (kornfield and mellman ) . it has been shown that m pr are concentrated within clathrin-coated buds in the trans-golgi (rijnboutt et al., ; klumperman et al., ) and there is evidence suggesting that they are then delivered to an early part of the endocytic pathway, most probably the tr-containing endosome (ludwig et al., ; m resse and hoflack, ) . previous work on ii (guagliardi et al., ; pieters et al., ; zachgo et al., ; romagnoli et al., ) also suggest that entry into the endocytic pathway is via the tr-containing endosome. our data show that the cytoplasmic domain of ii contains two signals recognized in the trans-golgi that are the same or overlap with the internalization signals recognized by clathrin-coated domains at the plasma membrane. this relationship implies that the signals active in the trans-golgi are recognized by clathrin-based sorting machinery. it is also apparent, however, that for direct transfer of ii from the trans-golgi to the endocytic pathway to operate with full efficiency, additional signal information within the transmembrane region is required. the ii transmembrane region also increases the efficiency of trafficking from the plasma membrane to the degradative endocytic compartment. golgi retention signals have previously been localized to the transmembrane region (machamer and rose, ; swift and machamer, ; munro, ; nilsson et al., ; wong et al., ) . however, there is no precedent for the transmembrane region of an integral membrane protein influencing sorting in the trans-golgi or trafficking along the endocytic pathway. our evidence shows that ii, like the m pr (kornfield and mellman, ), enters the tr-containing endosome from both the trans-golgi and the plasma membrane. however, the data also indicate that the ii-tr chimeras which enter directly from the trans-golgi are delivered to a degradafive compartment more efficiently than those which enter from the plasma membrane. our recent observations on the tr-contalning endosome in cef (hopkins et al., ) show that it is a morphologically complex compatmient and suggest that it consists of at least two interconnected subcompartments in which internalized trs are processed at different rates. receptors entering the tr-containing endosome can either be recycled rapidly (tee in rain) or remain for more prolonged periods (t~r in min) before returning to the surface. the data on ii-tr chimeras can thus be explained by proposing that most of the chimeras which enter from the cell surface travel via the rapidly recycling pathway and that on each cycle only • % of them are sorted towards the lysosome. direct transfer from the trans-golgi, though more efficient, probably occurs via the subcompartment of the endosome which lies within its immediate vicinity in the pericentriolar area and through which tr are processed more slowly. identification of a sorting signal in the ii transmembrane region that complements the non-tyrosine-based sorting signals in the ii cytoplasmic tail provides a plausible explanation for two earlier observations. pieters et al. ( ) showed that the ii cytoplasmic tall was sufficient to target a heterologous protein, influenza neuraminidase, to the endocytic pathway. however, they noted significant differences between the localization of the ii-neuraminidase chimera and ii within the endocytic pathway and concluded that other sorting information may be contained within the ii transmembrane region and/or extracellular domain. in a second study, nilsson et al. ( ) reported that an ii chimera containing the cytoplasmic tail of - , galactosyltransferase, an enzyme normally localized to the trans-golgi by a retention signal within its transmembrane region, paradoxically had a subcellular distribution similar to wild-type ii (p ). the galactosyltransferase cytoplasmic tall contains a dileucine sequence which may function as a sorting signal and when complemented by the ii transmembrane region signal promote similar trafficking to wild-type ii. finally, dintzis and pfeffer ( ) observed that the m pr cytoplasmic tall was not sufficient to target the epidermal growth factor receptor to prelysosomes and suggested that additional sorting information in the m pr transmembrane region and/or extracellular domain may be required. the identification of the ii sorting signals required for targeting mhc class h molecules to the compartment where they encounter antigen may have practical implications since, in principle, it should be possible to target endogenously synthesized recombinant proteins containing ii sorting signals to this compartment for degradation and selective presentation by mhc class ii to t helper cells. abbreviations used in this paper: cef, chicken embryo fibroblasts; ct, cytoplasmic tail iicr, the cytoplasmic tail of ii ii transmembrane region m pr, mannose- -phosphate receptor mch class h-associated invariant chain contains a sorting signal for endosomal compartments the reaction of ferric salts with transferrin defective major histocompatibility complex class ii assembly, transport, peptide acquisition, and cd + t cell selection in mice lacking invariant chain expression role for intracellular proteases in the processing and transport of class ii hla antigens lysosomal acid phosphatase is transported to lysosomes via the cell surface transferrin receptor internalization sequence yxrf implicates a type i turn as the structural recognition motif for endocytosis intracellular class ii hla antigens are accessible to transferrin-neuraminidase conjugates internalized by receptor-mediated endocytosis the preparation and use of gold probes the mannose -phosphate receptor cytoplasmic domain is not sufficient to alter the cellular distribution of a chimeric egf receptor iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment immunogenic peptides bind to class ii mhc molecules in an early lysosomal compartment transport of the lysosomal membrane glycoprotein lgpl (lgp-a) to lysosomes does not require appearance on the plasma membrane intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma a cells internalization and processing of transferrin and the transferrin receptor in human carcinoma a cells trowbridge. . in migrating fibroblasts recycling receptors are concentrated in narrow tubules in the pericentriolar area and then routed to the plasma membrane of the leading lameua vectors and genes for improvement of animal strains nonacylated human transferrin receptors are rapidly internalized and mediate iron uptake role of the human transferrin receptor cytoplasmic domain in endocytosis: localization of a specific signal sequence for internalization a his-leu-leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent marmose -pbosphate receptor is necessary for the lysosomal enzyme sorting function the cytoplasmic taft of the marmose -phosphate/insulin-like growth factor-h receptor has two signals for lysosomal enzyme sorting in the golgi new procedure for dna transfection with polycation and dimethyl sulfoxide differences in the endosomal distributions of the two mannose -phosphate receptors primary structure of the gene for the murine ia antigen-associated in variant chains (ii). an alternatively spliced exon encodes a cysteine rich domain highly homologous to a repetitive sequence in thyroglobulin the biogenesis of lysosomes rapid and efficient site-specific mutagenesis without phenotypic selection invariant chain targets hla class ii molecules to acidic endosomes containing internaiized influenza virus a novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of cd chains lysosomal membrane dynamics: structure and interorganellar movement of a major lysosornal membrane glycoprotein cycling of the integral membrane glycoprotein, lep , between plasma membrane and lysosomes: kinetic and morphological analysis intracelhilar transport of class ii mhc molecules directed by invariant chain distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells a specific transmembrane domain of a coronavirns el glycoprotein is required for its retention in the golgi the pathway and targeting signal for delivery of the integral membrane glycoprotein lep to lysosomes phosphorylation of the cation-independent mannose -phosphate receptor is closely associated with its exit from the trans-golgi network transferrin receptors promote the formation of clathrin lattices sequences within and adjacent to the transmembrane segment of a- , -sialyltransferase specify golgi retention. embo (eur time course of intracellular associations, processing, and cleavages of ii forms and class ii major histocompatibility complex molecules the membrane spanning domain of - , -galactosyltransferas¢ specifies trans golgi localization lysosomal targeting of limp ii membrane glycoprotein requires a novel leu-lle motif at a particular position in its cytoplasmic domain biosynthesis of the human transferrin receptor in cultured cells four la invariant chain forms derive from a single gene by alternate splicing and alternate initiation of transcription/translation targeting ofa lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phoaphatase is necessary and sufficient for targeting to lysosomes segregation of mhc class ii molecules from mhc class i molecules in the golgi complex for transport to lysosomal compartments intracellular transport and localization of major histocompatibility complex class h molecules and associated invariant chain the mhc class iiassociatext invariant chain contains two endosomal targeting signals within its cytoplasmic tail identification of subcellular compartments involved in biosynthetic processing of cathepsin d invariant chain association with hla-dr molecules inhibits immunogenic peptide binding stable surface expression of invariant chain prevents peptide presentation by hla-dr cell surface hla-dr-invariant chain complexes are targeted to endosomes by rapid internalization relationship between invariant chain expression and major histocompatibility complex class ii transport into early and late endocytic compartments dna sequencing with chainterminating inhibitors intracelhilar distribution of the mhc class ii molecules and the as-social~l invariant chain i) in different cell lines a new method of preparing gold probes for multiple-labelling cytochemistry two forms of the la antigenassociated invariant chain result from alternative initiations at two in-phase augs a golgi retention signal in a membrane-spanning domain of coronavirus e protein dna sequence analysis with a modified bacteriophage t dna polymerase insulin elicits a redistribution of transferrin receptors in t -l edipocytes through an increase in the rate constant for receptor externalization invariant chain distinguishes between the exogenous and endogenous antigen presentation pathways a study of positive staining of ultrathin frozen sections human cell surface glycoprotein related to cell proliferation is the receptor for transferrin signal-dependent membrane protein trafficking in the endocytic pathway mice lacking the mhc class h-associated invariant chain exposure of k cells to anti-receptor monoclonal antibody okt results in rapid redistribution and enhanced degradation of the transferrin receptor accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail the -residue transmembrane domain of ~-galactoside o~ , -sialyltransferase is sufficient for golgi retention human major histecompatibility complex class ii invariant chain is expressed on the cell surface a block in degrad-tion of mhc class h-associated invariant chain correlates with a reduction in transport from endosome carrier vesicles to the prelysosome compartment we thank adele gibson for expert technical assistance and albert tousson for helpful discussions and advice on the immunofluorescence experiments. the lep monoclonal antibody used in these studies was obtained from key: cord- - c slfbp authors: nan title: effect of caffeine and reduced temperature ( degrees c) on the organization of the pre-golgi and the golgi stack membranes date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: c slfbp in the present study we have dissected the transport pathways between the er and the golgi complex using a recently introduced (kuismanen, e., j. jantti, v. makiranta, and m. sariola. . j. cell sci. : - ) inhibition of transport by caffeine at degrees c. recovery of the golgi complex from brefeldin a (bfa) treatment was inhibited by caffeine at reduced temperature ( degrees c) suggesting that caffeine inhibits the membrane traffic between the er and the golgi complex. caffeine at degrees c did not inhibit the bfa-induced retrograde movement of the golgi membranes. further, incubation of the cells in mm caffeine at degrees c had profound effects on the distribution and the organization of the pre-golgi and the golgi stack membranes. caffeine treatment at degrees c resulted in a selective and reversible translocation of the pre- and cis-golgi marker protein (p ) to the periphery of the cell. this caffeine-induced effect on the golgi complex was different from that induced by bfa, since mannosidase ii, a golgi stack marker, remained perinuclearly located and the golgi stack coat protein, beta-cop, was not detached from golgi membranes in the presence of mm caffeine at degrees c. electron microscopic analysis showed that, in the presence of caffeine at degrees c, the morphology of the golgi stack was altered and accumulation of numerous small vesicles in the golgi region was observed. the results in the present study suggest that caffeine at reduced temperature ( degrees c) reveals a functional interface between the pre-golgi and the golgi stack. t ransport inhibitors have been important tools in revealing the existence of functionally distinct compartments within the cell. site-specific perturbation of the endomembrane architecture often results in the accumulation of transported proteins and membranes at specific steps along the transport pathways. in many cases the use of inhibitors has made it possible to identify functions and operating components resident in the sites of question. reduced temperature has been widely used to dissect transport steps in the exocytotic transport pathway (for review see saraste and kuismanen, ) . specifically, incubation at ~ blocks the transport at the pre-golgi level (saraste and kuismanen, ) and has led to the characterization of p , a resident protein in the pre-and cis-golgi compartments (saraste et al., ) . the depletion of cellular atp has been shown to inhibit distinct steps in vesicular transport (balch et al., ; tartakoff, ) . the important role of calcium in vesicular traffic between the er and the golgi compartment has become evident from in vitro studies (beckers and balch, ; sambrook, ) . also, the perturbation of intraceuular ca +-levels blocks the movement of secretory proteins out from the rough er (ix)dish and kong, ). in addition, lowered extraceuular ph has been reported to arrest the transport of influenza virus hemagglutinin at the pre-golgi level (matlin et al., ) . the central role of gtp in the vesicular transport has become evident from in vitro studies where the nonhydrolyz-able gtp-analogue, gtp- ,-s, has been used to inhibit erto-golgi transport both in mammalian and yeast ceils (beckers and balch, ; ruohola et al., ) . recent localization of the small gtp-binding proteins rablb and rab to the membranes responsible for the er-to-golgi transport has further emphasized the role of gtp as a regulator of er-to-golgi membrane traffic (chavrier et al., ; plutner et al., ) . interestingly, there is also evidence for the participation of trimeric gtp-binding proteins in the regulation of membrane traffic (stow et al., ; barr et al., ) , thus revealing a link between signal transduction and the processes in the intracellular membrane traffic (for review see balch, ; barr et al., ) . okadaic acid, a phosphatase inhibitor, has been shown to inhibit er-to-golgi transport (davison et al., ) and result in the fragmentation and dispersal of the golgi apparatus (lucocq et al., ) , further linking mechanisms of signal transduction and the endomembrane traffic. brefeldin a (bfa) ~ (hiirri et al., ) has been a frequently used transport inhibitor in mammalian cells. bfa was first reported to block the transport of vesicular stomatitis virus g protein (takatsuki and tamura, ) . later it has been shown that bfa treatment results in the fusion of the golgi membranes with the er (doms et al., ; lippincott-schwartz et al., , a strous et al., ) . the almost immediate effect of bfa is to detach at least two of the coat components-/ -cop ) and a -kd protein (narula et al., )-from the golgi stack, allowing golgi membranes to form tubular extensions along microtubules that finally fuse with the er . this effect of bfa has been proposed to reveal a normally occurring retrograde membrane traffic from the golgi apparatus to the er (doms et al., ; lippincott-schwartz et al., ; klausner et al., ) . bfa has also been reported to have effects on the organization of the endocytic compartment; tgn and endosomes form large tubular structures after a bfa treatment (wood et al., ; lippincott-schwartz et al., b; reaves and banting, ) . we have previously shown that mm caffeine affects the intracellular transport of semliki forest virus (sfv) membrane glycoproteins . in this study we have characterized the temperature-dependent effect of caffeine on the distribution and organization of the golgi complex membranes. at ~ caffeine is shown to translocate p , a marker for the pre-and cis-golgi compartments, to the periphery of the cell. in contrast, under the same conditions a golgi stack marker, mannosidase ii (man ii), remains perinuclearly located. the redistribution of membranes induced by caffeine is clearly different from that induced by bfa. further, caffeine at reduced temperature did not inhibit the retrograde movement of golgi stack membranes observed in bfa-treated cells. taken together the results in the present study suggest that mm caffeine at reduced temperature can be used to dissect the interface between the cis-golgi and the golgi stack. the antibodies were kind gifts from the following persons: polyclonal antibodies to/s-cop (eage) (duden et al., ) were from drs. r. duden (embl, heidelberg, germany) and t. kreis (university of geneva, geneva, switzerland); polyclonal antibodies to man ii were from drs. m. farquhar (university of california, san diego, ca) and k. moremen (massachusetts institute of technology, cambridge, ma); monoclonal antibodies to man ii ( -kd protein) were from drs. b. burke (harvard medical school, boston, ma) and g. warren (imperial cancer research fund, london, uk) (burke et al., ) ; polyclonal antibodies to p were from dr. j. saraste (university of bergen, bergen, norway) (saraste and svensson, ) ; and polyclonal antibodies to sfv glycoproteins el and e were from dr. j. pe~nen (university of helsinki, helsinki, finland). tritc-and fitc-conjugated secondary antibodies to rabbit or mouse igg were purchased from dako a/s, glostrup, denmark and peroxidaseconjugated fab fragments to rabbit igg were purchased from biosys, compiegne, france. bhk- cells were grown on -mm-diam tissue culture dishes in dmem (gibeo brl, roskilde, denmark) in the presence of % fcs (gibeo brl), iu/mi of penicillin, and #g/ml of streptomycin (penstrep) (gibco brl). for immunofiuorescence experiments the cells were grown on -mm-diam microscope coverglasses. incubations in temperatures other than ~ were carried out in water baths in nahco -free mem (gibeo brl) supplemented with mm hepes and penstrep as described earlier (kuismanen and saraste, ) . the temperature-sensitive mutant tsq ofsfv (sfv ts- ) was used for all virus infections (keranen and k~rifiinen, ; saraste et al., ) . before infections, the cells were washed once with pbs and twice with serum-free mem (gibeo brl). infections were carried out for h at ~ before the cells were shifted to the restrictive temperature . ~ . depending on the experiment, the cells were then shifted to the appropriate chase temperature as indicated in the text. for [ s]methionine pulse-labeling experiments, the bhk- cells were infected as described above. before labeling, the cells were grown for min in a methionine-free mem (gibco brl) whereafter they were pulsed for rain with #ci of [ s]methionine/dish. after the appropriate chase times, the cells were washed once with ice-cold pbs followed by solubilization for rain on ice with net buffer ( mm tris, ph . , mm edta, % triton x- , . m nac , and mm pmsf). immunoprecipitations and endoglycosidase h (endo h) analysis were carried out as described earlier . the samples were analyzed under reducing conditions in a % sds-page according to laemmli ( ) . the gels were prepared for fluorography using amplify tm (amersham, aylesbury, uk). the film used for autoradiography was fuji rx (fuji photo co. ltd., tokyo, japan). at the end of the incubations, the cells were fixed for rain at room temperature with % paraformaldehyde in . m phosphate buffer, ph . , followed by three washes with pbs. the cells were then permeabilized for min with . % triton x- in pbs at room temperature followed by three washes with pbs containing . % bsa. the primary antibodies were always incubated for rain at ~ whereafter the cells were washed with pbs containing . % bsa. incubation conditions for the second antibodies were the same as for the primary antibodies. finally, the cells were washed and mounted in % glycerol, ph . , on microscope objective glasses and viewed with zeiss lab fluorescence microscope with x oil immersion objective. film used for photography was tmax asa (eastman kodak co., rochester, ny). the quantitation of the immunofluorescence experiments was performed by photographing random fields of the coverglasses, counting the cells in the photographs, and by observing the distribution of man ii in the counted cells. for every time point at least cells were counted. to study the intracellular distribution of p , the cells were fixed with a paraformaldehyde-lysine-periodate fixative (mclean and nakane, ) . the immunoelectron microscopy procedure followed was essentially that described by brown and farquhar ( ) . in conventional electron microscopy the cells were fixed for h at room temperature with a fixative containing % glutaraldehyde and % paraformaldehyde in mm cacodylate buffer, ph . , and postfixed with reduced % oso for h on ice. all samples were embedded in lx- (ladd research industries inc., burlington, vt) and were viewed with a jeol jem- ex transmission electron microscope operated at an acceleration voltage of kv. we have previously shown that at ~ mm caffeine blocks efficiently the movement of newly synthesized sfv glycoproteins out from the er . normally, in the absence of caffeine at ~ protein transport occurs to the level of the tmns-golgi (matlin and simons, ; saraste and kuismanen, ) . the exact figure . determination of the temperature threshold allowing sfv e to reach the golgi in the presence (a) or absence (b) of mm caffeine. bhk- cells were infected with sfv ts- temperature-sensitive mutant, labeled with [ s]methionine and chased in the presence or absence of mm caffeine at , , , , , , , and ~ for h. the cells were then solubilized, immunoprecipitated with monospecific polyclonal antibodies to sfv el, treated with endo h, and run in a % sds-page under reducing conditions. from a it can be observed that sfv e remains endo h sensitive in the presence of mm caffeine up to c. e becomes gradually resistant to endo h treatment at c and at higher temperatures, indicating the arrival of e to the golgi stack. in b the sensitivity of e to endo h treatment in the absence of caffeine at different temperatures is shown. in the absence of caffeine, e becomes endo h resistant already at ~ (b). p, pulse. temperature threshold that allows sfv e membrane glycoprotein to reach the golgi stack in the presence of caffeine was analyzed by following the maturation of sfv el-linked glycans to the endo h-resistant form. we also carried out immunofluorescence experiments to analyze the exact temperature that allows sfv glycoproteins to exit the er. these experiments together should further reveal whether caffeine at reduced temperature could arrest the er-to-golgi traffic in possible intermediate steps. bhk- cells were infected with sfv ts- , shifted to the restrictive temperature, labeled at h min after infection with [ s]methionine for min and chased for h at , , , , , , , and ~ with or without mm caffeine. e glycoproteins were then immunoprecipitated and analyzed for their sensitivity to endo h. in the presence of mm caffeine, sfv el-glycoproteins remained endo h sensitive up to ~ (fig. a) . at ~ and at higher temperatures e reached the golgi stack as indicated by the increasing amount of endo h-resistant form of e ( fig. a) . in control cells ( fig. b) , e reached the golgi stack already at ~ as indicated by the maturation of the proteins to the endo h-resistant form. in fig. b, it can be seen that e does not become completely resistant to endo h treatment even at ~ this, however, is a constant finding for sfv ts- and has been reported and discussed earlier (saraste and kuismanen, ; gahmberg et al., ) . since the acquisition of endo h resistance indicates only that e has reached the golgi stack, we next studied whether sfv glycoproteins can exit the er at temperatures between c and ~ and whether they are observed to accumulate in intermediate elements between the er and the golgi compartment. bhk- cells were infected with sfv ts- for h, shifted to restrictive temperature ( . ~ and grown there for h. the cells were then shifted to , , , , , , and c, and chased for h in the presence or absence of mm caffeine. to be able to follow the movement of presynthetized e and e , the chase mediums contained t~g/ml of cycloheximide. in cells incubated at , , , and ~ in the presence of caffeine, the sfv glycoproteins remained in a reticular structure ( fig. , b, c, d, and e, respectively) identical to the er labeling observed in the control cells fixed at the restrictive temperature ( . ~ (fig. a) . at ~ only a few cells showed perinuclear vesicular labeling (fig. f, arrow) in addition to er labeling. at ~ the number of cells displaying a perinuclear pattern of labeling increased significantly compared with the situation at ~ (fig. g) . as was previously reported , the sfv glycoproteins accumulated in perinuclear structures at ~ in the presence of caffeine (fig. h) . in control cells, the glycoproteins were transported to the trans-golgi region already at c as described earlier (marlin and simons, ; saraste and kuismanen, ) , and at higher temperatures increased labeling of the plasma membrane was observed (data not shown). these biochemical and morphological experiments suggest that in the presence of mm caffeine, sfv e glycoproteins are retained in the er up to c, whereas at c they reach the golgi stack. the experiments may further indicate that sfv glycoproteins are able, at least in some degree, to escape to peripheral vesicular structures already at c when still possessing clearly endo h-sensitive glycans. however, to further clarify this issue, morphological colocalization experiments with a marker to the pre-golgi membranes will be necessary. the observed inhibition of er-exit of sfv glycoproteins could be a result of not only the inhibition of membrane traffic, but also other factors, such as inhibition of proper folding and oligomerization, known to control the er-exit of individual proteins (rose and doms, ; helenius et al., ) . to test the effect of mm caffeine and ~ on the movement of cellular proteins, we followed the recovery of golgi membranes from bfa treatment using as a marker man ii, a resident golgi membrane protein. bfa has recenrly been shown to cause the redistribution of man ii and other resident golgi proteins as well as golgi membranes to the er in a reversible manner (doms et al., ; fujiwara et al., ; lippincott-schwartz et al., , a strous et al., ) . bhk- cells were treated for min with #g/ml of bfa at ~ to ensure complete translocation of man ii to the er. to follow the movement of man ii out of the er, bfa was removed with several washes of preconditioned medium ( ~ in the presence or absence of mm caffeine. thereafter, the cells were chased for , , and min at ~ with (fig. , d, f, and h, respectively) or without (fig. , c, e, and g, respectively) caffeine, and then fixed and prepared for immunofluorescence. in fig. a, the distribution of man ii is shown before any drug treatments. in cells treated for min with bfa at c, man figure . the exit of sfv membrane glycoproteins e and e from the er in the presence of mm caffeine at different temperatures. bhk- ceils infected with sfv ts- were incubated for min in the presence of mm caffeine and #g/ml of cycloheximide at (b), (c), (d), (e), (f), (g), and ~ (h), fixed and processed for immunofluorescence, a shows the distribution of glycoproteins at the restrictive temperature . ~ at temperatures , , , and ~ (b, c, d, and e, respectively), sfv glycoproteins can be found in the er. at c (f), a few cells possess also vesicular labeling ~ arrow). at ~ in more than half of the cells, sfv glycoproteins can exit the er and accumulate to perinuclear structures (g). at ~ virtually all cells show perinuclear localization of el (h). bar, /~m. ii is translocated efficiently (in % of the cells) to the er (fig. b) . as the cells were incubated for min in a caffeine-free medium at ~ (fig. c) , man ii appears in peripheral and more central elements (in % of the cells), and also in the er. after a -min chase in caffeine-free medium at ~ the er was efficiently emptied and man ii was found in a few peripheral elements, but was mainly perinuclearly located (fig. e) (in % of the cells). after min in caffeine-free medium, man ii was found perinuclearly located with no detectable labeling in the er (fig. g) (in % of the cells). when the cells were chased after the removal of the bfa in the presence of mm caffeine at ~ for min, man ii failed to exit the er (fig. d) (in % of the cells). after a - (fig. f ) or -rain (fig. h) chase in the presence of caffeine, man ii was still located in the er (in and % of the ceils, respectively). these results show that mm caffeine and ~ inhibited efficiently the movement of man ii, a resident golgi membrane protein, out from the er. this may further suggest that caffeine at reduced temperature inhibits the recovery of the golgi from the bfa treatment and thus inhibits the membrane traffic from the er to the golgi complex, figure . mm caffeine at ~ inhibits the movement of man ii out from the er. a shows the normal distribution of man ii in bhk- cells. bhk- cells were treated with bfa ( #g/ml) at ~ for rain (b) or treated with bfa for min followed by washout with a medium supplcmented with or without mm caffeine at ~ the cells were then chased for , , or rain at ~ in the absence (c, e, and g, respectively) or presence of mm caffeine (d, f, and h, respectively), fixed, and labeled with antibodies to man ii. incubation of the cells in the presence of caffeine at ~ inhibits the exit of man ii from th er after the removal of the bfa (d, f, and h). in control cells at ~ already rain after the bfa removal, man ii can be seen to be located in peripheral elements and in some degree in the er (c). after min, man ii is mainly in perinuclear elements (e). after a -rain chase in a caffeine-free medium at ~ man ii is located perinuclearly (g). bar, #m. since caffeine at ~ inhibited the er-to-golgi membrane traffic, it was of great interest to study whether mm caffeine would also inhibit the golgi-to-er traffic. recently, the fungal antibiotic bfa has been used to study the retrograde transport mechanism from the golgi complex to the er (doms et al., ; lippincott-schwartz et al., , a fujiwara et al., ) . we therefore studied the effect of m m caffeine on the bfa-induced redistribution of golgi membranes at ~ using man ii as a marker. bhk- cells were treated for , , or min with #g/ml of bfa and #g/ml of cycloheximide at ~ in the presence or absence of m m caffeine. in fig. a the distribution of the golgi marker man ii is shown before any drug treatments. when the cells were treated with /zg/ml of bfa for min at ~ a characteristic er staining pattern was observed (fig. b) . when the cells were treated with #g/ ml of bfa for rain at ~ (fig. c) in % of the cells man ii was found in a perinuclear localization, and in % of the cells, man ii was found to be translocated partly or completely to the er. after a -min chase at ~ in the figure . the bfa-induced recycling of man ii from the golgi complex to the er is not inhibited by caffeine at "c. bhk- cells were incubated with bfa ( #g/ml) at ~ (b), with bfa ( ~g/ml) and mm caffeine at ~ for , , and min (d,f, and h, respectively) or with bfa ( /~g/rnl) only at "c for , , and min (c, e, and g, respectively), fixed, and processed for immunofluoreseence microscopy. in all cases /tg/ml of cycloheximide was supplemented to prevent further synthesis of man ii. normal distribution of man ii in bhk- cells (a). in b man ii is observed to label the er after a -rain bfa treatment at "c. in caffeinetreated cells at ~ the bfainduced transloeation of man ii to the er is not inhibited (d, rain;f, rain; andh, min). when compared with the control cells (c, min; e, rain; and g, rain) the bfa-induced disappearance of the perinuclear golgi labeling of man ii seems to happen more slowly in the caffeinetreated ceils than in the control cells. bar, ~m. presence of bfa only (fig. e) , in % of the cells man ii had a perinuclear localization and in % of the cells it was found only or partly in the er. as the chase was continued to min at ~ in the presence of #g/ml of bfa (fig. g) , man ii was still found in % of the cells in perinuclear elements and in % of the cells only or partly in the er. when the cells were treated with /zg/ml of bfa supplemented with mm caffeine at ~ for min (fig. d) , man ii was found in the er in % of the cells and in the perinuclear localization in % of the cells. after a -min chase in the presence of caffeine, man ii was located partly or completely in the er in % of the cells and was perinuclearly located in % of the cells (fig. f ) . when the chase was carried out for min (fig. h) , man ii was found in the er in % of the cells and perinuclearly located in % of the cells. these results show that, after min in the presence of caffeine, #g/ml of bfa is able to translocate man ii to the er . -fold slower compared with control cells. after rain, however, there is no significant difference between caffeine-treated and control cells in the number of cells showing label in the er. however, in caffeinetreated cells the disappearance of the perinuclear labeling of figure . caffeine-induced translocation of p to the periphery of the cells. bhk- cells were grown for min at "c in the presence (a and b) or absence (c and d) of mm caffeine, fixed, and double-labeled with antibodies to p (a and c) and man ii (b and d). the distribution of p can be seen to change dramatically after a caffeine treatment at "c. in a, p can be found scattered through the cytoplasma in contrast to control cells at ~ (c), where p is concentrated in the perinuclear region of the cell in addition to some labeling in peripheral vesicles. when the localization of p is compared with the localization of man ii in caffeine-treated and in control cells incubated at ~ p can be seen to colocalize only randomly with man ii in caffeine-treated cells (a for p and b for man ii), but to colocalize a great deal in control cells (c for p and d for man ii). also, the localization of man ii seems not to be affected by the caffeine treatment at ~ (b). bar, #m. man ii seems to occur more slowly than in the control cells at the -min time point by a factor of . , at the -min time point by a factor of . , and at the -min time point by a factor of . . thus, these results indicate that caffeine does not inhibit the effect of bfa, but it does appear to slow it down somewhat. interestingly, it seems that during a min chase at ~ both in caffeine-treated and in control cells the bfa-induced translocation of man ii reaches a steady-state situation after a -min incubation, whereafter no significant chances in the distribution of man ii in the cells seems to occur. these results suggest that even though caffeine inhibits the movement of membranes from the er to the golgi region, it does not inhibit the bfa-induced retrograde movement of golgi membranes to the er at ~ since mm caffeine at ~ did not prevent the bfa-induced retrograde movement of golgi membranes to the er, it was of interest to study the distribution of the pre-golgi and the golgi membranes under conditions where er-exit was inhibited. when the anterograde but not the retrograde membrane traffic between the er and the golgi is inhibited by caffeine, two things could happen to the golgi membranes: all the golgi membranes would recycle to the er as in the case of bfa or part of the golgi would do so. as markers for golgi membranes we used p protein, known to be localized in various cell types in the pre-golgi elements and in cis-golgi (saraste et al., ; saraste and svensson ; lahtinen et al., ) , and a -kd integral golgi stack membrane protein burke et al., ) which has recently been shown to be identical with man ii (baron and garoff, ) . bhk- cells were treated for , , or min with or without mm caffeine at ~ fixed, and labeled with a polyclonal antibody to p and a monoclonal antibody to man ii ( kd). surprisingly, after min at ~ in the presence of figure . the caffeine-induced translocation of p to the periphery of the cells is reversible. bhk- cells were incubated in the presence ( ~ caffeine) or absence ( ~ c control) of mm caffeine at ~ for rain. cells incubated at ~ with caffeine were further incubated rain without caffeine at ~ (reversion), fixed, and doubled-labeled with antibodies to p and man ii. no drug shows the normal distribution of p and man ii in bhk- cells. in reversion, the perinuclear localization of p , similar to that in ~ control, is restored. bar, #m. caffeine, p was found scattered throughout the cytoplasm (fig. a) whereas man ii still remained concentrated in a perinuclear localization (fig. b) . incubation for an additional or min showed similar distribution of p and man ii (data not shown) (see also fig. for a -rain incubation). in control cells, incubated for rain at ~ p and man i (fig. , c and d, respectively) were found to colocalize largely, except for the peripheral vesicles positive only for p . fig. , a and c, demonstrates that p is translocated away from its normal perinuclear localization at ~ in the presence of mm caffeine. in caffeine-treated cells, the colocalization of p (fig. a) and man ii (fig. b) oc-curred only rarely. even though the majority of the p protein seemed to move to the periphery of the cell in the presence of caffeine at ~ some p -positive vesicles still colocalized with the perinuclear elements also labeled with antibodies to man ii. this could partly be due to an apparent colocalization of not the same but closely adjacent structures. these results suggest that mm caffeine at ~ induces a translocation of p -positive, pre-golgi, and cis-golgi membranes to the periphery of the cells leaving man ii-positive membranes perinuclearly located. to study the reversibility of the caffeine-induced translocation of p , bhk- cells were treated first with mm caffeine at ~ for min, followed by several washes with noncaffeine medium. after incubation for an additional min in the absence of caffeine at ~ the localization of p was compared with that at normal temperature ( ~ and to that at ~ without caffeine. in addition, the localization of p was compared with that of man ii in the same cells. in untreated cells, p is located perinuclearly with some labeling in small peripheral vesicles and er as reported earlier (saraste and svensson, ) (fig. , no drug) . in cells treated with caffeine at ~ for min (fig. , ~ caffeine), the bulk of p becomes distributed throughout the cytoplasm while man ii still remains perinuclearly located. on the contrary, in control cells without caffeine at ~ p was concentrated to the golgi area and colocalized with man ii (fig. , ~ control) . the normal localization of p was restored when the cells maintained for min in the presence of mm caffeine at ~ were further incubated for min at ~ in a caffeine-free medium (fig. , reversion) . the observed reversible translocation of preand cis-golgi marker protein to the periphery of the cell and the unaltered distribution of a golgi stack marker protein suggest that caffeine at reduced temperature can be used to manipulate pre-and cis-golgi elements without affecting the localization of golgi stack membranes. because of the effective inhibition of membrane traffic out of the er at ~ and the translocation of p to the periphery of the cell in the presence of caffeine, it was of great interest to study whether the morphology of the golgi stack is affected under the conditions used. bhk- cells were incubated for rain in the presence or absence of mm caffeine at ~ fixed, and processed for electron microscopy. in caffeine-treated cells the morphology of the golgi stack was altered. the golgi regions of the cells were filled with small ( - -nm-diam) vesicles (fig. a) . in addition, some tubular membranous structures, possibly representing remnants of the golgi stack, were also observed (fig. a, arrow) . on the contrary, control cells incubated for min at ~ possessed typical golgi stacks (fig. b) . taken together, caffeine at ~ does not only inhibit the er-exit of proteins and translocate p to the periphery of the cells but also induces a change in the morphology of the golgi stack. to study the distribution of p in the morphologically altered golgi region in caffeine-treated ceils, we performed immunoelectron microscopy experiments. in control cells, maintained at ~ for min, p was found in cis-golgi and also in cytoplasmic tubulovesicular elements (saraste et al., ; saraste and svensson, ) (fig. b, arrows) . when the cells were incubated for min at ~ in the presence of mm caffeine the morphology of the golgi stack was altered as described above (fig. a) . only a few p -positive vesicles could be found in the golgi area (fig. a, arrows) , thus supporting the immunofluorescence finding where the majority of the p seemed to get translocated to the periphery of the cells while some of it remained also located near the nucleus. it has been shown that the pre-golgi markers p in normal rat kidney cells (saraste and svensson, ) and p (schweizer et al., ) in m cells ) get translocated to the periphery of the cell after a bfa treatment. the distribution and morphology of p positive elements in bhk- cells after incubation at ~ in the presence of mm caffeine resembled that observed earlier in bfa-treated cells. even though incubation at ~ in the presence of caffeine did not result in the movement of a resident golgi stack marker to the er (see fig. b) , it did cause profound effects on the organization of the golgi complex (see fig. a) . ~cop, a protein known to be associated with the pre-golgi and the golgi stack membranes (duden et al., ) is released rapidly from the golgi membranes in cells treated with bfa . we therefore tested whether caffeine would have an effect on the localization of e-cop. bhk- cells were incubated at ~ in the presence or absence of mm caffeine, fixed, and processed for immunofluorescence microscopy. in fig. a, the distribution of e-cop is shown in cells before any treatments. ~cop has a strong perinuclear localization and also the labeling of cytoplasmic vesicular elements is observed (fig. a) . when cells were incubated at ~ for min in the absence of caffeine (fig. b) , the distribution of /~-cop was essentially the same as in control cells without any treatments (fig. a) . a -min incubation at ~ with mm caffeine did not have any detectable effect on the distribution of/~-cop (fig. c) . because of the strong perinuclear labeling of e-cop, possible changes in the distribution of pre-and cis-golgi elements were undetectable in the experiment. this result nevertheless suggests that bfa and caffeine affect the membranes of the golgi complex through different mechanisms. recent work by many laboratories has shown that the transport between the er and the golgi is mediated by transport vesicles. these transport vesicles appear to exit throughout the er and to concentrate perinuclearly along the microtubules in vivo (saraste and svensson, ) . only a few markers for these intermediate elements are known. two membrane proteins, p (saraste et al., ) and p (schweizer et al., ) , are localized to the intermediate elements and the cis-golgi. in addition, the kdel receptor, responsible for the retention of soluble er proteins (pelham, ; pelham, ) , is likely to be located at least in some degree to these membranes . there is accumulating knowledge on the com- in the golgi area in cells treated with caffeine at ~ bhk- ceils were incubated at ~ with (a) or without (b) mm caffeine, fixed, and processed for immunoperoxidase electron microscopy with antibodies to p & the alteration of the golgi morphology in caffeine-treated cells (a) can be observed. arrows mark the p -positive structures found in the golgi region both in caffeine-treated (a) and in control cells (b). in caffeine-treated cells (a) the amount of p - abeled membranes seems to be lower compared with that observed in control cells (b). bar, nm. figure . effect of caffeine at ~ on the localization of/ -cop in bhk- cells. bhk- cells were grown for min in the presence (c) or absence (b) of mm caffeine at ~ fixed, and processed for immunofluorescence with antibodies to fl-cop, a shows the normal distribution of fl-cop in bhk- cells. caffeine at reduced temperature does not seem to affect the distribution of fl-cop (c) compared with that in control cells incubated only at ~ (b) or to that observed in cells without any treatment (a). bar, #m. plexity of the molecular machinery involved in the er-to-golgi transport (for review see rothman and orci, ) , based on work with yeast mutants (novick et al., ) and cell-free transport assays (fries and rothman, ; balch et al., ) . in studies concerning the organization of the anterograde and the retrograde membrane traffic, the reversible effect of bfa on the organization of the golgi complex has provided important new information (pelham, ; klausner et al., ) . our previous report and the results in the present study suggest that caffeine is a new potential tool to dissect steps in the exocytic pathway. caffeine is known to inhibit the enzyme phosphodiesterase (butcher and sutherland, ) , thus resulting in elevated levels of camp inside the cells. forskolin, which is known to elevate camp levels inside cells, did not affect the movement of sfv glycoproteins (data not shown). more recently caffeine has been shown to release ca § ions from the intracellular stores other than the inositol triphosphate-sensitive store (burgoyne et al., ) . also the temperature dependence of the caffeine-induced inhibition of membrane traffic follows interestingly well the conditions used in studies of the gtp-mediated ca + release (gill et al., ) . if the effect of caffeine on the membrane traffic and on the calcium release are in fact related, this could indicate that the ability of the membranes in the er-to-golgi interface to recycle escaped, resident er proteins back to the er might be regulated by calcium. since, on the one hand, caffeine is known to affect the control mechanisms preventing cells in the s-phase to proceed in the mitotic cycle (schlegel and pardee, ; downes et al., ) , and, on the other hand, the onset of mitosis involves complex cascades of phosphorylations and dephosphorylations controlling the progress of cell division (lewin, ) , it could be possible that caffeine acts through the activation or deactivation of some kinase or phosphatase. also, it has recently been reported that okadaic acid, a phosphatase inhibitor, affects the er-to-golgi traffic (davidson et al., ) and the morphology of the golgi apparatus (lucocq et al., ) . in the present study, we have dissected the compartmental organization of the er-to-golgi interface using the transport inhibition exerted by mm caffeine at "c . the temperature threshold inhibiting the exit of sfv glycoproteins from the er was found to be - ~ at ~ sfv e reached the golgi stack as indicated by the endo h analysis. some variation, however, was observed in the temperature dependence between individual cells close to the threshold temperature. therefore, in other experiments, ~ in the presence of caffeine was used. to study whether the transport of cellular proteins is also affected, man ii was translocated with bfa to the er, whereafter bfa was washed away and the movement of man ii was followed in the absence or presence of mm caffeine at ~ as was the case with virus proteins, man ii was unable to exit from the er in the presence of mm caffeine at ~ during a -min chase. in contrast, during a -min chase in the control cells, er was efficiently emptied and man ii accumulated perinuclearly in the absence of caffeine at ~ it is therefore suggested that caffeine affects the movement of membranes between the er and the golgi complex. it is, however, necessary to follow the movement of other cellular proteins and, for example, lipids between the er and the golgi in the presence of caffeine at reduced temperature to obtain a clear picture of this matter. we have also tested that the er-exit inhibition by caffeine functions in sfv-infected normal rat kidney cells (data not shown), indicating that the observed effect is not specific for one cell type. an interesting question was whether caffeine and reduced temperature would inhibit also the bfa-induced retrograde movement of the golgi membranes. in the presence of mm caffeine at ~ bfa was able to redistribute at least part of the cellular man ii to the er with nearly similar kinetics than with bfa only at ~ interestingly, caffeine did seem to slow down the rate by which bfa treatment results in the disappearance of any recognizable golgi complex by a factor of ~ . . nevertheless, already after a -min chase in the presence of caffeine at ~ % of the cells showed labeling in the er and long tubular structures emanating from the golgi complex. these results show that caffeine does not inhibit the bfa-induced retrograde golgi-to-er transport, although the traffic in the opposite (er-to-golgi) direction, is inhibited. also, since caffeine at ~ did not inhibit the bfa-induced retrograde movement of golgi membranes, known to be inhibited by the gtp-,y-s (tan et al., ) , it seems unlikely that the effect of caffeine would be due to caffeine mimicking gtp- ,-s. electron microscopy study of the golgi structure revealed a vesicularization of the golgi stack in the presence of mm caffeine at ~ these small vesicles remained as tight clusters at the perinuclear site where the golgi complex is normally localized. this result is in agreement with the immunofluorescence observations with man ii. since the perinuclear localization of the golgi complex in the cell is dependent on the integrity of the microtubules (thyberg and moskalewski, ; kreis, ) , it is unlikely that caffeine at ~ would act through disrupting the microtubular network. this is further supported by the fact that also in caffeine-treated cells the bfa-induced tubular, microtubuledependent structures were able to form. one possible explanation for the caffeineinduced vesicularization of the golgi stack could be that when the transport from the er to the golgi complex is inhibited, while the vesicular recycling in the golgi stack is still occurring, the golgi stack ultimately becomes vesicularized. localization of marker proteins of the pre-golgi membranes and the golgi stack demonstrated an interesting, differential effect of caffeine on the distribution of these membrane compartments. the p -positive pre-and cis-golgi membranes lost their normal perinuclear localization while the localization of a golgi stack protein, man ii, remained unaffected. the normal distribution of p was restored when caffeine was removed. when the localization of p was studied with immunoelectron microscopy in caffeine-treated cells at ~ the typical tubulovesicular p -positive elements (saraste and svensson, ) were absent from the golgi region and p was found only to be localized in a few small vesicles associated with the clusters of vesicles in the golgi region. even though immunoperoxidase technique is by no means a quantitative method, when compared with the control cells, the number of p -positive membrane elements in the golgi region seemed to be lower in caffeine-treated cells at reduced temperature. the statement that p is translocated away from the perinuclear o-calization in caffeine-treated cells is based on the results obtained in the immunofluorescence experiments, where distribution of the bulk of p can be observed. overall, the immunoelectron microscopic finding is in agreement with the immunofluorescence result in fig. a. as suggested by the immunofluorescence experiments, the bulk of the p seems to be translocated away from the perinuclear localization in caffeine-treated cells. these resuits suggest that caffeine and reduced temperature are able to induce a selective retrograde translocation of pre-and cis-golgi membranes while the localization of the golgi stack membranes remains unaffected. this was further confirmed by the unaffected localization of/~-cop protein. possible explanation to the observed loss of the perinuclear labeling of p could be that in the presence of caffeine the pre-and cis-golgi elements are induced to move toward the periphery of the cell. another possibility is that as the membrane traffic from the er is inhibited and at least the bfa-induced retrograde movement of membranes is not inhibited, p could finally be recycled to the er, being unable to exit from there. whatever the solution turns out to be, caffeine seems to make it possible to manipulate the membranes operating in the er-to-golgi traffic in a novel way. interestingly, lewis and pelham ( ) have recently shown that, by overexpressing the ligand for the kdel receptor, the bulk of the receptor is translocated to the er. to obtain a more detailed picture of the effect of caffeine and reduced temperature on the membranes operating between the er and the golgi complex, it is necessary to study whether the distribution of other markers for these membranes such as p (schweizer et al., ) , corona virus e glycoprotein (machamer et al., ) , and rab (chavrier et al., ) , is also affected. caffeine at reduced temperature on the one hand inhibited the exit of proteins from the er and on the other hand induced a selective translocation of pre-and cis-golgi membranes to peripheral locations; this is in contrast to the bfa action which seems to translocate unselectively all golgi membranes from their normal perinuclear location (lippincott-schwartz et al., , a doms et al., ; fujiwara et al., ; strous et al., ) . further, caffeine did not detach h-cop from the golgi membranes nor did it translocate man ii to the er. thus, the effect of caffeine on the organization of the membranes between the er and the golgi complex is clearly different from that induced by bfa. the different effect of bfa and caffeine on the golgi complex would indicate that the golgi complex is composed of two subsets of homotypic membranes differentially sensitive to caffeine treatment. whether this is also reflected in the differences of the coat structures of these membranes remains to be seen. from g minor to g major reconstitution of the transport of protein between successive compartments of the golgi measured by the coupled incorporation of n-acetylglucosamine atp-coupled transport of vesicular stomatitis virus g-protein between the endoplasmic reticulum and the goigi mannosidase ii and the -kd golgispecific antigen recognized by monoclonal antibody fc are the same dimeric protein trimeric g-proteins of the trans-golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules trimeric g proteins and vesicle formation calcium and gtp: essential components in vesicular trafficking between the endoplasmic reticulum and golgi apparatus immunoperoxidase methods for the localization of antigens in cultured cells and tissue sections by electron microscopy distribution of two distinct ca +-atpase like proteins and their relationships to the agonist-sensitive calcium store in adrenal chromaffin cells a monoclonal antibody against a k golgi membrane protein adenosin y, '-phosphate in biological materials localization of lower molecular weight gtp-binding proteins to exocytic and endocytic compartments evidence for the regulation of exocytic transport by protein phosphorylation brefeldin a redistributes resident and itinerant golgi proteins to the endoplasmic reticulum dissociation ofa -kd peripheral membrane protein from the golgi apparatus is an early event in brefeldin a action caffeine overcomes a restriction point associated with dna replication, but does not accelerate mitosis t-cop, a kd protein associated with non-clathrin coated vesicles and the golgi complex, shows homology to/ -adaptin transport of vesicular stomatitis virus glycoprotein in a cell-free extract brefeldin a causes disassembly of the oolgi complex and accumulation of secretory proteins in the endoplasmic reticulum efficient transport of semliki forest virus glycoproteins through a golgi complex morphologically altered by uukuniemi virus glycoproteins ca + release from endoplasmic reticulum is mediated by a guanine nucleotide regulatory mechanism die konstitutiou von brefeldin a the endoplasmic reticulure as a protein-folding compartment isolation and characterization of temperature-sensitive mutants from semliki forest virus brefeldin a: insights into the control of membrane traffic and organelle structure role of microtubules in the organization of the golgi apparatus low temperature-induced transport blocks as tools to manipulate membrane traffic effect of caffeine on intracellular transport of semliki forest virus membrane glycoproteins cleavage of structural proteins during the assembly of the head of bacteriophage t characterization of a kda cis-golgi protein in pancreatic exocrine cells driving the cell cycle: m phase kinase, its partners, and substrates a human homologue of the yeast hdel receptor ligand-induee.d redistribution of a human kdel receptor from the golgi complex to the endoplasmic reticulure the erd gene determines the specificity of the luminal er protein retention system rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence of membrane cycling from golgi to er microtubule-dependent retrograde transport of proteins into the er in the presence of brefeldin a suggest an er recycling pathway forskolin inhibits and reverses the effect of brefeldin a on golgi morphology by a camp-independent mechanism brefeldin a's effects on endosomes, lysosomes and tgn suggest a general mechanism for regulating organelle structure and membrane traffic perturbation of cellular calcium blocks exit of secretory proteins from the rough endoplasmic reticulum antibodies to golgi complex and to endoplasmic reticulum okadaic acid induces golgi apparatus fragmentation and arrest of intracellular transport the el glycoprotein of an avian coronavirus is targeted to cis-golgi complex reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycolysation reduced extracellular ph reversibly inhibits oligomerization, intracellular transport, and processing of the influenza hemagglutinin in infected madin-darby canine kidney cells periodate-lysine-paraformaldehyde fixative. a new fixative for immunoelectron microscopy identification ofa -kd, brefeldin-sensitive protein on golgi membranes identification of complementation groups required for post-translational events in the yeast secretory pathway evidence that luminal er proteins are sorted from secreted proteins in a post-er compartment control of protein exit from the endoplasmic reticulum multiple targets for bfa rablb regulates vesicular transport between the endoplasmic reticulum and successive golgi compartments perturbation of the morphology of the trans-oolgi network following brefeldin a treatment: redistribution of a tgnspecific integral membrane protein regulation of protein export from the endoplasmic reticulum molecular dissection of the secretory pathway reconstitution of protein transport from the endoplasmic reticulum to the golgi complex in yeast: the acceptor golgi compartment is defective in the sec mutant the involvement of calcium in transport of secretory proteins from the endoplasmic reticulum pre-and post-oolgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the golgi complex distribution of the intermediate elements operating in er to golgi transport semliki forest virus mutants with temperature-sensitive transport defect of envelope proteins antibodies to rat pancreas golgi subfractions: identification of a -kd cis-golgi protein caffeine-induced uncoupling of mitosis from the completion of dna replication in mammalian cells identification, by a monoclonal antibody, of a -kd protein associated with a tubulo-vesicular compartment at the cis-side of the golgi apparatus a heterotrimeric g protein, g~_ , on golgi membranes regulates the secretion of a heparan sulfate proteoglycan in llc-pk~ cells brefeldin a induces a microtubule-dependent fusion of galactosyltransferase-containing vesicles with the rough endoplasmic reticulum brefeldin a, a specific inhibitor of intracellular translocation of vesicular stomatitis virus g protein: intracellular accumulation of high-mannose type g protein and inhibition of its cell surface expression retrograde transport from the golgi region to the endoplasmic reticulum is sensitive to gtp'rs temperature and energy dependence of secretory protein transport in the exocrine pancreas microtubules and the organization of the golgi complex brefeldin a causes a microtubule-mediated fusion of the trans-golgi network and early endosomes many thanks go to drs. j. saraste and k. hedman for advice to j. j/intti with the immunoelectron microscopic technique. we thank jorma wartiovaara, head of the department of electron microscopy, university of helsinki, for letting us use the facilities of his department. virpi m/ikiranta and mervi lindman are acknowledged for excellent technical assistance. jaakko saraste, mikael rehn and camilla schalin-j~intti are acknowledged for critically reading the manuscript.this study was supported by the academy of finland, the alfred kordelin foundation, the finnish cultural foundation, magnus ehrnrooth foundation, and the university of helsinki. key: cord- -wyipzwo authors: gleeson, paul a.; teasdale, rohan d.; burke, jo title: targeting of proteins to the golgi apparatus date: journal: glycoconj j doi: . /bf sha: doc_id: cord_uid: wyipzwo the golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. resident golgi proteins must have localization signals to ensure that they are targeted to the correct golgi compartment and not swept further along the secretory pathway. there are a number of distinct groups of golgi membrane proteins, including glycosyltransferases, recyclingtrans-golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. recent studies indicate that there are a number of different golgi localization signals and mechanisms for retaining proteins to the golgi apparatus. this review focuses on the current knowledge in this field. the survival of a cell depends on maintaining the integrity of the intracellular organelles. this feat is achieved by highly selective sorting and accurate transport of proteins to their correct destinations. over the past few years the dissection of the molecular machinery for targeting and localization of proteins, in particular proteins of the secretory pathway, has been studied vigorously. defined sequence motifs have been identified on proteins which can act as 'address labels'. the golgi apparatus represents the 'hub' of the secretory pathway where intense membrane traffic is controlled. this organelle not only co-ordinates the sorting of newly synthesized proteins but is also responsible for the control of posttranslational modifications, in particular glycosylation. a fundamental question currently being addressed in cell biology is how the golgi apparatus is organized to achieve these demanding functions and how it maintains its structural integrity in spite of the intense membrane traffic which enters and leaves this organelle. this review will focus primarily on our understanding of the molecular signals and mechanisms for the retention of resident golgi proteins. the golgi apparatus is a highly complex and dynamic organelle, which has been difficult to define in three-dimensional terms [ ] . it consists of a number of (reflecting in part the cholesterol content), ph, and most importantly in the populations of resident proteins which they contain. however, detailed biochemical characterization of the individual cisternae is lacking as the current methods are inappropriate to allow their purification. newly synthesized proteins are transported sequentially from the er to the golgi and then to their final destination. the transport of newly synthesized proteins from the endoplasmic reticulum to golgi cisternae, between adjacent cisternae within the golgi stack, and from golgi cisternae to various destinations is mediated by vesicles shuttling between donor and recipient compartments. vesicles bud from one compartment and then target and fuse with the next compartment [ , ] . an increasing number of structural and regulatory components have been identified which are involved in the orchestration of the complex and intriguing processes of budding, specific targeting, docking and fusion [ ] [ ] [ ] . some of the components of this machinery are localized only to golgi membranes and are thought to be specific for membrane transport through and from the golgi apparatus. the restricted location of these components indicates the presence of specific localization signals. until recently it was widely believed that, if correctly folded, newly synthesized proteins are transported from the endoplasmic reticulum, through the golgi stack to the tgn without the requirement for a specific transport signal [ , ] . however, the concept of 'bulk flow' of membrane proteins from the er has recently been challenged by the finding that vesicular stomatitis virus g glycoprotein is significantly concentrated during export from the er [ ] . nonetheless forward transport, at least from the golgi apparatus to the cell surface, appears to constitute a signal-independent or default pathway. despite this extensive flux of proteins, it is imperative that the golgi apparatus maintains the set of resident proteins which define its unique structural and functional properties. thus, it would appear that newly synthesized golgi membrane proteins must stop at the correct cisterna, or subcompartment, and be prevented from being swept into transport vesicles that bud from the dilated rims of the cisternae. clearly, specific localization signals are required for retention of proteins which reside in the golgi apparatus. what do we know about the sorting signals and mechanisms for the localization of non-golgi proteins within the secretory and endocytic pathways? a number of sorting signals have been found associated with the cytoplasmic domains of membrane proteins [ ] . for example, short tyrosine-containing peptide motifs found on cytoplasmic domains direct the sorting of proteins from the plasma membrane via the receptor mediated endocytosis pathway [ ] and the transcytosis of basolateral proteins to the apical surface of certain polarized cells [ ] , while a dileucine-containing peptide motif directs the transport of man- -p receptors from the tgn to the late endosomes [ , ] . these cytoplasmic domain sorting signals mediate interactions with coat structures of budding vesicles and thereby allow the selective vesicular transport of these membrane proteins between a variety of compartments [ ] . much progress has been made in defining the retention signals for resident er proteins. targeting signals have been identified for both soluble and membrane-bound proteins residing in the er. a specific retention signal, comprising the carboxy terminal sequence kdel/hdel, has been identified for a number of resident soluble er proteins [ , ] , and a receptor for this retention sequence has been identified [ ] [ ] [ ] . retention of these soluble er proteins is mediated by a receptor-based salvaging mechanism, whereby escaped kdel-bearing proteins are retrieved from a post-er compartment by a recycling kdel receptor [ ] . for membrane proteins of the e r a double lysine motif (kkxx) at the cytoplasmically exposed carboxy terminus of certain type i membrane proteins has been shown to specify er residence [ ] . for type li membrane er proteins, a related double arginine motif at the cytoplasmically orientated amino terminus has been identified [ ] . interestingly, the localization of ergic- (p ), a type i membrane protein of the intermediate compartment or cgn, requires a kkxx er retention motif, again suggesting that the cgn may be an extension overall, the localization signals of non-golgi proteins are hydrophilic motifs located on either the cytoplasmic or luminal domains of the protein, and some of these signals have been shown to interact specifically with receptor molecules or with protein coats of budding vesicles. it is now clear from recent studies that there are a number of distinct types of golgi localization signals. based on these localization signals and other biochemical features the resident proteins of the golgi apparatus can be divided into five groups (table ) . they are all membrane proteins. interestingly no soluble resident golgi proteins have been identified within the lumen of the golgi apparatus, which probably indicates that the mechanisms for retaining proteins to this organelle are restricted to membraneassociated proteins. the five groups are described individually as the mechanism of golgi localization may be unique for each group. the golgi apparatus plays a key role in the glycosylation of newly synthesized membrane and secreted proteins [ , ] . based on the exquisite specificities of the currently defined glycosyltransferases [ , ] , the synthesis of all the known carbohydrate chains of glycoconjugates must require in the region of - different glycosyltransferase enzymes distributed throughout the golgi stack. however, very little is known about the structural organization of these integral membrane enzymes within the membranes of the golgi cisternae and the signals which define their localization within the golgi are only now beginning to be defined. a number of glycosyltransferases have restricted distributions within the golgi apparatus, notably /~ , galactosyltransferase (/? , galt) (trans-golgi) [ ] [ ] [ ] , :~ , sialyltransferase (e , st) (trans-golgi and trans-golgi network) [ , ] and n-acetylglucosaminyltransferase i (glcnacti) (medial-golgi) [ , , ] . furthermore, simultaneous immunogold localization of /~i, galt and gtcnacti in the same golgi apparatus, confirms that these enzymes have distinct, though overlapping, distributions [ ] . from the purification tables ofgolgi glycosyltransferases, it is clear that individual transferases constitute only a minor percentage of the proteins of the cisternae in which they reside. for example, glcnacti constitutes about ~ of medial-specific golgi membrane protein in rabbit liver [ ] . however, in view of the estimated number of golgi glycosyltransferases, collectively the glycosyttransferases of each golgi cisterna may represent a very significant proportion, if not the bulk, of the resident membrane proteins. numerous mammalian glycosyltransferases have been cloned and sequenced (see reviews [ ] [ ] [ ] [ ] ). individual glycosyltransferases are highly conserved across species, for example rabbit glcnacti shares ~o and ~ amino acid sequence identity with human and mouse glcnacti, respectively [ ] . but comparison of the amino acid sequences between the glycosyltransferases has revealed only isolated cases of sequence similarity. for example, there is a high degree of sequence similarity between blood group a and b glycosyltransferases [ ] , which are products of two alleles, and between a number of e ( ) fucosyltransferases [ ] . furthermore, a conserved motif has been identified in the catalytic domain of cloned sialyltransferases (the 'sialylmotif') [ ] . however, these examples of sequence similarity are the exceptions and, overall, little sequence similarity has been detected between different glycosyltransferases. this is dramatically illustrated by a lack of obvious amino acid similarity between the sequences of four different glcnac transferases involved in the synthesis of the outer antennae of complex n-glycans, namely glcnacti [ , ] , glcnactii [ ] , glcnactiii [ ] , and glcnactv [ ] . one would expect there to be structural similarity between the catalytic sites of these glcnac transferases but this has not been detected from their amino acid sequences. thus, comparison of the primary structures of golgi glycosyltransferases has not revealed a potential golgi localization motif. there is, however, a striking similarity in the domain structure of these golgi enzymes. all golgi transferases cloned to date are nin/cou t (type ii) membrane proteins containing a single hydrophobic membrane-spanning domain ( - amino acids) which also serves as a non-cleavable signal sequence. each has a short n-terminal cytoplasmic domain (many have less than amino acids), and a large carboxyl-terminal catalytic domain situated in the lumen of the golgi apparatus. the catalytic domain is linked to the transmembrane domain by a loosely defined 'stem' region which may play a role in positioning the catalytic domain away from the lipid bilayer facilitating access to the substrate. over the past years a number of groups have attempted to identify the targeting signal responsible for the localization of gtycosyltransferases. three glycosyltransferases have been extensively examined, namely cd, st, /~i, galt, and glcnacti. these enzymes are residents of the tgn, tgn/trans-gotgi, and medial-golgi respectively. a common strategy has been employed by all groups to identify a putative golgi retention signal(s) by analysing the localization, in transfected mammalian cells, of hybrid molecules containing limited sequences derived from golgi glycosyltransferases. in all cases, the membrane-spanning domains of the golgi glycosyltransferases have been shown to direct, at least partial localization of hybrid molecules to the golgi apparatus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . indeed it has been further demonstrated that the transmembrane domain of/?i, galt and glcnacti can specifically localize hybrid proteins to the trans and medial cisternae, respectively [ , ] . however, a number of studies have shown that sequences flanking the transmembrane domain also play auxiliary roles in mediating golgi localization [ , [ ] [ ] [ ] . these studies all agreed that the transmembrane domain plays a central role in the targeting and localization of resident golgi glycosyltransferases. this was an unexpected finding as localization signals up until then were hydrophilic glycopinion mini-review regions of the cytoplasmic or luminal domains. the involvement of a hydrophobic stretch of amino acids in targeting indicated a unique mechanism for the localization of these resident golgi proteins. these initial studies were based to a large extent on the premise that a discrete region, or motif, was responsible for the golgi localization signal. further studies have indicated that, although the transmembrane domain is relevant, the situation is far more complex than at first appreciated. figs - summarize graphically the regions of glycosyltransferases that have been examined and their ability to direct reporter molecules to the golgi apparatus. collectively, a large number of constructs have now been analysed. figs - include many of these constructs, however, it is by no means all-inclusive. those selected are the most instructive and highlight the complexity of the situation. there is considerable variability in the results obtained between groups, even when comparing the same glycosyltransferase. for example, wong et al. [ ] reported that the transmembrane domain of e , st resulted in very efficient golgi localization of a hybrid molecule, whereas a hybrid construct of munro [ ] , containing the equivalent c~ , st domain, resulted in considerable leakage to the cell surface ( fig. ) . site-directed mutagenesis of residues of the transmembrahe domain of // , gait, in the context of hybrid molecules, suggested that uncharged polar residues are critical for the ability of these hydrophobic domains to mediate golgi retention [ ] . however, a number of other studies have indicated that considerable alterations can be made to the transmembrane domain of the native enzymes without abolishing gotgi retention. for example, colley et al [ ] showed that sequential replacement of - amino acid blocks of the transmembrane domain of e , st had no effect on golgi localization. further, munro [ ] made the striking demonstration that the transmembrane domain of an e , st hybrid protein (containing the stem and tail of e , st) can be totally replaced by a poly-leucine sequence of similar length without adversely affecting golgi retention. munro [ ] also reported that the length of the polyteucine segment appeared to be important in maintaining efficient golgi localization as a transmembrane domain of leucine residues showed leakage to the cell surface. in contrast to this apparent length requirement, dahdat and colley [ ] replaced the amino acid transmembrane domain of e , st with the long amino acid transmembrane domain from influenza neuraminidase without any apparent disruption of the retention signal. the difficulty in defining the structural elements associated with transmembrane domains in golgi localization has been highlighted by a recent study by low et al. [ ] who demonstrated that swapping the transmembrane domains of two cell surface proteins resulted in hybrid molecules which either accumulated in the golgi or were retarded in transport through the golgi apparatus. gol jeell smc~ace (m) o~ ~ ce~ surface (n,w) -_ ~. t aa i stem aa gotgi ~w) ce~ surface (lv~ go~ce~ surface (d) thus, although both native proteins are transported efficiently to the cell surface, swapping the transmembrane domains of these two proteins altered golgi to cell surface transport. these investigators concluded that the hydrophobic transmembrane domain in relation to its charged flanking sequences is important in transport from the golgi apparatus to the cell surface. for both ct , st and glcnacti it is clear that regions flanking the transmembrane domain can augment the efficiency of golgi localization. for example, additional sequences from the stem region and the cytoplasmic tail increase the efficiency of golgi localization of ~ , st and glcnacti [ , , ] , although the tail and/or stem of ~ , st and glcnacti alone is not capable of retaining a reporter molecule to the golgi - , , ] , and removal of the stem region from wild type c~ , st [ ] or / , gait [ does not disrupt golgi localization. although the potential of the stem region has been addressed in a number of studies, the potential role of the catalytic domain has been overlooked in most studies. yet, removal of the cytoplasmic tail and stem, and considerable alteration of the signal/anchor domain, still allowed hybrid ~ , st molecules containing the catalytic domain to be golgi localized [- , ] . it should be noted that the membrane flanking sequences, comprising short stretches of charged residues, were maintained in the latter constructs which may also be an important factor in localization. the contribution of the membrane flanking sequences of /~i, galt to golgi localization is not known. comparison of the results of all these studies is not straightforward and there are a number of factors which may account for the apparent lack of agreement between them. first, as yet there is no direct evidence that glycosyltransferases localized to different golgi subcompartments are retained by identical mechanisms. there may be subtle differences between the localization signals which specify residency in medialand trans-cisternae and in the tgn. second, in the majority of studies sequences involved in golgi localization have been identified by their ability a. third, the definition of the transmembrane domain has varied from group to group; in some cases the transmembrane domain has been defined as a stretch of hydrophobic residues, excluding charged residues necessary for anchoring membrane proteins within the lipid bilayer [ , ] , whereas in other studies, two or three charged amino acids on either side of the hydrophobic stretch have been included in the sequence defined as the transmembrane domain [ ] . fourth, the appearance of hybrid or mutant glycosyltransferases at the cell surface has been frequently used as a measure of disruption of golgi localization. however, the majority of groups have only used fluorescence microscopy to compare levels of cell surface expression. fluorescence microscopy is relatively insensitive and comparisons are at best qualitative. only a few studies have employed the more sensitive and quantitative technique of flow cytometry to compare levels of cell surface expression between constructs. fifth, the expression levels of the hybrid constructs vary between and within studies. we believe this to be a critical factor in assessing these results. whereas many groups have shown that the native gtycosyltransferases can be expressed at very high levels without saturation of the golgi retention mechanism [ , , , ] [ ] . of expression have been observed between different constructs. in addition, for any one construct there is a wide range of expression within one transfection experiment, and indeed dahdal and colley [ ] noted that surface expression of some hybrid proteins appeared to be related to the level of expression in the cells. in our studies on fll, galt, we have observed that a construct expressed transiently in cos cells showed a different intraceltular distribution to that of the same construct stably expressed in mouse l cells. replacement of the amino acid transmembrane domain of /?i, galt with the amino acids from the transferrin receptor resulted in abundant cell surface expression in cos cells, and with very little detected in the golgi region, whereas in stable l cells, which expressed the hybrid molecules at a to -fold lower level, substantial amounts of the hybrid molecules were specifically retained within the golgi apparatus [ ] . clearly, stable clones expressing low levels of the hybrid molecules are likely to be more informative. sixth, several groups have identified glycosyltransferase sequences which are capable of conferring golgi localization upon reporter proteins, but have neglected to assess the role played by these sequences within the context of the full length enzyme. strategies involving reporter proteins are useful for determining the minimum sequence requirements for golgi localization of hybrid molecules. however, it does not necessarily follow that a sequence which is sufficient to confer golgi localization upon a reporter molecule is the only sequence involved in retention of the native enzyme. this point was illustrated earlier with the golgi localization of glycosyltransferases bearing substituted transmembrane domains (figs - ) . furthermore, most of the studies which have made substitutions of the native enzyme have not assessed the effect of those substitutions on enzyme activity, thus it is unclear whether the structure of the luminal catalytic domain has been perturbed in these studies. in our recent study on glcnacti [ ] we have attempted to address many of these problems and have assessed the relative contribution of the cytoplasmic tail, transmembrane domain, and catalytically active luminal domain in medial-golgi localization. stable l cell clones expressing hybrid molecules were generated, and clones which expressed equivalent amounts of hybrid proteins were selected for analyses. all hybrid molecules expressing the luminal domains of glcnacti were catalytically active, inferring a native structure for this domain. cellular localization was assessed by fluorescence microscopy, immuno-electron microscopy and flow cytometry. overall our study showed that each of the three gicnacti domains contributes significantly to medial-golgi localization. soluble, catalytically active forms of /?i, galt and ~ , st, which lack the cytoplasmic tail, transmembrane domain, and luminal stem region, have been detected in body fluids and are thought to be derived from the membrane-bound forms by proteolytic cleavage [ - . when the cytoplasmic tail, transmembrane domain and luminal stem region of either /~ , st or /~i, galt are replaced by a cleavable signal sequence, the resulting truncated enzymes are also rapidly secreted from transfected cells [ , ] . from these data it has been argued that the catalytic domains of glycosyltransferases do not contain golgi retention signals. however, it is entirely possible that the luminal catalytic domain can only function in golgi retention if it is anchored to the membrane. at this stage the relative contribution of the stem and catalytic domains in the localization of glycosyltransferases is unresolved. overall, it is most unlikely that golgi retention is determined by a discrete and continuous sequence motif or peptide segment, but rather localization of golgi glycosyltransferases could be mediated by interactions spanning the entire length of the molecule. what are the possible mechanisms for the compartmentspecific localization of the membrane-bound glycosyltransferases? it is unlikely that localization of glycosyltransferases involves a simple receptor-ligand interaction where the receptor is fixed in the golgi cisternae, as over-expression of wild-type transferases does not result in saturation of the retention mechanism [ , - , , ]. an alternative glycopinion mini-review possibility is that escaped golgi glycosyltransferases are retrieved from post-golgi compartments, as with soluble er proteins. however, experimental evidence seems to argue against a retrieval system for golgi glycosyltransferases. wong et al. [ ] have demonstrated that ~ , st leaked to the cell surface is not retrieved back to the golgi apparatus. also we [ ] have demonstrated that/~i, galt which has escaped golgi retention undergoes a post-translational modification, probably in the tgn, before appearance at the cell surface; as the golgi-localized /? , gait does not accumulate this modification, a retrieval system would appear unlikely to play a dominant role. what could be the basis of an active golgi retention mechanism? it has been suggested by a number of investigators that retention of golgi glycosyltransferases could be mediated by the formation of protein aggregates within the membranes of the correct golgi cisternae [ ] [ ] [ ] . this model proposes that such oligomers or aggregates of glycosyltransferases would then be excluded from entry into vesicles for forward transport. although an attractive hypothesis, the evidence for aggregation remains largely indirect. recent elegant experiments performed by nilsson et al. [ ] have shown that the addition of an er retention motif to the glcnacti cytoplasmic tail not only causes glcnacti to localize to the er but also partially retains another medial-golgi enzyme, namely ~mannosidase ii, within the er. furthermore, burke [ ] has demonstrated co-precipitation of glcnactii activity, another medial-golgi enzyme, using antibodies specific to glcnacti. as the amino acid sequences of the glcnacti and glcnactii transferases are not related, a likely explanation is the association of enzymes which occupy the same golgi cisternae. warren and colleagues have coined the term 'kin recognition' to denote this self-association of golgi enzymes [ ] . the proposed aggregation of golgi glycosyltransferases is also consistent with earlier observations that the majority of glcnacti and/~i, galt exist as high molecular weight material following detergent extraction of tissue [ , ] . how could the three domains of a glycosyltransferase play a role in aggregation? the fact that each domain of glcnacti is required for complete golgi retention implies that all three domains may be involved in the lateral interactions which lead to aggregate formation. for example, the transmembrane domains of resident golgi proteins may mediate homo-or hetero-dimerization via protein protein interactions along uncharged polar faces of c~-helixes, predicted for some of the glycosyltransferases [ ] , or along one face of the c~-helix containing a leucine zipper, as predicted for glcnacti [ ] . such dimers may form prior to their arrival in the golgi, as indicated by the results of er retention of glcnacti/manii [ ] . these homo-or hetero-dimers may then be induced to interact, within the correct golgi microenvironment, through their large luminal domains, resulting in a two-dimensional aggregate ( fig. ) . aggregation may be induced by differences within the golgi cisternae, such as ph and calcium concentration. this model differs somewhat from that of warren's group which proposes that the golgi enzymes form homo-dimers (via their stem regions) and interact via their transmembrane domains with different neighbours to generate linear hetero-oligomers. ~-mannosidase ii has been shown to be a disulphide-bonded dimer, but there is no evidence of stable covalent dimer formation for any of the golgi glycosyltransferases. the first report of a purified membrane-bound form of glycosyltransferase, namely /~i, galt, indicates that no disulphide bonded dimers exist [ ] , contrary to an earlier suggestion [ ] . in addition, warren's model of linear heteroaggregates cannot readily explain the efficient golgi retention of hybrid molecules containing a transmembrane domain of a glycosyltransferase and a reporter molecule known to be a monomer in the native state, such as ovalbumin [ , , ] . retention of such hybrid molecules could only occur at the ends of the iinear aggregates, via their transmembrane domains, and would effectively cap these linear structures resulting in only a very minimal number of hybrid molecules retained in the golgi apparatus. finally, the cytoplasmic tail of the glycosyltransferases may be necessary for either transmembrane-mediated dimerization or, as proposed by slusarewicz et al. [ ] , may interact in a salt-dependent manner with a putative intercisternat matrix. consistent with this proposal, the differences in solubility of wild-type gicnacti and the glcnacti hybrid proteins indicate that golgi localized molecules may exist in a different physical state from their cell surface counterparts [ ] . an interaction of the glycosyltransferases with the intercisternal matrix (the "golgi glue'), either directly or indirectly, would ensure that the aggregates are immobilized within the golgi membranes and so are excluded from transport vesicles. clearly an aggregation model of retention may involve many additional components and further biochemical analysis is now required. a model of golgi localization also needs to account for the presence of soluble catalytically active forms of fil, galt and ~ , st which have been detected in body fluids. the retention model proposed above could allow the release of soluble catalytic oligomers from the golgi aggregate by proteolytic cleavage, with the subsequent dissociation of the oligomers to monomers. gotgi membranes differ in lipid composition from the er and plasma membranes. such lipid differences may be important in mediating interactions between the transmembrane domains of glycosyltransferases. a lipid mediated mechanism of protein sorting has been proposed by bretscher and munro [ ] who have suggested that the typically shorter transmembrane domains of golgi proteins may interact selectively with the low cholesterol bilayers of golgi membranes and be excluded from the thicker, cholesterol-sphingotipid enriched bitayers of post-golgi membranes. a protein-lipid interaction is compatible with the observations that the length of the hydrophobic stretch coupled with the adjacent flanking residues is important in gotgi retention, rather than the actual amino acid sequence. the models discussed above are by no means mutually exclusive and it is possible that the golgi retention mechanism includes both a protein-lipid interaction (via the transmembrane domains of the proteins) as well as protein-protein aggregation. based on the aggregation model of golgi retention outlined above, wild type glycosyltransferases expressed in transfected cells may be retained within golgi cisternae as a consequence of self aggregation, or by virtue of their ability to interact or 'dock' to existing aggregates within the golgi apparatus of the mouse cells. self aggregation, as opposed to docking, represents a potentially non-saturable means of retention, consistent with the many reports that glycosyltransferases expressed in heterologous cell lines do not leak to the plasma membrane even when expressed at vastly elevated levels [ , - , , ] . on the other hand, hybrid constructs would have a reduced capacity to self-aggregate, due to insufficient domains, and may be retained by interacting or 'docking' to existing aggregates within goigi membranes, either through their luminal domain or transmembrane domain (fig. ) . this would be a more readily saturable means of retention, with only a finite number of exposed, endogenous molecules available as 'docking' sites. the fact that golgi localized glcnacti hybrid proteins, including those which lack the glcnacti cytoplasmic tail, are predominantly localized to medial-golgi cisternae is in agreement with this proposal [ ] . thus, golgi localization of hybrid molecules probably reflects the minimum structure(s) required to 'dock' with endogenous golgi aggregates. this model would also help to explain the discrepancies between studies as the expression levels of the hybrid molecules would be an important factor in the efficiency of golgi localization. the high level of conservation of individual glycosyltransferases across species is also consistent with structural constraints imposed by such an aggregation model. a conserved structure would be required in order to preserve the many interactions with neighbouring enzyme molecules of a heteroaggregate within the golgi compartment. if golgi glycosyltransferases have evolved with a fundamental requirement for such inter-molecular interactions, it would also explain the conservation of the retention mechanism across species and also the ability of an animal glycosyltransferase to be apparently correctly housed in the golgi apparatus of plant cells [ ] . while most viruses mature at the plasma membrane, a limited number of viruses acquire their envelopes by budding into intracellular compartments. viruses which assemble from golgi membranes include, coronavirus, bunyavirus and pox virus [ , ] . viral budding from the golgi apparatus is probably determined by the targeting of one or more viral glycoproteins to the golgi membranes. indeed, a number of viral proteins have been shown to be independently targeted to the golgi apparatus, including the m glycoproteins of an avian coronavirus [ ] and a related murine coronavirus [ , ] , the e and e spike glycoproteins of rubella virus [ ] and the g glycoprotein of punta tora virus [ ] . as a consequence of the specific localization of these viral glycoproteins they represent useful tools for the study of protein targeting to the golgi apparatus. the m (formerly called el) glycoprotein of the avian coronavirus, infectious bronchitis virus (ibv), has been shown to be localized specifically to the cis-golgi cisternae [ ] . in contrast to the type ii membrane orientation of the glycosyltransferases, the ibv m gtycoprotein contains a short glycosylated amino-terminal domain, three membrane spanning domains and a carboxy-terminat cytoplasmic tail. only the first of the three membrane spanning domains of m glycoprotein of ibv is required to retain this protein in the golgi [ ] . furthermore, this membrane spanning domain is sufficient to confer golgi localization upon a plasma membrane localized protein [ ] . thus, as for the gtycosyltransferases of the medial and trans-cisternae and the tgn, the transmembrane domain of a resident protein of the cis-cisternae has also been implicated in retention. extensive mutagenesis showed that four polar residues in the first m transmembrane domain were critical for golgi retention of a hybrid protein with the vsv g glycoprotein [ ] . these four polar residues are predicted to form an uncharged polar face along one side of an c~-helix, which has potential to be involved in protein-protein interactions and mediate oligomer formation. indeed, aggregation has been shown to correlate with retention of this m hybrid protein in the golgi apparatus [ ] . these investigators demonstrated that the appearance of sds-resistant aggregates of the m hybrid protein correlated with golgi localization, whereas mislocatized transmembrane domain mutants do not form oligomers. the aggregates have not been biochemically characterized but it is possible that they include endogenous golgi proteins. however, sds-resistant oligomers of the native m glycoprotein were not detected in this study [ ] , thus the relationship between aggregate formation of the m hybrid molecule and golgi retention of the native m protein remains unclear. in contrast to the findings for the m glycoprotein of ibv, machamer et al. [ ] in the past few years it has become apparent that there is a distinct set of resident gotgi proteins in the tgn of mammalian cells, and the late golgi of yeast, that have features associated with their localization which are distinct from the golgi glycosyltransferases [ ] [ ] [ ] . these differences are associated with the structure of the proteins. the group includes the mammalian tgn / [ ] and furin [ ] , and the yeast proteolytic enzymes kexlp, kex p, and dipeptidyl aminopeptidase a (dpap a) (for review see [ ] ). in contrast to the golgi glycosyltransferases, tgn / , furin, kextp and kex p are type i membrane proteins, however, membrane orientation is not a distinguishing characteristic of the group as dpap a is a type ii membrane protein. in contrast to the golgi glycosyltransferases, the cytoplasmic tail of all these proteins is essential for golgi localization and, in addition, a retrieval signal plays a role in defining residence of these proteins to the tgn or late golgi. tgn / is a heterodimeric membrane protein complex which cycles between the tgn and the cell surface [ , [ ] [ ] [ ] . tgn / has been shown to interact with cytosolic proteins and may be involved in the formation of exocytic vesicles from the tgn [ , [ ] [ ] [ ] . a number of groups have demonstrated that the tetrapeptide sequence yqrl, within the amino acid cytoplasmic tail of tgn , is both necessary and sufficient to target this membrane protein to the tgn [ ] [ ] [ ] . this tyrosine-containing motif also acts as an internalization motif from the plasma membrane, via interaction with clathrin-coated pits. recently this tyrosine motif has been shown to lie within an ~-helix, and not a tight /~-turn conformation which is typical of other tyrosine-containing internalization motifs [ ] . there is evidence that individual amino acids around the tyrosine of the tgn / internalization motif could signal different intracellular locations. for example, mutation of the yqrl sequence to yqdl abrogated tgn localization of tgn but did not affect internalization [ ] . recently furin, a membrane associated subtilisin-like protease, has been shown to be concentrated in the tgn [ ] . like tgn / , furin also cycles between the cell surface and tgn. sequences of the cytoplasmic tail of furin are required for tgn targeting, and a potential tyrosine motif has been identified [ , ] . on the other hand, potential tyrosine motifs for internalization appear to be absent in the cytoplasmic tails of gotgi glycosyltransferases. the yeast proteins dpap a, kex p and kexlp are all integral membrane proteins with cytoplasmic tails of about amino acids. these cytoplasmic tails are required for retention of these enzymes in the late golgi since deletions in the tail reduce the efficiency of retention [ ] [ ] [ ] . the retention signals within the cytoplamic tails of these proteins have been identified and are very similar to the proposed general motif for clustering into clathrin-coated pits of animal cells (see [ , ] ). deletion of the golgi retention signal, or over-expression of these proteins, results in mislocalization to the vacuolar compartment. this initially surprising finding has led to the conclusion that the default destination for membrane proteins in the yeast secretory pathway is the vacuolar compartment and not the plasma membrane. studies on the yeast vps mutants suggest that dpap a may leak from the late golgi and is transported, via the default pathway, to a post-golgi/pre-vacuolar compartment [- ] . the cytoplasmic localization signals of these escaped dpap a moiecules then mediate retrieval back to the late golgi; in the absence of the cytoplasmic tail golgi localization signals these membrane proteins would continue to be transported along this default pathway to vacuoles. thus there are clear similarities in the mechanism of golgi localization of these yeast proteolytic enzymes and mammalian tgn / and furin. the kdel receptor resides in the cgn and possibly throughout the entire golgi stack [ , ] . the kdel receptor is predicted to have six or seven transmembrane domains [ ] . empty receptors do not recycle back to the er, however, after binding to ligand the ligand-receptor complex is then returned by retrograde transport to the er [ ] . thus, this receptor is likely to have signals for goigi localization. however, mutational analysis of the kdel receptor, although defining structural features associated with ligand binding and retrograde transport, revealed very little about the nature of the putative golgi localization signal [t ]. there are a number of structural membrane proteins and proteins associated with the machinery of vesicular transport that are localized specifically to the golgi apparatus, for example/~-cop [ ], rab and tab [ ] , p [ ] , p [ ] , heterotrimeric g proteins [ , ] , sec [ ] and the actin binding protein, comitin [ ] (table ) . these are not integral membrane proteins as they do not have transmembrane domains, but rather are peripheral membrane proteins associated with the cytosolic face of golgi membranes. some of these components recycle between a cytosolic pool and golgi membranes. in general very little is known about the gotgi localization signals for these peripheral membrane proteins. there is evidence that the carboxy-terminat region of the gtp binding protein g,n is required for golgi membrane binding [ ] . membrane association of rab proteins requires the geranylgeranylation of one or two c-terminal cysteines [ ] as well as a localization signal to define the organelle-specificity. the hypervariable c-terminus of rab proteins has been implicated in localization [ ] , although a recent study on rab indicates that efficient localization of this rab protein to golgi membranes requires both n-terminal and c-terminal domains [ ] . the identification of the precise nature of the targeting signals and the mechanism of localization of these peripheral membrane proteins will be important to the understanding of the organization of the golgi apparatus and vesicular transport. it is now apparent that the localization of resident golgi proteins includes more than one mechanism. for some late golgi membrane proteins a retrieval system operates to recycle proteins from post-golgi compartments. on the other hand, golgi glycosyltransferases appear to be actively retained within golgi membranes; there is no evidence that glycosyltransferase molecules which have leaked from the golgi apparatus can be retrieved. from many 'cut and paste' experiments it is apparent that the localization of glycosyltransferases does not involve a discrete retention signal but may be dependent on many interactions spanning the length of the molecule. furthermore, there is increasing evidence to suggest that retention of glycosyltransferases involves the formation of aggregates within the golgi apparatus. the challenge now is to biochemically characterize these aggregates, to identify any associated molecules that may be important in mediating retention, and to identify the conditions which induce aggregation. this will require the development of novel strategies to allow the isolation and biochemical analyses of individual golgi compartments, in particular the lipid composition, the organization of the resident proteins within golgi membranes, and the nature of interactions with the intercisternal matrix. thus, the problem now is understanding the biogenesis of golgi membranes themselves. the enzymes of biological membranes (martonosi an this work was supported by grants from the national health and medical research council of australia and the australian research council. key: cord- - ircruag authors: pugsley, anthony p. title: later stages in the eukaryotic secretory pathway date: - - journal: protein targeting doi: . /b - - - - . - sha: doc_id: cord_uid: ircruag nan the secretory pathway of eukaryotic cells comprises a succession of compartments, the secretory organelles, through which proteins pass en route to their final destinations. although each secretory organelle has its own special characteristics, a number of basic features are common to all of them. some proteins pass more or less unimpeded through the entire length of the secretory pathway, whereas others are retained in secretory organelles. this raises two of the fundamental questions which will be addressed in this chapter, namely, what determines whether a protein will be taken out of circulation at a particular step in the pathway, and how tight is the separation between secretory organelles? ultrastructure and cell fractionation studies indicate that there is little or no physical link between the rer and the golgi. furthermore, specific proteins are known to reside in individual compartments of the golgi apparatus. as we shall see, the physical separation of golgi enzymes is also indicated by the succession of posttranslational modifications to which secretory proteins are subjected, by in situ immunocytochemistry, and by the separation of golgi-derived vesicles containing different en zymes. how then do secretory proteins move between these compart ments, and is the secretory pathway a continuous gradient of secretory organelles or are they functionally and structurally independent? a feature common to all stages of the secretory route beyond the rer is that proteins move between secretory organelles in specific classes of transport vesicles. consequently, soluble secretory proteins never come into direct contact with the outer face of the organelle to which they are being targeted and therefore can play no direct part in sorting. integral membrane proteins usually have segments exposed on the cytoplasmic face of the transport vesicle which could be recognized by receptors on the target organelle. microinjected antibodies recognizing the c-terminal, cytoplasmic tails of plasma membrane proteins can prevent their trans port to the cell surface ( , ) , but this is probably due to antibodyinduced changes in protein conformation which make the protein incom petent for transport along the secretory pathway, rather than to inhibition of receptor-secretory protein interactions. in this chapter, we will consider three different ways in which sorting of secretory proteins might occur: (i) all secretory proteins have signals which target them successively through the secretory organelles and then on to their final target; some proteins remain in different organelles because they lack the signal necessary for targeting to the next organelle in the pathway. (ii) secretory proteins only have sorting signals for the last stage in the secretory pathway; some proteins have retention signals which pre vent them joining the bulk flow through the secretory pathway, thereby causing them to be retained in specific organelles. (iii) as (ii), except that secretory organelle proteins pass through the secretory pathway in the bulk flow and are recycled via signaldependent counterflow. if either of the last two models are correct, then molecules devoid of retention or sorting signals should travel through the secretory pathway as part of the bulk flow. the rate at which molecules are transported out of the cell will thus be determined by the rate at which they diffuse to sites within the rer and golgi where transport vesicles are formed and depart en route to the next compartment in the chain. this flow rate has recently been measured with the n-glycosylation acceptor tripeptide asn-tyr-thr (section v.b.i). if this tripeptide contains a radioiodinated tyr residue, its progress through the secretory pathway, which it seems to enter by diffusing across the rerm, can be followed by chromatography and autoradiography. wieland et al. ( ) found that the tripeptide was gly cosylated in the er (this prevented it from leaking back into the cytosol), then trimmed by mannosidases located in the golgi (section v.d), and finally secreted into the medium. tripeptide was detected in the medium after - min, depending on the cell type, which is considerably faster than the time required for the transport of most secretory proteins through the secretory pathway. thus, bulk flow is very efficient, implying that diffusion through the lumen of the er and golgi can occur relatively easily and that there is massive, vectorial movement of vesicles between the organelles and the cell surface. the way in which this result influences our understanding of protein sorting in the secretory pathway is discussed in following sections. soluble and integral membrane secretory proteins fold once they have crossed the rerm; they are also often covalently modified. the follow ing sections deal with the types of modifications which occur in the er and their role, if any, in protein sorting. palmitoylation and phosphatidyl inositol modification of secretory proteins are discussed in section iii.f. .c; only their role in protein transit through the secretory route will be discussed here. most secretory proteins are nitrogen (n)-glycosylated in the rer. be cause we are primarily concerned with the effects of glycosylation on protein targeting, only general details of the glycosylation reactions will be given here. a. the sequence of reactions shown in fig. v .l, which is common to both yeasts and higher eukaryotes, results in the addition of a (glucose) -(mannose) -( j /v-acetylglucosamine) complex onto asn residues in the sequence asn-x-ser or thr (the acceptor peptide, in which x can be any i i i i amino acid except possibly proline or aspartate). isolated tripeptides can act as acceptors only when the two extremities are blocked. longer ac ceptor peptides are more rapidly glycosylated ( ). as shown in fig. v .l, the entire complex is assembled before it is attached to the asn residue in the lumen of the rer. at least some sugars cross the rerm as intermediates complexed with the long-chain lipid dolichol, whereas others may cross the rerm as nucleotide '-diphosphate-sugar complexes. however, it is by no means certain that the complex is assembled entirely within the lumen of the rer. for example, there is no evidence that gdp-mannose can be trans ported across the rerm, leading to the proposal that the five mannose residues are added via gdp-mannose donors on the cytoplasmic face of the rerm, whereas later modifications, and possibly earlier modifica tions, occur in the lumen. this presupposes that the dolichol-pp-(nagn) complex can "flip" from the lumen to the cytoplasmic face and then back again once the five mannose residues have been added [see ( ) for further discussion of the topology of glycosylation reactions in the rer]. the preferred donor lipid-carbohydrate complex is the com plete complex shown in fig. v.l ( ) . however, truncated versions lacking glucose and even mannose residues can act as donors in vitro, and under-glycosylated complexes can act as donors in vivo in yeasts ( ) and protozoa ( ) . variations in the mannose content of transferred oligosaccharides have also been reported ( ). the transfer of the first n-acetylglucosamine (nagn) residue from the udp complex to the dolichol, is inhibited by the antibiotic tunicamycin, which therefore blocks all n-glycosylation. this provides a valuable tech nique for studying the role of n-glycosylation in protein traffic. the yeast (s. cerevisiae) gene (alg ) coding for the rerm-associated, tunicamycin-sensitive enzyme (udp-nagn : dolichol-p-transferase) was cloned by virtue of its ability to rescue tunicamycin-treated cells when present on multiple copy number plasmids ( ). null mutations in alg are lethal. mutants affected in other stages in the yeast glycosylation pathway have been isolated by mannose suicide selection (section ii.f.l.b). only muta tions blocking the earliest stages in the pathway are lethal; incomplete dolichol-linked oligosaccharides containing a minimum of four mannose residues allow normal growth, presumably because they can be attached to secretory proteins if the full-length lipid-oligosaccharide complex is absent (see above) ( ). the complete oligosaccharide chain is transferred onto the asn accep tor site in the lumen of the rer. n-glycosylation is generally thought to occur as the polypeptide is being threaded through the rerm, while it is still in its unfolded state. however, some acceptor sites are not glyco-sylated. this may be because they rapidly become inaccessible within the structure of the folded polypeptide, although other explanations are possi ble ( , ) . an unexplained anomaly is that some er membrane glyco proteins have glycosylated residues exposed on the cytoplasmic face of the er ( ). one explanation could be that additional nagn transferases are present on the cytoplasmic face of the smooth er, where these glyco proteins are predominantly located. the initially homogeneous oligosaccharide is processed immediately fol lowing its attachment to the polypeptide chain, initially by the removal of glucose residues by one or more glucosidases. glucose trimming of glyco sylated vesicular stomatitis virus (vsv) g protein has been reported to occur cotranslationally ( ). further processing steps are different in yeasts and in animal cells. in s. cerevisiae cells, a single al- -linked mannose residue is replaced by αϊ- mannose. a mutation in the gls gene coding for α - glucosidase does not affect removal of the mannose residue or subsequent chain elongation, whereas removal of the ninth al- -linked mannose residue may be essential for outer chain elongation, which occurs in the golgi (section v.d.i) ( ). further processing of animal cell glycoproteins also occurs in the golgi, although further man nose trimming of resident er proteins and reglycosylation may both oc cur in the er ( , ). c. ser and thr residues on secretory proteins of yeasts and possibly other fungi can be o-glycosylated in the er ( ). the process is less well characterized than n-glycosylation but seems to involve the direct trans fer of mannose residues from dolichol- -mannose onto acceptor amino acids. studies with acceptor oligopeptides suggest that no particular se quence is required around the modification site ( , ). further process ing of the mannose residue occurs in the golgi apparatus ( ) (section v.d. ). yeast secretory proteins may be both n-and o-glycosylated. most studies indicate that o-glycosylation occurs exclusively in the golgi apparatus (section v.d. ). secretory proteins apparently fold spontaneously as they are extruded .across the rer membrane ( , ) . however, disulfide bridge forma tion is probably catalyzed by protein disulfide isomerase (pdi), which is loosely associated with the rerm ( , ). the formation of soluble protein complexes occurs shortly after synthesis ( ), but trimerization of vsv g protein and influenza virus hemagglutinin (ha), both of which are integral membrane proteins, occurs only - min after synthesis ( , , , ) and involves the selection of polypeptides from a ran dom pool of prefolded monomers rather than from a restricted pool of monomers synthesized on a single polysome ( ). kreis and lodish ( ) suggest that this delay may be due to the segregation of pdi in a late "compartment" of the er. according to the papers discussed above, oligomerization of g and ha occurs just before they leave the er, - min after synthesis. this view is challenged, however, by yewdell et al. ( ) , who found that mono clonal antibodies specific for oligomerized ha reacted only with proteins in the golgi apparatus, and not with ha in the er (see next section). a possible explanation for this ambiguity is that ha monomers fold and trimerize in the er and are then transported to the golgi apparatus, where further modifications alter the conformation of the ha trimers to produce the antigenic sites recognized by the antibody used by yewdell et al. different proteins transit through the secretory pathway together, but the rates at which they are secreted may vary considerably ( , , , ) . the site at which the secretion lag is most prominent seems to be transit from the rer to the golgi ( ). it has been suggested that this delay might reflect the need for secretory proteins to interact with specific receptors in the rer for transport to the golgi and that carbohydrates might form part of the recognition signal ( , , ). alternatively, proteins may be retained in the er until they have folded correctly; different folding kinetics may result in different retention times in the er ( , ). indeed, exit from the er of one of the proteins studied by fitting and kabat ( ) coincided with a partial proteolysis event, and polypeptides were "selected" for exit from a random pool of "new" and "old" proteins. furthermore, studies discussed in the preced ing section showed that oligomerization of virus-encoded membrane pro teins occurs just before they are transported to the golgi. if secretory proteins are indeed retained in the er until they are folded and oligomerized in the correct way, then exit-incompetent proteins should either remain indefinitely in the er or be secreted at very much reduced rates. this could explain why genetically altered or hybrid pro teins are sometimes translocated into the rer without difficulty and yet do not transit further through the secretory pathway ( , , ) , and why incomplete glycosylation or glucose trimming and failure to process signal peptides sometimes affect secretion kinetics ( , , , ) . even minor sequence changes have been reported to affect protein con formation and exit from the er ( ). what happens to incorrectly folded proteins in the er? results from numerous studies indicate that they stay in the er ( , ) or are de graded either in the er ( a) or in the lysosome ( ). these proteins do not seem to precipitate in large aggregates. instead, a specific, major er protein seems to bind to some incorrectly folded proteins, thereby pre venting their exit from the er. haas and wabl ( ) first detected this protein (bip) complexed with immunoglobulin heavy chains synthesized in the absence of light chains (heavy and light chains are only transported to the golgi as complexes) ( ), and it was subsequently found to be complexed with incorrectly oligomerized or monomeric ha ( , ) , nonglycosylated invertase (in vitro in dog pancreatic microsomes) and incorrectly folded prolactin ( ), and recombinant human factor vii in chinese hamster ovary cells ( ). it was not detected in association with aggregated vsv g protein ( ) or with incorrectly folded (mutant) class i histocompatibility antigen ( ). the dissociation of secretory protein-bip complexes requires atp ( ), which might explain part of the atp requirement for protein movement along the secretory pathway, but be cause atp is usually present in cell ly sates, some bip complexes (such as g-bip) may dissociate during extraction. bip has also been shown to associate with nascent polypeptides as they enter the lumen of the er ( ). two interesting features of bip are that its synthesis is stimulated by glucose deprivation and that it is structurally related to a heat shock protein ( , ). glucose starvation is likely to reduce glycosylation, thereby increasing the proportion of incorrectly folded secretory proteins in the er. studies have confirmed the idea that the extent of glycosylation can affect the association between secretory proteins and bip, as well as their rate of secretion ( ). the accumulation of misfolded (mutant) pro teins in the er also increases bip synthesis ( ), possibly because the cell senses that its store of bip has been sequestered into protein com plexes. thus, synthesis of bip may be increased according to require ments ( ), but studies on the "proofreading" or quality control role of bip are still at an early stage. a different, cytoplasmic quality control protein may be responsible for proofreading cytoplasmic domains in transmembrane proteins to ensure that they too are correctly folded and oligomerized ( a). protein conformation, rather than any specific structural feature such as glycosylated residues, is thus likely to determine whether a protein is competent to leave the er. this feature is illustrated by studies on the effects of glycosylation on vsv g protein. machamer et al. ( ) used site-directed mutagenesis to replace the glycosylated asn residues. nonglycosylated g protein was transported from the er to the golgi but did not reach the cell surface. when only one of the asn residues was de leted, the protein reached the cell surface; thus g protein with one glyco sylated asn residue can be transported through the entire secretory path way. g protein export became temperature-sensitive when new glycosylation sites were added ( ). kotwal et al. ( ) noted that some natural g protein variants have only one oligosaccharide whereas others have none at all and suggested that compensatory changes in primary structure may allow nonglycosylated protein to fold into a secretion-com petent conformation. thus, glycosylation is probably needed for correct protein folding but is not directly implicated in the formation of secretion competence signals. at least one secreted protein, ovalbumin, is not gly cosylated. the er contains a large number of proteins, some of which it shares with the contiguous nuclear membrane ( ). they include proteins involved in secretory protein translocation through the rerm, signal peptidase, ribophorins, bip, enzymes involved in lipid synthesis and in protein glycosylation, and cytochrome p . the characterized proteins are similar to other secretory proteins: they often have signal peptides ( , , ) , are glycosylated, and may be located in the lumen or in the rerm. rothman ( ) has argued that it may be physically impossible for the er to prevent the escape of endogenous proteins, and particularly mem brane proteins, to the cis golgi. he cited two observations in support of the idea that these proteins could leave the rer as part of the bulk flow through the secretory pathway, and then recycle from the golgi to the er: (i) some er proteins are detected in significant amounts in vesicles derived from the cis golgi when cells are fractionated ( ). (ii) bulk lipid flow out of the er necessitates efficient recycling, possi bly from the cis golgi, which could provide "carriers" for the recy cling of er proteins. most er proteins are, however, almost completely excluded from the golgi ( , ). furthermore, er proteins are not terminally glycosyla ted ( , ), which means that they do not reach the medial or trans golgi (section v.e). mannose residues on some er proteins are not trimmed beyond the stage catalyzed by er mannosidase ( , , ), but a lysosomal protein carrying an er retention signal (see below) is phosphorylated by nagn phosphotransferase, indicating that it reaches the early cis golgi ( ). significantly, the phosphate groups are not modi fied by nagn phosphodiesterase, which may be located in a different, er-distal golgi compartment (table v.l). warren ( ) has proposed that er proteins are salvaged from an intermediate compartment, the socalled transitional element (section v.c), located between the er and the cis golgi. recycling of "escaped" endogenous er proteins, rather than receptormediated retention in the er, is the currently favored model for the specific accumulation of proteins in the er ( ). it is not clear whether soluble and er membrane proteins are both subject to the same retention mechanism, but studies on the rates of diffusion of membrane proteins in the er indicate that rerm proteins are more restricted than bip and that the mobility of bip devoid of its er retention-salvage signal (see below) cannot be distinguished from that of normal bip ( ). several studies have sought to determine the nature of the er reten tion-salvage signal(s). the rotaviral type ii er membrane protein vp was found to be secreted when the two potential n-terminal transmem brane segments were deleted ( ), suggesting that retention-salvage de pended on a membrane anchor domain. however, subsequent studies showed that the entire n-terminus was absent from mature vp ( ) and thus that some other feature must account for vp retention in the er ( c). although deletion of the c-terminal transmembrane and cyto plasmic tails of the type i er membrane protein Ε of adenovirus also causes the protein to be secreted, deletion of the last eight residues (fidekkmp) of the c-terminal, cytoplasmic tail alone causes the protein to appear on the cell surface, suggesting that the c-terminus contains the er retention-salvage signal. furthermore, cell-surface interleukin re ceptor protein β chain was converted into a resident er protein following fusion of fidekkmp to its cytoplasmic c-terminus ( ). the cytoplas- mically exposed signal could be recognized by "salvage receptors" in the transitional element. these studies on the Ε protein are reminiscent of earlier, seminal work on the soluble er protein bip which resulted from the observation that three soluble proteins in the lumen of the er, including bip, had the same four residues, lys-asp-glu-leu (kdel), at their c-termini. munro and pelham ( ) found that if this sequence was deleted or extended, bip was secreted into the medium. this suggests that kdel is the er reten-tion signal and that it must be at the extreme c-terminus, which is pre sumably the last segment of the polypeptide to fold and may therefore be exposed on the surface of the protein. in a complementary study, dna coding for kdel was fused to the end of a cdna clone coding for secreted lysozyme. the resulting hybrid protein was retained in a perinu clear region probably corresponding to the er ( ). further studies are required to determine whether kdel is the "uni versal" er retention signal. preliminary studies suggest that some mam malian er proteins have the sequence rdel at the c-terminus, whereas yeast er proteins have the sequence hdel ( a). hdel or kdel is presumably recognized either by an endogenous er membrane protein, which could anchor soluble proteins in the er, or, more likely, by the recycling receptor located in the transition element or cis golgi. indeed, a putative hdel receptor gene (erd ) has recently been identified in yeast cells ( , a). it is not clear what triggers the release of bip from its receptor once it is recycled to the er. β-glucanase is a typical lysosomal protein; small but significant amounts of it, however, are retained in the er through a specific interac tion with an endogeneous er protein, the esterase egasyn. studies by medda et al. ( ) indicate that / -glucanase-egasyn interaction is blocked by inhibitors of esterase activity, leading to β-glucanase secretion (rather than sorting to the lysosome). whether this is a physiologically significant mechanism for retaining proteins in the er remains to be de termined. only about % of egasyn is complexed with / -glucanase ( ), so perhaps it also binds to other resident er proteins. it would be inter esting to see whether egasyn itself has a c-terminal kdel-like sequence. as discussed above, secretory proteins do not move to the cis golgi until they attain an exit-competent state, and some proteins are specifically retained in the er, probably by recycling of escaped proteins. both nor mal secretory proteins ( ) and exit-incompetent proteins have been reported to accumulate in a specific region of the smooth er, the vacuolar transitional element ( , ), from which vesicles either migrate to and fuse with the cis golgi ( ) or coalesce to form new golgi cisternae ( ). specific membrane proteins [e.g., the product of the sec gene in yeasts ( a)] may be required to form this specialized domain of the er and hence directly or indirectly assist vesicle formation and protein transport to the golgi. secretory pathway "shuttle" vesicles are difficult to isolate because ( ) also showed that atp depletion caused secretory pro teins to accumulate in transitional elements, but this study did not distin guish between energy requirements for protein folding and for vesicular transport from the transitional element to the golgi. a different in vitro assay was developed by haselbeck and schekman ( ) to study protein movement from the er to the golgi in yeast cell extracts. their assay uses donor er vesicles derived from a strain carry ing the secl ts mutation, which blocks protein movement from the er, and the mnnl mutation, which prevents terminal (golgi) mannosylation of secretory proteins. invertase accumulated in the er grown at the restric tive temperature was mannosylated after transfer to recipient golgi from wild-type cells. the transfer efficiency was low, however, possibly be cause invertase accumulated at the restrictive temperature did not be come exit-competent at the permissive temperature in vitro, or because recipient golgi were saturated. as in the mammalian system, protein transfer was atp-dependent and required soluble cofactors including sec protein ( b) as well as proteins on the surface of the recipient golgi. a similar but more efficient reconstitution system using gently lysed yeast-cells has recently been developed ( a). results obtained with this system indicate the probable requirement for gtp in er to golgi traffic. secretory proteins are subjected to further chemical modification as they transit through the golgi. these processes are discussed in the following sections (except palmitoylation, which was covered in section iii.f. .c); their significance with regards to compartmentalization of the golgi appa ratus and their role in protein targeting will be discussed in later sections of this chapter. in section v.b.i, we saw that the basic oligosaccharide core on asn residues is trimmed by glucose-and mannosidases before secretory pro teins leave the er. following their arrival in the golgi, mannose residues on secretory proteins may be processed in one of two ways, depending on whether they are targeted to the lysosome. soluble lysosomal proteins carry phosphorylated mannose residues which function as lysosomal sorting signals (section v.g. ). they un dergo specific mannose phosphorylation catalyzed by one or two nacetylglucosamylphosphotransferases and v-acetylglucosamine- -phosphodiester-a-n-acetylglucosaminidase (phosphodiesterase) in different cis golgi compartments ( a). sequential action of these enzymes results in the transfer of tv-acetyl glucosamine- -phosphate from udp-nagn to any one of five mannose residues in the oligosaccharide core, followed shortly afterwards by the removal of the jv-acetylglucosamine to expose the phosphomannosyl group ( ). goldberg and kornfeld ( ) found three partially phosphorylated peptides in β-glucuronidase, indicating that lysosomal enzymes may not be uniformly phosphorylated. lysosomal enzymes presumably contain signals which are recognized by the phosphorylating enzymes ( ). deglycosylated (endoglycosidase Η-treated) lysosomal cathepsin d is not a substrate for the phosphoryl ation reaction in vitro, but it inhibits phosphorylation of intact lysosomal enzymes. proteolytic fragments of glycosylated cathepsin d are also not phosphorylated, and do not inhibit phosphorylation when they are dephosphorylated, indicating that the "phosphorylation signal" is probably not a linear sequence of amino acids ( ). frog oocytes are also able to recognize the phosphorylation signal of human cathepsin d ( ). renin is closely related to cathepsin d and yet is secreted by mammalian cells, presumably because it lacks the phosphorylation signal. however, renin produced in oocytes is phosphorylated, remains intracellular, and is de graded (presumably in the lysosome) ( ). this suggests that renin has a phosphorylation signal which is recognized by amphibian but not by mam malian phosphorylating enzymes. outer chains on lysosomal and nonlysosomal secretory proteins of com plex eukaryotes may be further trimmed by golgi mannosidase i to leave five mannose residues on the core oligosaccharide. further modifications involve the addition of an n-acetylglucosamine residue by n-acetylglucosaminyltransferase i, further removal of two mannose residues by golgi mannosidase ii, fucosylation of the innermost tv-acetylglucosamine by fucosyltransferase, and the addition of galactose, n-acetylglucosamine, and sialic acid residues by appropriate transferases (fig. v. ). oligosaccharides of both lysosomal and nonlysosomal proteins may be further modified by sulfation of mannose and n-acetylglucosamine resi dues and o-acetylation of sialic acid residues ( ). outer chain modification in yeasts is markedly different. golgi mannosyltransferases extend the basic (man) core oligosaccharide to produce large mannan structures typical of many yeast mannoproteins, which may carry as many as mannose residues ( ). the single o-linked mannose residue on yeast glycoproteins (section v.b.l.c) may be further modified by the addition of up to four more mannoses transferred from gdp-mannose ( , blocked in sec mutants, which prevent secretory proteins from reaching the golgi ( ). in complex eukaryotes, hydroxyl groups of ser or thr residues are oglycosylated by enzymes thought to be located exclusively in the golgi ( , ). acceptor octapeptides can be o-glycosylated. v-acetylgalactosamine is the primary sugar in o-linked oligosaccharides; further resi dues of galactose, sialic acid, fucose, ^-acetylgalactosamine, and nacetylglucosamine can then be added. lipid intermediates are not in volved in o-glycosylation, and the reaction is not inhibited by tunicamy cin ( ), but it is not known whether other sugar transferases are in volved in both n-and o-glycosylation. glycosylated and unglycosylated secretory proteins are major substrates for tyrosine sulfation ( ). tyrosynylprotein sulfotransferase is enriched in golgi-derived membrane fractions and has its active site oriented to ward the golgi lumen ( ). the sulfate donor is '-phosphoadenosine phosphosulfate. almost all tyrosinated proteins have an acidic residue, a glycine or proline residue, and no cysteines, basic residues, extended secondary structure, or n-glycosylation sites close to the modified tyro sine, but there does not appear to be a strict consensus sequence around the modification site ( , ). inhibition of tyrosine sulfation is reported to retard the exit of a secretary protein from the tgn ( b). many secretory proteins undergo secondary proteolytic processing fol lowing removal of the signal peptide. the best characterized examples of this class of processed secretory proteins are α factor and killer toxin of yeasts. the α-factor peptide is present four times in the pro-a-factor protein which reaches the golgi apparatus. this probably represents a way of reducing "shipping costs" since α factor itself may be too small to be efficiently transported to the rerm and it would be inefficient to pad the polypeptide out with redundant sequences ( ). julius et al. ( ) found that pro-a-factor processing was blocked by sec mutations, which prevented protein movement through the golgi, and that mature α factor was normally present in secretory vesicles en route to the cell surface. thus, processing occurs in the golgi. some viral coat proteins such as influenza virus ha are also proteolytically processed in the trans-golgi network (tgn) or trans golgi. processing of mammalian prohormones, which is similar to that of pro-α factor, occurs in secretory granules budding from the trans golgi [see section v.g. .c and ( )]). the four -residue-long α-factor peptides in pro-α factor were found to be separated by - residues (lys-arg-glu-ala-asp-ala-glu-asp) ( ). this suggests that at least one processing protease has trypsin-or chymotrypsin-like activity. killer toxins are processed at similar sites ( , ). in fact, the enzyme which performs this initial processing step, the product of the kex gene, is a ca + -thiol protease which cleaves between basic residues. the enzyme is inhibited by anti-αΐ-trypsin and can correctly process mammalian proalbumin ( ). strains mutated at kex secrete unprocessed pro-α factor ( ). the product of the ste gene, a membrane-associated aminopeptidase ( ), processes the nterminal tetrapeptide, and further processing of the c-terminus is per formed by kex -encoded carboxypeptidase ( ). other secretory pro teins produced by different species of yeasts may also be processed by one or more of these enzymes ( b). results from a number of experimental approaches (summarized below) show quite conclusively that individual golgi cisternae are separate, bio chemically and functionally distinct entities organized according to a very strict pattern and that secretory proteins progress in a synchronized wave from one end of the stack of cisternae to the other en route to their final destinations. according to these data, golgi stacks must contain at least three distinct cisternae (generally referred to as cis, medial, and trans according to their orientation with respect to the er). heterogeneity within these "domains" indicates that the actual number of cisternae is probably higher than three (see table v .l, fig. v . ) ( ). cells normally have one or a very limited number of golgi stacks. the stacks break up into clusters of many vesicles during mitosis, apparently to ensure equal partitioning of golgi components to daughter cells, although the number of golgi clusters produced is far in excess of that required for this pur pose. golgi breakdown presumably involves membrane fission, so each cluster of vesicles contains cis, medial, and trans components ( ). pro tein secretion is usually shut down during mitosis or meiosis, but the yeast s. cerevisiae continues to process and secrete invertase during mitosis, implying that the golgi fragments remain active ( ). in general, results from different analyses give a coherent picture of the cis to trans organization of golgi cisternae (see table v . ), on the basis of which a simple map can be drawn (fig. v. ) . the segregation of modify ing enzymes presumably allows prosthetic groups to be added or removed according to a strict sequence and prevents competition between process ing enzymes which could act on the same substrate ( ). table v .l is five, but the actual number of golgi cisternae may be higher than that shown. nagn, iv-acetylglucosamine; tgn, trans-golgi network. early studies showed that one or two cis (er-proximal) golgi cisternae were preferentially stained during prolonged exposure to osmium [pre sumably due to strong reducing conditions in these cisternae ( )]. sub sequent histological and cytochemical tests showed that many enzymes involved in protein glycosylation and other reactions, as well as some proteins with unknown function, were not evenly distributed through the golgi stock but were compartmentalized in one or two cisternae (table v .l). the techniques employed include immunocytological detection of proteins using specific antibodies, enzymatic cytochemical reagents for specific enzymes, and the detection of lectin-specific sugar residues on terminally modified glycoproteins in transit through the golgi (table v .l). as noted by farquhar ( ), some studies with different cell lines give conflicting results, suggesting that the organization of the golgi stack may vary depending on cell type and function. intermediates in the secretion pathway can be detected by pulse-chase experiments in which oligosaccharides or other prosthetic groups are la beled by the metabolic incorporation of radioactive precursors. the se quence in which processing occurs can thus be determined and correlated with the location of processing enzymes in the golgi cisternae. golgi enzymes involved in n-and o-glycosylation, sulfation, and palmi toylation of secretory proteins can be separated by density gradient cen trifugation of golgi-derived vesicles, which apparently differ in density due to differences in cardiolipin content ( ). enzymes identified in cis golgi by kinetic and histological or cytochemical tests are generally found in heavier golgi membrane fractions, and density is now widely used to define the approximate location of golgi proteins in the stack (table v .l). the ionophore monesin slows or arrests intra-golgi transport and inhibits late (trans) golgi functions, and can thus be used to distinguish between early and late golgi processing events. monesin also causes secretory proteins to accumulate in medial or late golgi compartments, which be come distended and vacuolated, aiding their identification by cytochemi cal methods ( , , ). however, as discussed by dunphy and rothman ( ), studies on the effects of monesin in different cell types often give conflicting results and do not necessarily indicate the precise site of secretory protein accumulation or processing. oligosaccharides on secretory proteins are only cleaved by endoglycosi dase Η before they are processed by nagn transferase i and mannosi dase ii. these enzymes are probably located in medial golgi cisternae. thus, endoglycosidase Η-sensitivity provides a simple test for determin ing whether secretory proteins have reached these cisternae ( ). rothman et al. ( , ) demonstrated that pulse-labeled vsv g protein present in the golgi of a cell lacking a particular modification enzyme can be modified upon fusion with a cell producing the modifying enzyme. the golgi cisternae from donor and recipient cells did not fuse, and the g protein, rather than the modifying enzyme, was transferred from one cisterna to the other. these observations have implications for the mecha nisms of protein movement between cisternae (section v.f) but, like in vivo kinetic experiments, may also indicate the sequence in which pros thetic groups are added or removed ( having established that secretory proteins are progressively processed as they migrate through the golgi stacks, we now turn our attention to how the proteins themselves migrate between cisternae, and how golgi pro teins can be specifically retained within specific cisternae. of all of the models explaining intra-golgi movement of secretory pro teins ( ), only that invoking shuttle vesicles fits the experimental data showing that golgi cisternae are distinct entities. numerous proteincoated vesicles are produced by golgi stacks in vitro under conditions which favor the intra-golgi movement of secretory proteins (see below) ( , ). these uniformly sized vesicles were shown to contain at least one secretory protein, vsv g protein, indicating that they could be bona fide intra-golgi shuttle vesicles ( ). similar vesicles were also found around the golgi apparatus in situ ( , ) . furthermore, different se cretory proteins destined to different locations and exported or secreted at different rates were present in the same vesicles ( ), which agrees with the idea that secretory proteins are not segregated from each other during intermediate stages in the secretory pathway. the protein coat on these vesicles is distinct from the clathrin-containing coats present on endocytic and lysosomal vesicles and on budding secretory granules (see section v.g. ). the only regions of the golgi apparatus which have pro tein coats are those from which the golgi shuttle vesicles bud. a number of different conditions prevent intra-golgi movement of secre tory proteins in vivo and in vitro. the ionophore monesin (section v.e. ) probably prevents movement though the trans cisternae by disrupting a proton gradient maintained by an atp-dependent protein pump present in golgi membranes ( ), thus causing the ph of the normally acidic trans golgi cisternae to rise. saraste and hedman ( ) demonstrated that mi gration between different golgi cisternae was blocked at different critical temperatures and suggested that this might be caused either by atp depletion or by changes in membrane fluidity. atp may be required to maintain the acidic ph of trans cisternae, and vesicle fission and fusion, which must occur at the cisternal membranes, can probably only occur when membrane lipids are in a "fluid" state above the phase transition temperature. atp and soluble cytosolic factors including a -kda, n-ethylmaleimide-sensitive protein ( a) are required in both early and late stages of intra-golgi movement in vitro ( , , a, ) and in permeabilized cells ( ). intriguingly, soluble yeast cell extracts can replace endogenous cy toplasmic components in a mammalian cell-derived assay system for in tra-golgi movement of proteins ( ), raising the possibility of using ex tracts from yeast sec mutants blocked at different stages in the secretory pathway to define the role of the corresponding wild-type gene products in intra-golgi transport. indirect evidence based on the effects of gtp analogs suggests that gtp is also needed for intra-golgi movement of secretory proteins ( ), and studies with antibodies against a yeast gtp binding protein indicate that a similar protein is apparently located in the golgi apparatus in multicellular eukaryotes ( ). the significance of this observation is not understood, but gtp and gtp binding proteins (g proteins) may be involved in maintaining vectorial movement of shuttle vesicles or in vesicle recycling (see below). surface components on golgi membranes are also required for intra-golgi transport ( , , ). a recep tor may recognize the protein coat on the golgi transport vesicles. balch et al. ( ) considered that the migration of secretory proteins from one cisterna to another depends on three separate events: (i) secretory proteins are primed to make them competent or available for transport. priming probably involves the migration of secretory proteins to regions of the cisternae where budding occurs, princi-pally at the outer rims. this step might depend on interactions be tween the secretory protein and receptors migrating to the budding areas or may rely on free diffusion within the cisternal membrane or lumen. segregation may depend on signals generated by processing enzymes in individual golgi cisternae, but most experiments in which processing inhibitors have been used do not support this idea and show instead that processing is nonessential for secretory pro tein targeting. some mutationally altered secretory proteins, how ever, transit normally through the early stages of the secretory path way and yet are not transported through the golgi ( , ). perhaps secretory proteins must fold into a particular conformation to be competent for movement between golgi cisternae. the accu mulation of such abnormal proteins in the golgi may cause it to become distended, but this does not drastically affect intra-golgi movement and secretion of other secretory proteins ( ). (ii) vesicles move from one cisterna to another. vesicle migration be tween golgi cisternae may be either vectorial or random. vectorial movement is more compatible with traditional views on the strict sequence of events in secretory protein processing and is supported by the observation that the likelihood of forward transfer is at least five times greater than that of lateral movement in golgi of fused cells ( , ) . this implies that there are receptors on the surfaces of golgi cisternae which recognize vesicles budding from the pre ceding cisterna in the chain. nonetheless, the topology in the golgi complex is well maintained, possibly because the cytoskeleton pre vents cisternae from coming into direct contact. another puzzling feature of vesicular intra-golgi transport is that small transport vesi cles would be expected to diffuse away from the golgi complex. the cytoskeleton may restrict the movement of golgi vesicles and may even play a more positive role in directing vesicles between cister nae. however, although movement along microtubules has been well documented for large organelles ( ) and may be important in the sorting of some proteins leaving the golgi complex (section v.g. ), there is no evidence that it could play more than a minor role in the movement of vesicles over the very short distances which separate golgi cisternae ( ). furthermore, cell fusion studies by rothman et al. ( ) show that inter-golgi movement of vsv g protein can occur, indicating either fusion of the two golgi com plexes or, more likely, that movement between golgi cisternae is dissociative; i.e., the vesicles do indeed diffuse into the cytoplasm. (iii) vesicle fusion with the membrane of the acceptor cisterna and re lease of vesicle contents into the lumen of the cisterna occurs. this model for intra-golgi transport fits many experimental observa tions, but further studies are required to define clearly the steps involved. for example, atp may be required for vesicle fission and fusion, as well as for reducing the ph in trans golgi compartments, but this has not been proven, and the nature and role of some of the cytosolic components required in in vitro assays have yet to be determined. further work is needed to define how proteins find their way to budding regions of the golgi membrane and what triggers vesicle formation and vesicle fusion with acceptor membranes. how are proteins specifically retained in individual golgi cisternae? we saw earlier that receptor-dependent recycling of er proteins from the cis golgi or an intermediate compartment could explain how these proteins remain almost entirely in the er (section v.b. ). golgi residents proba bly also have signals which are recognized by some kind of receptor. indeed, coronavirus el membrane glycoprotein appears to have a golgi retention signal in one of its transmembrane domains ( ). however, recycling of escaped proteins is a far less attractive model for explaining how golgi proteins are retained than it is for the case of er proteins. one possibility, proposed by pfeffer and rothman ( ) , is that the membranes of golgi cisternae have two domains: a fluid domain, close to the budding rims, and an immobile phase, in which endogenous membrane proteins are anchored. a protein would require a signal to associate with a recep tor in the immobile phase or to become anchored to it, whereas all other proteins would enter the mobile membrane phase or the bulk phase of the cisternal lumen. pfeffer and rothman point out that this model reduces the number of proteins which need to have signals for routing through or retention in the golgi, because fewer proteins are retained in the golgi than transit through it. cytoskeletal structures, including possibly the cytoplasmic matrix, which "glues" the cisternae together, were proposed to limit movement in the immobile phase. an alternative idea, also con sidered by pfeffer and rothman, is that endogenous golgi proteins inter act to form patches which, by virtue of their size, are too small to fit into transport vesicles. secretory proteins transit through the golgi apparatus and arrive in the trans cisternae together. the trans golgi compartment is therefore the point at which the different branches of the secretory pathway diverge. the following sections deal with the site at which sorting occurs, the ways in which proteins are sorted, and what happens when vesicles carrying secretory proteins arrive at their destinations. as its name suggests, the trans-golgi network (tgn, also called golgi endoplasmic reticular lysosomes or gerl) is the most distal compart ment of the golgi apparatus (relative to the rer). it differs from the golgi cisternae in that it has a distended, reticular appearance rather than that of a flattened dish. early morphological studies suggested that the tgn was a reticular adjunct of the golgi specifically involved in lysosome biosynthesis [lysosomal enzymes were originally thought to bypass the golgi ( )]. the tgn is now known to be distinct from endosomes or lysosomes. endocytosed horseradish peroxidase does not accumulate in the tgn ( ), although some endocytosed proteins may be recycled to the cell surface via the trans golgi and especially via the tgn ( ) (see chapter viii). the tgn probably contains tyrosinyl sulfotransferase ( ), acid phosphatase, sialotransferase, and galactosyltransferase ( , ) (table v .l, fig. v. ) , although tgn-derived vesicles are diffi cult to distinguish from those derived from the trans cisternae. the tgn also marks the site at which assembled clathrin, one of the proteins which coat some secretory and endocytic vesicles, appears along the secretory pathway (see section v.g. ). the tgn is the most acidic golgi compartment, although the ph is almost certainly not as low as in secretory granules ( ) or in lysosomal sorting vesicles, in which low ph causes the dissociation of lysosomal proteins from the mannose- -phosphate receptor ( ) (see section v.g. ). furthermore, influenza virus hemagglutinin, which is activated at low ph, reaches the cell surface as a nonactivated form, indicating that it does not spend an appreciable period (more than min) in a compartment with a ph of less than ( ). different secretory proteins accumulate together in the tgn ( , ), which can become further distended when the load of secre tory or lysosomal proteins increases ( ). transport of secretory pro teins from the tgn is blocked at °c, and numerous protein-coated and naked vesicles accumulate as buds on the surface of the tgn ( ). different types of vesicles seem to be involved in sorting secretory pro teins into different branches of the secretory pathway. thus, different classes of soluble secretory proteins may be segregated into different domains of the tgn according to their interaction with specific receptors (fig. v. ). although this model does not explain how receptor proteins note that constitutive default sorting is not receptor-mediated and that sorting into the regulated secretory branch of the pathway may result from protein aggregation and granule formation rather than receptor interactions. tgn, trans-golgi network; l, lysosome; pl, prelysosome. segregate into these domains, it does serve to illustrate the possible mech anisms involved in receptor-dependent segregation and packaging secre tory proteins discussed in the following sections. default sorting is the final stage in the secretion of proteins which lack specific sorting (lysosomal, vacuolar, or polarity) signals and which are not accumulated within specific secretory storage granules of the regu lated branch of the secretory pathway, i.e., those which are constitutively secreted (fig. v. ) . (note, however, that some constitutively secreted proteins carry sorting signals.) immunocytochemical studies show that proteins secreted by the default pathway accumulate in secretory vesicles which do not have protein coats ( , ). plasma membrane proteins are transported to the cell surface in the same vesicles as secreted proteins ( , , ) . ( ) (see section ii. f. ) required atp and was sensitive to trypsin, implying that at least one cytoplasmic or vesicle-cytoplasmic membrane surface protein is involved. atp might be needed for fusion between the vesicle and cytoplasmic mem brane. saccharomyces cerevisiae strains carrying a temperature-sensitive mu tation in the sec gene accumulate large numbers of secretory vesicles. these were purified by walworth and novick ( ), who found them to contain three dominant proteins ( kda, - kda, and kda) to gether with invertase, the secretory marker protein. these three proteins were made during the period in which the vesicles accumulated, i.e., after secretion had been shut down at the nonpermissive temperature. the -kda protein was the most abundant protein in the lumen of the secretory vesicles, whereas the other two proteins were membrane-associated and had cytoplasmic domains which could interact with the cytoskeleton, the cytoplasmic membrane, or cytoplasmic components ( ). it should be noted, however, that exocytosis in s. cerevisiae might be considered as polarized rather than default sorting because secretory vesicles are di rected towards the growing bud rather than being randomly distributed over the entire cell surface (section v.g. and fig. ii. ) . the sec gene was cloned and sequenced by salminen and novick ( ), who found it to be homologous to gtp-binding ras regulatory proteins of higher eukaryotes. whether this is significant for the role of sec in secretion is unclear, especially since it is not known whether gtp is required for exocytosis in yeasts. recent studies show that sec protein binds gtp ( ). overexpression of sec suppresses the effects of mutations in three other sec genes, but strains carrying sec ts muta tions rapidly become secretion-defective at the nonpermissive tempera ture, suggesting a direct role in secretion. the cytoplasmic and secretory vesicle membrane-associated sec product does not itself appear to be a secretory protein because its predicted primary sequence does not in clude a potential secretory routing signal. one possibility raised by salminen and novick is that the c-terminal cysteine residue of sec is acylated, as are the gtp-binding ras proteins and that the acyl groups anchor the polypeptide in the membrane, but this could not be confirmed directly. another gtp binding protein, ypt (which is % homologous to sec ) was detected close to the bud as well as in ill-defined structures (possibly the er and golgi) of s. cerevisiae cells ( ). mutations in the ypt gene affect several stages in the secretory pathway, but ypt pro tein probably plays an indirect role in protein transport as a result of its role in ca + regulation ( ). the plasma membrane of epithelial cells is divided into two distinct do mains. the apical surface, which may have microvilli, is oriented toward the outside (e.g., lumen of the intestine), whereas the basolateral surface is on the inside, facing the basolateral surface of other cells or resting on an extracellular matrix of basal lamina (fig. v. ) . the two membrane domains have distinctly different lipid contents in their outer leaflets [the apical membrane has a higher glycolipid and cholesterol content, and a lower phosphatidylcholine content than the basolateral membrane ( )], although the lipid contents of their inner leaflets may be identical (inset to fig. v. ). this implies that only the outer leaflets of the two membranes are separated by tight junctions, morphologically distinct structures rich in nonbilayer lipids and containing unique proteins which form the junction between apical and basolateral surfaces and probably prevent the movement of outer leaflet lipids and proteins between them, as well as acting as ion gates between adjacent cells (fig. v . ) ( , , , ( ) ( ) ( ) ) . there may also be differences in basal and lateral membrane composition. cell-cell interactions are required for effi cient formation of the basolateral surface of polarized cells, but not for sorting to the apical zone ( ). adjacent cells may be held together by desmosomes. the major breakthrough in studies on protein sorting in polarized cells ( ). in general, the steady-state distribution of polarized membrane proteins indicates that sorting of basolateral pro tein is highly efficient (> % fidelity) ( ), whereas apical proteins may be found in significant amounts in the basolateral membrane. however, the surface area of the apical membrane is usually much smaller than that of the basolateral membrane, which means that the fidelity of apical tar geting is actually quite high (about %) ( ). basolateral and apical sorting seem to represent two distinct pathways, but most polarized cells also secrete some proteins from both surfaces, implying that neither basolateral nor apical sorting are default pathways ( , ). furthermore, lysosomal proteins are secreted by the default pathway from both apical and basolateral surfaces when lysosomal sort ing is blocked ( ). this may not be the case in caco- cells in which high-level basolateral secretion of normally nonpolarized lipoproteins im plies that the basolateral sorting predominates over all other sorting path ways, including the default pathway ( , a). do polarized cells sort and direct proteins directly to their target mem branes, or are they all first randomly targeted to both domains, or specifi cally to one or other domain, and then transcytosed? although some studies designed to answer this question have been conducted with en dogenous proteins, the most detailed results come from studies on the basolaterally targeted vsv g protein and the apically targeted influenza virus hemagglutinin (ha) in mdck cells. furthermore, it is through derivatives of these proteins that most recent studies have attempted to identify apical and basolateral targeting signals (see next section). the results of pulse-chase experiments combined with a trypsin sensi tivity assay and tests with anti-ha antibodies applied to the basal surface of mdck cells led matlin and simons ( ) to conclude that ha was targeted directly to the apical surface. conversely, pfeffer et al. ( ) showed that pulse-labeled g protein went directly to the basolateral mem brane, where it could be detected with specific monoclonal antibodies. another approach to studying the sorting of ha and g proteins is to ''freeze" them in the golgi apparatus by cooling the cells to °c (which specifically blocks protein exit from the tgn) or to use cells infected with viruses carrying temperature-sensitive mutations in the ha or g genes at the restrictive temperature. the bulk movement of these proteins can thus be followed by immunocytochemistry when the cells are restored to the permissive temperature. rindler et al. ( ) found that gts protein was transported directly from the golgi to the adjacent lateral cell sur face, whereas wild-type ha accumulated at °c was sorted directly to the region of the apical surface closest to the tgn. similarly, pfeiffer et al. ( ) found that g protein accumulated in the tgn went to the baso lateral membrane times faster than to the apical surface when the cells were warmed to °c. these studies are compatible with the idea that apical and basolateral proteins go directly to their respective target mem branes. different results were reported by bartles et al. ( ) , who found that endogenous apical proteins in hepatocytes appeared first in the baso lateral membrane and were subsequently redistributed to the apical sur face by transcytosis (section viii.a. .b). whether this result indicates the existence of totally different mechanisms for sorting apical proteins in kidney and liver cells could be tested by expressing hepatocyte genes for apical proteins in mdck cells. sorting of polarized secreted and membrane proteins is assumed to de pend on sorting signals (probably signal patches) [see section i.d. and ( )] present in the sorted proteins and on their interaction with receptors. sorting presumably occurs in the tgn, where at least one set of cognate receptors are probably located. to date, the primary approach used to locate sorting signals has been to introduce major sequence alterations including the deletion of cytoplasmic or transmembrane domains from the ha, g, or other polarized viral glycoproteins, or to create hybrids be tween them. even these relatively unsophisticated studies give conflicting results. for example, mcqueen et al. ( ) and roth et al. ( ) found that either deleting the transmembranous and cytoplasmic tail of ha (to make a soluble, secreted protein) or replacing them with the correspond ing regions of g did not affect apical targeting and concluded that the apical sorting signal was located in the n-terminal, extracytoplasmic do main of ha. gonzalez et al. ( ) reported, however, that truncated ha was secreted by the default pathway and concluded that the sorting signal was in the c-terminal transmembranous or -amino-acid cytoplasmic domains. there is also disagreement concerning the location of the g protein basolateral sorting signal, which, according to the deletion and ha gene fusion studies of paddington et al. ( ) and gonzalez et al. ( ) is located in the -amino-acid, c-terminal cytoplasmic domain. stephens and compans ( ) mcqueen et al. ( ) have suggested that the origin of these conflicting results may be the fact that recombinant genes are not stably expressed in mdck cells. their studies ( , ) were carried out shortly after infec tion by recombinant viruses and are, they claim, more likely to reflect the true sorting pathway of the recombinant gene product. other groups may in fact be studying the sorting of hybrid or truncated proteins which have been further modified by genetic selection for more stable cell lines. an other argument in favor of the idea that sorting signals are in the extracy toplasmic domain is that this part of the signal is initially localized in the lumen of the tgn, where sorting is presumably initiated, and could there fore bind to hypothetical sorting receptors which segregate sorted pro teins from those destined for default export or secretion. these receptors may themselves have cytoplasmic domains recognized by receptors on the basolateral or apical surfaces. furthermore, such a system could han dle both secreted and exported (membrane) proteins. however, there is some evidence, based on the effect of nh c (or ph) on basolateral sorting of laminin and heparin sulfate proteoglycan, that soluble and membrane proteins may be sorted by different mechanisms ( ). clearly, more work remains to be done before we can identify polarity sorting signals with any precision. one of the difficulties may be that these signals are probably not linear sequences of amino acids and are therefore less amenable to gene fusion studies, which have been so useful in identi fying routing signals. even very subtle conformational changes may alter sorting signals and render them nonfunctional; this could explain why prevention of glycosylation with tunicamycin may cause normally polar ity-sorted proteins to enter the default pathway ( ). another aspect of the work on polarity sorting signals which needs to be pursued is the identity of receptors and the stage at which sorted proteins dissociate from them. sorting receptors are presumably re cycled. little is known about how the vesicles are targeted to different regions of the plasma membrane. one possibility is that sorting of lipids ( fig. v. ) is involved ( , a) . alternatively, sorting vesicles could interact with components of the cytoskeleton, along which they are driven by atp-dependent motors attached to microfilaments; further receptor-ligand interactions might complete the sorting process when vesi cles arrive at the plasma membrane ( ). although there is no evidence that microfilaments are required for default sorting of secretory proteins ( ), rindler et al. ( ) have reported that colchicine and other microtubule-disorganizing agents abolished specific apical sorting of ha and caused influenza virus to bud from both cell surfaces in polarized cells. salas et al. ( ) obtained the opposite result, however, and neither group observed any effect on basolateral targeting of vsv g protein. rogalski ( ), however, found that agents which caused microtubule disassembly caused random sorting of g protein. thus, the role of the cytoskeleton in the targeting of polarity-sorted secretory proteins in complex eukaryotes' proteins remains unclear. a temperature-sensitive mutation in the s. cerevisiae actin gene results in abnormal exocytosis and bud formation at the nonpermissive temperature, suggesting that actin filaments may direct secretory vesicles to the growing bud in yeast cells ( ). there is evidence to show that the cytoskeleton may be important in maintaining polarized distribution in complex eukaryotic cells ( ). a polarized atpase has recently been shown to bind directly to ankyrin, a protein which is known to link an integral erythrocyte membrane protein to spectrin and actin of the erythrocyte cytoskeleton ( ). thus, ankyrin might link the atpase to microfilaments and thereby maintain its polar ized distribution. two features distinguish regulated exocytosis from other branches of the secretory pathway: (i) protein release only occurs when the cells are stimulated by secretagogues (e.g., cyclic amp or ca + ). (ii) proteins accumulate within the cell before their release. proteins accumulate in a special class of secretory vesicles called secre tory granules, wherein protein concentrations reach such high levels that they become electron-dense and can be clearly identified by electron mi croscopy. the secretory pathway is restricted to certain cell types (e.g., endocrine and exocrine cells, mast cells), and only certain types of pro teins (examples include hormones, albumin, and some degradative en zymes including proteases and lipases) enter into the pathway. another feature of the regulated secretory pathway is that proteins may be pro-teolytically processed before release or as they are released from the cell, although this feature is also found in some constitutively secreted pro teins. regulated and constitutive branches of the secretory pathway can coexist in the same cell ( , , ) , implying that proteins destined for storage in secretory granules must be sorted from other secretory proteins. elec tron microscopic studies show that both classes of secreted proteins tran sit through the golgi cisternae together and are segregated in the tgn, where proteins destined for the regulated branch of the pathway condense into specific areas coated with the protein complex clathrin (see figs. v. and ; see also section v.g. ) ( , ). the clathrin coat is subse quently removed as the condensing granules bud from the tgn and ma ture ( ) (see fig. v. ). these areas probably represent the sites at which secretory granules mature and are released from the tgn. it should be noted, however, that some proteins which are normally se creted by the regulated pathway may 'escape" and be secreted 'consti tutively" ( ). it remains to be determined whether this is due to incorrect sorting or to low-level, secretagogue-independent secretion from storage granules, as suggested by studies by von zastrow and castle ( ). bur gess and kelly ( ) propose that this "spillover" secretion may be due to inefficient sorting in the specific cell lines tested, since rhodes and halban ( ) observed much more efficient sorting into the regulated pathway. efficient segregation of proteins into regulated and constitutive branches of the secretory pathway implies that the former have sorting signals (see section v.g. .b for an alternative explanation). the exis tence of these signals was suggested by moore and kelly ( ), who transfected a pituitary tumor cell line with a hybrid gene comprising the ' end of the vsv g protein and the ' end of the gene for human growth hormone. the hybrid protein was diverted into the regulated secretory pathway, indicating that the constitutive pathway had been bypassed due to sorting signals present in the growth hormone part of the hybrid. there are no obvious sequence similarities between proteins secreted by the regulated pathway, implying that the sorting signal is probably a patch signal rather than any identifiable linear stretch of amino acids. several proteins secreted via the regulated pathway are proteolytically processed prior to their release from the cell (see below). this raises the possibility that the sorting signal could reside in that part of the secretory polypep tide which is eventually cleaved off. this possibility was tested by bur gess et al. ( ) , who found that deleting dna coding for the propeptide part of trypsinogen had only a minor effect of the targeting of the enzyme into secretory granules. thus, they concluded, there must be at least one sorting signal in the mature part of the trypsinogen. one surprising feature of regulated pathway sorting is that the putative sorting signal seems to be universal. moore et al. ( ) , for example, found that human proinsulin was packaged into secretory granules in mouse adrenocorticotropic hormone (acth)-secreting cells, that it was correctly processed (see below), and that its release from these cells was stimulated by the same secretagogues that stimulated acth release. similar results were obtained in studies on the expression of human kid ney renin dna in the same cell line ( ). fibroblast l cells, however, which do not have the regulated pathway, secreted (unprocessed) proin sulin via the constitutive pathway ( ). receptors localized in specific domains of the tgn membrane may segregate proteins into the regulated branch of the secretory pathway, but this has not been proven, and other factors may be important. for exam ple, treatment with the weak base chloroquinone causes acth to be secreted by the constitutive pathway, indicating that low ph is required for sorting into the regulated pathway ( ). low ph in condensing secre tory granules may dissociate proteins from their receptors, which can then be reused (but see section v.g. .b). another feature of secretory granules which appears to have been largely overlooked is that proteases involved in post-tgn processing of secretory proteins must also carry sorting signals. it remains to be seen whether the membrane content of secretory granules differs significantly from that of vesicles of the consti tutive branch of the secretory pathway. in certain exocrine cells, the concentration gradient of a secretory protein between the rer and secretory granules can be as high as ( ). the dense core of aggregated protein is sometimes seen to be separated from the membrane of the secretory granule ( ) and may remain intact when the membrane is removed ( ) or upon exocytosis ( , ) . although receptor-mediated sorting into specific regions of the tgn may assist the condensation process, proteins secreted by the regulated pathway may aggregate spontaneously and be packaged into secretory granules when they reach a critical size. thus, the packaging of g protein-growth hor mone hybrids into secretory granules discussed above ( ) may be due to the presence of "aggregation" sequences in the growth hormone segment of the polypeptide, rather than to the presence of a specific sorting signal. the low ph of the condensing granule ( , ) may be important for protein aggregation, since aggregates dissociate at high ph ( ). pfeffer and rothman ( ) suggest that this could explain the failure of chloroquinone-treated cells to package secretory proteins into secretory gran ules (see above). secretory granules of exocrine (hormone-secreting) cells remain acidic during maturation, but those of endocrine (enzyme-secret ing) cells return to neutral ph as they mature ( ). atp is also needed for secretory granule formation. there are conflicting views as to whether different secretory proteins cosegregate and coaggregate into the same secretory granule. detailed studies by fumagalli and zanini ( ) revealed that bovine growth hor mone and prolactin could be present either in different aggregates in the same secretory granule or in mixed aggregates in the same granule or in pure aggregates in different granules. the ratios of the three type of granules varied from animal to animal. similar results were reported by mroz and lechene ( ) , who showed that the enzyme content of individ ual secretory granules derived from single cells from the same gland can vary enormously. the simplest interpretation of these data seems to be that segregation is a random process and that the formation of mixed or pure aggregates depends on the local concentration of the respective pro teins and on their preference for forming homo-rather than heteroaggregates. packaging of proteins into different secretory granules, however, might permit their release to be stimulated by different secretagogues, but there is only limited evidence for such a phenomenon at the level of individual cells ( , , a) . many of the proteins secreted by the regulated branch of the secretory pathway are proteolytically processed and activated, usually in secretory granules. processing often involves the proteolytic removal of an n-terminal propeptide and can be mimicked by exogenous proteases such as trypsin ( ). alternatively, short spacer peptides may be removed from polyprotein precursors ( ). in the latter cases, cells in different tissues can process the precursors to give different "mature" forms. this is the case for prosomatostatin, which is processed to a -amino-acid form by cells in the gut and to a -amino-acid form by brain and pancreatic cells ( ). islet tissue from angler fish pancreas contains at least two proteases which process prosomatostatin to give products of different lengths. one of these proteases can also process proinsulin ( ) . other examples of processing of heterologous secretory proteins ( , , ) indicate the existence of only a limited number of processing proteases, which may also be found in some cell types which do not have a regulated secretory pathway ( , ) . when secretory proteins which would normally use the regulated pathway are produced in cells which do not have the regu-lated pathway, however, they are constitutively secreted as unprocessed (pro-) forms ( ). the site of proinsulin processing was determined cytochemically by orci et al. ( , ) , using monoclonal antibodies specific for mature insulin. fully processed insulin was first detected in clathrin-coated vesi cles budding from the tgn, and subsequently in naked granules. process ing was coincident with condensation and acidification and was inhibited at higher ph, indicating that the two proinsulin-processing proteases have low ph optima ( ). therefore, this and other proteolytic processing steps probably occur in a late "compartment" of the tgn. although propeptides may not play a role in protein sorting, they may prevent enzymes such as proteases from folding into active conforma tions prior to their release, thereby protecting secretory granules from endoproteolytic attack. proinsulin and other prohormones may be loosely membrane-associated ( , ). in these cases, bridge sequences may contribute to the patch signal which shunts proteins into budding secre tory granules. relatively little appears to be known about the events which accompany protein release from secretory granules. the general view seems to be that secretagogues directly, or more likely indirectly, stimulate fusion between the granule membrane and the cytoplasmic membrane, resulting in the release of granule contents to the outside of the cell. secretagogues bind to specific cell-surface receptors and promote ca + influx, which seems to be intimately related to the fusion event ( ). gtp binding proteins also seem to be involved in generating the signal which stimu lates secretion ( ), and microtubules may play a minor role in directing storage granules to the cell surface ( ). inhibitors of metalloprotease and dipeptide protease substrates inhibit exocytosis, suggesting that pro teolytic cleavage of a membrane protein may be essential for exocytosis. mundy and strittmatter ( ), who found that metalloprotease activity was highest in the plasma membrane, propose that proteolysis may un mask the active site on a fusogenic membrane protein. breckenbridge and aimers ( ) have recently studied exocytosis-associated changes in membrane capacitance in a mouse mast cell mutant with enlarged secretory granules. small fluctuations in capacitance preceded larger increases, which themselves preceded granule swelling and the release of a fluorescent tracer dye from the lumen of the granule. the large increase in capacitance probably results from productive fusion be tween the plasma and granule membranes ( ), whereas capacitance "flutters" may represent nonproductive membrane association. the most plausible interpretation of these data is that release of secretory granule contents is preceded by the formation of a narrow channel, the fusion pore, ( ) between the two membranes, leading eventually to the open ing of the granule membrane to the outside of the cell and the subsequent swelling and dissociation of the granule contents and their release as soluble proteins. lysosomes (in animal cells) and vacuoles (in plant and fungal cells) con tain most of the cells' degradative enzymes, which function not only in general "housekeeping" but also in the degradation of endocytosed mate rial (chapter viii). the following sections review the evidence for lysoso mal and vacuolar sorting signals, special features of the sorting pathways, and differences between the lysosomal and vacuolar routes. specific sorting of soluble lysosomal enzymes is determined by mannose- -phosphate (m p) residues on n-linked core oligosaccharides (section v.d.i.a). two receptors have been identified. the major m p receptor ( kda, formerly called the -kda receptor) was detected predomi nantly in the cis golgi compartment ( ), leading to the proposal that the lysosomal pathway diverged from the main secretory pathway at the cis end of the golgi stack rather than in the tgn. this idea is incompatible with the observation that some lysosomal proteins are terminally pro cessed by enzymes located in medial and trans golgi compartments. other cytological studies indicate that m p receptors are located in the tgn, as well as throughout the golgi stack ( ), in coated vesicles and the plasma membrane ( , ) (see below for explanation), and in a golgi-proximal vesicular structure ( ), but not in lysosomes. thus, ly sosomal enzymes probably do transit through the tgn, possibly already complexed with their receptor, although farquhar ( ) argues strongly in favor of multiple lysosomal sorting pathways and in particular for sorting from the cis golgi in certain cell lines. thus, the site of accumulation of m p receptors along the secretory pathway may have little relevance for lysosomal enzyme sorting. two m p receptors ( kda and kda) have been identified and characterized. the receptor activity of the -kda protein, but not that of the -kda protein is cation-independent, and the -kda receptor recognizes only phosphomonoesters whereas the -kda protein also binds methylphosphomannosyl residues ( ). the -kda protein ap pears to be present in most mammalian cell lines tested so far ( ), but the distribution of the -kda receptor has not been determined. some mutant cell lines lack the -kda m p receptor yet still target lysosomal enzymes normally, which suggests that both receptors are involved in lysosomal sorting ( ). studies with such cell lines revealed another difference between the two receptors, however. although part of the cellular pool of both receptors is located on the cell surface, only cells with the -kda protein can endocytose secreted lysosomal proteins ( ). this "defect" in the -kda protein appears to be due to a failure to bind the ligands, since antibodies against the -kda protein are endocytosed normally (see chapter viii for more details on endocytosis). thus, the -kda protein seems to be specifically involved in the sorting of endogenous lysosomal enzymes. the genes for both m p receptors have been cloned and sequenced. although the predicted sequence of the two gene products are generally different, the two proteins have a region of moderate sequence similarity in their lumenal domains ( , ) which dahms et al. ( ) propose could be the m p binding domain. the sorting of the lysosomal enzymes cathepsin c and cathepsin d was studied directly by lemansky et al. ( ) , who found that lysosomal enzyme precursors occurred only in coated vesicles. proteolytically ma tured forms were found in lysosomes. schulze-lohoff et al. ( ) also observed the transient accumulation of one of these enzymes in coated vesicles, which, they proposed, are specifically involved in the sorting of lysosomal proteins from the secretory pathway. lemansky et al. ( ) devised procedures which allowed vesicles derived from the secretory pathway to be separated from those derived from the endocytotic path way. they found that both classes of vesicles contained precursor forms of cathepsin. these studies have two profound implications: (i) the fact that vesicles involved in direct sorting of cathepsins to the lysosome contain clathrin implies that they had passed through the tgn, the first site along the secretory pathway at which clathrin is detected (section v.g.i). (ii) the fact that some lysosomal enzymes are "fished" out of the sur rounding medium and retargeted to the lysosome implies that some lysosomal enzymes are incorrectly sorted, probably into the consti tutive secretory pathway (see also section v.d. .a). higher levels of incorrect sorting occurs in nh -cl-treated cells, probably because low ph is required to dissociate m p from its receptor in the prelysosome ( another interesting observation concerning the m p receptor is that it specifically binds to one of the components (the -kda accessory pro tein) of the clathrin cage which coats the sorting vesicles ( ) (see be low). this may have particular relevance for the sorting of lysosomal enzymes because different classes of clathrin-coated vesicles appear to have different types of accessory proteins ( ). coated vesicles almost certainly do not transport proteins directly into lysosomes. instead, the vesicles are targeted to endosome-like reticular organelles (prelysosomes or secondary endosomes), where the receptor probably dissociates and recycles back to the golgi cisternae. this organ elle is also the site to which endocytosed lysosomal proteins are targeted ( , ) ( fig. v. and section viii. a. .b). a different class of vesicles may complete the transport of lysosomal enzymes once they have dissoci ated from their receptor, but von figura and hasilik ( ) consider it more likely that there is a gradual transition from tubular prelysosomes to lyso somes proper. although m p is undoubtedly the major, and in some cells the only, sorting signal on lysosomal enzymes, some lysosomal proteins do not have m p residues. owada and neufeld ( ) found that a human liver cell line devoid of iv-acetylglucosamine-l-phosphotransferase [and there fore unable to phosphorylate mannose residues (see section v.d.i.a)], still targeted some lysosomal enzymes correctly with, at most, only slightly reduced efficiency. these cells may have a completely m p-independent system for sorting lysosomal enzymes. all lysosomal membrane proteins are also devoid of m p residues. therefore, some lysosomal enzymes may have membrane-associated intermediates which are sorted to the lysosome together with authentic lysosomal membrane proteins. barriocanal et al. ( ) used immunocytochemistry to follow the fate of three lysosomal membrane proteins which they detected in lysosomes, the golgi apparatus, and coated and uncoated vesicles in the region of the tgn. they found that oligosaccharide modifications were not required for lysosomal targeting (although they may be required to protect against proteolysis). thus the sorting pathway for these proteins remains to be determined; they could be sorted completely independently of lysosomal enzymes or could be colocalized to the same coated vesicles by an m pindependent receptor and then segregated from recycling vesicle mem brane components in the prelysosome. green et al. ( ) have recently found that newly synthesized lysosomal membrane proteins appear in lysosomes with the same kinetics as newly synthesized plasma membrane proteins appear at the cell surface, making it unlikely that the former pass via the plasma membrane en route to the lysosome. although vacuoles are the functional equivalents of lysosomes in animal cells, the sorting of vacuolar enzymes is completely independent of m p receptors. this was demonstrated most simply by the fact that tunicamy cin treatment did not affect vacuolar protein targeting ( , ). how ever, at least one vacuolar protein does have phosphorylated mannose residues. most of the work on the sorting of vacuolar proteins has concentrated on the identification of sorting signals in yeast vacuolar proteins. early studies showed that vacuolar proteases were proteolytically processed in two distinct stages, the first of which corresponded to the removal of a signal peptide ( ). the second processing step is catalyzed by a vacuo lar protease, proteinase a, which removes an additional n-terminal poly peptide segment, the propeptide, from other vacuolar enzymes. protein ase a is also autoactivated by the same mechanism ( , ). the second processing step is blocked by certain sec mutations, which cause secre tory proteins to accumulate in the rer or golgi apparatus, whereas mutations which affect the final stage of the secretory pathway from the golgi to the cell surface do not affect vacuolar protein sorting or process ing ( ). this implies that the golgi is the site of sorting of vacuolar and secreted or plasma membrane proteins in yeast cells. gene fusion studies conclusively demonstrated that propeptides are vacuolar sorting signals. bankaitis et al. ( ) and johnson et al. ( ) found that at most n-terminal residues of preprocarboxypeptidase y (cpy), including the -residue propeptide, could target the normally secreted enzyme invertase into the vacuole. similar results were obtained with proteinase a-invertase hybrids ( ). vails et al. ( ) subse quently found that mutations affecting the sequence of the procpy pro peptide caused the enzyme to be secreted in an inactive form. there appears to be no sequence similarity between the propeptides of different yeast vacuolar enzymes, even though genetic studies described below suggest that they are sorted into the vacuole via a common pathway. part of the propeptide may be required to maintain vacuolar enzymes in an inactive form until they reach the vacuole and may also maintain the precursors in a competent conformation for transport through the secre tory pathway. the overproduction of vacuolar proteinase a ( ) and of cpy-inver-tase hybrids ( ) causes them to be secreted into the medium, suggesting that some component of the vacuolar sorting pathway (e.g., a receptor) had been saturated. these observations led to the development of tech niques for selecting mutants which secreted cpy or cpy-invertase with out overproduction (section ii.f.l.b). mutations in over different genes (called vpl or vpt) have been identified ( , a, ) . the extent of the sorting defect varied in different mutants: some of them, for exam ple, did not affect the targeting of proteinase a, and none of them affected the sorting of the vacuolar membrane protein α-mannosidase, which is presumably sorted to the vacuole by an alternate pathway ( ). it is unlikely that any of the mutations affected protein retention in the vacuole because vacuolar enzymes were not terminally processed, and the kinet ics of cpy secretion were comparable to those of a normally secreted protein. the characterization of the vpt or vpl gene products and their localization in the cell could provide revealing insights into the mecha nisms of vacuolar protein targeting, but at present we can only speculate on their roles. obvious candidates are the propeptide receptor, vacuolar or golgi atpases which might produce a low ph environment necessary for sorting or receptor dissociation, or proteins involved in vesicle fission and fusion. much less work has been done on plant vacuolar proteins. tague and chrispeels ( ) found that the plant vacuolar storage protein phytohemagglutinin was targeted mainly to the vacuole when its structural gene was expressed in yeast cells. this protein does not have a cleavable propeptide (it does have a signal peptide, which was at least partially processed in yeast cells), which means that the vacuolar plant sorting signal which is recognized by the yeast vacuolar sorting pathway is lo cated in the mature part of the phytohemagglutinin polypeptide. mrna coding for a second plant storage protein, globulin p, has been microinjected into frog oocytes, which secreted the protein into the medium ( ). this confirms that plant vacuolar proteins are bona fide secretory proteins and that plant cells have a special branch of the secretory pathway which shunts storage proteins into vacuoles. as we have seen, protein-coated vesicles are involved in transporting secretory proteins at various stages of the secretory pathway. some vesi cles (e.g., lysosomal sorting vesicles and immature secretory granules), are coated with a protein complex called clathrin, which also coats endocytotic vesicles (chapter viii). other secretory vesicles (e.g., those me diating protein transport through the golgi) have a different type of pro tein coat ( ). clathrin, which is composed of equimolar amounts of heavy and light chains, forms a three-layered cage which envelops vesicles in a shell with fibrous interconnections, which give it mechanical strength and stability. the vesicle membrane is thought to have receptors which anchor the clathrin cage to the surface via a number of ancillary assembly proteins which probably act as bridges ( , ) . different assembly proteins are found in different classes of clathrin-coated, tgn-derived, and endocytic vesicles, suggesting that they might contribute to their respective specificity for particular membrane targets ( ). the clathrin coat probably prevents intimate contact between fusing membranes and must thus be removed to allow fusion to occur. this may explain, for example, the disappearance of the clathrin coat from matur ing secretory granules (section v.g. .b) and the presence of lysosomal protein precursors in naked as well as coated vesicles. vesicles coated with other proteins are also presumably uncoated to allow fusion to oc cur. the uncoating of clathrin-coated vesicles is mediated by the atpdependent cytosolic "uncoating" protein, which remains attached to the released clathrin ( ). soluble clathrin retains its typical triskelion con formation. uncoating protein is a member of a highly conserved group of stress proteins and may be related to hsp heat shock proteins involved in other stages in secretory protein transport and in mitochondrial protein import (sections iii.c. and vlb. ). the significance of this observation remains to be determined ( , ), but one possibility is that the atpase (hsp ) is required to activate an uncoating enzyme which is already present in the clathrin complex. the action of uncoating protein must be triggered in some way to pre vent it from destroying protein coats on immature vesicles or on coated buds, but the nature of the signal remains to be determined. the require ment for atp for uncoating activity could in part explain the observed requirement for the same nucleotide during the transport of proteins be tween different stages of the secretory pathway if a similar activity is required to remove other, nonclathrin coats. attempts to determine the role of clathrin in protein targeting in yeast cells have given ambiguous results. yeasts are known to have clathrin, and coated vesicles have been observed, but it is not known whether clathrin coats vesicles involved in secretory protein targeting ( ). the gene for the clathrin heavy chain was independently cloned by two groups who used it to inactivate the chromosomal gene to study the effect of the absence of clathrin on cell growth and protein secretion. payne and schekman ( ) and payne et al. ( , a) reported that their mutants grew somewhat more slowly than wild-type cells, secreted invertase at a slightly reduced rate, and were partially defective in prepro-a-factor pro-cessing. the mutants accumulated unusual vacuoles, vesicles, and golgiderived structures. these results suggested that the absence of clathrin did not completely impair plasma membrane growth and protein secretion but that there was nevertheless a reduced rate of transit of secretory proteins through the later stages of the secretory pathway. different results were obtained using exactly the same approach by lemmon and jones ( ). they found that cells lacking the clathrin heavy chain were not viable unless they also carried a suppressor mutation. even with this mutation, the cells grew slowly, were larger and rounder, had an unusual granular appearance, tended to aggregate in liquid culture, and were poly ploid. these results suggest that the absence of clathrin is highly detri mental to yeast cells, making it difficult to determine whether clathrin plays a specific role in protein secretion in this organism. the secretory pathway is almost certainly the route by which the vast majority of secreted and plasma membrane proteins are exported by eukaryotic cells. however, there is increasing evidence that some plasma membrane and secretory organelle proteins may reach their final destina tions directly rather than via the secretory pathway. examples of this class of proteins include ras-like gtp binding proteins such as sec ( ) and ras ( ) of the yeast s. cerevisiae and similar proteins from complex eukaryotes ( ), mating pheromones in yeast and some fungi ( , , ) , capsid proteins of picornaviruses ( ), and src proteins of rous sarcoma and other transducing viruses ( ). most if not all of these proteins are fatty acylated. two different amino acids seem to be modified: n-terminal glycines, which are substrates for myristoyl coa protein v-myristoyltransferase ( ), and c-terminal cysteines in raslike proteins ( ). these cys residues are reported to be palmitoylated, but a farnecyl residue has been found in basidiomycete pheromones ( ). the absence of the fatty-acylated amino acid disrupts membrane associa tion of ras and src proteins ( , , , ) , indicating that fatty acids probably anchor these proteins in their respective membranes. it remains to be determined how fatty-acylated proteins actually cross the plasma membrane (as in the case of the fungal pheromones) or what determines their specificity for certain membranes. a further example of a secreted protein which does not have a secretory routing signal is interleukin , but very little appears to be known about how this protein crosses the plasma membrane. there is general agreement concerning the events which lead to the sort ing of secretory proteins into different terminal branches of the secretory pathway, as illustrated in fig. v. , but we are clearly a long way from understanding exactly what directs proteins to their specific targets. patch signals are undoubtedly necessary for sorting soluble proteins (other than lysosomal enzymes) into the various branches of the secretory pathway, but these will be difficult to identify by gene fusion techniques. at present, we have no clear idea of the extent to which the sorting vesicles have different membrane contents, but it seems probable that specific groups of membrane proteins (lysosomal, secretory granule, apical, and basolateral) accumulate at different sites in the membrane of the tgn from which sorting vesicles bud. this specialization is presumably also determined by protein-protein interactions, but the possibility that other interactions (e.g., protein-lipid) might be involved should not be over looked. another interesting observation is that atp seems to be required at almost every stage in the secretory pathway. atp has been proposed to act in a variety of ways, including acidification of secretory organelles and vesicles, phosphorylation of receptors or ligands, protein folding and "proofreading," activation of cytoskeletal motors, and vesicle uncoating activity. there is increasing evidence that gtp and gtp binding proteins (g proteins) are also involved at several stages in the secretory pathway. gtp binding proteins are also known to be involved in the generation of other intracellular signals, such as the activation or inactivation of adeny late cyclase, the activation of cyclic gmp phosphodiesterase, and the control of phospholipase c action ( ). by analogy, gtp binding pro teins may act as signals or to activate receptors or ligands to ensure vectorial transport through the secretory pathway ( ). constitutive and regulated secretion of proteins progress in unravelling pathways, of golgi traffic assembly of asparagine-linked oligosaccharides the sorting of proteins to the plasma membrane in epithelial cells biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi protein localization and membrane traffic in yeast lysosomal enzymes and their receptors key: cord- -xsuialnn authors: kellokumpu, sakari; hassinen, antti; glumoff, tuomo title: glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized date: - - journal: cell mol life sci doi: . /s - - - sha: doc_id: cord_uid: xsuialnn glycosylation is the most common and complex cellular modification of proteins and lipids. it is critical for multicellular life and its abrogation often leads to a devastating disease. yet, the underlying mechanistic details of glycosylation in both health and disease remain unclear. partly, this is due to the complexity and dynamicity of glycan modifications, and the fact that not all the players are taken into account. since late s, a vast number of studies have demonstrated that glycosyltransferases typically form homomeric and heteromeric complexes with each other in yeast, plant and animal cells. to propagate their acceptance, we will summarize here accumulated data for their prevalence and potential functional importance for glycosylation focusing mainly on their mutual interactions, the protein domains mediating these interactions, and enzymatic activity changes that occur upon complex formation. finally, we will highlight the few existing d structures of these enzyme complexes to pinpoint their individual nature and to emphasize that their lack is the main obstacle for more detailed understanding of how these enzyme complexes interact and function in a eukaryotic cell. in eukaryotes, the majority of glycans are synthetized in specialized organelles, the endoplasmic reticulum (er) and the golgi apparatus. together they harbor dozens of functionally distinct glycosyltransferases (and glycosidases) that sequentially add (or remove) a single sugar residue at a time to (or from) the growing oligosaccharide chain [ , ] . as an example, fig. shows the schematic representation of the n-glycan processing steps that take place in the er and the golgi apparatus of eukaryotic cells. this generally accepted view, however, fails to explain how this sequence of enzymatic reactions is orchestrated to guarantee faithful synthesis of thousands of different glycans without any template, in the presence of enzymes that compete for the same substrate and/acceptor protein and also localize in the same golgi sub-compartment [ ] . preservation of fidelity is important, as even a single change in the linkage type can have a drastic effect on glycan's d structure and thus, also for its normal functions in a cellular context. as of now, it turns out that one answer to that puzzle has been lying around for the last years, but remained mainly unrecognized. namely, several different studies carried out during the late s and early s already showed that glycosyltransferases tend to exist as enzyme complexes in the cells. one of the first examples of these was the observation that a soluble lactose synthase (ls, ec . . . , for enzyme names and definitions, see table ) typically found in bovine milk consists of two the term ''complex'' is used in this review to define a functional assembly of two or more similar (homomer) or dissimilar (heteromer) glycosyltransferases that interact with each other and in the case of the latter, typically act sequentially during glycan synthesis. protein components, galt-i (encoded by the b galt gene) and a-lactalbumin [ ] [ ] [ ] [ ] [ ] . this interaction lowered the k m of galt-i transferase for glucose, enabling it to use glucose as an acceptor for lactose synthesis. schwarz et al. [ , ] were the first to identify complexes that consist of two dissimilar glycosyltransferases. by using immunoprecipitation, they showed that a xylosyltransferase (xylt) and a galactosyltransferase (galt), enzymes that initiate the synthesis of chondroitin sulfate, interact directly with each other. similar results were also obtained by fishman [ ] between n-acetylgalactosaminyl transferase (gal-nact) and galactosyltransferase (galt)) that synthetize neural glycolipids. although no direct interaction between the two enzymes was demonstrated in this study, it was found that the endogenous acceptor, i.e., the product of the galnact, was a far better substrate for the galt than an exogenously added glycolipid. it also had an order of magnitude lower k m than the latter. in addition, ivatt [ ] suggested that n-acetyllactosamine synthesizing enzymes (glcnact, galt) form a complex upon their co-adsorption into the same liposome (in contrast to separate liposomes), as the endogenously generated reaction intermediate was used as the preferential substrate for the reaction. these observations were suggested to be consistent with the facilitated passage of the intermediate glycoprotein within the complex itself and also beneficial for glycosylation, as complex formation likely provides a means to increase the fidelity of glycan synthesis by preventing intervention by competing enzymes. since then, a number of other biochemical and cell biological studies including recent live cell imaging methods have provided compelling evidence for the existence of glycosyltransferase complexes in all eukaryotes and also bacteria. the current data now shows that such note that some glycosylation (addition of n-acetylglucosamine) takes place also in the cell's cytoplasm. right a schematic cartoon showing the sequential processing of n-glycans by glycosyltransferases in the er and the golgi. the enzymes (glycosyltransferases and glycosidases) involved are traditionally thought to function separately one after the other by adding or removing sugar residues one at the time in a specified order to and from the growing oligosaccharide chain glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized complexes are prevalent and exist in most-if not allglycosylation pathways found in eukaryotic cells. in the next paragraphs, we will review the data gathered from yeast, plant and animal cells on these glycosyltransferase complexes one pathway at the time. we will focus mainly on their mutual interactions, rather than to their complexes with other proteins, unless the interaction is critical for the activity of a given enzyme. in the latter part of the review, we will also discuss both structural and biochemical data on the few complexes whose enzymatic activities change upon complex formation, or whose d structures have already been determined, just to provide a first glimpse on how these enzymes interact and how complex formation regulates the enzymatic activity of the complex constituents. we apologize the bacterial community for not including any bacterial data in this review, and also those whose data may have escaped our attention. yeast n-glycosyltransferase complexes the yeast n-glycosylation pathway shows a high degree of pathway conservation with higher eukaryotes, involving similar oligosaccharide-dolichol precursor synthesis, glycan transfer to nascent proteins by an oligosaccharyltransferase (ost) complex, and removal in the endoplasmic reticulum (er) of the three glucoses and the central-arm a , -linked man from the newly transferred glc man glcnac [ , , [ ] [ ] [ ] . the first steps to form glcnac -pp-dolichol glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized involves stepwise addition of two glcnacs by glcnac- phosphate transferase (algt ) and alg p/alg p udp-glcnac-transferases (fig. ). these first three enzymes have been shown both by immunoprecipitation and gel filtration analyses [ ] to form a hexameric complex with a stoichiometry of : : and with native molecular weight of * kda. recently, alg was shown to be the central unit and able to organize the formation of this threeenzyme glycosyltransferase complex [ ] . the next enzymes in the row, the er mannosyltransferases alg , alg and alg that add three more mannose residues to form the man glcnac -pp-dol intermediate, have similarly been shown to form complexes with each other. both genetic and biochemical evidence indicates that alg interacts with itself, alg , or with alg [ ] . thus, the two heteromeric alg -containing complexes differ from one another in that one complex contains alg and the other contains alg . both of these complexes were found to be functionally important, as missense mutations affecting the activity of alg , but not its assembly with alg or alg , exhibited a dominant negative phenotype. thus, dolichol-linked nglycan precursor synthesis on the cytoplasmic side of the er membrane appears to involve three main enzyme complexes, one formed by alg p/alg p/alg p and the other two either by alg /alg or alg /alg . this arrangement likely ensures that each mannose residue will be linked correctly to the dolichol-linked precursor glycan structure even in the presence of several other competing mannosyltransferases on the cytosolic surface of the er membrane. after flipping to the er lumen by the rft protein [ ] , four additional mannose residues and three glucose residues are further added to man glcnac -pp-dol intermediate. enzymes responsible for these additions involve the alg / alg /alg mannosyltransferases [ ] , which add the four mannoses to form the man glcnac -dolichol structure. this is then followed by addition of three glucoses that are added by alg , alg and alg glucosyltransferases, but it is currently unclear whether these enzymes form complexes with each other as well. this core oligosaccharide is next attached to a specific asparagine residue (consensus motif asn-x-ser/thr) in a polypeptide chain by the subunit oligosaccharyl-transferase complex [ ] [ ] [ ] [ ] [ ] . the glc -man glcnac core structure is subject to trimming by aglucosidases i and ii, yielding glc man glcnac and glc man glcnac structures, the latter of which serves for er quality control by er chaperones erp , calnexin and calreticulin [ ] [ ] [ ] . the glucose and the terminal mannose residue in the middle branch of the oligosaccharide chain are removed by a-glucosidase ii and er mannosidase i, respectively, before transport of the man glcnac to the golgi. upon arrival in the yeast golgi, up to mannose residues are added to an outer chain of an n-glycan of secretory pathway proteins such as invertase, and the 'mannan' structural proteins of the cell wall. in contrast to mammalian n-glycan processing in the golgi (fig. ) , this 'hypermannose' structure consists of a long backbone of a , -linked residues with a , -linked branches which usually terminate in a , -linked residues [ ] . after och p has attached the first a , -linked mannose to the core structure, two distinct polymerase complexes, m-pol i (which consists of van p/mnn p) and m-pol ii (hoc p/ mnn p/mnn p/anp p/mnn p) then synthetize the long mannan backbone [ ] , while the branching of this backbone is accomplished by the sequential actions of mnn p, mnn p and mnn p. some of the branches also receive a phosphomannose that is added by mnn p and mnn p. all these proteins or their complexes were found to have mannosyltransferase activity in vitro. n-glycosyltransferase complexes in mammals the processing of n-glycans in the golgi apparatus of higher eukaryotes differs markedly from that of yeast. high mannose glycans synthetized in the er are extensively processed to form hybrid and complex type n-glycans ( fig. ) by a defined set of glycosyltransferases and glycosidases in each cell type (stanley et al. chap . in: [ ] ). similar to the er-localized glycosyltransferases, these golgi enzymes also tend to form complexes with each other. the first example of these was galt-i, which has been shown to form high molecular weight oligomers and/ or homo(di)mers in isolated membrane preparations prepared from mammalian cells [ , ] . oligomerization was thought to have a role in golgi retention [ ] . similar oligomers or homodimers have since been detected also with glcnact-v, st gal-i, fuct-i, fuct-iii and fuct-vi [ ] [ ] [ ] [ ] [ ] [ ] [ ] . by utilizing bimolecular fluorescence complementation (bifc) and fluorescence resonance energy transfer (fret) approaches, we have also recently confirmed that not only galt-i but also the other main human golgi n-glycosyltransferases tested (glcnact-i, ii, galt-i, st gal-i, st gal-iii) form homomers in live cells [ , ] . the first heteromeric glycosyltransferase complexes were detected by using an er-re-location assay developed by the nilsson group [ , ] . they showed that glcnact-i, fused with a dibasic er-retention signal derived from the cytoplasmic tail of the p invariant chain, was able to relocalize endogenous mannosidase ii or glcnact-ii (but not galt-i) from the golgi to the er, suggesting a direct interaction between these medial-golgi enzymes. based on their data, the authors suggested the new golgi ''kin recognition'' model for medial-golgi enzymes, which serves their correct targeting to the golgi. later on, the interaction between glcnact-i and glcnact-ii has also been confirmed by using an immunoprecipitation approach [ ] . enzymes such as b gnt- and b gnt- involved in the elongation of specific branch structures of multi-antennary n-glycans with polylactosamine have also been shown to form a complex with each other [ , ] . moreover, cross-testing of all the potential interactions between the main n-glycosyltransferases by using bifc and fret approaches, we have shown that they all form heteromeric complexes with each other [ , ] . the medial-golgi enzymes glcnact-i and glcnact-ii were found to form one such complex that may also contain other medial-golgi glcnac transferases and mannosidase ii. the other complexes consist of trans-golgi enzymes, galt-i and st gal-i or galt-i and st gal-iii. crosstesting potential interactions between n-and o-glycosyltransferses [ ] did not reveal any interacting partners, suggesting that complexes form only between sequentially acting enzymes within the same glycosylation pathway. very recently, the bifc approach was used to show interactions between tyrosylprotein sulfotransferases tpst and tpst [ ] with the result that these enzymes formed complexes not only with themselves, but also with stgalt-i, producing either tpst /st gal-i or tpst / st gal-i heteromers. a different type of interaction takes place between glcnact-i and gnt ipl (mgat d) protein in that the latter has no known enzymatic activity and is expressed at high levels in the testicular germ cells [ ] . the functional relevance of this inhibitory interaction has been shown to be in the down-regulation of the synthesis of hybrid and complex type n-glycans and thereby to increase attachment of developing sperm cells to nourishing sertoli cells. plant n-glycosyltransferase complexes complex type n-glycans in plants are essential for plant development and defense mechanisms. however, the glycans themselves are somewhat different from mammalian n-glycans. for example, the proximal core n-acetylglucosamine in plants is substituted by an a , fucose (instead of a , fucose in mammals) and the b-mannose of the core is substituted by a bisecting b , xylose (a bisecting b , n-acetylglucosamine in mammals). in addition, b , galactose and a , fucose linked to the terminal n-acetylglucosamine of plant n-glycans form the lewis a (le a) oligosaccharide structure instead of b , linked galactose followed by a sialic acid in mammals. recently, it has been shown [ , ] that a number of plant n-glycosyltransferases also form complexes with each other. by using the two-photon fret-flim approach, it was shown that arabidopsis thaliana golgi a-mannosidase i, nicotiana tabacum b , -n-acetylglucosaminyltransferase i, arabidopsis golgi a-mannosidase ii (manii), and arabidopsis b , -xylosyltransferase, form both homodimers and heterodimers, whereas the late-acting arabidopsis b , -galactosyltransferase (galt ) and arabidopsis a , -fucosyltransferase, do not. however, galt was found to interact with the medial-golgi manii. this observation may reflect slight differences in the organization of the golgi cisternae between plants and mammalian cells, as the latter do not show any such interactions between medial-and trans-golgi enzymes tested thus far [ , ] . collectively, these data show that most n-glycosyltransferases in yeast, plant and mammalian cells form both enzyme homomers and a variety of functionally relevant enzyme heteromers between sequentially acting glycosyltransferases in each glycosylation pathway (see table for a collected list the known enzyme complexes described in this review). enzymes that function in different glycosylation pathways are expected to form distinct complexes with other enzymes in a pathway dependent manner. an important question that needs to be answered is whether heteromerization is an inherent property of all glycosyltransferases or only those that operate at critical points such as at initiation, branching, and termination of glycan chains. mucin type o-galnac glycosylation o-glycosyltransferases catalyze addition of sugar residues to hydroxyl groups of serine or threonine amino acids in proteins. most common o-linked glycans are mucins, and * different galnacts exist to initiate their synthesis using different protein acceptors. this is then followed by chain elongation to form different core structures which are further processed to form a number of different glycan structures. enzymes that form these core structures are well known and have also been tested for their ability to form complexes in live cells. as with n-glycosyltransferases, all the core forming transferases and two sialyltransferases [ ] were found to form homomeric complexes in the cells (see table ). this is consistent with the earlier notion that the core b , n-acetylglucosaminyl-transferase (c gnt-i) forms disulfide bonded dimers [ ] . in addition, it has been shown that the active c galt- (t-synthase, core synthase, b -galactosyltransferase) is a dimer and does not bind its specific folding chaperone cosmc, unlike the unfolded and inactive enzyme [ , ] . other potential homomeric and heteromeric interactions between o-glycosyltransferases themselves were recently tested by the dynamic fret approach. similarly to n-glycosyltransferases, it was found that all these enzymes form enzyme homomers [ ] . in addition, the initiating galnact- and c galt required for the synthesis of the t(f)-antigen were shown to form a hetoromeric complex with each other. the same galnact was also found to interact with the core and core forming glycosyltransferases (c gnt-i, glcnact-i/glcnact-ii n-glycan branching h. sapiens [ , , ] galt-i/st gal-i and galt-i/st gal-iii termination of n-glycans h. sapiens [ , ] b gnt- /b gnt- synthesis of polylactosamine h. sapiens [ , ] tpst /st gal-i, tpst /st gal-i tyrosylprotein sulfation/sialylation h. sapiens [ ] glcnact-i/gnt ipl inhibition of n-glycan branching m. musculus [ ] galnact- /c galt- , -/c gnt- , -/c gnt- ssi, ssiia, and sbeiia/b amylopectin synthesis and branching t. aestivum/z. mays [ , ] c gnt-i). cross-testing of other potential interactions between the nine o-glycosyltransferases tested did not reveal any other enzyme heteromers in the cells [ ] . other o-linked glycans drosophila and mammalian fringe proteins possess a fucose-specific a , n-acetylglucosaminyltransferase activity that initiates elongation of o-linked fucose residues attached to epidermal growth factor-like sequence repeats of notch. although direct evidence is missing, it has been suggested that fringe proteins function as dimers [ ] . o-mannose containing proteins were originally identified in yeast already in the s. their typical tetra-saccharide structure consists of siaa - galb - glcnacb - manser/thr. they are now known to constitute one-third of all o-linked glycans in the brain of higher organisms, but are also abundant in the muscle tissue, in which the protein a-dystroglycan is the main acceptor for o-linked mannose addition. a defect in its o-mannosylation/glycosylation is associated with walker-warburg syndrome, an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy [ , ] . the enzymes responsible for adding the o-mannose and nacetylglucosamine are catalyzed by o-mannosyltransferase (pomt ) and (pomt ). neither pomt nor pomt have enzymatic activity alone, but when co-expressed, omannosyltransferase activity was recovered [ , ] . this suggests that the activity of pomt and pomt requires physical interaction of these enzymes in vivo. it is not known whether pomgnt , which adds b , -linked nacetylglucosamine to o-mannose-containing glycoproteins and glycopeptides forms complexes with itself, later enzymes in the pathway or with pomts themselves. in addition to mammalian enzymes, yeast o-mannosyltransferases have also been shown to form complexes with each other. members of the pmt subfamily pmt p and pmt p were found to interact with members of the pmt subfamily, forming complexes between pmt p/pmt p, pmt p/ pmt p or pmt p/pmt p [ ] . on the other hand, pmt p (a member of the pmt subfamily) was found to form and function as a homomer. finally, proteins with collagen domains are modified by a disaccharide, glca - galb, which is assembled on hydroxylysine or hydroxyproline residues. the first step in the pathway is the obligate hydroxylation of either lysine or proline by lysyl-and prolylhydroxylases prior to addition of glucose (and galactose). glycosylation takes place before the formation of the triple helix, and may control the rate of triple-helix formation and, thus, also the size of the collagen fibrils [ ] . mice lacking the lysylhydroxylase isoform (lh ) fail to glycosylate collagen iv properly, causing embryonic lethality and deposition of misfolded collagen in the er [ ] . this single polypeptide-containing enzyme has all three enzyme activities needed for collagen glycosylation, and has been shown to exist as a homodimer on the luminal side of the er membrane [ , ] . glycosphingolipids form the main class of glycolipids. their synthesis starts in the er, and is completed by distinct glycolipid glycosyltransferases operating in the golgi apparatus [ , , ] . the first step in this series of reactions involves addition of either galactose or glucose to ceramide to form galactosyl-or glucosylceramide (galcer, glccer). galcer and other galactolipids are seldom extended to larger glycans. in more complex vertebrate glycosphingolipids, the glccer is typically extended first with b-linked galactose to give lactosylceramide (galb - glcbcer) which is then further extended to generate a series of neutral ''core'' structures classified as ganglioseries (galb - galnacb - galb - glcbcer), neolactoseries (galb - glcnacb - galb - glcbcer), lacto-series (galb - glcnacb - galb - glcbcer), globo-series (gala - galb - glcbcer), and isoglobo-series (gala - galb - glcbcer). of these, the ganglio-series are the most common, being highly concentrated in the brain and being rich in sialic acids, making them negatively charged and thus important in the processes of neural signaling [ ] . the neolacto-series in turn are most common in the hematopoietic system; lacto-series are prominent in secretory organs while the globo-series are most abundant in erythrocytes [ ] . the synthesis of ganglio-series glycosphingolipids involves first b galt , which adds the b , -linked galactose to ceramide yielding lactosylceramide (laccer i.e. galb - glcbcer). this is then further modified mainly (with the exception of neutral ga ''core'' structure by b galnt ) to acidic gangliosides by siat and siat a to form mono-, di-or tri-sialylated gangliosides (gm , gd and gt gangliosides, respectively). their synthesis is further elaborated to a-, b-and c-series by b galnt n-acetylgalactosaminyltransferase and by b galt galactosyltransferase. several of these enzymes have been shown to form complexes with each other. for example, both gm and gd synthases have been shown to form homodimers [ , ] . enzyme activity measurements performed by bieberich et al. [ ] suggested an enzyme complex also between st (siat a, also named st sia , table ) and galnact (b galnt ) but not with st (siat ). complex formation between siat a and b galnt was confirmed both by co-immunoprecipitation and by fret measurements. similarly, by using co-immunoprecipitation and fret microscopy, maccioni et al. were able to show that the gm , gd , and gt synthases (b galt , siat and siat a, respectively) form a multi-enzyme complex in the proximal golgi [ ] [ ] [ ] , whereas the gm , gd and gt synthase (b galnt and b galt ) form a distinct complex in the distal golgi (tgn). currently, it is not known whether similar complexes form also between enzymes that extend these sugar structures with sialic acid, fucose or glca. some of these enzymes (e.g., siat , siat ) may also operate in other glycosylation pathways as well. yeast and plant glycolipids although lactosylceramide is the most common glycosphingolipid in vertebrates, it is rare in other eukaryotes. for example, it is substituted by manb - glc-cer and glcnacb - glc-cer in invertebrates by inositol- -o-phosphorylceramide in yeast, and by glcnaca - glcaa - -myo-inositol- -o-phosphorylceramide in plants. no direct evidence exists at the moment if the enzymes that synthesize these glycolipids form similar complexes to those described above for mammalian glycolipid synthesizing enzymes. the golgi apparatus is also responsible for the synthesis of cell surface and extracellular matrix proteoglycans. the polysaccharide side chains (glycosaminoglycan) added to various proteoglycans include keratin sulfate, chrondroitin sulfate, dermatan sulfate and heparin or heparan sulfate [ ] . they all consist of disaccharide repeats made of either glucuronic acid (glca), iduronic acid (idoa), gal-nac, glcnac or gal. they are also variably sulfated, linear, and often up to sugar residues long attached to serine residues of various proteins via a common tetrasaccharide primer (xyl-gal-gal-glca). an exception is keratansulfate, which utilizes n-(ks i) or o-linked (ks ii) sulfated poly-n-acetyllactosamine structures ([galb - glcnacb - ] n ) that are otherwise identical to those found on n-and o-linked glycoproteins. thus, the enzymes responsible for keratan sulfate synthesis, synthesize other glycans as well. by using electrophoretic mobility assays and crosslinking studies of the membrane-bound glcat- , ouzzine et al. [ ] have shown that the membrane-bound glcat-i, an enzyme that adds glucuronic acid residue onto the above trisaccharide primer, forms enzymatically active disulfidelinked homodimers. in addition, mccormick et al. [ ] have shown that ext and ext (both of which have glca-and glcnac-transferase activities and contribute to heparan sulfate biosynthesis [ ] ) form not only enzyme homomers but also heteromers both in yeast and mammalian cells. heteromerization also augmented their enzymatic activities in cell lysates. moreover, of the five heparan sulfate biosynthetic enzymes, galactosyltransferase i and glucuronosyltransferase i (required for the formation of the linkage region), glcnac n-deacetylase/nsulfotransferase , uronosyl -epimerase, and uronosyl -o-sulfotransferase), pinhal et al. [ ] were able to show that the last two of these (uronosyl -epimerase and uronosyl -o-sulfotransferase) interact with each other based on the er relocation assay developed by nilsson's group [ ] . izumikawa et al. [ ] also demonstrated multiple interactions between chondroitin synthase- (chsy- ), chsy- (chondroitin sulfate synthase , css ), and also between chondroitin-polymerizing factor (chpf) and glucuronyltransferase ii (csglca-t). hyaluronan, a glycosaminoglycan defined by the disaccharide unit (glcnacb - glcab - ) n that is neither sulfated nor covalently linked to any protein, is rather exceptionally synthesized at the plasma membrane in various cell types by transmembrane enzymes called hyaluronan synthases. by using co-immunoprecipitation, fret measurements and the proximity ligation assay, tammi et al. [ ] also recently were able to show that each of the three different hyaluronan synthases (has , has , has ) interact either with itself or with each other, forming heteromeric has /has , has /has and has /has complexes. a vast array of other proteins is known to be modified with various glycans such as n-acetylglucosamine (glcnacazylation) and c-linked mannose. these modifications are in many cases also accomplished by glycosyltransferases complexed with either soluble or membrane proteins, which however, do not possess known transferase activity, and therefore, do not represent the main focus of this review. chitin synthesis in s. cerevisiae, the b , -linked nacetylglucosamine polymer, chitin, is synthesized by a family of specialized chitin synthases encoded by chs , chs and chs genes. each of these have special role in the synthesis of septum, lateral cell walls and the bud neck. whether all these enzymes form oligomers is currently mostly unclear, but it has been shown that at least the chs p isoform forms a complex with chs p/skt p protein [ ] . in the tobacco hornworm (manduca sexta), the chs- has a molecular weight of kda, thrice the calculated molecular weight of the monomer, suggesting it exists as a trimer [ ] . cellulose synthesis plant cell walls are composed mainly of high-molecular-weight polysaccharides, proteins, and lignins. cellulose is the most abundant linear polysaccharide present and consists of up to thousands of b , -linked d-glucose units. these are synthesized in plants by a plasma membrane-localized cellulose synthase complex that forms - nm diameter symmetrical rosettes each with six subunits [ ] . the core components of this complex in higher plants are a family of ten cesa proteins [ ] [ ] [ ] . genetic and biochemical evidence has shown that three unique cesa isoforms are required for both primary cell wall and secondary cell wall cellulose synthesis. in arabidopsis thaliana, these are cesa , cesa , and cesa and cesa , cesa , and cesa , respectively [ , ] . the remaining cesas (cesa , cesa , cesa , and cesa ) are likely involved in tissue-specific processes and are partially redundant with cesa [ ] . intriguingly, these enzymes were also shown to homodimerize prior to formation of larger cesa oligomers [ ] , similar to n-glycosyltransferases found in mammalian cells [ ] . very recently, a split-ubiquitin membrane yeast twohybrid system demonstrated interactions between the four primary cesas (cesa , cesa , cesa , cesa ) and three secondary cesas (cesa , cesa , cesa ) but also between the primary cesas and secondary cesas in a limited fashion. further functional analysis of transgenic lines showed that cesa could partially rescue irx (ce-sa ) null mutants, resulting in complementation of the plant growth defect and cellulose content deficiency [ ] . the interactions between these enzymes were shown to utilize both disulfide bonds and non-covalent interactions [ ] . pectin and pectic arabinan synthesis plant cell wall pectic polysaccharides are complex carbohydrates that are synthesized by low-abundance, golgi membrane-bound biosynthetic enzymes. arabidopsis galacturonosyltransferase (gaut) is an a , -galacturonosyltransferase (galat) that synthesizes homogalacturonan, the most abundant pectic polysaccharide. it has been shown that gaut functions in a protein complex with the homologous gaut protein [ ] . in addition, golgi localization of gaut is dependent on the formation of the gaut / gaut complex. other yet unknown protein components were also found to co-immunoprecipitate with the gaut / gaut , but their functional relevance in pectin synthesis remains obscure. similarly, pectic arabinan, consisting of a , -linked l-arabinofuranosyl residues to which other larabinofuranosyl residues are attached via a , and a , linkages, form a comb-like arrangement, and are synthetized by arad and its close homolog arad . by using bimolecular fluorescence complementation, fret and nonreducing gel electrophoresis, harholt et al. [ ] showed that arad and arad are localized in the same golgi compartment and form intermolecular homo-and heterodimers when expressed transiently in nicotiana benthamiana. xyloglucan synthesis xyloglucan is the major hemicellulosic polysaccharide in the primary cell walls of dicotyledonous plants and has important structural and physiological functions in plant growth and development. in arabidopsis, the golgi-localized , -b-glucan synthase, cellulose synthase-like c (cslc ), and three xylosyltransferases, xxt , xxt , and xxt , are responsible for the synthesis of the xyloglucan backbone. by using bimolecular fluorescence, complementation and in vitro pull-down assays chou et al. [ ] have shown that at least two of these enzymes cslc and xxt form homomeric complexes. heteromeric complexes were also detected between xxt /xxt , xxt /xxt , and xxt /cslc . the same authors also showed very recently that three additional enzymes (mur , xlt and fut ) involved in the xyloglycan synthesis form complexes between fut and mur , xlt , xxt or xxt . xlt also interacts with xxt , but mur does not [ ] . they also showed that fut , xxts and cslc also form disulfide-linked enzyme homomers, while the formation of the heteromers does not involve covalent interactions. in vitro pull-down assays indicated that in the fut /mur and fut /xxt interactions are mediated by the catalytic domains of these enzymes. starch synthesis amylose is a major polysaccharide of starch, making up approximately - % of the structure. the other main component of starch is amylopectin, which is branched and makes up to - % of the structure. previous studies have indicated that starch synthases form high molecular weight complexes [ ] [ ] [ ] . co-immunoprecipitation experiments and affinity chromatography assays with recombinant proteins showed that starch synthase i (ssi), ssiia, and sbeiib (starch branching enzyme) form complexes with each other. all interactions were enhanced by atp and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. the authors proposed that, during amylopectin biosynthesis, ssi and ssiia form the core of a phosphorylation-dependent glucan-synthesizing protein complex, which then recruits sbeiib. differences in stromal protein complexes mirrored the complement of the starch synthesizing enzymes detected in the starch granules, suggesting that the complexes have a functional role in starch biosynthesis. the vast majority of golgi localized glycosyltransferases are type ii membrane proteins (fig. ) , and thus, have a short n-terminal cytoplasmic domain, a single-pass transmembrane domain (tmd), a stem domain and a c-terminal catalytic domain facing the golgi lumen [ , , ] . based on current evidence, all these domains have been shown to link glycosyltransferases together. for example, homodimerization of many enzymes such as galt-i and st gal-i has been shown to be mediated by disulphidebonds located in their transmembrane domains [ , , , , , ] . qian et al. [ ] showed that a conserved cys in the transmembrane region of rat st gal-i is required for dimerization, as a cys ala variant remained monomeric on sds-page. this does not, however, exclude the presence of other non-covalent interactions involved in homodimer formation. in fact, our domain swapping experiments combined with fret measurements have shown that cys deletion mutants still interact in the fret assay via their catalytic domains [ ] . other cys variants made ( total) probably reflect misfolding rather than breakage of monomer-monomer contacts, given that the mutants localize into the er (unpublished observations) or are used as sialyl motifs l and s within the catalytic domain of each st gal-i monomer [ , ] . some plant glycosyltransferases also seem to utilize the same system for complex formation [ , , ] . similarly, glycolipid-synthesizing enzymes galnact and galt have also been shown to interact via their n-terminal cytosolic and transmembrane domains [ , ] . in addition to disulphide bonds, both the stem and catalytic domains of glycosyltransferases have been shown to be responsible for the heteromeric interactions between mammalian medial-golgi enzymes glcnact-i and manii [ , ] . our previous domain swapping experiments showed that nearly all the main human n-and o-glycosyltransferase interactions are mediated by interactions between the catalytic domains of these enzymes [ ] , the only exception was galt-i whose interactions either with itself or with st gal-i seemed to involve mainly the cytoplasmic, transmembrane and/or the stem domain ( n-terminal amino acids). moreover, arabidopsis cisand medial-golgi enzymes [ ] seem also to utilize aminoterminal cytoplasmic-transmembrane-stem regions (cts) for complex formation. atmodjo et al. [ ] in turn showed that arabidopsis golgi galacturonosyltransferases form heteromers via both covalent and non-covalent forces between their catalytic domains. by using bifc and pull down assays, chou et al. [ ] have provided data recently to show that xyloglucan xylosyltransferases (xxt) not only form both homo-and heteromeric complexes via covalent and non-covalent bonds between the catalytic domains, but that the interaction surfaces are likely different depending on the xxt isoforms in the complex. the observed difference in the ph sensitivity of the n-and oglycosyltransferase heteromers, relative to enzyme homomers [ ] , also suggests that the interaction surfaces needed for the self and non-self-binding are likely different. collectively, the observation that all different domains are utilized for complex formation depending on the enzyme(s) in question does not necessarily reflect contradictory findings, but rather, high sequence diversity of this class of enzymes and the use of distinct interaction surfaces in each case to link enzymes together. this would guarantee sufficient specificity for the interactions so that only relevant enzymes have a chance to interact. sequence diversity at the amino acid level among glycosyltransferases may thus be an evolutionally driven phenomenon that likely has helped to organize glycosyltransferases into distinct and functionally relevant units. complex formation has been thought to be beneficial for glycosylation in several respects. firstly, it has the potential to regulate the enzymatic activity of the complex constituents. previous studies have shown that although enzymes can be active as monomers, homomers and heteromers [ ] , yet there is clear evidence that some enzymes also become activated upon complex formation. the best-known example of this is the heparin sulfate synthesis by ext and ext glycosyltransferases. when expressed alone, these enzymes localize predominantly to fig. most of the golgi localized glycosyltransferases are type ii membrane proteins with a short n-terminal cytoplasmic domain, a * amino acid a-helical tm-domain, a stem domain and a c-terminal globular catalytic domain in the lumen of the secretory pathway the er and have only moderate activity [ ] . however, when co-expressed, they localize to the golgi and form more active enzyme heterodimers, as demonstrated by the increased levels of cell-associated glca and glcnac. similarly, studies both in yeast and cos- cells showed that while both ext and ext enzymes have glcat and glcnact activity, their activities were markedly ( - fold, table ) augmented upon their co-expression [ ] . the increase of glycosyltransferase activities was demonstrated also using only ext and ext catalytic domains, suggesting that they are sufficient to generate the heteromeric functional enzyme. yet, there is still some controversy that relates to the exact functional role of ext , as it has very low activity. one possibility is that it may help folding and/ or transport of ext to the golgi [ ] . in addition, coexpression of polylactosamine synthesizing enzymes b gnt- and b gnt- has also been shown to increase the activity of both of these two enzymes in vitro [ ] . recent data from our laboratory has also shown that galt-i and the st gal-i activities are both increased by roughly . fold (relative to homodimers) upon their co-expression [ ] . it is notable that in the case of galt-i, the observed increase was not due to any trivial change in the amount of the enzyme protein itself nor of acceptor substrates, but rather to its interaction with the later enzyme in the same pathway. furthermore, synthesis of gm and gd glycolipids by sialyltransferases sial-t and sial-t has been shown to result in near . -fold higher sial-t activity upon their co-transfection in cho-k cells, relative to single transfected cells [ , ] . this activity increase was also not found to be due to the appearance of sial-t gene transcription activators or the stabilization of the sial-t protein, but rather, to the activation of the sial-t enzyme itself due to the formation of gal-t /sial-t /sial-t multi-enzyme complex. complex formation may also modulate the enzymatic activity of glycosyltransferases in a different manner. for example, ganglioside gm synthase [ ] was found to be more active as a homodimer than as a monomer, whereas st gal-i [ ] monomer was more active than the homodimer. unfortunately, however, it remained unclear to what extent, if any, heteromerization alters the activity of these enzymes (relative to enzyme homomers and monomers) in vivo and in vitro. another example of an inhibitory interaction is the recently identified glycosyltransferase-like protein gnt ip-l [ ] , which is known to interact specifically with the medial-golgi enzyme glcnact-i. even though the gnt ip-l protein does not seem to possess any measurable glycosyltransferase activity, its binding causes almost complete inhibition of glcnact-i, which in turn down-regulates the synthesis of complex and hybrid n-glycans and helps developing sperm cells to remain attached to sertoli cells and further differentiate into mature sperm cells. similar interactions of glycosyltransferases with proteins that do not possess any enzymatic activity can modulate the enzymatic activity also by other means. as an example of this type of effect is the c galt- (t-synthase) and cosmc, a specific folding chaperone that helps the enzyme to fold correctly [ , ] . another example is the dpm synthase, an enzyme that is needed for the synthesis of dolichol-phosphate-mannose (dpm), and consists of three non-homologous proteins, dpm , dpm and dpm . of these, dpm and dpm are needed to keep the catalytically active dpm bound to the er membrane and active, as otherwise it would move to the plasma membrane [ ] [ ] [ ] [ ] [ ] . in addition, dpm may enhance the binding of the acceptor dolichol-phosphate and thereby keep dpm more active [ ] . moreover, crosstalk between dpm synthase and n-acetylglucosaminyl -phosphate transferase has been shown to increase the activity of both enzymes [ ] . complex formation between gpi-n-acetylglucosaminyltransferase and dpm has also been found to increase the transferase activity of the former by threefold [ ] . collectively, these observations suggest that the activity of the glycosyltransferases in many cases is modulated by complex formation. an increase in enzyme activity likely reflects co-operative functioning of the interacting enzymes in glycan synthesis and may involve substrate channeling through the complex and/or changes in the affinity of donor or acceptor substrates. both of these are necessary prerequisites for faster processing and synthesis of a glycan chain. complex formation may also be inhibitory to allow down-regulation of some glycosylation reactions for regulatory purposes. galt-i/st gal-i yes (moderate) yes . [ ] sial-t /sial-t yes (moderate) yes . [ , ] a heteromer activity vs. monomer/homomer glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized other surprises are likely to come when more work are accomplished with other glycosyltransferase complexes. secondly, complex formation likely helps to preserve fidelity during glycan synthesis. although direct evidence is still lacking, complex formation is generally thought to provide a means to prevent intervention by potentially competing enzymes, which otherwise would be able to use the same sugar as an acceptor for glycan extension. thus, by linking relevant (sequentially acting) enzymes together, complex formation would guarantee that correct sugar and linkage type would be used to extend the glycan chain. in this scenario, enzyme complexes could act as a kind of ''templates'' for each specific glycosylation step. the mucin-type core o-glycosylation represents an example of this. it involves several core structure forming enzyme complexes that can potentially compete with other enzymes that use the same galnac as an acceptor, such as sialylation of the initiating galnac to form the tn-antigen. normally, the presence of a preformed heteromeric core complex such as that between ppgalnact- and c galt-i is expected to prevent such competing reactions from occurring, thus favoring the synthesis of a complex-specific core structure (e.g. t-antigen, the product of the ppgal-nact- /c galt-i complex). in the absence of such a complex, such as in cancer cells [ ] , competing enzymes such as the st galnac-i can function and prematurely sialylate the initiating o-linked galnac. consistent with this scenario, cancer cells typically express both tn-and sialyl-tn antigen at much higher levels than non-malignant cells (see [ ] and references therein). moreover, we showed that a golgi ph increase inhibits the formation of this heteromeric ppgalnact- /c galt-i complex and also the normal mucin type o-glycosylation likely by affecting both the core synthesis and its extension to more complex o-glycans. nevertheless, direct proof is needed and requires the construction and/or testing mutant enzymes which preserve their activity but cannot interact with their relevant partner. thirdly, complex formation may also modulate glycan synthesis by altering enzyme's substrate or acceptor specificity, or its polymerizing capacity. lactose synthase complex is a good example for the first case. lactalbumin binding to galt-i enables the enzyme to use glucose as an alternate acceptor [ ] . on the other hand, chondroitin sulfate polymerizing glucuronyltransferase (chondroitin synthase- ) is an example of the case where the polymerizing capacity of an enzyme is changed upon complex formation. this enzyme has been shown to form multiple enzyme complexes that consist of distinct chondroitin synthase family members [ ] . depending on the complex, distinct sized chondroitin sulfate chains were produced. in addition, they exhibited distinct, but overlapping acceptor substrate specificities towards two synthetic acceptor substrates. the same situation may also be true with hyaluronan synthesis where different complexes between has , has and has have been shown to exist recently [ ] . fourthly, the observed ph sensitivity of the enzyme heteromers, but not of homomers [ ] , is important to notice, and reflects their formation in different cellular compartments. the latter were shown to form in the er whereas the former are now known to assemble in the acidic golgi environment [ ] . this result emphasizes that homomers and heteromers are not competing enzyme species, but rather represent inter-dependent membrane constituents that undergo constant organelle micro-environment-dependent transitions between two physical states during their suggested recycling between the early secretory compartments (fig. ) . such transitions indeed take place between the er and the golgi [ ] , and provide a simple means to localize the most active enzyme species (heteromers) in the golgi where these enzymes are known to operate, and thereby can increase the glycosylation potential of the golgi. the absence of enzyme heteromers in acidification and glycosylation -defective cancer cells is in accordance with this view. nevertheless, an important question that remains is whether divalent cations needed for the enzymatic activity of several glycosyltransferases have any role in complex formation. it needs also to be emphasized that not all enzymes have been shown to form such heteromers. thus, the possibility that some enzymes may exist and also function as monomers and/or homomers cannot be excluded at this point. finally, complex formation has been suggested to have a role also in the correct targeting of glycosyltransferases to the golgi and to its various sub-compartments. this possibility was raised in the early s by showing that oligomerization correlated with the golgi localization of various membrane proteins including coronavirus m protein and various glycosyltransferases [ , , , , ] along with the ''kin recognition'' hypothesis [ ] . these observations raised an idea that golgi enzymes form complexes large enough not to fit into the forming transport carriers, leading to their retention in the organelle. however, the now generally accepted ''cisternal maturation'' model is not in accord with this oligomerizationmediated golgi retention hypothesis, as it assumes that the golgi enzymes continuously recycle between golgi cisternae and the er. in fact, it has been shown by using the frap (fluorescence recovery after photobleaching) approach that glycosyltransferases in general [ ] as well as their homomers and heteromers [ ] remain as mobile golgi membrane constituents that recycle between the first two secretory compartments. thus, even though some enzymes are mislocalized after experimental golgi ph increase [ ] , it is currently not clear whether complex formation itself is a determinant for golgi retention or retrieval. further studies with explicitly monomeric and oligomeric glycosyltransferases will help answer to this pertinent question. there are approximately known glycosyltransferases which, according to their dna sequence, are categorized into distinct families. classification is based on amino acid sequence comparisons and differences in activated donor and specific acceptor molecules in forming glycosidic bonds. crystal structures exist for glycosyltransferases representing families including families with at least one human enzyme structure solved. yet, nearly all the existing crystallographic structures represent only the globular catalytic domains of enzyme monomers. they all also fall into one of the three main fold types (gt-a, gt-b and cst ii fold), depending on the relative positions of a-helices and a central b-sheet in each fold [ ] . the main impact of all these structures is that they have helped to resolve both donor and acceptor glycan binding modes as well as their catalytic mechanisms (for excellent reviews, see [ ] [ ] [ ] [ ] [ ] ). here, instead, we focus on the few enzymes whose structures either appear in the crystals, or have been resolved, as complexes. volkers et al. [ ] recently published apo-and ligandbound crystal structures of human st siaiii sialyltransferase active in polysialylation. the two enzyme monomers in the asymmetric unit are related by near twofold symmetry (fig. ) . analysis by using the pisa server [ ] showed that the dimer interface area & Å is well in the range typically observed for functionally relevant protein complexes and the presence of such a dimer was also experimentally detected in solution. st siaiii structure represents a prime example of a glycosyltransferase homodimer with well plausible assembly: the dimer formation places both active sites on the same side of the dimer and within a distance from each other, which makes various functional scenarios possible. the structure of human a , -fucosyltransferase, fut [ ] , is another glycosyltransferase crystal structure exemplifying a homodimer. the fut catalytic domain resembles the gt-b fold, but has an embedded sh domain (a common interaction domain) in the c-terminus, which does not take part in the binding interface, however. the crystallized protein structure contains also a large part of the stem domain, which is disordered in the crystal structure. the beginning of the catalytic domain forms two long a-helices (fig. ) , which serve as the dimerization interface forming a compact four-helix bundle with a buried area of Å . in this structure the catalytic centers of the two fut monomers are also placed on the same side of the dimer, but not facing the membrane, and also on opposite ends of the dimer. vicinity of the n-terminal ends of the catalytic domains would enable a scenario where the stem and the transmembrane domains contribute to dimer formation. the other homodimeric complexes of glycosyltransferases present in crystal structures represent variations of the two structures discussed above. there are differences in the interaction surfaces, both in their volume and the structural details that characterize these interactions. bovine b , -galactosyltransferase (galt-i) [ ] appears a dimer with the two catalytic domains making contact through eight hydrogen bonds within a total contact area of Å [ ] . the interaction area is sufficiently large to represent a functionally relevant interaction. two structures of an engineered dual-specificity blood group a and b antigen glycosyltransferase aa(gly)b by [ ] were solved using two different crystals with different molecular packing (crystal contacts) and having either one or two molecules per asymmetric unit (fig. ) . despite these differences, the homodimer is assembled in either case exactly in the same way, indicating that the assembly represents a functionally relevant dimer possibly formed prior to exposing the protein sample to the crystallization screening. human b , -glucuronyltransferase i (glcat-i, fig. ) [ ] and glcat-p [ ] both represent physiologically relevant homodimers with % of the total surface area being buried in the dimer interface (glcat-i) and nearly identical residues forming the interface also in the glcat-p homodimer. interestingly, the homodimer assembly is highly different from that of the blood group a and b antigen glycosyltransferase aa(gly)b complex [ ] . while the latter has the active sites on opposite ends of the dimer, in glcat-i and glcat-p dimers they are next to each other on the same side of the homodimer. the few cases discussed here show how little is known in structural detail on the formation and nature of glycosyltransferase complexes. from this data, it is apparent that such complexes can form in various ways and there is no one or two models that fit all. it is to be noted that all glycosyltransferase structures represent the catalytic domains with either complete or partial omission of the stem region from the crystallized constructs. therefore the involvement of the fig. homomeric glycosyltransferase complex structures representing functionally relevant case studies. top left st siaiii sialyltransferase homodimer (gray cartoon model) [ ] shows how the active sites come relatively close to each other and face the golgi membrane. n-termini of the crystallized catalytic domains are situated on both ends of the dimer with the first amino acid residue (trp ) indicated with space filling atoms and completed to a type ii membrane protein model with schematic stem-and membrane spanning domains (black line and blue rectangle) included. positions of the active sites are indicated with a catalytic residue his . top right the structure of a , -fucosyltransferase fut [ ] is shown. the first residues in the crystallized construct, leu , come close to each other suggesting a possible assembly of the stem-and membrane spanning domains. active sites are indicated by catalytic residues arg . bottom left engineered dual-specificity blood group a and b antigen glycosyltransferase aa(gly)b homodimer [ ] also shows the active sites (represented by trp ) on the ends of the dimer, but interestingly the n-termini are located on opposite sides of the dimer: the left one (ser ) towards the viewer and the right one (val ) on the back (indicated by a short dashed line and an arrow). bottom right human b , -glucuronyltransferase i (glcat-i [ ] and glcat-p appear to have their active sites (gln ) buried deeper in the end of each monomer and n-termini (met ) located parallel with them, perpendicular to the membrane. protein data bank entries bo , de , zgf and fgg were used, respectively, to draw the figure. it should be noted that n-termini in these figures are those made visible in the structures, and that the stem-and transmembrane regions can be assembled in different ways, and are represented only schematically in the pictures stem region and possibly also the transmembrane domain in taking part in the dimerization cannot be excluded. these observations emphasize that structure determinations of also functionally important heterodimers at atomic detail are necessary for detailed understanding on how the complexes interact and how they co-operatively function in glycan synthesis. it is not clear if the catalytic domains-for which the structures already exist-are sufficient in each case to establish stable homo-or heterodimers. attempts to resolve these issues are thus necessary and should take into account the following important considerations. firstly, crystallization conditions may fail to simulate the conditions of the golgi lumen important for heteromer formation. this is especially important for the ph sensitive interactions of some of the glycosyltransferase heteromers, though they have been shown to resist solubilization but only under proper experimental conditions [ , ] . secondly, it is clear that the homomeric and heteromeric complexes are transient and dynamic owing to their constant and microenvironment dependent transitions between less active homomers and more active heteromers during their recycling within the early secretory compartments. this may in fact be behind the difficulties in defining functionally relevant or irrelevant interactions. thirdly, the trials in which the complex constituents are combined in a test tube does not necessarily guarantee that a complex will form. rather, they may require conditions that exist only in the living cell. fourthly, structures or structural models of any glycosyltransferase dimers or higher complexes must fulfill the expectation that the active sites are not buried within the interaction surface, and also that the active sites are situated in such a manner that the binding of donor and acceptor molecules is feasible. a wealth of information now has accumulated and shows that glycosyltransferases in general tend to form functionally relevant complexes in live cells, as in some cases, it has already been shown that the formation of such complexes, especially between sequentially acting enzymes, either augments or diminishes the enzymatic activity of the complex constituents. yet, the main impact of their existence in most glycosylation pathways in all eukaryotes is that they expose a new level of regulation of glycan synthesis. they also seem to provide the ''molecular template'' for critical points in glycan synthesis by linking relevant enzymes together and thereby, preventing competing reactions from occurring. evidence already exist that their absence in diseases such as cancers alters glycosylation of cell surface glycans, a phenomenon that often has fatal consequences on human wellbeing. due to the individual nature of these complexes, future work should focus on detailed molecular characterization of the complexes, clarification of the interaction surfaces by mutagenesis and on resolving their d structures as well as functional consequences on glycan synthesis. this would help understand better how their interactions are regulated by organelle micro-environmental factors (such as ph) in both normal cells and cancerous cells in which such complexes are rare. clarifying these issues will help uncover important cell and glycobiological questions related to the golgi apparatus and its' functioning as a glycosylation device and as a central station for protein delivery to post-golgi compartments and beyond. attachment of terminal n-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the golgi stack assembly of asparagine-linked oligosaccharides mapping the distribution of golgi enzymes involved in the construction of complex oligosaccharides resolution of a soluble lactose synthetase into two protein components and solubilization of microsomal lactose synthetase the isolation and identification of the b protein of lactose synthetase as alpha-lactalbumin the role of alpha-lactalbumin and the a protein in lactose synthetase: a unique mechanism for the control of a biological reaction the role of alpha-lactalbumin in lactose synthetase the interaction of -lactalbumin and the a protein of lactose synthetase biosynthesis of chondroitin sulfate: interaction between xylosyltransferase and galactosyltransferase biosynthesis of chondroitin sulfate: immunoprecipitation of interacting xylosyltransferase and galactosyltransferase normal and abnormal biosynthesis of gangliosides regulation of glycoprotein biosynthesis by formation of specific glycosyltransferase complexes role of n-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation protein n-glycosylation along the secretory pathway: relationship to organelle topography and function, glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized protein quality control, and cell interactions translocation of lipid-linked oligosaccharides across the er membrane requires rft protein hetero-oligomeric interactions between early glycosyltransferases of the dolichol cycle alg organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis physical interactions between the alg , alg , and alg mannosyltransferases of the endoplasmic reticulum the dolichol pathway of n-linked glycosylation stt , a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo oligosaccharyltransferase: a complex multisubunit enzyme of the endoplasmic reticulum dimeric organization of the yeast oligosaccharyl transferase complex structure of the mammalian oligosaccharyl-transferase complex in the native er protein translocon structural basis of substrate specificity of human oligosaccharyl transferase subunit n /tusc and its role in regulating protein n-glycosylation dissecting glycoprotein quality control in the secretory pathway quality control in the endoplasmic reticulum n-glycan processing in er quality control glycoprotein biosynthesis in yeast multi-protein complexes in the cis golgi of saccharomyces cerevisiae with a- , -mannosyltransferase activity the consortium of glycobiology editors post-translational modifications distinguish cell surface from golgi-retained beta , galactosyltransferase molecules. golgi localization involves active retention golgi retention mechanism of beta- , -galactosyltransferase. membrane-spanning domaindependent homodimerization and association with alpha-and beta-tubulins a disulfide-bonded dimer of the golgi beta-galactoside alpha , -sialyltransferase is catalytically inactive yet still retains the ability to bind galactose trafficking and localization studies of recombinant alpha , -fucosyltransferase vi stably expressed in cho cells medial golgi but not late golgi glycosyltransferases exist as high molecular weight complexes. role of luminal domain in complex formation and localization the critical role of the stem region as a functional domain responsible for the oligomerization and golgi localization of n-acetylglucosaminyltransferase v. the involvement of a domain homophilic interaction location and mechanism of alpha , -sialyltransferase dimer formation. role of cysteine residues in enzyme dimerization, localization, activity, and processing different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation importance of cys, gln, and tyr from the transmembrane domain of human alpha / fucosyltransferase iii for its localization and sorting in the golgi of baby hamster kidney cells golgi n-glycosyltransferases form both homo-and heterodimeric enzyme complexes in live cells functional organization of the golgi nand o-glycosylation pathways involves ph-dependent complex formation that is impaired in cancer cells kin recognition. a model for the retention of golgi enzymes kin recognition between medial golgi enzymes in hela cells characterization of a novel galactose beta , -n-acetylglucosaminyltransferase (beta gn-t ): the complex formation of beta gn-t and beta gn-t enhances enzymatic activity activation of beta , -n-acetylglucosaminyltransferase- (beta gn-t ) by beta gn-t . possible involvement of beta gn-t in increasing poly-nacetyllactosamine chains in differentiated hl- cells heterodimers of tyrosylprotein sulfotrans-ferases suggest existence of a higher organization level of transferases in the membrane of the trans-golgi apparatus a testis-specific regulator of complex and hybrid n-glycan synthesis time-resolved fluorescence imaging reveals differential interactions of n-glycan processing enzymes across the golgi stack in planta golgilocalized enzyme complexes for plant cell wall biosynthesis highly conserved cysteines of mouse core b , -n-acetylglucosaminyltransferase i form a network of disulfide bonds and include a thiol that affects enzyme activity the endoplasmic reticulum chaperone cosmc directly promotes in vitro folding of t-synthase tight complex formation between cosmc chaperone and its specific client non-native t-synthase leads to enzyme activity and client-driven dissociation molecular genetic analysis of the glycosyltransferase fringe in drosophila missense mutations in beta- , -nacetylglucosaminyltransferase (b gnt ) cause walker-warburg syndrome mutations in ispd cause walker-warburg syndrome and defective glycosylation of alpha-dystroglycan mutations of the pomt gene found in patients with walker-warburg syndrome lead to a defect of protein o-mannosylation demonstration of mammalian protein o-mannosyltransferase activity: coexpression of pomt and pomt required for enzymatic activity members of the evolutionarily conserved pmt family of protein o-mannosyltransferases form distinct protein complexes among themselves secretion and assembly of type iv and vi collagens depend on glycosylation of hydroxylysines reduction of lysyl hydroxylase causes deleterious changes in the deposition and organization of extracellular matrix identification of amino acids important for the catalytic activity of the collagen glucosyltransferase associated with the multifunctional lysyl hydroxylase (lh ) dimerization of human lysyl hydroxylase (lh ) is mediated by the amino acids - structure, organization, and function of glycosphingolipids in membrane cellular and molecular biology of glycosphingolipid glycosylation regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases physical and functional association of glycolipid n-acetyl-galactosaminyl and galactosyl transferases in the golgi apparatus ganglioside glycosyltransferases organize in distinct multi-enzyme complexes in cho-k cells glycosylation of glycolipids in the golgi complex the dxd motif is required for gm synthase activity but is not critical for nucleotide binding gm alpha , -sialyltransferase (gd synthase): protein characterization and sub-golgi location in cho-k cells structure/function of the human ga beta , -glucuronosyltransferase. dimerization and functional activity are mediated by two crucial cysteine residues the putative tumor suppressors ext and ext form a stable complex that accumulates in the golgi apparatus and catalyzes the synthesis of heparan sulfate the ext /ext tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis enzyme interactions in heparlan sulfate biosynthesis: uronosyl -epimerase and -o-sulfotransferase interact in vivo identification of chondroitin sulfate glucuronyltransferase as chondroitin synthase- involved in chondroitin polymerization: chondroitin polymerization is achieved by multiple enzyme complexes consisting of chondroitin synthase family members fluorescence resonance energy transfer (fret) and proximity ligation assays reveal functionally relevant homo-and heteromeric complexes among hyaluronan synthases has , has and has an interactional network of genes involved in chitin synthesis in saccharomyces cerevisiae purification of an active, oligomeric chitin synthase complex from the midgut of the tobacco hornworm evidence for an intramembrane component associated with a cellulose microfibrilsynthesizing complex in higher plants molecular analysis of cellulose biosynthesis in arabidopsis immunogold labeling of rosette terminal cellulosesynthesizing complexes in the vascular plant vigna angularis the cellulose synthase superfamily genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in arabidopsis features of the primary wall cesa complex in wild type and cellulose-deficient mutants of arabidopsis thaliana interactions among three distinct cesa proteins essential for cellulose synthesis glycosyltransferase complexes in eukaryotes: long-known, prevalent but still unrecognized organizational interplay of golgi n-glycosyltransferases involves organelle microenvironment-dependent transitions between enzyme homo-and heteromers functional analysis of complexes with mixed primary and secondary cellulose synthases elucidating the mechanisms of assembly and subunit interaction of the cellulose synthase complex of arabidopsis secondary cell walls galacturonosyltransferase (gaut) and gaut are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex arad proteins associated with pectic arabinan biosynthesis form complexes when transiently overexpressed in planta xyloglucan xylosyltransferases xxt , xxt , and xxt and the glucan synthase cslc form golgi-localized multiprotein complexes protein-protein interactions among xyloglucan-synthesizing enzymes and formation of golgi-localized multiprotein complexes analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions multimeric states of starch phosphorylase determine protein-protein interactions with starch biosynthetic enzymes in amyloplasts golgi localization of glycosyltransferases: more questions than answers structures and mechanisms of glycosyltransferases disulfide bonds of gm synthase homodimers elevated golgi ph impairs terminal n-glycosylation by inducing mislocalization of golgi glycosyltransferases the structure of human alpha- , -sialyltransferase receals the binding mode of complex glycans enzymatic basis for n-glycan sialylation: structure of rat alpha , -sialyltransferase (st gal ) reveals conserved and unique features for glycan sialylation spatial organization and stoichiometry of n-terminal domain-mediated glycosyltransferase complexes in golgi membranes determined by fret microscopy the role of the membrane-spanning domain and stalk region of n-acetylglucosaminyltransferase i in retention, kin recognition and structural maintenance of the golgi apparatus in hela cells a unique molecular chaperone cosmc required for activity of the mammalian core b -galactosyltransferase the exostosin family: proteins with many functions organization of the synthesis of glycolipid oligosaccharides in the golgi complex glycosyltransferase complexes improve glycolipid synthesis a homologue of saccharomyces cerevisiae dpm p is not sufficient for synthesis of dolichol-phosphate-mannose in mammalian cells dpm regulates biosynthesis of dolichol phosphate-mannose in mammalian cells: correct subcellular localization and stabilization of dpm , and binding of dolichol phosphate human dolichol-phosphate-mannose synthase consists of three subunits, dpm , dpm and dpm n-glycans in cell survival and death: crosstalk between glycosyltransferases initial enzyme for glycosylphosphatidylinositol biosynthesis requires pig-p and is regulated by dpm alpha-lactalbumin (la) stimulates milk beta- , -galactosyltransferase i (beta gal-t ) to transfer glucose from udp-glucose to n-acetylglucosamine. crystal structure of beta gal-t x la complex with udp-glc a golgi retention signal in a membrane-spanning domain of coronavirus e protein oligomerization of a membrane protein correlates with its retention in the golgi complex diffusional mobility of golgi proteins in membranes of living cells glycosyltransferases: structures, functions, and mechanisms structural insight into mammalian sialyltransferases recent structures, evolution and mechanisms of glycosyltransferases recent structural and mechanistic insights into post-translational enzymatic glycosylation structure-function relationships of membrane-associated gt-b glycosyltransferases structure of human st siaiii sialyltransferase provides insight into cell-surface polysialylation inference of macromolecular assemblies from crystalline state crystal structure of mammalian alpha , -fucosyltransferase, fut crystal structure of beta , -galactosyltransferase complex with udp-gal reveals an oligosaccharide acceptor binding site protein-protein crystal-packing contacts structures of a human blood group glycosyltransferase in complex with a photo-activatable udp-gal derivative reveal two different binding conformations heparan/chondroitin sulfate biosynthesis. structure and mechanism of human glucuronyltransferase i structural basis for acceptor substrate recognition of a human glucuronyltransferase, glcat-p, an enzyme critical in the biosynthesis of the carbohydrate epitope hnk- acknowledgments the authors would like to thank emil aaltonen foundation for financial support. we would also like to give our warmest thanks to an experienced japanese female researcher who shared her early findings on glycosyltransferase complexes with us, but whose name we unfortunately cannot track anymore. key: cord- -wasdp gh authors: morré, d. james; minnifield, nita; paulik, mark title: identification of the °c compartment of the endoplasmic reticulum in rat liver and cultured hamster kidney cells date: - - journal: biol cell doi: . /j. - x. .tb .x sha: doc_id: cord_uid: wasdp gh summary— in many systems transfer between the endoplasmic reticulum and the golgi apparatus is blocked at temperatures below °c. in virus‐infected cells in culture, a special membrane compartment is seen to accumulate. our studies with rat liver show a similar response to temperature both in situ with slices and in vitro with isolated transitional endoplasmic relticulum fractions. with isolated transitional endoplasmic reticulum fractions, when incubated in the presence of nucleoside triphosphate and a cytosol fraction, temperature dependent formation of vesicles occurred with a q( ) of − but was apparent only at temperatures greater than °c. a similar response was seen in situ at °c and °c where fusion of transition vesicles with cis golgi apparatus, but not their formation, was blocked and transition vesicles accumulated in large numbers. at °c and below and especially at °c and °c, the cells responded by accumulating smooth tubular transitional membranes near the cis golgi apparatus face. with cells and tissue slices at °c neither transition vesicles nor the smooth tubular elements accumulated. those transition vesicles which formed at °c were of a greater diameter than those formed at °c both in situ and in vitro. the findings show parallel responses between the temperature dependency of transition vesicle formation in vitro and in situ and suggest that a subpopulation of the transitional endoplasmic reticulum may be morphologically and functionally homologous to the °c compartment observed in virally‐infected cell lines grown at low temperatures. reduced temperature has been shown to affect posttranslational processing and secretion in a number of cell systems. tartakoff [ ] demonstrated that at °c secretory proteins of the exocrine pancreas accumulated in pre-golgi apparatus transition vesicles, while at temperatures of °c or greater, progress through the golgi apparatus and into condensing vacuoles occurred. secretion of igm and the appearance of hla- on the cell surface decreased gradually in lymphoid cells as the temperature was reduced from °c to °c but no intracellular accumulation was observed [ ] . histamine secretion from mast cells also decreased markedly at temperatures below °c [ ] . with hepatic cells, secretory p~oteins were blocked in a pre-golgi apparatus compartment at °c, but at °c these proteins were exocytosed normally [ ] . this was similar to rat pancreatic cells where, at °c, most of the • correspondence and reprints. this article is dedicated to the memory of professor pierre favard. labeled secretory proteins remained in the endoplasmic reticulum while at °c the medial golgi region was reached [ ] . these and other data, especially with the intracellular transport and surface expression of protein in virusinfected cells h, , , , , , ] has led to the concept of a °c compartment. this pre-golgi apparatus compartment would differ from the bulk of the endoplasmic reticulum and would presumably be located in the secretory pathway between the transitional endoplasmic reticulum and the golgi apparatus. despite the considerable evidence for the existence of such a compartment based on the interruption of processing and transport of secretory and membrane proteins, the data on the morphological identification of such an intermediate compartment are more limited and somewhat conflicting. this study was undertaken to utilize quantitative morphometric methods to identify that endomembrane compartment of. rat liver which may accumulate at temperatures below °c and in cells in culture for comparison. studies were then conducted with isolated transitional endoplasmic reticulum fractions from rat liver to determine if a similar mechanism of control by temperature of transition vesicle formation could be duplicated in the cell-free system. male holtzman rats were sacrificed and livers removed. unless stated otherwise, slices of liver averaging about . x . x . mm were cut by hand with a razor blade as described [ ] . slices were incubated in phosphate-buffered saline at the temperatures indicated for or rain and fixed directly with osmium tetroxide. cell culture bhk (baby hamster kidney) cells were grown in mem (eagles minimum essential medium) supplc;,~ented with fetal calf serum, ° triptose phosphate, u/ml penicillin and tz~/mi streptomycin [ . all temperature experiments were carried out in constant temperature incubators (± . c). the incubators were housed in a refrigerated room ( "c) to ensure that the room temperature never exceeded that of the chambers. equilibration of culture media, membrane preparatinn and liver slices to the temperature of the chambers occurred rapidly in a matter of a few minutes. fixations were at the same temperature as the incubations. cells or isolated fractions were fixed after temperature incubation in . °/ glutaraldehyde in . m sodium phosphate, ph . followed by post fixation in osmium tetroxide in the same buffer. dehydration was through an acetone series with embedment in epon [ ] . thin sections were observed and photographed using a philips em electron microscope. endoplasmic reticulum !ractions were isolated as described [ ] . livers were homogenized in vol of a medium containing . mm tris maleate, ph . , . m sucrose, mm mgci, and % dextran for sec with a polytron st operated at , rpm. the homogenates were centrifuged for rain at , g to remove nuclei, plasma membrane fragments and golgi apparatus, and the supernatant diluted : with homogenization medium was centrifuged at , g to remove mitochondria. the supernatant containing the endop!asmic reticulum was layered onto a discontinuous sucrose gradient consisting of . , !. and . m sucrose layers. after centrifugation at , g for min, the membranes from the . m sucrose/sample interface were withdrawn using a pasteur pipette and pelleted by centrifugation for rain at , g as the starting fraction for temperature incubation in a'reconstituted system capable of in vitro formation of transition vesicles (see below). part-rough, part-smooth transition elements of endoplasmic reticulum were isolated as described above and incubated in the presence of nucleoside triphosphate and cytosol at either °c or °c. in addition, the complete incubation systems contained an atp regenerating system of mm hepes-koh, mm kci, . mm mg acetate, pm atp, /~m utp, mm creatine phosphate and iu/ml creatine phosphokinase (final ph . ) [ , . part-rough, part-smooth fractions of the transition endoplasmic reticulum fraction from rat liver when resuspended in atp, atp regenerating system and c ~osol, were observed to contain = nm bebs and vesicles reminiscent of transition vesicles produced in situ ( fig. ; see nowack et al. [is] for a comparison). these vesicles appeared to vary in number as a result of incubation at different temperatures. to quantitate the numbers of vesicles present, the preparations were pelleted onto nitrocellulose filters, prepared for electron microscopy, and the pellets were photographed through their entire cross sectional area. all vesicles were counted in representative /~m areas. only circular profiles with solid interiors representing filled vesicles rather than cross sections of hollow tubules, and in the approximate size range to be transition vesicles were scored as transition vesicles. many were still connected to the transitional endoplasmic reticulum membranes by a narrow stalk (fig. , inset) . the numbers of putative transition vesicles present in the resuspended membranes was approximately . membrane profiles per membrane profiles at °c (fig. ) . with increasing temperature, the number of additional vesicles followed a hyperbolic function of increase to approximatively double at °c with qi of increase of about over the temperature range °c to °c. by extrapolation, the temperature dependent formation of nm vesicles below °c was insignificant. the effect of temperature on transition vesicle production in situ was examined with liver slices as well. the morphology of the golgi apparatus of the slices at °c (figs. a and a) was representative of that observed both at °c and at °c. golgi apparatus with characteristic stacked cisternae were present at all temperatures so that golgi apparatus zones could be readily located. over the temperature range °c to °c, transition vesicles were sparse. however, transition elements were well developed and numerous tubular smooth elements of the endoplasmic reticulum were evident within the golgi zone adjacent to the c/s golgi apparatus face. these tubular smooth-surfaced membranes were more evident after rain of incubation (fig. a ) than after rain of incubation (fig. a) . with slices incubated at °c (fig. b ) or °c ( fig. b and c) , the most obvious morphological areration from the appearance at cc was an i~crease in the number of transition vesicles. especially with rain of incubation, the vesicles accumulated in clusters, usually near the cis golgi apparatus face between the c/s golgi apparatus cisternae and the transitional endoplasmie reticulum. the tubular transitional membranes were still present at both ~c and °c but were less evident than at °c or °c. at °c, the appearance of the golgi apparatus zone was changed markedly from that at °c or °c ( fig. c and d ). the number of transition vesicles was reduced compared to °c and those vesicles present were no longer present in clusters. additionally, the tubular smooth membrane elements present throughout the golgi apparatus zone at temperatures of °c and below were much less in evidence. with further increases in temperature, the numbers of transition vesicles appeared to be increased slightly but the tubular elements of the endoplasmic reticulum did not reappear within the golgi apparatus zone. overall, the numbers of transition vesicles present at the cis golgi apparatus face showed an increase with increasing temperature to an optimum at °c ( rain) or °c ( rain) with an abrupt decline in transition vesicle numbers between °c and °c (fig. ) . as with the isolated fractions (fig. ) , the numbers of transition vesicles did not appear to increase until temperatures exceeded °c. neither did they accumulate in large numbers at temperatures above °c presumably through release of the temperature block at temperatures below °c that allowed for their formation but retarded their fusion with the golgi apparatus. the distribution of diameters of transition vesicles present in situ at °c differed from those present in vitro with isolated transition elements at °c (fig. ) . those formed in vitro exhibited a larger diameter of /~m compared to ~m in ~itu. comparing vesicles formed in situ at °c with those formed at c (fig. ) , the diameters ~so were shifted to larger values in the distribution profile ,~.z~d the average diameter was increased to /~m. a similar increase in vesicle diameter was seen with the transition vesicle formed in vitro from /~m at °c to /~m at °c. since most previous investigations of temperature effects on transition~ endoplasmic reticulum have been with cultured cells, studies were carried out with bhk cells under identical conditions of temperature incubations used for liver slices. at °c (fig. a) , the general appearance the use of inhibitors to interrupt the flow of membrane~ between the endoplasmie reticulum and the golgi apparatus might be expected to have effects similar to low temperatures. therefore, bhk cells were treated with substances to block membrane flow. potassium cyanide blocks secretion, presumably by reducing the cellular energy charge, but did not result in an accumulation of transition vesicles (fig. b ) compared with control preparations (fig. a) with , or rain of incubation. in contrast, mm coc! conditions known to block vesicular flow in other systems and axonal transport (see discussion), did produce morphological changes very similar to those observed with the low temperature block (fig. c) . numbers of transition vesicles were approximately doubled relative to the control preparations and the peripheral cytoplasm contained numerous tubular transition elements of the endoplasmic reticulum similar in appearance to those observed with incubation at °c (fig. a) . these morphological changes were observed with incubation times with mm coci as short as rain and the alterations after rain (not illustrated) of incubation were similar to those after rain of inct:bation (fig. , fig. c ). using part-rough, part-smooth transitional element fractions isolated from rat liver, nowack et al. [ ] demonstrated the temperature dependent transfer of mereof the golgi apparatus zone of the bhk cells was similar to that for liver. transition vesicles present in the cytoplasm adjacent to the cis golgi apparatus face were numerous and accompanied by abundant tubular transitional elements in the peripheral cytoplasm. with cells incubated at °c, both the tubular transitional elements of the endoplasmic reticulum and the numbers of transition vesicles were reduced markedly (fig. b) . at higher temperatures, e.g. °c, the appearance was similar to that at °c except that the numbers of transition vesicles may have increased slightly. cells incubated at temperatures below °c were similar in appearance to those incubated at °c except for small variations in numbers of transition vesicles (fig. ) . results with rain of incubation (fig. ) and min of incubation (not illustrated) were comparable. quantitation of vesicle numbers of transition vesicles associated with golgi apparatus stacks of bhk cells gave results (fig. ) similar to those observed for liver slices (fig. ) . the r, lost characteristic feature of these data was the precipitous drop in number of transition vesicles between °c and °c. thereafter, with increasing temperature, the number of vesicles present increased slightly. as with liver cells, the average diameter of the transition vesicles in bhk cells at °c was .+ /zm but increased to + ~m at c. . the appearance at °c is similar to that at °c except that transition vesicles appear to be more numerous. bar= . /~m. brane from transitional endoplasmic reticulum donor that was prepared from liver slices labeled with [ c]leucine to unlabeled golgi apparatus acceptor. transfer was -fold greater at °c than at °c commensurate with a ql of for the overall transfer process. little or no transfer was observed at °c compared to °c even in the presence of the complete incubation system containing atp, atp regenerating system and cytosol. in the present study, the transition element fractions from rat liver exhibit temperature dependent formation of vesicles similar to that observed in situ. the formation of = - /~m transition vesicle-like structures over and above those initially present began at temperatures of °c and above. the origin of at least a major fraction of these vesicles was from the transition endoplasmic reticulum vesicles present in the fraction. many of the vesicles re- mained attached to larger endoplasmic reticulum vesicles by a narrow stalk (fig. ) . the attachment of the vesicles during tissue homogenization is ruled out by the absolute dependency of their presence upon atp [i ] . specificity is also shown by the absence of such vesicles from incubated preparations consisting only of conventional rough endoplasmic reticulum collected from gradient fractions of density greater than those yielding the responsive transitional membrane elements [ ] . in situ, transitional endoplasmic reticulum of liver slices incubated at different temperature conditions behaved similarly. over the range °c to °c, there was little evidence of transition vesicle formation. however, between °c and °c not only were transition vesicles formed but they accumulated as shown by others, presumably due to an inability of the vesicles to fuse with the cis golgi apparatus at temperatures of °c or below [ , , , , ] . quite abruptly, betwcen °c and °c, the number of transition vesicles declined as the temperature block that prevented vesicle fusion with the golgi apparatus was relieved [ , , , ] . interestingly, not only was there an effect of temperature on transition vesicle numbers but on the distribution of diameters as well. at °c in situ, the distribution of transition vesicle diameters was shifted in the direction of larger diameters such that their overall average diameter was increased from to /~m. for these measurements, the transition vesicles were identified not only from their characteristic appearance but from their spatial location within the cytoplasm. in situ measurements from structures other than transition vesicles would have been largely eliminated. when a figure . --effect of coci and kcn on transitional endoplasmic reticulum of bhk cells. a, control cells incubated in the absence of inhibitor. b, incubation for rain in the presence of kcn. the appearance is similar to that of control cell,. c, incubation with i mm coci, for rain. numerous tubular transition elements and transition vesicles are present in the golgi apparatus zone similar to those seen at °c. bar= . ~m. similar analysis was carded out with those vesicles scored as transition vesicles in the in vitro incubated fractions, the average diameters were greater both at °c and at °c. however, even in vitro, the temperature effect on vesicle diameter, although small, was still evident. the average vesicle diameter at °c was /am while the average diameter at °c was fro. thus the response of transitional elements of the endoplasmic reticulum from liver to temperature in vitro paralleled that observed with liver slices in situ. temperature-dependent formation of transition vesicles occurs primarily at temperatures above °c, becom~.ng significant at approximately °c. the vesicle diameter was greater at °c than at °c both in situ and in vitro with the isolated fractions. a °c compartment intermediate between the endoplasmic reticulum and the golgi apparatus has been reported for cultured cells infected with temperature sensitive virus strains [ , , , ] . in order to attempt to define a °c compartment for liver, parallel experiments were carried out with bhk cells incubated at different temperatures in normal culture medium. as with liver cells, both at °c and at °c, transition vesicles accumulated. however, at temperatures of °c and below, there was also an accumulation of smooth tubular elements of the endoplasmic reticulum in the peripheral cytoplasm surrounding the golgi apparatus most often in near proximity to the cis face. we suggest this structure to be morphologically and structurally homologous to the °c compartment of virus-infected cells [ ] . the smooth tubular endoplasmic reticulum elements seen with liver slices at temperatures of °c and below and especially at °c and °c also appear to be structurally homologous to the °c compartment seen in cultured cells. while the formation of a °c compartment by the isolated membrane fractions is not immediately obvious, preliminary indications are that the stalked vesicles or that some of the membrane blebs still attached to endoplasmic reticulum fragments seen in isolated preparations exposed to atp and cytosol but not incubated at temperatures of °c and above may be the equivalent structures. both cyanide and cobalt have been reported to inhibit the transfer step between endoplasmic reticulum and golgi apparatus [ ] . cyanide presumably blocks through its ability to deplete the energy charge of the cell whereas how cobalt inhibits is not known. with bhk ceils, cyanide did not result in vesicle accumulations. at least in the cell-free [ ] and perforated [ , ] cell systems, vesicle formation and transfer shows an absolute energy dependence. thus, an accumulation of vesicles under conditions of energy depletion would not be expected. in contrast to cyanide, the cobalt treatment resulted in increased numbers of transition vesicles with the bhk cells rcsembling the response to °c. similar results could not be shown with liver due to the extreme cytotoxicity of liver slices to the same cobalt concentrations and treatment conditions that resulted in transition vesicle accumulations with tile cultured cells. cobalt ions were selected initially for comparison to low temperature because of their reported ability at mm to inhibit fast axonal transport [ ] . previous findings suggested that cobalt was acting at a rate-limiting step in the initiation of axonal transport perhaps at a site where calcium or zinc might be involved in the initial coupling of protein to the transport. reconstitution of transport from the endoplasmic reticulum to the golgi complex using semi-intact ceils semiintact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the golgi complex effect of reduced temperature on glycoprotein (ig, hla) processing and transport in lymphoid cells folding, trimerization and transport are sequential events in the biogenesis of influenza virus hemagglutinin the effects of low teml~ratures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes exit of newly synthesized membrane proteins from the transcisterna of the golgi complex to the plasma membrane inhibition of fast axonal transport of [~h]protein by cobalt ions tunicamycin resistant glycosylation of a coronavirus glycoprotein: determination of a novel type of viral glycoprotein sorting and coordinate induction of three endoplasmic reticulum proteins containing the kdel retention signal temperature dependence of mast cell histamine secretion improvements in epoxy resin embedding methods reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation transition vesicle formation in vitro golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices intracellular membrane flow: reconstitution of transition vesicle formation and function in a cellfree system isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to golgi apparatus cisternae of rat liver pre-and post-golgi vacuoles operate in the transport of semliki forest virus membrmke glycoproteins to the cell surface temperature sensitive steps in the transport of secretory proteins through the golgi complex in exocrine pancreatic cells temperature and energy dependence of secretory protein transport in the exoc~ne pancreas replication of coronavirus mhv-a in sac(-) cells: determination of the first site of budding of progeny virions site of addition of n-acetylgalactosamine to the e glycoprotein of mouse hepatitis virus-a ) interferon production by inactivated newcastle disease virus in cell cultures and in mice this work was supported in part by a grant from the national institutes of health hd . we thank charles bracker for use of electron microscope facilities, dorothy werderitsh for excellent technical assistance and olympia mejia for help with preliminary experiments. the participation of angela downham and victoria vucich in a portion of the study is gratefully acknowledged. key: cord- -ht ry i authors: nan title: sorting within the regulated secretory pathway occurs in the trans- golgi network date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: ht ry i bioactive peptides cleaved from the egg-laying hormone precursor in the bag cell neurons of aplysia are sorted into distinct dense core vesicle classes (dcvs). bag cell prohormone processing can be divided into two stages, an initial cleavage occurring in a late golgi compartment, which is not blocked by monensin, and later cleavages that occur within dcvs and are blocked by monensin. prohormone intermediates are sorted in the trans-golgi network. the large soma-specific dcvs turn over, while the small dcvs are transported to processes for regulated release. thus, protein trafficking differentially regulates the levels and localization of multiple biologically active peptides derived from a common prohormone. . abbreviations used in thispaper: asw, artificial sea water; dcv, dense core vesicles; elh, egg-laying hormone; hd, half-distance; ile, isoleucine; phe, phenylalanine; tgn, trans-golgi network. et al., ) . while the mechanism of this sorting is still anknown, proposals have been made both for an aggregation event whereby the condensation of neuropeptides excludes other proteins from the forming dcv (kelly, ) , and a receptor-mediated sorting event (burgess and kelly, ; pfeffer and rothman, ; chung et al., ) analogous to lysosomal sorting where a specific signal (mannose- phosphate) targets proteins through a recycling receptor (griffiths et al., ) . sorting within the regulated secretory pathway has recently been demonstrated both in somatomammotrophs (fumagalli and zanini, ; hashimoto et al., ) and in the bag cell neurons of aplysia californica . in the bag cells, different products of the egg-laying hormone (elh) precursor are localized to separate classes of dcvs. peptides from the carboxy-terminal side of the elh precursor are found in a class of small dcvs which are rapidly transported to the neuronal processes, while peptides from the amino-terminal side of the precursor are found both in a class of large dcvs that are restricted to the cell body and a distinct class of small dcvs which are transported to processes (fig. ) . this sorting event has important physiological consequences as the different regions of the elh prohormone contain peptides with unique biological activities (kupfermann, ; rothman et al., a,b; mayeri et al., ; kauer et al., ; brown and mayeri, ) . in this paper, we investigate the cellular pathway of protein trafficking within the bag cells using em autoradiography and the carboxylic acid ionophore monensin. the bag cells have two features that facilitate these studies; (a) the elh prohormone is the major protein produced by the bag cells and accounts for up to % of the translated protein (berry and arch, ; scheller et al., ) ; (b) an asymmetric distribution of amino acids across the prohormone fig. ) allows the selective labeling of the sorted carboxy or amino terminal-derived peptides. in this study, we use these properties to selectively follow the flow of neuropeptides derived from the elh precursor through the secretory pathway. the results suggest possible mechanisms that underlie sorting within the regulated secretory pathway. - -g aplysia were obtained from sea life supply (sand city, ca). bag ceil clusters were isolated and equilibrated in artificial sea water (asw; mm naci, mm kci, mm mgci , mm mgso , mm caci , and mm tris, ph . ) at °c for rain. the clusters were then incubated in isoleucine ([ h] ile) or l-[ , , , , - h]phenylalanine ([ h]-phe)(amersham corp., arlington heights, il) ( mci/ml for a -rain pulse; mci/mi for a -rain pulse) for either or min and then chased with asw plus mm phe, asw plus mm ile, or isotonic li plus mm phe for the -h chase at °c. the chase was stopped by placing the clusters into a fixation solution consisting of % glutaraldehyde, % paraformaldehyde, % dmso, . m sucrose in pbs ( . m nac , . mm kci, mm na po , . mm kh po , ph . ). occasionally, % acrolein was also included in the fixation solution. the tissue was fixed for - h at room temperature (occasionally overnight at °c). the clusters were rinsed with pbs, desheathed, and osmicated with % oso in pbs for h followed by four washes in ddh and an additional h in % aqueous uranyl acetate. the clusters were then step-wise dehydrated to % ethanol followed by propylene oxide before infiltrating and embedding with epon-araldite. after polymerization, thin sections ( - nm) were cut on a microtome (reichert jung, vienna, austria) and collected on formvar-coated nickel grids. the grids were then lead stained, carbon coated, and covered with emulsion l ; ilford ltd., basildon, essex, england) using the loop technique (caro and van tubergen, ; caro, ; williams, ) . the emulsion was exposed in the dark at °c from d to mo depending on the experiment and then developed by incubating in microdol x (eastman kodak co., r~chester, ny; rain), stopped in % acetic acid, fixed with % sodium thiosulfate ( rain), and washed in dvh . grids were then visualized using a transmission electron microscope ( ; philips electronic instruments, inc., mahwah, nj). micrographs were photographed at , x and then printed to give a final magnification of , x. this size was chosen as the smallest magnification that small clear vesicles around the golgi apparatus are clearly visible. pictures were not taken at grid coordinates but rather over concentrations of grains. this was necessary due to the large size of the bag cells ( /~m table i . quantitation of em autoradiography in diameter) and the small area of the cells in which one could find grains. therefore, all results are given in percentage of grains in each compartment rather than density of radioactivity in each compartment. the micrographs were digitized (gp digitizer; science accessories corp., southport, ct) and analyzed using a computer-implemented maximum likelihood algorithm (miller et al., ) . an important parameter in this program is the approximate error due to the spread of radioactivity, which depends on the thickness of the section and the emulsion. we calculated all time points at half-distance (hd) values (saltpeter and bachmann, ) of , , and am. the calculations are fairly insensitive to this parameter, and the data in table i are calculated with an hd of run, as this value matched our estimates for section and emulsion thickness. the largest variations observed at different hd values occur between the cytoplasm, er, and small mature vesicles. all of these compartments have large areas and low densities and are situated in close proximity to each other, making it difficult to asign counts between these compartments. the largest change is seen at the ile -rain pulse plus -h chase time point, when, with an hd of am, the percentage of counts in small mature vesicles and cytoplasm are . and . %, respectively, as opposed to . and . % with an hd of am. all other changes were minor in comparison, and in the compartments important in these studies-small and large immature vesicles, clear vesicles, and golgi apparatus-the results between calculations at three hd values differ by no more than % in any experiment. there were nine compartments entered during digitization of the micrograph (er, golgi apparatus, golgi-associated sacs [usually situated on the cis side of the goigi apparatus], dcvs, immature dcvs, clear vesicles, mitochondria, lysosomes, and nucleus). all nonlabeled parts of the micrographs defaulted to cytoplasm. mature and immature dcvs were then divided into small and large classes using a size cutoff (area = , nm , based on a diameter of nm; fisher et al., ) to make a total of compartments. the areas of labeled (center of at least one grain within the vesicle) and unlabeled large vesicles were also calculated to determine if phe-labeled small immature dcvs gradually increased in size to form large phe-labeled immature dcvs. at the earliest time point that significant radioactivity is observed in large immature vesicles (phe -rain pulse plus rain chase), the average area of labeled large vesicles was actually larger than the average area of unlabeled large vesicles (l. x) and exceeded the cutoff size by a large margin (average diameter = am). immature vesicles were defined by either (a) membrane extensions (bulge or sac; see fig. ); (b) irregularities in shape; (c) extremely ruffled membranes; or (d) connection to a tubule or golgi stack. it is important to note that the percentage of immature vesicles is probably an underestimate, as serial sections through bag cells often reveal sacs on vesicles or connections to tubules that are absent in other sections. for the phe -rain pulse plus % chase experiment, the iysosome compartment was divided into mottled dcvs and clear lysosomes. for all experiments other than phe -rain pulse plus -h chase, the digitization was done blindly without knowledge of the time point or the label used in the experiment. most time points include data from more than one application of emulsion. data was pooled from experiments as long as the average number of grains per micrograph was within %. for the phe -min pulse plus -h chase experiment, results are a weighted average of two different sets of data. the standard deviations in table i are the result of simulation experiments (miller et al., ) to evaluate the error inherent in the quantitation. in these experiments, new grains for each micrograph were generated using the assumptions inherent in the algorithm (a poisson distribution of grains in a compartment and a gaussian error vector between the location of the radioactivity and the center of the simulated grain). the maximum likelihood calculation was then repeated for the simulated grains. five simulations were done for each time point, and the standard deviation of these simulations is presented in table i . in several cases, the original value was not within one standard deviation of the simulation average, although all values were within two standard deviations. this occurred mainly in the small mature and clear vesicle compartments and is probably caused by the inhomogeneity of these compartments. for example, the clear vesicle compartment contains transport vesicles from er to goigi apparatus, from one golgi slack to another, and from golgi apparatus to plasma membrane as well as parts of the tgn, primary lysosomes, and other compartments that cannot be differentiated by morphology. the simulation treats these as homogeneous compartments, while the grains may have been coming from a small subset of these compartments. therefore, the simulations arrive at estimates different from the original computation. the small mature vesicles at early time points are usually found close to the golgi apparatus and thus do not share the distribution of the majority of small mature vesicles, again causing differences between the simulation and the original experiment. other errors, such as sampling bias and digitation errors, are not included in the standard deviations and probably add to the total error in the experiment. it is difficult to quantitate these errors and they are presumed to be minor contributions compared with the errors due to the quantitation procedure. simulations were also used to evaluate the possibility that the high percenlage of grains in clear vesicles resulted from their proximity to both golgi apparatus and small immature vesicles. at three time points (phe rain pulse plus -min chase, ile -min pulse plus -rain chase, and phe -rain pulse plus -rain chase), the counts in clear vesicles were assigned proportionally to golgi apparatus and small immature vesicles, and a total of simulations were carried out with these new densities. in these simulations, the maximum value of counts ever seen over clear vesicles is %, and thus we conclude that the high values seen over this compartment at these time points is not due to their proximity to other compartments that contain radioactivity. one source of concern is the relative incorporation and washout of [ h]ile and [ h]phe. as our results are mainly based on comparing the flow of the different regions of the prohormone, as opposed to comparing the absolute position at a time point, we do not feel that possible differences in incorporation and washout would affect our results. the relatively slow washout of accumulated [ h]phe probably accounts for the difference in absolute timing observed between the experiments with -and -rain pulses of [ h]phe. bag cell clusters were isolated and fixed in % glutaraldehyde, % paraformaldehyde, % dmso, . m sucrose in . m na cacodylate buffer, ph . , for min at °c. the sheath was then dissected from the cluster, and the cluster was washed in . m na acetate, ph , and incubated in . % cytidine monophosphate (sigma chemical co., st. louis, mo) or glycerophosphate (sigma chemical co.) and . % lead nitrate in . m na acelate, ph , for rain at °c (novikoff et al., ) . results were similar for both of the labels, and control preparations without substrate showed no lead precipitate. clusters were washed with . m na cacodylate, ph . , osmicated in % oso in . m na cacodylate, ph . , for h, and then dehydrated and embedded as described above. thin sections ( - nm) were cut and stained with % uranyl acetate followed by lead nitrate before visualization on a transmission electron microscope ( ; philips electronic instruments, inc.). bag cell clusters or atrial gland pieces were incubated in #m monensin (sigma chemical co.) for h in asw with % ethanol at °c. control experiments with asw and % ethanol alone showed no effect on the bag cells. after the -h preincubation, clusters were labeled with tritiated amino acids as described above. all incubations and washes were done in the presence of #m monensin and % etoh or % etoh alone. the clusters were then processed for em immunohistochemistry (fisher et ai., ) , autoradiography (described above), or sds-nrea gels . in the monensin em autoradiography ~periment, quantitation was similar to that already described, but different compartments were used. the compartments measured in the monensin experiment were er, vacuoles, vacuoles with densities, densities in vacuoles, dense core vesicles, mitochondria, iysosomes, nucleus, and cytoplasm. a separate compartment, vacuolar membranes, was calculated in the entry algorithm. as the vacuoles curve through the thickness of the section, a -nm distance was used to define the limit of the membrane in these calculations. also, for quantitating monensin experiments, micrographs were taken at , x magnified to , x. clusters were labeled as described . the gels were quantitated by densitometry of the x-ray film. figure . proteolytic processing of the elh prohormone. a schematic of the -amino acid elh prohormone is shown including a -amino acid signal sequence (horizontal black bars). four biologically active bag cell peptides, the c~,/ , and , bag cell peptides as well as elh, are shaded. vertical bars are sequences of basic residues used as proteolytic processing sites. the first cleavage of the prohormone occurs at a tetrabasic sequence arg-arg-lys-arg, and the pathway then proceeds as shown, leading to the final set of product peptides shown . the peptides amino terminal to the first cleavage are packaged into one set of vesicles, and the peptides carboxy terminal to this cleavage are packaged in a distinct vesicle class. above the schematic are the positions of the peptides used to raise antisera used in immunohistochemical experiments . the top of the figure shows the distribution of the amino acids-leucine (l), isoleucine (i), and phenylalanine (f)-within the prophormone's sequence. elh precursor must occur after the initial cleavage of the prohormone. to determine the intracellular site of various cleavages, we have correlated the extent of prohormone endoproteolytic cleavage with the location of the molecule within the secretory pathway. bag cell clusters were labeled with [ h]ile to label the carboxy-terminal, elh-containing side of the prohormone or [ h]phe to label the aminoterminal, bag cell peptide-containing side of the prohormone. acid acetone extracts were then fractionated on sds-urea polyacrylamide gels (fig. , a , further cleavage of the initial amino-terminal intermediate (f ; fig. a) occurs more slowly than that of the initial carboxy-terminal intermediate ( ; fig. b) . monensin is an ionophore that blocks the proteolytic cleavage of a number of neuropeptide prohormones and causes them to accumulate in golgi-related compartments (crine and dufour, ; orci et al., ) . presumably, the cleavage is inhibited either by blocking transport of the hormone to the site of cleavage or by disrupting necessary ionic gradients. in contrast to other systems, initial cleavage of the elh prohormone still occurs after a monensin block; however, all subsequent cleavages are inhibited, and the two initial processing intermediates accumulate in monensin-treated cells (fig. c) (yates and berry, ) . this is not a peculiar effect of monensin in aplysia tissue, as all cleavages of the atrial gland prohormone are blocked after incubation with monensin ( fig. c) . therefore monensin divides elh processing into two distinct steps: the initial cleavage, which occurs before the monensin block, and all subsequent cleavages. to identify the site of the monensin block, we examined monensin-treated bag cells in the electron microscope. as in other systems, monensin causes a specific enlargement of golgi-related compartments while leaving other cellular compartments such as the nucleus, er, mitochondria, lysosomes, and mature dcvs relatively unchanged (tartakoff, ) . in the bag cells, some of these vacuoles contain dense core material, and these can be divided into two general types: (a) vacuoles with densities that are homogeneous in appearance ( %) and (b) vacuoles that contain two different types of densities ( %; fig. a) . examination of the densities with immunoelectron microscopy reveal thathomogeneous dense cores contain intermixed carboxy-terminal (elh) and amino-terminal (peptide k) immunoreactivity, but, as illustrated in fig. b, when two separate densities - ) . the clusters were then extracted with acid acetone and fractionated on sds-urea gels. (neweomb et al., ) . the bands are marked according to the nomenclature outlined in fig. . the initial cleavage is occurring at the -min chase and is complete after h. (c) after pretreatment for h with /~m monensin plus % etoh (lanes and ) or asw plus % etoh (lanes and ), bag cell clusters (lanes i and ) or atrial gland fragments ( mm x mm; lanes and ) were pulsed for h with [ h]leucine and chased for h (bag cells) or h (atri .al glands) before acid acetone extraction. under these conditions, the initial cleavage of the bag cell prohormone is completed in monensin, but all further cleavages of the intermediates are blocked (lane ). in contrast, the atrial gland precursor accumulates in monensintreated cells (lane ). other bag cell clusters labeled with [ h]leucine were assessed by hplc analysis (newcomb and scheller, ) , and all radioactivity was found in the first intermediates, demonstrating more conclusively that no further cleavages of the f or intermediate occur in the monensin experiment (data not shown). are observed the immunoreactivity of the different dense cores is segregated. to demonstrate that these densities actually represent the site of blockage, the monensin-treated bag cells were pulse chased with either [ h]leu, [ h]phe, or [ h]ile and processed for em autoradiography. as evident in fig. monensin blocks transport at various sites in different systems but almost always blocks transport somewhere within the golgi apparatus (gritliths et al., ; johnson and schlessinger, ; orci et al., ) . osmium, a specific marker for the cis-golgi compartment (friend and murray, ) , labels a subset of vacuoles but does not label vacuoles that contain densities (data not shown), suggesting the monensin block is beyond this point in the secretory pathway. the presence of sorted densities suggests that segregation occurs at this stage of prohormone processing and that the intermediates may have intrinsic abilities to aggregate selectively. these results must be interpreted cautiously as the disruption of ionic and ph gradients in monensin-treated cells may cause perturbations in the sorting process. in pulse-chase studies using either ph]ile (carboxy-terminal) or [ h]phe (amino-terminal), counts flow through the secretory pathway from er to golgi apparatus to granules (table i) . after a -min pulse with phllle, a large number of grains are seen over the golgi apparatus ( fig. a and table i). since at this time point no processing has occurred (fig. b) , one can conclude that cleavage does not occur early in the golgi apparatus. both amino and carboxy terminal-associated radioactivity are transported through the golgi stacks and enter small immature dcvs (defined by membrane extensions, connection to tubules, or nonspherical morphology) and clear vesicles after a -min pulse and -min chase (fig. b, fig. , and table i ). this is the time point correlated with the initial cleavage of the prohormone (fig. , a and b) . after a -min pulse and -h chase with [ h]phe, grains are found predominantly over large immature dcvs (fig. c, fig. , and table i) , while grains are found predominantly over small immature dcvs at this time point when [ h]ile is used (fig. d, fig. , and table i ). therefore, we can conclude that after a -min pulse and -h chase, the two regions of the elh prohormone have been sorted into distinct compartments. the em autoradiographic results are a strong independent confirmation of immunohistochemical studies demonstrating that large dcvs contain more immunoreactivity from amino terminal-derived (phe) peptides, while the majority of small dcvs contain more immunoreactivity from carboxy terminal-derived (lie) peptides kreiner et al., ) . to look more closely at the movement of the aminoterminal intermediate from small to large immature vesicles, a number of experiments were done using -min pulses of [ h]phe (table i ). the movement from small to large dcvs occurs quickly (largely between and min of chase), and a large percentage of counts are also seen in clear vesicles at these time points. the vectorial flow of the amino-terminal portion of the precursor appears to be from golgi apparatus to clear vesicles and small immature dcvs then to large immature dcvs. the flow of the amino-terminal intermediate from small to large immature vesicles could arise by (a) distinct aminoterminal specific small vesicles increasing in size or (b) movement of the amino-terminal intermediate from small immature vesicles containing carboxy-terminal intermediates to a distinct class of large immature vesicles. we favor the second possibility due to the fact that we do not see a gradual increase in the size of phe-labeled immature vesicles the bag cell soma, some of which contain dense cores. the small and large arrows identify two dense cores within a single vacuole that appear to be of different composition. about - % of the densities in vacuoles had this appearance. (b) immunoelectron microscopic analysis of monensin-treated bag cells. bag cell sections were labeled with a rabbit anti-peptide k and a rat anti-elh primary antibody followed by a -nm colloidal gold anti-rabbit and -nm colloidal gold anti-rat secondary antibodies. immunoreactivity within the dense cores is segregated; the small arrow points to peptide k immunoreactivity, and the large arrow points to elh immunoreaetivity. (see materials and methods) but do observe an abrupt change from small to large phe-labeled vesicles. furthermore, small immature vesicles containing amino-terminal peptides that are contiguous with the golgi apparatus are not observed, although carboxy-terminal peptide immunoreactive small immature vesicles are detected . it is possible that features of immature vesicles may result from their association with the tgn. a standard marker for the tgn is acid phosphatase (grifiiths and simons, ) , and, as illustrated in fig. , a-c, both small and large immature vesicle membranes are ringed by the lead phosphate reaction product. immature vesicles have also been shown to contain acid phosphatase in a number of other systems (smith and farquhar, ; novikoff et al., ; hand and oliver, ) . fig. , a-c, shows a series of micrographs from a set of serial sections; the structure marked by the small arrow actually attaches to the proximal large immature vesicle (fig. a) and in the next section attaches to a small immature vesicle (fig. b) . many other attachments between acid phosphatase-containing vesicles are seen in serial sections, suggesting that the immature vesicles are to some extent still connected to the tubular portion of the tgn. mature small and large vesicles, as well as er and other golgi stacks, do not stain with acid phosphatase, while lysosomes stain intensely. double label experiments which combine em autoradiography with acid phosphatase treatment formally demonstrate that the immature dcvs contain acid phosphatase activity (fig. d) and are therefore part of the tgn. interestingly, one of the large vesicles in this micrograph contains only patches of acid phosphatase reaction product. this is a common occurrence and suggests that the acid phosphatase is removed in patches from immature vesicles, as opposed to gradually fading in intensity. acid phosphatase is an enzyme destined for transport to endosomes and lysosomes. the appearance of acid phosphatase reaction product in patches suggests that lysosomal proteins are removed, not only from clear regions of the tgn, the journal of cell biology, volume i , but also from membranes of immature vesicles. immature vesicles often contain patches of clathrin, although the function of this clathrin is unclear. this clathrin has been proposed to be involved in the budding of regulated secretory vesicles (orci et al., (orci et al., , or recycling of protein from immature vesicles to the golgi apparatus (tooze and tooze, ) . lysosomal enzymes are most probably removed via clathrin-coated vesicles from the tgn (campbell and rome, ; grifliths and simons, ; von figura and hasilik, ) , suggesting that the function of clathrin patches on immature vesicles may be to facilitate sorting of lysosomal proteins. the above results suggest that sorting occurs as the aminoterminal intermediate moves from small immature vesicles and clear vesicles to large immature vesicles, all of which are defined by acid phosphatase cytochemistry as part of the tgn. furthermore, we have demonstrated the site ofprohormone cleavage to be the clear vesicles, small immature vesicles, or golgi stacks. yet, at a time when the prohormone is intact ( -min pulse; fig. b) , counts are located within the golgi stacks (fig. a) , suggesting that the cleavage of the prohormone must occur after this point, either in a late golgi stack or, more likely, the tgn. (table i) quantitative hplc studies indicate that the carboxyterminal (ile) final product peptides are present at three-to eightfold higher steady-state levels than the amino-terminal (phe) final peptides ( fig. ; fisher et al., ) . the large dcvs are not transported to the processes (kreiner et al., ) and quantitative immunoelectron microscopy studies suggest that the different levels of peptides could be due to the selective degradation or release of these large vesicles . to examine this question we chased [ h]phe-labeled bag cell clusters for longer times to observe the eventual fate of the large dcvs. after h of chase, many grains are observed in vesicles with heterogeneous cores that we refer to as mottled dcvs (table i) . the appearance of the core is quite variable, ranging from vesicles that are mostly clear but contained patches of dense material (fig. a) to dense dcvs with nonhomogeneous patches (fig. b) . loss of the homogeneous dense core suggests the peptides are being degraded. few grains are observed over large membranous lysosomes or multivesicular bodies. other interesting results from this time point include the movement of grains from immature to mature large vesicles and an increase in the number of grains in small mature vesicles (table i). coupled with the lack of constitutive release of large vesicle contents (fisher, j., unpublished data) , these results suggest that the asymmetry in peptide steady-state levels is generated through a degradative pathway. our results suggest a model for sorting within the regulated secretory pathway (fig. ) . correlating a biochemical assay of processing with em autoradiography predicts cleavage of the elh prohormone in a late golgi compartment. further support for this model comes from subcellular fractionation studies of bag cells, where the initial cleavage is shown to occur in a light fraction enriched in mannosidase ii activity (a golgi marker) and well separated from the dcvs (sweet, a., j. m. fisher, w. s. sossin, r. newcomb, and r. h. scheller, manuscript submitted for publication). the initial cleavage is not blocked by monensin, which does block the formation of mature granules. the protease that cleaves the tetrabasic site may be a tgn resident protein whose activity triggers the condensation and sorting of the processing intermediates in this compartment. alternatively, processing enzymes with different ph activation profiles or different affinities for the various cleavage sites may explain the distinct subcellular sites of endoproteolytic cleavage in the bag cells. multiple enzymes with different ph profiles have been proposed to differentiate between the two cleavages of the insulin precursor (davidson et al., ) . after the initial cleavage event, the elh-containing carboxy-terminal intermediate condenses in one region of the tgn (small immature vesicles) and the amino-terminal bag cell peptide-containing intermediate condenses in another region of the tgn (large immature vesicles). this model is consistent with immunocytochemical studies that demonstrate that small immature vesicles connected to the tgn contain largely carboxy-terminal immunoreactivity. these regions of the tgn may be connected by tubules, or transport between them may occur through vesicles. how are these intermediates segregated before formation of a dense core? after a -min chase both amino and carboxy terminal-associated grains are found over small imma- schematic diagram for prohormone processing and sorting in the bag ceils is described fully in discussion. a solid line represents the carboxyterminal intermediate; a dotted line represents the amino-terminal intermediate; a dotted line attached to a solid line represents the elh prohormone; smaller lines represent the carboxy-terminal final product peptides; and dots represent the amino-terminal final product peptides. l indicates an organelle of degradative function (endosome or lysosome). direct connection between the part of the tgn containing large immature vesicles and small immature vesicles is putative and this is represented by a dotted line. shading represents acid phosphatase-positive membranes. the small amino-and carboxy-terminal vesicles will be transported to processes for regulated release, where they are present at a carboxy-toamino terminal ratio of : . ture vesicles and clear vesicles. although the compartment of clear vesicles is obviously heterogeneous, the location and the time course of counts moving into these vesicles is most consistent with their arising from sections through the tgn. the lack of grains located over clear vesicles in other em autoradiographic studies of neuropeptide transport (saltpeter and farquhar, ) may be due to an earlier peptide condensation event in that system. amino terminal-associated radioactivity then decreases in clear vesicles and small immature granules and increases in large immature granules. it is the selective movement of the amino-terminal intermediate to large immature granules and/or the selective retention of the carboxy-terminal intermediate in small immature granules that underlies sorting within the regulated pathway. these results suggest a flow of the amino-terminal intermediate through the tgn from an early region that conrains small immature vesicles to a later region that contains large immature vesicles. later cleavages occur in vesicles after leaving the tgn, presumably due to activation of enzymes by the acidification of this granule (orci et al., ; anderson and orci, ) ; these cleavages are blocked by monensin. we propose that the small amino terminal-containing vesicles that are transported to processes arise from the larger immature vesicles, although the evidence supporting this idea is not yet conclusive. in support of this idea, a higher proportion of processing intermediate immunoreactivity is found in the large vesicles (kreiner et al., ) . furthermore, in [ h]phelabeled experiments, a high concentration of autoradiographic grains is seen in mature small dcvs only after a -h chase but not at earlier time points (table i) . the large vesicles appear to have a short half-life since > % of the counts associated with these vesicles appear in profiles that appear to be fated for degradation after a -h chase. the molecular mechanism by which the two processing intermediates are segregated from each other is still an open question. one appealing possibility is that the carboxyterminal intermediate selectively condenses in the early region of the tgn, leaving the amino-terminal intermediate soluble. perhaps different ionic conditions or different accessory proteins (chung et al., ) located in the late region of the tgn would allow the amino-terminal portion of the prohormone to condense at this site. a differential timing of condensation has also been proposed to explain the separation of prolactin and growth hormone in somatomammotrophs (fumagilli and zanini, ) . the aggregated carboxy-terminal portion of the precursor may be prevented from traveling to the late compartment either through steric hindrance of aggregates moving through small tubules, the inability to be packaged into small transport vesicles, or a specific association with an immobile membrane-bound receptor. alternatively, movement of the amino-terminal portion of the precursor may be mediated through the actions of a membrane-bound recognition system. in support of this model, the bag cell amino-terminal intermediate (f ) is associated with membranes at a time point ( -min pulse and -h chase) when the carboxy-terminal intermediate (i ) is not membrane associated (fisher, j., manuscript in preparation; illustrated in fig. ) . a selective association of the amino-terminal intermediate with a membrane-bound receptor would allow a vesicular sorting mechanism similar to that proposed for lysosomal enzymes. the amino-terminal bag cell peptides act locally to modulate the electrical activity of abdominal ganglion neurons and in an autocrine fashion to regulate the excitability of the bag cells (rothman et al., a; kauer et al., ; brown and mayeri, ) . the carboxy-terminal elh acts both on nearby neurons (mayeri et al., ) and through the circulation at peripheral targets as a hormonal substance (kupfermann, ; r~thman et al., b). thus, while the polyprotein motif ensures cosynthesis, the relative levels of these two sets of substances are regulated by the proteolytic cleavage, packaging, and targeting described above. recent studies indicate that non-elh precursor-related bag cell vesicle proteins are also selectively sorted ). these latter molecules may play a role in selective transport or release of different vesicle types. other neurons that express the elh prohormone ( a view of acidic intracellular compartments activation of neurosecretory cells enhances the synthesis of secretory protein positive feedback by autoexcitatory neuroendocrine bag cells of aplysia constitutive and regulated secretion of proteins coated vesicles from rat liver and calf brain contain lysosomal enzymes bound to mannose- -phosphate receptors a common source of ditficulty in high resolution autoradiography high resolution autoradiography two neuronal populations in the head ganglia of aplysia californica with egg-laying hormone-like immunoreactivity molecular sorting in the secretory pathway effects of monensin on the processing of proopiomelanocortin in the intermediate lobe of the rat pituitary intranrganellar calcium and ph control proinsulin cleavage in the pancreatic b cell via two distinct site-specific endopeptidases multiple neuropeptides derived from a common precursor are differentially packaged and transported osmium impregnation of the golgi apparatus in cow anterior pituitary, growth hormone and prolactin can be packaged into separate granules in the same cell nearophysin biosynthesis: conversion of a putative precursor during axonai transport the trans-golgi network: sorting atthe exit site of the golgi complex dissection of the golgi complex. i. moneasin inhibits the transport of viral membrane proteins from medialto trans-golgi cisternae in baby hamster kidney cells infected with semliki forest virus the mannose -phosphate receptor and the biogenesis of lysosomes the role of the goigi apparatus and gerl in secretory granule formation in acinar cells of the~rat exorbital lacrimar gland sorting of three secretory proteins to distinct secretory granules in acidophilic ceils of cow anterior pituitary vesicular stomatitis virus and sindbis virus glycoproteins transport to the cell surface is inhibited by ionophores alpha bag cell peptide directly modulates the excitability of the neurons that release it pathways of protein secretion in eukaryotes localization of aplysia neurosecretory peptides to multiple populations of dense core vesicles, jr large dense cored vesicles are enriched in neuropeptide processing intermediates in the aplysia bag cells stimulation of egg laying: possible neurocndocrine function of bag cells of abdominal ganglia ofaplysia californica proteolysis in neuropeptide processing and other neural functions nonsynaptic characteristics of neurotransmission mediated by egglaying hormone in the abdominal ganglion ofaplysia in situ hybridization to study the origin and fate of identified neurons maximum-likelihood estimation applied to electron microscopic autoradiography proteolytic processing of the aplysia egg-laying hormone and r - neuropeptide precursors processing of the elh precursor in the bag cell neurons of aplysia goigi apparatus, gerl, and lysosomes in neurons of rat dorsal root ganglia studied by thick section and thin section cytochemistry perrelet. . a clathrin-coated golgi-related compartment of the insulin secreting cell accumulates proinsulin in the presence of monensin proteolytic maturation of insulin is a post-golgi event which occurs in acidifying clathrin-coated secretory vesicles the transmost cisternae of the golgi complex: a compartment for sorting of secretory and plasma membrane proteins biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi primary structure and neuronal effects of alpha bag cell peptide, a second candidate neurotransmitter encoded by a single gene in bag cell neurons of aplysia egg-laying hormone: direct action on the ovotestis of aplysia autoradiography high resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary a single gene encodes multiple neuropeptides mediating a stereotyped behavior lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland a bag cell neuron-specific antigen localizes to a subset of dense cored vesicles in aplysia californica cellular and molecular biology of neuropeptide processing and packaging perturbation of vesicular traffic with the carboxylic ionophore monensin clathrin-coated vesicular transport of secretory proteins during the formation of acth-containing secretory granules in att cells an antibody specific for an endoproteolytic cleavage site provides evidence that proopiomelanocorticotropin is packaged into secretory granules in att- cells before its cleavage sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells lysosomal enzymes and their receptors preparation of em autoradiographs subcellular sites of processing of precursors of neurosecretory peptides in the bag cells of aplysia: inferences from the effects of monensin, fccp, and chloroquine key: cord- - sjlf authors: elbein, alan d. title: the use of glycosylation inhibitors to study glycoconjugate function date: - - journal: cell surface and extracellular glycoconjugates doi: . /b - - - - . - sha: doc_id: cord_uid: sjlf nan physiological processes ( , ) . the fact that these many different systems have complex carbohydrates as a common theme has given rise to the term "glycobiology," which has been defined as "a study of the structure and function of the carbohydrate modifications on different types of macromolecules" ( ) . complex carbohydrates are found attached both to proteins and to lipids. the glycoproteins produced by eucaryotic cells can have either n-linked oligosaccharides or o-linked oligosaccharides, and frequently both types of structures are found on the same protein ( ) . in addition, the number of oligosaccharide chains attached to the protein can vary from only one to many chains. in the n-linked types (described in more detail below), the carbohydrate is attached to the amide nitrogen of asparagine residues that occur in the tripeptide sequences, asn-x-ser (thr)(ll). however, this sequon is not sufficient for glycosylation to occur since many -asn-x-ser(thr)-sequences that are found in pro teins are not glycosylated. it appears that the conformation of the protein around this sequence and perhaps its exposure are critical factors in addition of carbohydrate. on the other hand, in the more common types of o-linked structures such as the mucins, the oligosac charides are attached by o-glycosidic bonds that usually involve galnac linked to the oh groups of serine or threonine residues, but there is no known amino acid sequence necessary for glycosylation ( ) . in terms of lipids, the best known and most common types of glycolipids are the sphingolipids, which contain oligosaccharide chains com posed of some of the following sugars: glucose, galactose, glcnac, galnac, sialic acid, and fucose. these sugars may be present in various configurations and combinations and are linked to a ceramide that is made up of fatty acid and sphingosine. these compounds are of considerable importance to medicine since certain gangliosides have been shown to be receptors for diphtheria and cholera toxins, while some cerebrosides are involved in bacterial attachment to animal cells. since a number of current reviews have covered these compounds in detail, they will not be considered here ( , ) . a more recently de scribed type of glycolipid is the glycosyl phosphatidylinositol (gpi) type, in which a glycan of mannose and glcnac (and sometimes other sugars such as galactose) is attached to the membrane phospholipid phosphati dylinositol. these gpi molecules appear to be important in anchoring many proteins to the membrane ( , ) , and some of the inhibitors of nlinked oligosaccharide assembly also affect the synthesis of the glycan portion of these anchors. they will be considered in more detail in a later section. although a considerable amount of information is available concern ing the structures of n-linked oligosaccharides and gpi glycans, there is relatively little data concerning the function of the carbohydrate portion of these molecules. it appears that protein (or lipid) glycosyla tion has two major roles: to modulate or change the biochemical proper ties of these molecules, including such parameters as bioactivity, fold ing, stability or turnover, immunoreactivity, and so on; and to serve as a determinant in molecular recognition events, such as lysosomal enzyme targeting, parasite or symbiote binding or adhesion to host cells, attachment of toxins or other biologically important ligands to cell surfaces, and so on ( ) . in most cases, it has been somewhat difficult to demonstrate a func tional role for the carbohydrate portion of the glycoprotein. one obvious way to do this would be to obtain the particular glycoprotein without its carbohydrate and then compare the biological and biochemical prop erties of the unglycosylated form with those of the normal glycoprotein. there are a number of ways to produce glycoproteins without their associated carbohydrate, especially in the case of the n-linked glycopro teins. for example, one can remove the n-linked oligosaccharides with endoglycosidases, such as endoglucosaminidase h (endo h), which cleaves "high-mannose" oligosaccharides between the two internal glcnac residues ( ) , or peptide glycosidase f, which hydrolyzes most n-linked oligosaccharides at the glcnac to asparagine bond ( ) . the problem with this approach is that many n-linked oligosaccharides are resistant to cleavage by these enzymes when the protein is in its native state. therefore, it is necessary to denature the protein in order to obtain complete removal of the n-linked oligosaccharides, making this method untenable for functional studies. another problem with this approach is that removing the carbohydrate after the protein has been synthesized and has achieved its proper conformation may be quite different physiologically than having the protein synthesized in the cell in the absence of carbohydrate. a second possibility is to produce the glycoprotein of interest in a cell system in the presence of the antibiotic tunicamycin ( ) . this compound inhibits the glcnac- -p transferase that catalyzes the first step in the "dolichol cycle," thereby preventing the formation of lipidlinked oligosaccharides ( ) . this inhibition usually results in a com plete blockage in n-linked glycosylation ( ) . the difficulty with this approach is that many glycoproteins may be improperly folded or be come insoluble when they are synthesized without any n-linked carbo hydrate. as a result, physiological studies again become of questionable value. a third alternative for preventing glycosylation is to use site-directed mutagenesis to modify the asn-x-ser sequon(s) that is glycosylated to one that is a "non-acceptor" sequon ( , ) . the beauty of this method is that one can modify one glycosylation site at a time (assuming that there is more than one) and determine how an alteration at that site affects function. or one can modify any number of these sites at the same time and compare the various proteins. two potential obstacles are ( ) the gene coding for the protein of interest must be available and ( ) one must make the assumption that the change in the amino acid sequence does not itself affect protein conformation. this methodol ogy has provided some valuable insights into the role of asparaginelinked glycosylation. however, if all n-linked oligosaccharide sequons are modified, then the same problem of improper folding or solubility that one finds with tunicamycin would also apply here. another possi bility is to modify glycosylation rather than prevent it, so that the resulting n-linked glycoprotein contains an altered oligosaccharide structure. one can then determine if and how this alteration affects the biological activity of a given glycoprotein. there are several ways to modify n-linked oligosaccharide structures. for example, the protein of interest can be produced in a mutant cell line that has a defect in one of the biosynthetic enzymes. cell lines that are missing various glycosyltransferases or processing glycosidases have been isolated, and several excellent reviews have described the various mutants and the techniques for their utilization ( , ) . in this case also, one must have the gene for the glycoprotein of interest in order to introduce it into the mutant cell line for synthesis of the appropriate protein. the carbohydrate structure can also be modified with selected inhibi tors that block the activity of key enzymes in the processing pathway, so that synthesis is stopped at a premature step and the various types of complex chains cannot be formed. such inhibitors and their sites of action are the main topics of this chapter. one disadvantage on the use of such inhibitors in in vivo studies is that all n-linked glycoproteins are affected, and therefore it may be difficult to relate changes in physiology to an effect on the specific glycoprotein of interest. in fact, one criticism that can be levied at the use of inhibitors in most biological systems (and also as chemotherapeutic agents) is that absolute specific ity is seldom if ever achieved. of course in the above case, if the altered glycoprotein can be isolated from the inhibited (or mutated) cell and its activity compared with the normal protein, either in vitro or in another system, then this criticism can be overcome. one final method for altering carbohydrate structure is to disrupt or alter the normal transport of glycoproteins from the er through the various golgi stacks to their ultimate location. since it is now clear that certain of the oligosaccharide trimming reactions occur in the er, whereas others occur in the cis-golgi and still others in the medial golgi, etc., the basis of this approach is to prevent the protein from being transported to that region where the enzymatic activity is located. if one can somehow prevent the glycoprotein from reaching the specific compartment that houses that activity, then that reaction should not occur. there are several valuable compounds that have such properties and these drugs will be discussed in a later section. these types of inhibitors have been extremely useful tools for helping us to understand the mechanism(s) of protein targeting. in terms of the glycan portion of the gpi anchor, a number of recent studies have demonstrated that various inhibitors that act on the re linked oligosaccharides also inhibit some of the reactions in anchor formation. these inhibitors are providing useful information on the biosynthetic pathway and should also be helpful for functional studies. however, at this stage, we still do not know enough about the synthesis of the glycan portion of the gpi anchors, nor do we really understand their function. this chapter will deal with various types of inhibitors that affect gpi anchor formation, as well as those that act on the re linked glycoproteins. ii . the most common types of glycoproteins that are present in eucaryotic cells, as cell surface proteins, membrane proteins, and secretory proteins, are those having oligosaccharides attached to the amide nitro gen of asparagine (n-linked or asparagine-linked) ( ) . the oligosac charide portion of these molecules may have a variety of different structures that fall into one of the general groups shown in fig. . these are referred to as high-mannose, complex, or hybrid types of oligosaccharides. it can be seen from the figure that each of these structures contains the same basic core region (shown by the boxed-in area) composed of the oligosaccharide sequence manal, (manal, ) man) , glcnacj , glcnac. in the high-mannose structures, this core region is elongated by as many as six additional mannose residues (i.e., a total of nine mannose units) attached in al, , al, , and al, linkages, as shown by the typical structure presented in fig. . however, some high-mannose oligosaccharides may have fewer than six additional mannoses, since some trimming can occur during biosynthesis (see section v). on the other hand, in the complex types of oligosaccharides, the pentasaccharide core region can be extended with a number of different substitutions. shown in the figure is a typical biantennary type of oligosaccharide, containing two of the trisaccharide sequences, neuaca , gal/ , glcnac/ , . on the other hand, some complex chains may have three (triantennary) or four (tetraantennary) of these trisaccharide sequences, as well as various other substitutions such as al, -linked l-fucose on the innermost glcnac or sulfate residues attached to this glcnac ( ) . various other alterations may also occur, such as al, -linked fucose units on the outer glcnac residues, polylactosamine chains, a / , -bisecting glcnac residue attached to the βlinked mannose and so on, as well as various glycosidic linkages such as a , -linked sialic acid rather than a , -linked sialic acid, etc. al-though the exact number of different types of complex chains is not clear, it has been estimated that there are probably several hundred known structures ( ) . the final type of n-linked oligosaccharide is the hybrid structure, which still contains a manal, (manexl, ) branch on the al, -mannose arm, but also has a type of complex chain on the al, -branch. most hybrid chains also apparantly have a bisecting glcnac attached in / , -linkage to the β-linked mannose, and this substitution may play a role in the control of further processing reactions. the biosynthesis of all of these n-linked oligosaccharides involves two independent and sequential pathways that occur in the endo plasmic reticulum (er) and golgi apparatus of the cell. in the first series of reactions, membrane and secretory proteins that are under going synthesis on membrane-bound polysomes become glycosy lated by transfer of a common oligosaccharide having the structure glc man (glcnac) from its lipid-linked saccharide form, i.e., glc man (glcnac) -pp-dolichol, to specific -asn-x-ser (thr)-sequences on the protein ( ) . this pathway is referred to as the "dolichol cycle" and is depicted in fig. . a series of glycosyltransferases that are associated with the er membrane sequentially add the sugars glcnac, mannose, and glucose to the lipid carrier dolichyl-p to give the above-mentioned lipid-linked oligosaccharide donor. to study this pathway in animal cell culture systems, one can add one of the radioactive sugars, i.e., [ h]glucosamine, [ h]galactose (as a tag for the glucose residues), or [ h]mannose, to the medium and examine the incorporation of the label into lipid-linked saccharides. these intermediates are easy to extract from the cells by a sequential extraction procedure, first with chc :ch h:h ( : : ), which ex tracts the lipid-linked monosaccharides and smaller sized lipid-linked oligosaccharides, and then extraction of the remaining residue with chc :ch h:h ( : : ) to obtain the lipid-linked oligosaccharides that contain the larger sized oligosaccharides. the intact lipid-linked monosaccharides can usually be identified by tlc, whereas the larger oligosaccharides from the lipid-linked oligosaccharides are usually re leased by mild acid hydrolysis and characterized by gel filtration on bio-gel p- or by hplc ( , ) . thus, the first enzyme in the biosynthesis of n-linked oligosaccha rides transfers glcnac-l-p from udp-glcnac to dolichyl-p to form dolichyl-pp-glcnac ( , ) . this enzyme is the site of action of the very widely used glycosylation inhibitor tunicamycin ( ) (discussed later). the glcnac- -p transferase has been purified to homogeneity from bovine mammary glands ( ) , and the gene for this enzyme was recently cloned and expressed is cos cells ( ) . the next glcnac transferase adds the second glcnac, also from udp-glcnac, to dolichyl-pp-glcnac to form glcnac/ , glcnac-pp-dolichol. this en zyme has recently been purified to homogeneity from pig aorta ( ) . the next five sugars to be added are mannose residues and are do nated via gdp-mannose. as indicated below, two lines of evidence have demonstrated that gdp-mannose, rather than dolichyl-pmannose, is the mannosyl donor for these additions: (a) a mutant cell line that is missing the dolichyl-p-mannose synthase can still form the manal, manal, manal, (manal, )man i , glcnaci , glcnac-ppdolichol but is not able to further elongate it ( ) ; and (b) amphomycin, an inhibitor of the in vitro formation of dolichyl-p-mannose ( ), does not prevent the formation of the above man (glcnac) -pp-dolichol, but it does block further elongation of this intermediate ( ) . the βmannosyl transferase that adds the first mannose to the dolichol inter mediates has recently been purified to homogeneity from pig aorta, and an azido-labeled [ p]gdp-mannose was used to identify the subunits of this protein ( ) . several of the α-mannosytransferases have also been partially purified from liver ( ) ( ) ( ) ( ) , and these enzymes require gdp-mannose rather than dolichyl-p-mannose as the manno syl donor. however, these proteins have not yet been purified to homo geneity so that their detailed properties or their orientation in the er membrane is not known. once the man (glcnac) -pp-dolichol is formed, it can be further glycosylated by the addition of four more mannose residues and then three glucose units ( ) . these mannose residues all are believed to be donated via dolichyl-p-mannose (because of the topology of the oligosac charide in the er lumen and the results with mutants missing the dolichyl-p-mannose synthase) ( ) , whereas the glucosyl donor is dolichyl-p-glucose ( ) . most of these enzymes have not been isolated or studied in any detail, and therefore little is known about their properties. however, the enzyme that adds the first mannose from dolichyl-p-mannose (i.e., the sixth mannose to the lipid) has been par tially purified from aorta extracts and its substrate specificity was determined. for activity, this enzyme required dolichyl-p-mannose as the mannosyl donor and man (glcnac) -pp-dolichol as the mannosyl acceptor ( ) . likewise, the enzyme that adds the first glucose from dolichyl-p-glucose to man (glcnac) -pp-dolichol was purified to near homogeneity from pig aorta and its substrate specificity and oligosac charide acceptor specificity were determined. while a number of oligo saccharide structures linked to dolichol could serve as glucose ac ceptors, they all required the manal, manal, mana:l, -branch for this activity ( ) . the enzyme(s) that adds the last two glucoses has not yet been purified. the role of the glucose units in the biosynthesis of n-linked oligosac charides is presumably to facilitate the transfer of oligosaccharide from lipid to protein. that is, the oligosaccharyltransferase that catalyzes this final step in the dolichol cycle recognizes or prefers glucosecontaining lipid-linked oligosaccharides as the oligosaccharide donor ( , ) . in fact, studies with microsomes showed that glucosecontaining lipid-linked oligosaccharides were much more efficient oli gosaccharide donors than were glucose-free lipid-linked oligosaccha rides. thus, this last step of the dolichol cycle occurs in the er and the oligosaccharide is transferred to specific asparagine residues on the protein, as the polypeptide is being elongated on er-bound poly somes ( ) . the oligosaccharyltransferase has recently been purified to apparant homogeneity from liver ( ) . once the oligosaccharide chain(s) is transferred to the protein, and while the protein is still undergoing synthesis in the er, two membrane-bound and highly specific glucosidases remove all three glucoses from the glc man (glcnac) . the processing pathway for re linked oligosaccharides is outlined in fig. . thus, glucosidase i re moves the outermost al, -linked glucose to give a glc man (glc-nac) -protein ( ), whereas glucosidase ii removes the next two al, -linked glucoses to give the man (glcnac) -protein ( ) . glucosi dase i has been purified from calf ( ) and porcine ( ) liver, from bovine mammary glands ( ) , from mung bean seedlings ( ) , and from saccharomyces cerevesiae ( ) . this enzyme has a ph optimum in the neutral range ( . - . ), does not work with p-nitrophenyl-a-d-glucopyranoside, and is quite specific for al, -glucosidic linkages. the mam malian enzyme is a tetramer with subunits of about kda, whereas the yeast enzyme has subunits of about kda. in s. cerevesiae, the glsl mutation results in cells that lack glucosidase i and produce glyco proteins that maintain the glc man (glcnac) structure ( ). how ever, this alteration in structure has no effect on secretion of the glyco proteins, in contrast to mammalian cells in which such an alteration had profound effects (see section on glucosidase inhibitors). a cho mutant cell line has been isolated by virtue of its resistance to l-pha, and this mutant cell, called lec , has been shown to lack glucosidase i ( ). these cells were found to synthesize the g protein of vsv having hybrid and high-mannose oligosaccharides, as well as complex chains. glucosidase ii was initially identified in microsomal fractions of rat liver ( ), calf liver ( ) , and yeast ( ) and was shown to re move both of the al, -linked glucoses from glc man (glcnac) to form man (glcnac) . however, removal of the outermost al, -linked glucose is quite rapid (£ / = min ) as compared with cleavage of the innermost glucose (t / = - min). although glucosidase ii also can catalyze the cleavage of aryl-a-glucosides such as p-nitrophenyl-a-glucopyranoside, it appears to be fairly specific for al, -linked glucoses since its activity is inhibited by the al, -linked glucose disaccharide nigerose, but not by other α-linked glucose disaccharides (i.e., kojibiose, maltose, isomaltose, or trehalose) ( ) . glucosidase ii from pig kidney was re ported to have a subunit molecular mass of kda and to be a highmannose glycoprotein ( ) , whereas the enzyme from mung beans was composed of two identical glycoprotein subunits of kda, also with high mannose oligosaccharides (some of which probably also contain some glucose residues) ( ) . in some animal systems, however, the glucosidase ii had quite different subunit molecular weights of around kda ( , ) . the localization of the enzyme has also been studied and may vary, depending on the tissue, from being concentrated in rough er (rer) and smooth er (ser) in pig hepatocytes ( ) to post-golgi apparatus structures in pig kidney tubular cells ( ) . the best substrates for assaying these processing glucosidases are radiolabeled glc man glcnac and glc man glcnac. these substrates can be prepared by incubating animal cells (or virus-infected cells) in the presence of the processing inhibitors castanospermine (discussed later), to prevent the removal of glucose residues, and deoxymannojirimycin, to prevent the removal of al, -linked mannoses. [ h]galactose is then added to the cell cultures to label the glucose moieties in the newly synthesized glycoproteins. the glycopeptides resulting from pronase digestion of the glycoproteins are isolated by gel filtration on columns of bio-gel p- , and these glycopeptides are then treated with endo h to obtain the [ h]glucose labeled glc man glcnac. this oligo saccharide can then be treated with a glucosidase i preparation that is free of glucosidase ii and α-mannosidases to obtain [ h]glc man glc nac. for assay of the processing glucosidase activities, the previously mentioned radiolabeled substrates are incubated with the appropriate enzyme preparations, and the release of labeled glucose is measured by a concanavalin a-sepharose binding assay ( ) . the removal of three glucose residues from the glc man (glc nac) -protein results in the formation of a high-mannose man (glcnac) -protein, which can then be further processed to other highmannose or complex types of oligosaccharides (as seen in figs. and ). early studies on the processing pathway indicated that there were two or three "neutral" α-mannosidase activities involved in trimming the n-linked oligosaccharide, and these activities were distinct from those of the lysosomal α-mannosidase ( ) . as a result of increasing interest in this area of research, a number of new α-mannosidase activi ties have been described and current work is aimed at trying to under stand how these various enzymes function in the removal of α-linked mannoses from eucaryotic cell glycoproteins. in fact, one of the im portant reasons for having a number of specific mannosidase inhibitors (see section v) is to be able to use them to distinguish among various mannosidase activities, as well as to determine what happens to pro cessing and function of a given glycoprotein when a specific a-mannosidase is inhibited. a well-written review deals with the various amannosidases ( ), so these enzymes will be only briefly covered here. an er α-mannosidase that removes a single al, -linked man nose from man (glcnac) to give a specific man (glcnac) isomer has been purified from yeast ( ) . this enzyme is inhibited by the pro cessing inhibitor deoxymannojirimycin, requires ca + for activity, and does not work with aryl-a-mannosides. the gene for the enzyme has been isolated and codes for a -kda protein with three nglycosylation sites, a ca + binding consensus sequence, and a noncleavable signal sequence near the Ν terminus that acts as a transmembrane type ii domain ( ) . a ca + requiring al, -mannosidase was also puri fied from rabbit liver and this enzyme shows considerable homology ( % identity and % similarity) to the yeast enzyme ( ) . however, the rabbit liver enzyme can remove most if not all of the al, -linked mannoses from man (glcnac) , and it requires either nonionic deter gents or phospholipids for activity ( ) . two other ca + requiring amannosidases have been purified from calf ( ) and pig ( ) livers, and these enzymes differ from the rabbit enzyme in some of their properties. these enzymes also do not work with the aryl-a-mannopyranosides as substrates. the pig liver enzyme was localized by immunological techniques and found to be distributed in rer, ser, transitional ele ments of the rer, and vesicles that engage in transportation between the er and the golgi, but it was not in the golgi ( ) . actually, the first neutral α-mannosidase to be reported was named mannosidase i and this enzyme was isolated from the golgi apparatus of eucaryotic cells ( ) . this α-mannosidase has catalytic properties very similar to those of the ca + -dependent mannosidases, but it apparantly does not need ca + , although this is not certain because the requirement was not tested in the presence of edta ( ) . the mannosi-dase i was resolved into two distinct proteins by chromatographic meth ods and these two forms were termed mannosidase ia and ib. ia was purified to homogeneity and shown to be a tetramer composed of kda glycoprotein subunits ( ) . presumably ia and ib have similar substrate specificities, but it is not clear if they are derived from the same gene. once the four al, -linked mannose units are removed, a medial golgi glcnac transferase (called glcnac transferase i) adds a glcnac from udp-glcnac to the mannose that is attached to the β-linked mannose in al, -linkage ( ) . this glcnac transferase was purified over , -fold to homogeneity from liver ( ) and the enzyme has been sequenced and cloned ( ) . the glcnac is apparantly a recognition signal for another highly specific α-mannosidase, called mannosidase ii, that cleaves the al, -linked and al, -linked mannoses from the glcnac-man (glcnac) -protein to give a glcnac/ , manal, [manal, ]manj , glcnac) , glcnac-protein ( ) . in spite of this substrate specificity, mannosidase ii does act on the aryl-a-mannopyranosides such as p-nitrophenyl-a-d-mannopyranoside. mannosidase ii was purified to homogeneity from rat liver ( , ) and from mung bean seedlings ( ) . both enzymes migrate on sds gels as proteins of about kda, although the sequence for the murine enzyme predicts a mw of kda for the deglycosylated protein. both enzymes are also glycoproteins and the murine enzyme has been shown to be a type ii transmembrane glycoprotein with a single transmembrane region and a luminally oriented catalytic site ( ) . there are several other α-mannosidases that are distinct from the above enzymes based on their sensitivity to processing inhibitors and to catalysis of high-mannose oligosaccharides. however, the relative role of these enzymes in the glycoprotein processing pathway remains to be established. one of these enzymes is a cytosolic and neutral amannosidase from rat liver ( ) . although it was originally thought that this enzyme was a proteolytic product of an er a-mannosidase, it now appears that both the cytosolic and the er α-mannosidase are related to an α-mannosidase that was isolated from rat brain and that can cleave al, -, αϊ, -, and al, -linked mannose residues ( ). this last enzyme can cleave a man (glcnac) structure to a man (glcnac) . the difference between the er liver enzyme and the brain enzyme is that the former enzyme is also active with aryl-a-mannosides, whereas the latter is not. two other α-mannosidases that can cleave al, -, al, -, and al, -linked mannoses have been purified from rat sperm ( ) and rat liver ( ) . another type of hydrolytic enzyme that is also proposed to be involved in processing is an endo-a-mannosidase that can cleave a glcal, man disaccharide from glc man (glcnac) (or a glc man from glc man (glcnac) , etc.) ( ) . it is interesting to note that the man (glcnac) that results from the action of this enzyme is a different isomer from the man (glcnac) that is formed by the action of the er α-mannosidase or the yeast α-mannosidase ( ) . this endomannosidase appears to be a resident of the golgi apparatus and may function to trim oligosaccha rides on glycoproteins that have escaped the er still retaining one or more glucose residues. as shown in fig. , following the trimming of all of the glucose residues and a number of mannose units from the n-linked oligosaccha rides, various glycosyltransferases act to add different sugars, such as glcnac, galactose, sialic acid, fucose, etc., to the core n-linked struc ture to form many different types of complex chains ( ) . since there are no inhibitors available for these glycosyltransferases, they will not be considered further in this review. glycosyl phosphatidylinositols are a recently discovered class of glycolipids that have been shown to anchor many proteins (and other mole cules such as heparan sulfates) to biological membranes via covalent linkages. recently, the chemical nature of these molecules has been deduced, but the complete structure is still elucidated only for a few of these anchors. the characterization of these glycolipids has involved compositional analyses using gc-ms in combination with specific chemical treatments and sequential digestion with exoglycosidases, analysis of glycosidic linkages by methylation analyses, and gel filtra tion and tlc of intact glycans and their degradation products ( ) . the best known and most thoroughly detailed gpi anchors are those of trypanosoma brucei and rat thy- . although these structures show some differences, they do have a conserved linear region shown in . general structure of the glycosylphosphatidylinositol that serves as a mem brane anchor for many cell surface proteins. fig. to be composed of - -ethanolamine-p- -manal, mana:l, manal, glcnal, -inositol ( ) . in the case of the variable surface antigen (i.e., vsg) of trypanosomes, there are a number of galactose residues attached to the first mannose, whereas in thy- there may be a galnac and/or an ethanolamine-p attached to this mannose ( ) . also these structures may differ in the number of fatty acid resi dues attached to the inositol and also in the structure of the fatty acids. although some of the details of the biosynthesis of the glycan portion of these anchors are known, there are still considerable gaps in our understanding of the pathway. nevertheless, the steps in biosynthesis are proposed to occur as outlined in fig. . the sequence begins with the transfer of a glcnac from udp-glcnac to phosphatidylinositol to form glcnac-pi, which then undergoes deacetylation to form glcn-pi ( ) . these reactions have been demonstrated in vivo by labeling studies with h-glucosamine and in cell-free extracts incubated with udp-glcnac. three mannose residues are then added to the glcn-pi and these mannoses are all proposed to be derived from dolichyl-p-mannose, rather than from gdp-mannose (see section ix for experimental evidence). none of these enzymes has been purified and virtually nothing is known about how other sugars such as gal or glcnac are added. in addition, it is not clear when in the biosynthetic pathway the fatty acids are added to the pi, although there is now evidence that the glcn-pi must be acylated to serve as an acceptor for the first mannose ( ) . once the mannose units have been added, phosphatidylethanolamine serves as a donor of ethanolamine-p, which becomes attached via a phosphodiester bond to the position of the terminal mannose. the signal for gpi addition to the nascent protein resides in a short hydrophobic carboxy-terminal peptide that is removed and replaced by the gpi anchor ( ) . this addition of protein to the gpi apparantly occurs on internal membranes, but the details of these biosynthetic steps are not known at this time. probably the most widely used and most valuable inhibitor of nlinked glycosylation is tunicamycin. this compound is a nucleoside antibiotic that is produced by the bacterium streptomyces lysosuperificus and was initially found to inhibit the replication of a number of enveloped viruses ( ) . the structure of tunicamycin was elegantly elucidated by tamura and co-workers ( ) and is shown in fig. . the antibiotic contains a uracil moiety to which is attached, by an nglycosidic bond, an carbon aminodeoxydialdose called tunicamine. the tunicamine is itself substituted by having a glcnac attached to . the structure(s) of the various tunicamycin components. r refers to the differ ent fatty acids, designated i to x, that may be attached to the tunicamine portion of the molecule. its anomeric carbon in an o-glycosidic bond and a fatty acid linked in amide bond to the amino group. the fatty acid may vary in structure from c to c , may be saturated or unsaturated, and may be branched or unbranched. in fact, tunicamycin is actually produced as a complex that can be separated into as many as to peaks by hplc ( , ) , and the basis of this separation is the fatty acid moiety. interestingly enough, all of these peaks have biological activity, but those compo nents with the longer chain fatty acids appear to be more effective inhibitors in cell culture ( ) , possibly because they are taken up by the cells at a faster rate. the site of action of tunicamycin was initially demonstrated with a microsomal enzyme preparation from calf liver, and the antibiotic was shown to be a potent inhibitor of the enzyme that transfers glcnac- -p from udp-glcnac to dolichyl-p ( , ) . this reaction produces dolichyl-pp-glcnac, which is the first intermediate in the dolichol pathway. tunicamycin had the same site of action in plant microsomes ( ) and in microsomes from chick oviduct ( ) . in addition, tuni camycin is a very potent inhibitor with partially purified preparations of the udp-glcnac:dolichyl-p glcnac-l-p transferase from several animal tissues ( , , ) . this inhibitor also affects several microbial enzymes that are involved in the synthesis of peptidoglycan, i.e., the enzyme that forms undecaprenyl-pp-iv-acetylmuramoyl-pentapeptide ( ) , and the enzyme involved in production of undecaprenyl-pp-glcnac ( ) . however, tunicamycin had no effect on other glcnac transferases such as the enzyme that adds the next glcnac to dolichyl-pp-glcnac ( ) , the enzymes that add terminal glcnac residues to the n-linked oligosaccharide core ( ) , the phosphotransferase that adds glcnac- -p to high-mannose chains on lysosomal enzymes ( ) , and the trans ferase that adds glcnac to serine residues on nuclear proteins ( ) . it also had no effect on the formation of undecaprenyl-pp-galactose or undecaprenyl-pp-galnac ( ) , but at high concentrations it did inhibit formation of dolichyl-p-glucose ( ) . summing up these data, it appears that tunicamycin inhibits phosphotransferases that catalyze the translocation of a glcnac- -p from its udp derivative to a polyprenyl-p, but the sugar must be chirally related to glcnac. tunicamycin is probably a competitive inhibitor of the glcnac- -p transferase, but it binds so tightly to this enzyme that it has not been possible to reverse the inhibition by adding high concentra tions of udp-glcnac ( ) . it was suggested that this compound was a tight-binding reversible inhibitor and might be a substrate-product transition-state analog ( ) . tunicamycin has been used in many cell culture systems to try and assess the role of n-linked glycosylation in the function of various glycoproteins. probably the best test of whether tunicamycin is really effective in preventing glycosylation in cell culture is to determine whether it blocks the in vivo incorporation of labeled glucosamine into dolichyl-pp-glcnac or the incorporation of [ - h]mannose into lipidlinked oligosaccharides. to do this, one can incubate cell cultures in the presence of various concentrations of tunicamycin (usually . to μg/ml) for min to hr and then add the labeled glucosamine or mannose to these cells. after an appropriate period of labeling (deter mined experimentally for each cell type), the cell pellet is extracted with chc :ch h:h ( : : ) and then chc :ch h:h ( : : ) to obtain the lipid-linked monosaccharides and the lipid-linked oligo saccharides ( ) . one important point here is that the effective concen tration of inhibitor may vary widely depending on the cell system being used, and the optimum concentration needs to be determined in each case. in fact, it has been demonstrated that tunicamycin-resistant mu tants can be isolated and these mutant cells have amplified levels of the glcnac-l-p transferase ( ) . thus, it seems reasonable to assume that the level of this enzyme, and therefore the susceptibility of the cell to tunicamycin, may vary from cell to cell or from tissue to tissue. something else to consider when using tunicamycin for functional studies is that this compound may inhibit protein synthesis ( ) . for this reason, any effects on glycoprotein function should be thoroughly examined to be certain that they result from the absence of carbohy drate, rather than from the loss of certain proteins. for example, in some of the various studies, at tunicamycin concentrations of . to μ-g/ml, [ h] leucine incorporation into protein was inhibited from to %, while glycosylation was inhibited from to %. but again, the important point is that some cell types are much more sensitive to inhibitors than are other cell types. perhaps one control for some of these studies would be to use other inhibitors (i.e., cycloheximide or puromycin) that are known to block protein synthesis but do not affect carbohydrate synthesis or addition, or some other inhibitors that mod ify the structure of the carbohydrate chain, and compare the effects of these compounds with those of tunicamycin on the function of the particular glycoprotein. the results of producing glycoproteins in cells in the presence of tunicamycin vary considerably depending on the glycoprotein in ques tion. in terms of structural studies, tunicamycin at the appropriate concentration seems to prevent glycosylation of all n-linked glycopro teins, regardless of the biological system. however, in terms of the protein portion of the molecule, some glycoproteins are still synthesized at a normal rate and are still secreted even when they are not glycosyl ated ( ) . thus, no effect of this inhibitor on the secretion of procolla gen from chick embryo cells, secretion of a number of serum proteins from rat hepatocytes, interferon secretion by leukocytes or l cells, secretion of the enzyme / -iv-acetylhexosaminidase by fibroblasts, and so on was observed ( ) . the conclusion here is that a number of different types of cells and glycoproteins do not require their carbohy drate for either folding or conformation and appear to function normally in the absence of added carbohydrate. some proteins, however, are greatly affected by the absence of carbo hydrate. thus, a considerable decrease in the rate of secretion of ige and iga by mouse and rat plasma cells was observed in the presence of tunicamycin, as was the secretion of igm in hybridoma cells ( ) . one rather strange and interesting result that emerged from these studies was the observation that tunicamycin inhibited igm secretion in hybridoma cells, but did not affect the secretion of igg. these results led the authors to hypothesize that the differential effects of this inhibi tor were due to factors intrinsic to the respective heavy-chain polypep tides themselves, rather than to some property of the cells ( ) . it would seem likely that such factors are related to the conformation of the respective proteins and to the influence of the carbohydrate on protein structure and/or folding. not surprisingly, tunicamycin also has dramatic effects on some membrane receptors, such as the acetylcholine receptor of muscle cells ( ) , the insulin receptor ( ) , the receptor for low-density lipoproteins (ldl) ( ) , and the receptor for nerve growth factor ( ) , to cite only a few examples. in general, with all of these receptors, tunicamycin significantly decreased the number of receptor molecules at the cell surface, but did not change the total number of receptors in the cells or the binding affinity of those receptors that were present in the plasma membrane. in some of these studies, the effects of tuni camycin were reversible, and in one study with the insulin receptor, it appeared that the nonglycosylated receptor could become glycosylated after removal of the drug ( ) . that would suggest that in some cases, proteins might be glycosylated post-translationally. since there is some precedence for surface glycoproteins being endocytosed and recycled back through the golgi (and perhaps the er) ( ) , it is conceivable that glycosylation could occur on some exposed asparagine sequons. probably one of the reasons for the great variation in tunicamycin effects on protein function is that some glycoproteins require their re linked oligosaccharides for stability (or longevity), and in the absence of glycosylation these proteins are rapidly degraded. that is, the carbo hydrate may protect or somehow prevent the degradative enzymes from attacking glycosylated proteins. such has been suggested to be the case for fibronectin, acetylcholine receptor, influenza viral hemagglutinin, and various immunoglobulins, just to cite a few examples ( ) . in fact, in the influenza system, it was observed that when viral replication was examined in the presence of tunicamycin, the viral envelope glyco proteins could not be detected. however, when a protease inhibitor, such as tcpk or leupeptin, was added to the cells along with the tunicamycin, the viral proteins in their nonglycosylated form could be detected ( ) . these studies and others cited above all indicate that the influence of the n-linked oligosaccharides on the protein in question depends on its amino acid sequence, which in turn determines folding and conformation. thus, the carbohydrate may play a much greater role in affecting or maintaining the conformation of some proteins than that of others. one study that provides strong support for the hypothesis that the amino acid sequence of the protein determines what type of and how important a role the carbohydrate plays in the function of the glycopro tein was done using various temperature-sensitive mutants of vesicular stomatitis virus ( vsv) that were grown in the presence of tunicamycin. with one strain of vsv (called the san juan strain), tunicamycin prevented viral replication by more than % when the virus was grown at either or °c. however, with another strain (called the orsay strain), the drug inhibited viral replication by % when the virus was grown at °c, but there was only to % inhibition at °c. furthermore, when this virus was grown in tunicamycin at °c, the nonglycosylated viral envelope protein (g protein) was detected at the host cell surface, indicating that it was synthesized and targeted in the normal fashion. however, at °c, this protein could not be detected at the cell surface. finally, studies on the physical properties of these various nonglycosylated proteins also indicated that these proteins differed markedely in their solubility in detergents such as triton x- at the different temperatures ( ) . the probable explana tion for such results is that the carbohydrate has a significant role in providing a conformation that has increased solubility as well as stability. five other antibiotics have been described that are now known to be structurally and functionally related to tunicamycin. these antibiotics are streptoviridin ( ), mycospocidin ( ), antibiotic ( ), antibiotic mm ( ) , and corynetoxin ( ) . all of these com pounds have the same general structure as tunicamycin, but they differ in the nature of the fatty acids attached to the tunicamine. thus, in some cases, they may be identical to the various tunicamycin homologs discussed earlier. for example, the streptoviridin components have shorter chain fatty acids than the tunicamycins, as evidenced by the fact that their molecular weights range from to , whereas those of tunicamycins are from to ( , ) . on the other hand, hplc comparison of the mycospocidin components with those from the tuni camycin complex showed a number of identical components ( ) . the corynetoxins are an interesting group of tunicamycin-related com pounds that appear to be involved in or responsible for ryegrass toxicity. in this case, the developing seed heads of annual ryegrass may be invaded by a nematode which then causes a gall to be produced. some of the nematodes carry the bacterium corynebacterium rathayi, which produces the tunicamycin analog corynetoxin. the corynetoxins are apparantly toxic to sheep that eat the infected ryegrass ( ) . all of these compounds appear to have the same action as tuni camycin, i.e., to inhibit the glcnac-l-p transferase. detailed studies on the mechanism of action were done with streptoviridin and this inhibitor was compared with tunicamycin on the crude and partially purified preparations of the enzyme, as well as in cell culture systems ( ) . as indicated above for the various tunicamycin homologs, this group of analogs also showed differences in inhibitory activity, and those compounds with the longer chain fatty acids were more effective inhibitors in cell culture systems than those with shorter chain fatty acids. in addition, studies with cell-free extracts or partially purified glcnac- -Ρ transferase preparations showed that the streptoviridins had a lower affinity for the enzyme than did the tunicamycin complex ( ) . in fact, with this inhibitor it was possible to demonstrate competi tive inhibition with regard to udp-glcnac, because the affinity of the glcnac-l-p transferase for streptoviridin was considerably lower than that for tunicamycin. a number of other antibiotics and other compounds have been found to be useful inhibitors of some of the steps in the lipid-linked saccharide pathway. many of these inhibitors, especially the antibiotics, were initially discovered because they block the formation of bacterial cell wall polymers such as peptidoglycan or lipopolysaccharide. because the formation of these polymers also involves polyprenyl-linked saccharide intermediates, these compounds appeared to be likely candidates to test on the n-linked glycosylation pathway. for example, the antibiotics amphomycin and tsushimycin inhibit the transferase that adds phospho-n-acetylmuramoyl-pentapeptide to the growing peptidoglycan chains ( ) , whereas bacitracin inhibits the enzyme that catalyzes the dephosphorylation of undecaprenyl-pyrophosphate ( ) , the car rier lipid in cell wall biosynthesis. these antibiotics also inhibit the formation of n-linked oligosaccharide assembly as discussed later. amphomycin is a hydrophobic undecapeptide containing either isododecanoic or -anteisododecanoic acids attached to the n-terminal aspartic acid in an amide linkage ( ) . the structure of amphomycin is presented in fig. a . in cell-free extracts of aorta tissue, amphomycin blocked the formation of dolichyl-p-mannose from gdp-mannose, but it did not affect the transfer of mannose to lipid-linked oligosaccharides from either dolichyl-p-mannose or gdp-mannose ( , ) . thus, when microsomes were incubated with gdp-[ c]mannose in the presence of enough amphomycin to completely inhibit the formation of dolichyl-p-[ c]mannose, these extracts accumulated c-man (glcnac) -ppdolichol, but they were not able to elongate this lipid to larger sized oligosaccharide-lipids. on the other hand, when dolichyl-p-[ c]mannose was used as the substrate with these extracts, mannose was transferred to oligosaccharide-lipids that contained six or more mannose residues. these studies suggested that the first five mannose residues in the lipid-linked oligosaccharides were donated from gdp-mannose, whereas the next four mannoses came from dolichyl-p-mannose ( ) . similar results were obtained with oviduct tissue ( ) and embryonic liver ( ) . the isolation of a cho mutant cell line missing the dolichyl-p-mannose synthase clearly established that doli- chyl-p-mannose was the mannosyl donor for the last four mannose residues ( ) . in cell-free extracts of brain tissue, amphomycin inhibited the pro duction of dolichyl-p-glucose, dolichyl-p-mannose, and dolichyl-pp-glcnac, leading these investigators to suggest that amphomycin might form a complex with dolichyl-p and dolichyl-p derivatives ( ) . some evidence for such a complex between dolichyl-p and amphomycin has been obtained, although it is not clear how this relates to the inhibition of lipid-linked saccharide formation. amphomycin has also been used in various other experiments, such as one study that demonstrated that dolichyl-p was involved in sugar translocation across the er membrane ( ) , and in one case to show that dolichyl-p-mannose, rather than gdp-mannose, was involved in the elongation of man (glcnac) -ppdolichol ( ) . another lipopeptide antibiotic, called tsushimycin, has a mechanism of action similar to that of amphomycin ( ) . unfortu nately, these peptide antibiotics do not appear to be effective in cell cultures of eucaryotic cells, probably because they do not cross the plasma membrane. bacitracin is another lipopeptide antibiotic that has been useful in studying both bacterial cell wall biosynthesis and the synthesis of lipidlinked saccharides. the structure of this compound is presented in fig. b . bacitracin was shown to inhibit the synthesis of bacterial cell wall peptidoglycan by inhibiting the dephosphorylation of undecaprenylpyrophosphate, which is necessary for the reutilization of this carrier molecule ( ) . in animal cells, the specific site of inhibition by this antibiotic is not clear because it has been reported to act at several different points, including some of the mannosyltransferases that add mannose to the trisaccharide-lipid man/ (glcnac) -pp-dolichol, the glcnac- -p transferase that forms glcnac-pp-dolichol, and the glcnac transferase that forms glcnac-glcnac-pp-dolichol ( ) . it seems likely that the action of this inhibitor is similar to that of ampho mycin; i.e., it may bind various dolichyl derivatives under the right conditions and therefore may interfere with a variety of reactions in the "dolichol cycle." several other antibiotics that affect the synthesis of bacterial cell walls are also effective on the reactions described here. generally, these compounds have not been well defined structurally or functionally, but some of their inhibitory actions have been reported. the structure of diumycin is not known, but it was reported to inhibit the formation of dolichyl-p-mannose in a yeast membrane preparation, as well as the transfer of mannose from dolichyl-p-mannose to serine or threonine residues ( ) . in a membrane preparation from acanthamoeba, diu mycin blocked the transfer of mannose and glcnac to lipid-linked monosaccharides, but it did not affect the synthesis of dolichyl-pglucose ( ) . in fact, diumycin (and another antibiotic, showdomycin) has been used to add support to the hypothesis that dolichyl-p-mannose is a positive regulator of the glcnac- -p transferase that forms glcnac-pp-dolichol ( ) . thus, when either of these drugs was added to a microsomal preparation, it blocked the formation of dolichyl-pmannose from gdp-mannose and therefore prevented the stimulation of glcnac-pp-dolichol formation by gdp-mannose. flavomycin is an antibiotic of the moenomycin group of compounds. this drug interferes with the formation of lipid-linked saccharides in microsomes from pig brain ( ) . unfortunately, no other studies have been reported with this compound, so additional studies will be needed before we can understand its mechanism of action. the same applies to diumycin. showdomycin is a nucleoside antibiotic whose structure is shown in fig. . this compound has a maleimide group that reacts with sulfhydryls on proteins, and this reaction is probably the basis of the show domycin inhibition of lipid-linked saccharide formation. in cell-free extracts of aorta, the synthesis of dolichyl-p-mannose and dolichyl-p-glucose were both inhibited by this drug, but the formation of dolichyl-pp-glcnac was not affected ( ) . however, in volvox extracts, the syntheses of dolichyl-p-glucose and dolichyl-pp-glcnac were equally sensitive to showdomycin in the presence of triton x- , and this inhibition was reversed by the addition of dithiothreitol ( ) . as indicated earlier, showdomycin has also been used to study the regulation of lipid-linked saccharide formation by dolichyl-p-mannose. probably two of the earliest inhibitors of viral envelope formation to be described were glucosamine ( ) and -deoxyglucose ( ) . it is now known that glucosamine interferes with the formation of lipidlinked oligosaccharides and blocks protein glycosylation. this inhibi tion, however, requires intact cells and involves the metabolism of glucosamine probably to an intermediate such as udp-glucosamine ( ) , which may compete with udp-glcnac for various glcnac transferases. when mdck cells were incubated in the presence of mm glucosamine, there was a complete shift in the composition of the lipid-linked oligosaccharides from mostly glc man (glcnac) -ppdolichol in normal cells to man _ (glcnac) -pp-dolichol in inhibited cells. this shift to smaller sized oligosaccharide-lipids became even more pronounced when the concentration of glucosamine was raised to mm ( ) . the glucosamine effect could be reversed by removal of this amino-sugar from the culture medium, but the exact site or mechanism of action of this compound is still unknown. interestingly enough, another amino-sugar, mannosamine, also in hibits the formation of lipid-linked oligosaccharides, but the mecha nism of action of this compound is quite different from that of glucos amine. the inhibition by mannosamine was dose-dependent and resulted in a shift in the size of the oligosaccharide moieties of the lipid-linked oligosaccharides from glc man (glcnac) -pp-dolichol to lipids with man (glcnac) to man (glcnac) . the major species were oligosaccharides having five or six mannose residues. these studies demonstrated that mannosamine itself was incorporated into the lipidlinked oligosaccharides, mostly in place of the mannose that is in al, linkage to the β-linked mannose. this substitution appears to prevent the addition of the al, -linked mannose to this al, -linked mannosam ine ( ) . in the presence of mannosamine, a series of basic oligosaccha rides linked to dolichol were formed, and at least some of these oligosac charides were still transferred to protein. however, it is not clear whether these altered glycopeptides can be processed by the normal pathway or they accumulate in the cells as mannosamine and mannose containing structures. in terms of glucose analogs, such as -deoxyglucose, it is now clear that this sugar is metabolized in cells and is converted to udp- deoxyglucose and gdp- -deoxyglucose, as well as to dolichyl-p- deoxyglucose. the inhibition of glycoprotein synthesis by this deoxysugar appears to be due to the gdp- -deoxymannose (glucose), because mannose itself can reverse the inhibition ( ) . probably when deoxyglucose is incorporated into the lipid-linked oligosaccharides instead of the mannose at the -branch, it acts like mannosamine and prevents further extension of this chain because additional mannoses are linked in al, -bonds. these -deoxyglucose-containing oligosaccharides ap peared not to be transferred to protein in contrast to the situation with the mannosamine-containing oligosaccharides (from the lipid-linked oligosaccharides). a number of fluoro-sugars such as -deoxy- -fluoro-d-mannose, and -deoxy- -fluoro-d-mannose have also been shown to be inhibitors of glycosylation ( ) . it seems likely that the mechanism of action of these analogs is similar to that of -deoxyglucose. some animal cells, especially chinese hamster ovary (cho) cells, show a phenomenon that has been called "glucose starvation." thus, when glucose is removed from the medium, these cells can no longer produce the typical glc man (glcnac) -pp-dolichol that is the natural oligosaccharide donor for n-linked glycosylation. instead, such cells produce a truncated lipid-linked oligosaccharide that is a man (glcnac) -pp-dolichol (i.e., manal, manal, manal, [manal, ] man/ , glcnac/ , glcnac-) and then glucosylate this intermediate to produce a glc man (glcnac) -pp-dolichol ( ) ( ) ( ) . it appears that under glucose starvation conditions, the cells are not able to synthesize dolichyl-p-mannose, but the reason for this lesion is not clear because these cells still do make dolichyl-p-glucose and appear to glucosylate the oligosaccharides. in several cell lines, the effect of glucose starva tion was seen within min after the removal of glucose, as long as the cells were at low to moderate density, but at high density no effect was seen ( ) . the glucose starvation phenomenon could be overcome by the addition of glucose, but not the addition of galactose, glutamine, pyruvate, inositol, or glycerol. in rat hepatoma cells that were starved for glucose, a lower molecular weight form of a r acid glycoprotein was produced and this decrease in molecular weight was due not only to the truncated oligosaccharides, but also to fewer oligosaccharide chains on the protein ( ) . carbonyl-cyanide m-chlorophenylhydrazone (cccp) is an uncoupler of oxidative phosphorylation that causes cells to be depleted of energy. when this compound is given to various animal cells, it causes a similar situation to that seen with glucose starvartion; i.e., cells are unable to produce dolichyl-p-mannose, although they still synthesize dolichyl-pp-glcnac and dolichyl-p-glucose. these cells form a glc man (glcnac) -pp-dolichol and transfer this oligosaccharide to protein ( ) . on the other hand, in thyroid slices incubated with cccp, man . (glcnac) -pp-dolichol intermediates were produced, and a decrease in n-linked glycosylation was observed ( ) . in this study, other inhibi tors of respiration such as n or antimycin a caused the same effects. since some uncouplers of oxidative phosphorylation have been reported to disrupt the recycling of the glucose transporter, these compounds may limit the entry of glucose into the cells, thus mimicking the glucose starvation effect. the puzzle here is the same as that mentioned earlier; i.e., why is the formation of dolichyl-p-mannose inhibited, but not that of dolichyl-p-glucose? perhaps it could have something to do with the postulation that the dolichyl-p-mannose synthase is a regulatory en zyme and the protein itself undergoes phosphorylation under some conditions. a number of naturally occurring, sugarlike compounds in which the ring oxygen is replaced by a nitrogen have been isolated, and these alkaloids have been found to be potent inhibitors of various glycosidases. apparantly this nitrogen in the ring makes these compounds inactive from a metabolic standpoint, but they are still recognized by specific glycosidases and therefore function as useful enzyme inhibitors. because the enzymes involved in the early glycoprotein processing reactions are glycosidases, these inhibitors have played a significant role in studying the sequence of reactions of n-linked oligosaccharides, as well as the requirement for glycoprotein processing in many animal cell lines. many of these inhibitors have been isolated from plant sources, but the characterization of their structures and the demonstra tion of their biological activity have stimulated the chemical synthesis of a number of other analogues. the plant inhibitors are all alkaloids and fall into various chemical classes designated piperidines, pyrroli dines, indolizidines, and pyrrolizidines ( ) . castanospermine is an indolizidine alkaloid that is found in the seeds of the australian chestnut tree, castanospermum australe ( ) . its structure is shown in fig. . this alkaloid is a potent inhibitor of βglucosidase ( ) and also inhibits the glycoprotein processing glucosidases (i and ii), as well as various other α-glucosidases such as sucrase, maltase, and lysosomal a-glucosidase ( ) . because this compound structures of various glucosidase inhibitors. castanospermine and , -dihydroxymethyl- , -dihydroxypyrrolidine are inhibitors of both glucosidase i and glucosi dase ii, whereas australine is a much better inhibitor of glucosidase i than glucosidase ii and mdl is a more potent inhibitor of glucosidase ii. lentiginosine, shown for compari son, is an inhibitor of amyloglucosidase but is ineffective against the processing glucosidases. is such a strong inhibitor of α-glucosidases (such as sucrase and maltase) and therefore prevents the normal digestion of starch and sucrose, the seeds of this plant are toxic to animals and cause severe diarrhea ( ) . when various cultured animal cells are grown or incubated in the presence of this alkaloid, the processing of n-linked glycoproteins is blocked at the first step in the pathway (see fig. ), and the asparaginelinked glycoproteins have oligosaccharides mostly of the glc man _ (glcnac) structure. another glucosidase inhibitor is the polyhydroxylated piperidine analog nojirimycin, which corresponds to glucose in the pyranose ring form (see fig. ). this compound was originally isolated from streptomyces and was found to be a potent inhibitor of a-and β-glucosidases ( ) . chemical reduction of nojirimycin gave the more stable deoxynojirimycin, which is also a glucosidase inhibitor and which has subsequently been isolated from bacteria and plants ( ) . deoxynojirimycin causes the same general effects in cell culture as does castanospermine, although it is more active toward glucosidase ii than toward glucosidase i, and it does cause other alterations such as changes in the structures of the lipid-linked saccharides ( ) . another glucosidase i inhibitor that also is found in plants is the pyrrolidine alkaloid , -dihydroxymethy - , -dihydroxypyrrolidine ( ) . this compound, although considerably less effective than the six-membered ring structures described above (e.g., compare the structure in fig. with those of castanospermine and deoxynojirimycin), causes similar changes in the structures of the n-linked oligosaccharides. the effect on glycoprotein targeting and function of preventing the removal of glucoses from the n-linked oligosaccharides is quite dra matic. for example, when hepatocytes (hep-g cells) are incubated in the presence of deoxynojirimycin, the rate of secretion of a r antitrypsin was substantially diminished, whereas the rate of secretion of other n-linked glycoproteins such as ceruloplasmin and the c component of complement was only marginally altered. based on some preliminary cell fractionation studies, it was suggested that the presence of glucose on the oligosaccharides of certain glycoproteins might retard the move ment of these glycoproteins from the er to the golgi apparatus ( ) . similar results were obtained with fibroblasts and smooth muscle cells when the effect of castanospermine on the ldl receptor was examined. in this case, there was a significant decrease in the binding and endocytosis of ldl when arterial smooth muscle cells were upregulated to produce ldl receptors in the presence of castanospermine. the reason for this decreased binding was shown to be due to a reduced number of receptors at the cell surface rather than to a change in the affinity of receptor for its ligand. furthermore, the total number of ldl recep tors in the cell did not change in the presence of castanospermine, indicating that this drug did not inhibit the synthesis of the receptor, nor did it affect its degradation. cell fractionation studies designed to isolate various membrane fractions demonstrated a shift in the distri bution of ldl receptors within the cell compartments and indicated a decrease in receptors at the cell surface and a corresponding increase in receptors in the er-golgi membrane fractions ( ) . in the case of im- lymphocytes, castanospermine was used to study the biosynthesis and processing of the insulin receptor. when these cells were treated with this alkaloid, they showed a % decrease in cell-surface insulin receptors as demonstrated by the binding of iinsulin, as well as by na i-lactoperoxidase labeling and by crosslinking studies with i-insulin. these studies demonstrated that pro cessing, i.e., removal of glucose from the n-linked oligosaccharides, was not necessary for the cleavage of the insulin proreceptor, i.e., for protein maturation. however, as indicated above, the presence of glu cose appears to slow down the transport of glycoproteins from the er to the golgi apparatus ( ) . these data suggest the presence of a glucose receptor in the er that "holds up" or binds glucose-containing glycoproteins, or the data may indicate that some proteins undergo a conformational change when glucose is removed, and the newly altered protein is transferred from the er to the golgi at a more rapid rate. in the case of synthesis of the e glycoprotein of coronavirus, casta nospermine or deoxynojirimycin caused a drop in virus formation by logs and also caused a dramatic decrease in the appearance of e at the cell surface. interestingly enough, the e that was produced in the presence of these drugs was still acylated with a fatty acid, as was the control e , but the glycoprotein accumulated intracellularly in a compartment that was distinct from the golgi apparatus ( ) . could this compartment be the er? in another case, post-translational addition of palmitic acid was also not affected by glucosidase i inhibitors. the na channel of rat brain neurons is composed of a-and β-subunits that form a complex during maturation. the α-subunit is post-translationally modified by the addi tion of a palmitic acid, and the incorporation of labeled palmitate into this glycoprotein is blocked by the glycosylation inhibitor tunicamycin. however, the α-subunit formed in the presence of castanospermine is of lower molecular weight, does not contain sialic acid, and has a lower sulfate content, but it is similar to control α-subunit in its content of palmitic acid ( ) . the drug also did not prevent the covalent assembly of a-and / -subunits, nor did it affect the biological activity of the channel with regard to its binding of saxitoxin. the external glycoprotein of the aids-associated virus hiv is called gp and is a heavily glycosylated protein that is involved in the mechanism of attachment of hiv to the cd receptor on Τ lymphocytes and other susceptible cells. the gp interacts with target molecules on susceptible cells to cause the fusion of the cells, which is referred to as the formation of syncytia. both glucosidase i inhibitors (i.e., castanospsermine and deoxynojirimycin) caused a drastic inhibition of virus replication as well as of syncytium formation ( ) ( ) ( ) . because of these interesting results, castanospermine and deoxynojirimycin, as well as other glucosidase i and ii inhibitors and analogs of these various inhibitors, are being tested as potential anti-aids compounds. as shown in fig. , there are other compounds besides castano spermine and deoxynojirimycin, that also have activity against the α-glucosidases and that function as glycoprotein processing inhibitors. one of these compounds is the pyrrolidine alkaloid mentioned earlier and referred to as , -dihydroxymethyl- , -dihydroxypyrrolidine (dmdp), which has been found in several plants of the leguminosae family. in cell culture, dmdp gives the same type of oligosaccharide structure, i.e., glc man _ (glcnac) , as does castanospermine, although it is much less active as an inhibitor (i.e., higher concentrations are required) ( ) . nevertheless, it is an interesting inhibitor because it demonstrates that a six-membered ring structure is not absolutely required for inhibitory activity, and knowledge of such structures helps us to define the particular structural requirements that are necessary for such biological activity. along these lines, two other structures that are also interesting glucosidase inhibitors, partly because they are unusual and partly because they show more selectivity than those described thus far, are shown in fig. . australine is a tetrahydroxypyrrolizidine alkaloid that was isolated from the seeds of the tree c. australe (as is castanospermine). this compound has a unique substitution pattern (i.e., (lr, r, r, s, as)- -hydroxymethyl-l, , -trihydroxypyrrolizidine). this bicyclic alkaloid is a reasonably good, competitive inhibitor of the fungal amyloglucosidase (an aryl-glucosidase) and it also inhibits the glycoprotein pro cessing enzyme glucosidase i. however, in contrast to castanospermine and deoxynojirimycin, australine is a very poor inhibitor of glucosidase ii ( ) . this compound is the first glucosidase inhibitor to distinguish between these two processing glucosidases. thus, additional compounds such as australine that show increased specificity will be valuable to help us understand how these two glucosidases differ in their mecha nism of catalysis and also to determine the chirality and spacial config urations that are necessary for activity of various glycosidases. another inhibitor whose structure is shown in fig. is , -diamino- , -imino- - -(^-d-glucopyranosyl)-d-glycero-l-guloheptitol (mdl). this compound was chemically synthesized to mimic a disaccharide that would act as a transition state analog of the intestinal enzyme sucrase ( ) . as anticipated, mdl inhibited rat intestinal maltase, sucrase, isomaltase, glucoamylase, and trehalase at micromolar con centrations. interestingly enough, when tested on the glycoprotein pro cessing glucosidases, it was the opposite of australine in its specificity. that is, mdl was a much more effective inhibitor of glucosidase ii than it was of glucosidase i ( ) . in cell culture experiments, mdl caused the accumulation of glycoproteins having mostly glc man _ (glcnac) structures. the finding that australine and mdl are more specific inhibitors than castanospermine and deoxynojirimycin will hopefully stimulate a more intense search for other naturally occurring inhibitors with improved selectivity for glucosidases and mannosidases, and will also entice chemists to use their imagination to synthesize more novel compounds. the first glycoprotein processing inhibitor to be reported was swainsonine ( ) , which is described in more detail below. this indolizidine alkaloid was initially found to be an inhibitor of lysosomal and arylmannosidases ( ) , and was later shown to specifically inhibit the processing mannosidase ii ( ) . however, for ease of understanding, i will consider the various mannosidase inhibitors in the order in which they occur in the processing pathway rather than in the order in which they were first reported. the discovery of deoxynojirimycin and castanospermine as glycopro tein processing inhibitors indicated that a good glycosidase inhibitor should possess the following properties: (a) a pyranose ring structure with a nitrogen in the ring replacing the oxygen and (b) the stereochem istry of the hydroxyl groups in keeping with those of the particular sugar that is recognized by the glycosidase of interest. thus, it was reasonable to assume that a structure similar to deoxynojirimycin but with the mannose configuration (i.e., the -epimer) should be a good inhibitor of mannosidases. the -epimer of deoxynojirimycin was syn thesized chemically and was indeed found to be a potent inhibitor of the glycoprotein processing enzyme mannosidase i ( ) . surprisingly enough, deoxymannojirimycin d d not inhibit jack bean or lysosomal α-mannosidase, nor did it inhibit che processing mannosidase ii. in fact, this inhibitor demonstrates thai, it is dangerous to screen for new inhibitors using aryl-glycosidases as the source of enzymes or even using only a few of the processing enzymes, because the most useful and desirable processing inhibitors will be specific for a particular processing glycosidase and may not inhibit these other activities. the structures of deoxymannojirimycin, swainsonine, and other known mannosidase inhibitors are shown in fig. . since the time when many of the studies on deoxymannojirimycin were done, several other neutral α-mannosidases that have different specificities and are enzymes distinct from mannosidase i or mannosidase ii have been identified. some of these enzymes have been found to be resistant to inhibition by deoxymannojirimycin, but there are not enough mannosi dase inhibitors yet known to be able to specifically inhibit some of these activities. in addition, most of these other enzymes have not yet been purified, so they have not been tested against the various processing inhibitors. in animal cells, deoxymannojirimycin inhibited the golgi mannosidase ia/b and caused the accumulation of glycoproteins hav ing high-mannose oligosaccharides, mostly of the man . (glcnac) structure ( ) . this inhibitor, however, did not prevent the secretion of igd or igm in cell culture, although the glucose analog deoxynojiri mycin did ( ) . thus the presence of glucose on the oligosaccharides does have a significant effect on the intracellular transport of at least some glycoproteins, but the presence of high-mannose oligosaccharides rather than the normal complex chains does not appear to be a problem in terms of protein secretion. deoxymannojirimycin was also without effect on the formation and appearance of the g protein of vesicular stomatitis virus, the hemagglutinin of influenza virus, and the hla a,b, and c antigens, as well as on a r aeid glycoprotein and a r protease inhibitor ( ) . deoxymannojirimycin was used in one study to determine if mem brane glycoproteins were recycled through the golgi apparatus during the endocytic process. in this experiment, membrane glycoproteins were synthesized and labeled with [ h]mannose in the presence of deoxymannojirimycin so that the glycoproteins would all have highmannose structures. then the inhibitor and labeled sugar were re moved, and the oligosaccharide structure of the transferrin receptor was determined at various times and in conditions under which this receptor would be endocytosed. the initial oligosaccharides of the trans ferrin receptor were high-mannose structures because of the presence of deoxymannojirimycin during their synthesis, but during the chase, a small percentage of the recycled receptor underwent processing and attained complex types of oligosaccharide structures, indicating that some of the endocytosed glycoprotein was recycled through the cis-golgi compartments ( ) . however, the amount of glycoprotein that actually underwent modification during the recycling was quite small, indicating that, while some glycoprotein does recycle through the golgi, this is probably not a major route. the role of endoplasmic reticulum α-mannosidase was also examined in ut- cells, a cell line that overexpresses hmg coa reductase. the hmg coa reductase resides in the er of cells and is a glycoprotein that has high mannose oligosaccharides mostly of the man (glcnac) and man (glcnac) structures. previous studies indicated that "er" α-mannosidase was not inhibited by deoxymannojirimycin. thus, in the presence of deoxymannojirimycin, the initial trimming of the oligo saccharide chains of the reductase still occurred, but in this case, the major oligosaccharide structure on the protein was a man (glcnac) and the smaller sized oligosaccharides were not detected. these studies indicated that the er α-mannosidase was involved in the removal of the first mannose, but that other α-mannosidases were necessary for the removal of other mannose residues ( ) . another mannosidase inhibitor is l, -dideoxy-l, -imino-d-mannitol (dim). this inhibitor has a furanose ring structure rather than the pyranose rings of most of the other inhibitors. dim was synthesized chemically from benzyl-a-d-mannopyranose and was found to be a good inhibitor of jack bean α-mannosidase ( ) . it also inhibited the pro cessing of n-linked glycoproteins in cell culture when it was added to the culture medium of mdck cells. in those experiments, the major oligosaccharides formed on the cell glycoproteins were man (glcnac) structures in keeping with the hypothesis that dim inhibited the golgi mannosidase i ( ) . however, it is not known whether dim also inhib its the er mannosidase, because this activity does not appear to be present in mdck cells. furthermore, there is no indication that this inhibitor has been tested against this er mannosidase or other mannosidases, although it is active against mannosidase i. thus, in vitro stud ies with dim and a partially purified preparation of mannosidase i from rat liver showed that this compound inhibits the release of [ h]mannose from [ h]mannose-labeled man glcnac. dim is not nearly as effective as a mannosidase inhibitor as is swainsonine or kifunensine. however, it is much more readily available in large amounts because the chemical synthesis is quite simple. therefore, dim and its isomers should be valuable for animal studies, as well as for other experiments requiring large amounts of mannosidase inhibitor. kifunensine is an alkaloid, produced by the actinomycete kitasatosporia kifunense and corresponding in structure to the cyclic oxamide derivative of -aminodeoxymannojirimycin ( ) . kifunensine is a very weak inhibitor of jack bean α-mannosidase (ic = . χ " m ), but it is a potent inhibitor of the glycoprotein processing mannosidase i (ic = - χ " m). thus, this inhibitor is about times more effective against this enzyme than is deoxymannojirimycin. on the other hand, kifunensine had no effect on mannosidase i ( ) . using rat liver microsomes as a source of mannosidase activities, kifunensine strongly inhibited the golgi mannosidase i but it was probably without effect on the proposed er α-mannosidase. in cell culture with mdck cells, kifunensine gave rise to glycoproteins having man (glcnac) structures just as did deoxymannojirimycin, but the amount of kifunen sine needed for this effect was only about one-fiftieth as much as deoxy mannojirimycin. recently, a broad-spectrum α-mannosidase inhibitor was synthesized chemically as a mimic of the mannopyranosyl cation, which is the putative intermediate in the hydrolysis of mannopyranosides ( ) . this compound, whose structure is shown in fig. , is called mannonolactam amidrazone. this compound was found to be a more general mannosidase inhibitor than any of the other currently known com pounds and those shown in fig. . thus, the amidrazone not only inhibits the golgi mannosidase i (ic = μΜ) and mannosidase ii (ic = - nm), but also is the first compound that has been found to be a potent inhibitor of the soluble or er α-mannosidase (ic = μΜ). in addition, the amidrazone also inhibited both the aryl-amannosidase and the aryl-/ -mannosidase, although it was much more effective on the enzyme recognizing the α-linkage (ic = η m for jack bean α-mannosidase compared with μΜ for /^-mannosidase). in cell culture studies, the amidrazone completely prevented the forma tion of complex types of oligosaccharides and instead caused the forma tion of oligosaccharides having man (glcnac) and man (glcnac) structures. it is hypothesized that the reason that this compound is so effective against all of these mannosidases is that it is the first monosaccharide analog that mimics the true half-chair conformation of the cationic intermediate that is believed to be involved in the cataly sis of the α-mannose glycosidic bond. this compound should serve as a useful model to synthesize other more specific mannosidase inhibitors. as indicated above, the first glycoprotein processing inhibitor to be identified was swainsonine. the initial interest in this compound was due to the fact that it is found in plants that are toxic to grazing animals. in australia, the major plant having this alkaloid is called swainsona canescens ( ) , while in the united states, various plants of the genus astragalus contain swainsonine ( , ) . these plants present a major hazard to the livestock industry and cause severe neurological and skeletal problems in animals that feed on them. early studies on the inhibition of aryl-a-mannosidases by swainsonine sug gested that its potent inhibitory activity (ic = χ " m) resulted from the structural similarity of the protonated form of swainsonine to that of the mannosyl cation, since the glycosyl ion intermediate is probably formed during natural hydrolysis by glycosidases ( ) . lymphnodes of sheep that had injested these toxic plants contained high levels of mannose-rich oligosaccharides ( ) , which led to the postulation that swainsonine causes a "lysosomal-like" storage disease that is similar to that of human α-mannosidosis, both morphologically and biochemically. that swainsonine is actually the causative agent of these symptoms was indicated by feeding pigs either the whole toxic plant (i.e., astragalus) or swainsonine and showing that both sets of animals exhibited the same symptoms ( ) . in these animals, liver golgi mannosidase ii activity was significantly decreased, but strangely enough, a number of lysosomal hydrolases such as amannosidase, α-fucosidase, and ^-hexosaminidase increased in activ ity. in addition, the activity of various hydrolases in the plasma was also elevated. in sheep fed locoweed, various high-mannose oligosaccharides were found in the urine and the two major oligosaccharides were identi fied as man (glcnac) and man (glcnac) ( ) . the relative abun dance of the various oligosaccharides changed over the course of the feeding, but when the feeding was discontinued, the amount of urinary oligosaccharides declined rapidly and reached a baseline within about days. similar types of high-mannose oligosaccharides also accumu lated in rats and guinea pigs fed swainsonine, but there were some differences in the structures, especially the presence of one or two glcnac residues at and near the reducing terminus ( ) . these differ ences probably reflect the presence or absence of an endoglucosaminidase in some of these animals. swainsonine was the first compound that was shown to inhibit glyco protein processing ( ) . in the initial studies, swainsonine was added to the culture medium of mdck cells and was found to cause a great decrease in the amount of [ - h]mannose incorported into complex types of glycoproteins and a corresponding increase in incorporation of labeled sugar into oligosaccharides that were sensitive to digestion by endoglucosaminidase h. in influenza-virus-infected mdck cells, swainsonine caused the viral hemagglutinin, an n-linked glycoprotein with several complex chains, to migrate more rapidly on sds gels, and these changes were shown to be due to changes in the structure of the n-linked oligosaccharides ( ) . later studies with purified processing enzymes showed that the site of action of swainsonine was the golgi mannosidase ii and that this alkaloid was inactive toward the pro cessing mannosidase i ( ) . in keeping with this site of action, this indolizidine alkaloid also caused the formation of hybrid types of struc tures when it was placed in the culture medium of cells producing various glycoproteins such as the vsv g protein ( ) , fibronectin ( ) , and bhk cell surface glycoproteins ( ) . swainsonine has been used with a number of cell culture systems to try and determine the role of specific oligosaccharide structures in glycoprotein function. in most of these studies, swainsonine was found to have little effect on the glycoprotein in question, indicating that a partial complex oligosac charide (i.e., hybrid chain) is sufficient for functional activity, at least in tissue culture. for example, swainsonine did not impair the synthesis or export of thyroglobulin in porcine thyroid cells ( ) , nor did it affect surfactant glycoprotein a in type ii epithelial cells ( ) , h -dk histocompatability antigen in macrophages ( ) , or von willebrand protein in epithelial cells ( ) . it also did not affect the targeting or function of various membrane receptors, including insulin receptor ( ) , epidermal growth factor receptor ( ) , or the receptor for asialoglycoproteins ( ) . the inhibitor did, however, block the receptormediated uptake of mannose-terminated glycoproteins by macro phages. this blockage appeared to be due to the formation of hybrid types of glycoproteins on the macrophage surface, which then reacted with and bound the mannose receptors ( ) . swainsonine proved to be useful in demonstrating the sequence of addition of the various sugars in the n-linked oligosaccharide assembly pathway. thus, when fucosylation ( ) and sulfation ( ) of the influenza viral hemaggluti nin was examined in cells grown in the presence of swainsonine or castanospermine, it was found that the hemagglutinin did contain [ h]fucose and [ s]sulfate when the virus was raised in the presence of swainsonine, but not when virus was produced in the presence of castanospermine (or deoxymannojirimycin). these studies suggest that the fucosyltransferase and the sulfotransferase recognize the glcnac-man (glcnac) structure as the appropriate acceptor sub strate. some proteins do show a loss of function when they are produced in the presence of swainsonine. thus, the glucocorticoid stimulation of resorptive cells, which appears to involve the attachment of osteoblasts and related cells to bone, is inhibited or blocked by swainsonine ( ) . this drug also reduced the interaction of tyrpanosoma cruzi with peri toneal macrophages when either the host cells or the parasites were treated with swainsonine ( ) . also a dramatic decline in the ability of b melanoma cells to colonize the lungs of experimental animals was observed when the cells were incubated in swainsonine ( ) . treatment of lymphocytes with the mitogen concanavalin a stimulates them to proliferate, but this proliferation can be blocked by an immunosuppresive factor found in the serum of mice that contain the tumor sarcoma . the suppression caused by this factor is somehow over come by swainsonine, indicating that this alkaloid might have some chemotherapeutic value in immunosuppressive diseases ( ) . the turnover or degradation of endocytosed glycoproteins is also inhibited by swainsonine and this leads to the accumulation of these glycopro teins in the lysosomes of tissue culture cells incubated with this drug. the block here appears to be due to the inhibition of lysosomal mannosi dase (and perhaps other enzymes), indicating that the carbohydrate portion of these glycoproteins must be degraded before the protein ( ) . all of these studies suggest that swainsonine may have important effects on some cell systems and therefore it or some related compounds may have some chemotherapeutic value. however, additional studies will be required with these inhibitors. another inhibitor of mannosidase ii that was recently isolated from a fungus is called mannostatin ( ) . this compound is interesting because it is the only one of these inhibitors presently known that has an exocyclic nitrogen rather than a nitrogen in the ring. in addition, as seen in fig. , mannostatin has a thiomethyl group and a fivemembered ring. mannostatin was found to be a potent inhibitor of mannosidase ii (ic = nm) and jack bean mannosidase. in cell culture studies, mannostatin gave rise to the same types of hybrid structures that are observed in cells incubated in the presence of swasinsonine ( ) . however, the n-acetylated derivative of mannostatin was inactive against any of the mannosidases. although mannostatin is not any different in its site of action, or any more active than swain sonine, it is an interesting addition to the repertoire of inhibitors be cause it shows a heretofore unanticipated structure as a glycosidase inhibitor. this opens up a number of new possibilities for chemical synthesis of related structures or perhaps structures that combine some aspects of mannostatin and some aspects of other alkaloids. a number of laboratories have been developing methods for the syn thesis of the above alkaloids or their derivatives, as well as of other new inhibitors ( ) . so far, with the exception of mannostatin, all of the inhibitors have had a nitrogen in the ring in place of the oxygen, and at least three assy metric hydroxy groups. the substitution of the ring oxygen by a nitrogen apparantly renders the compound metabolically inert but does not prevent the recognition of the compound by glycosidases or perhaps by other carbohydrate recognizing enzymes. presumably the hydroxyl groups in these inhibitors have the same chirality as the sugar that is a substrate for the glycosidase to be inhibited. interestingly enough, these inhibitors do not necessarily need to have a six-membered ring structure to mimic the catalytic intermediate that is involved in the substrate recognition or hydrolytic step. in fact, swainsonine best fits as a mannose analog when it is in a staggered or half-chair conformation ( ) , and other pyrrolidine or pyrrolizidine ring structures may also assume similar conformations. molecular modeling studies with these compounds will help to under stand the mechanism of inhibition by some of these compounds. such modeling studies have been done with some of the mannosidase inhibi tors compared with the mannopyranosyl cation as the active intermedi ate of jack bean mannosidase ( ) . however, the synthesis or isolation of other and different inhibitors of these enzymes will add to this infor mation. the synthesis of lipid-linked oligosaccharides in animal tissues or by cultured animal cells has been shown to be inhibited by several inhibitors of protein synthesis ( ) . for example, when mdck cells were grown in the presence of cycloheximide or puromycin, both in hibitors of protein synthesis but by different mechanisms, there was a dose-dependent inhibition in the incorporation of [ - h]mannose into the lipid-linked oligosaccharides. however, these inhibitors did not affect mannose incorporation into dolichyl-p-mannose, nor did they block mannose incorporation from gdp-[ c]mannose into lipid-linked saccharides in cell-free extracts prepared from mdck cells or from porcine aorta. these results could be explained by one of the following postulations: (a) limitation in the amount of dolichyl-p available to serve as a carrier of the sugars or (b) feedback inhibition in the synthesis of lipid-linked saccharides caused by the accumulation of lipid-linked oligosaccharide resulting from lack of acceptor protein. however, since the synthesis of dolichyl-p-mannose was not inhibited, the feedback mechanism seemed more likely ( ) . additional studies showed that the inhibition by cycloheximide could not be overcome by adding exogenous dolichyl-p, even though addition of dolichyl-p to normal cells did stimulate the incorporation of mannose into lipid-linked saccharides. however, the specific site of inhibition (or feedback inhibition) by these inhibitors is not known. actinomycin, an inhibitor of rna synthesis, was also found to inhibit the incorpora tion of mannose into lipid-linked oligosaccharides, but again the exact site of inhibition was not determined ( ) . based on the above results, as well as additional studies on the levels of various intermediates in normal and inhibited cells, it was postulated that the mechanism of inhibition involved a feedback type of control that was caused by ele vated levels of gtp in the cells ( ) . in n-linked glycosylation, the consensus sequence that accepts the oligosaccharide chain is h n . . . asn-x-ser (thr) . . . , where x can be any amino acid except proline or aspartic acid. this sequence is, however, not sufficient for glycosylation to occur, and the conforma tion of the protein around this glycosylation site is apparantly also critical. statistical studies on a number of asparagine-linked glycopro teins have indicated that most of the asparagine residues that do be come glycosylated are located in regions of the polypeptide chain that favor the formation of / -turns. the importance of the threonine residue in the tripeptide sequence was examined by using a threonine analog, β-hydroxynorvaline, and determining its effects on cotranslational glycosylation in extracts of ascites cells. this analog inhibited the glycosylation of the α-subunit of human chorionic gonadotropin and the β subunit of bovine leutinizing hormone. this inhibition could be overcome by adding threonine, strongly suggesting that the analog was inhibiting by competing with threonine for incorporation into protein ( ) . in cultured fibroblasts, β-hydroxynorvaline caused the formation of a variety of cathepsin d molecules that differed from each other by having one, two, or no oligosaccharide chains on the protein. the nonglycosylated form of cathepsin was a minor component and it was rapidly degraded within min of its synthesis. however, those forms having one or two oligo-saccharide chains behaved normally, were segregated to the lysosomes, and underwent proteolytic maturation in the usual fashion ( ) . simi lar types of results were observed with respect to the effects of this analog on the formation of a r acid glycoprotein ( ) . another amino acid analog that has interesting effects on glycosyla tion is threo-/ -fluoroasparagine. this analog is toxic to asparaginedependent cells when aspartic acid is included in the culture medium. the actual mechanism of toxicity has not been determined but it is believed to be due to the incorporation of the analog into protein. at mm concentrations, this analog strongly inhibits glycosylation but this inhibition is blocked by adding asparagine, further strengthening the idea that the analog is incorporated. however, the erythro-analog was not an inhibitor of glycosylation ( ) . interestingly enough, when the above two amino acid analogs (i.e., β-hydroxynorvaline and threoβ-fluoroasparagine) were used to inhibit protein synthesis, they also inhibited the incorporation of radioactive mannose into lipid-linked oligosaccharides as demonstrated with cycloheximide and puromycin ( ) . it seems likely that the toxicity of fluoroasparagine is due to its preventing the cells from glycosylating their n-linked glycoproteins in much the same manner as tunicamycin. vii. as indicated earlier, dolichyl-p serves as an obligatory carrier in the glycosylation of n-linked glycoproteins. dolichyl-p is produced by the same initial pathway as that responsible for the synthesis of polyprenol units and, therefore, one would anticipate that inhibitors of polyprenol formation should also inhibit dolichyl-p formation. fur thermore, such inhibitors should also have a potent effect on the gly cosylation of asparagine-linked glycoproteins. since the formation of polyphenols utilizes the rate-limiting step catalyzed by the enzyme hydroxymethylglutaryl-co a reductase (hmg coa reductase), inhibi tors of this enzyme such as -hydroxycholesterol and compactin (or mevanolin) should also inhibit glycosylation. when smooth muscle cells, derived from aorta, are grown in the presence of -hydroxycholesterol, the incorporation of [ c]acetate into cholesterol and into dolichyl-p is inhibited by up to %, as is n-linked glycosylation. however, mevalonate incorporation into these lipids is unaffected, as expected. mevalonate, in fact, when added to inhibited cells, can overcome the inhibition with respect to n-linked glycosylation, presumably because it allows dolichyl-p to be formed ( ) . on the other hand, in mouse l cells, hydroxycholesterol also affected the synthesis of dolichol and cholesterol, but in this system, there were conditions that gave great fluctuations in cholesterol synthesis but only slight effects on dolichyl-p formation. the authors postulated that there were other points in the pathway that must control the levels of these two lipids (i.e., cholesterol and dolichol) ( ) . cholestyramineis a compound that when fed to animals causes an increase in the activity of hmg coa reductase and a great stimulation in the incorporation of acetate into cholesterol. however, it did not increase the incorporation of acetate into dolichyl-p ( ). on the other hand, feeding animals a diet high in cholesterol would be expected to suppress cholesterol synthesis at the level of the hmg coa reductase. however, this treatment caused a significant increase in the incorporation of mevalonate into dolichol and dolichol-linked oligosaccharides as well as increased activity of some of the glycosyltransferases that are required for glycoprotein synthesis ( ) . the explanation for these data is not clear, but perhaps inhibiting the cholesterol branch of the polyprenol pathway results in the accumula tion of certain intermediates that are key players in other branches of the biosynthetic pathway. compactin is a fungal product that is a competitive inhibitor of hmg coa reductase ( ) . compactin, and a number of chemically synthe sized derivatives of this structure (i.e., mevinolin), have been studied extensively because of their enormous chemotherapeutic potential in lowering serum cholesterol levels. in one study, when rat hepatocytes were cultured in compactin for hr, the synthesis of dolichyl-p was decreased by - %, suggesting that dolichyl-p was mainly synthe sized by the de novo pathway and that the ctp-mediated phosphoryla tion of dolichol was of limited use. however, another study ( ) indi cated that the dolichol kinase and the dolichyl-p phosphatase played key roles in regulating the cellular levels of dolichyl-p. when com pactin, at concentrations of - μΜ, was fed to sea urchin embryos, it induced abnormal gastrulation. this effect appeared to be due to an inhibition in dolichyl-p formation because these embryos also showed a decreased capacity to synthesize lipid-linked oligosaccharides and glycoproteins. furthermore the inhibitory effect of compactin on devel opment could be overcome by supplementing the embryos with dolichyl-p, but the addition of cholesterol or coenzyme q could not overcome the inhibition ( ) . similar results were obtained with mouse embryos, in which compactin and an oxygenated sterol arrested development at the -cell stage and left the blastomeres decompacted. here also, nglycosylation was effected and this affect could be overcome by adding mevalonate ( ) . during their synthesis and processing, the n-linked glycoproteins are transported from the initial site of synthesis in the endoplasmic reticulum of the cell through the various golgi stacks (i.e., from cis-golgi, through medial golgi, to trans-golgi) to their final destination in the plasma membranes, in lysosomes or in other locations in the cell. while the signals that direct these proteins to specific sites are known in some cases (e.g., the mannose- -phosphate receptor for lyso somal targeting has been elegantly described ( ) ), in other cases the signals are just beginning to become known. nevertheless, a number of compounds have been identified that perturb the transport or migra tion of proteins through the various compartments of the cell. most of these pertubants are ionophores that affect the concentrations of vari ous ions in some cellular compartments and may also affect the internal ph of these compartments. for example, the secretion of immunoglobu lins by plasma cells was markedly inhibited by monensin, and striking alterations in the ultrastructural appearance of the golgi apparatus were seen ( ) . the suggestion was made that depletion of ca + levels by this ionophore rendered the golgi vesicles incapable of fusing, which resulted in a disruption in the flow of glycoproteins from the er to the golgi ( ) . the transport of other proteins, such as fibronectin and procollagen, is also inhibited by ionophores ( ) , as is the transport of various viral envelope glycoproteins ( ) . in these experiments, membrane vacuoles could be visualized by electron microscopy, and large accumulations of procollagen and fibronectin could be seen in these vacuoles by immunofluoresence. in one interesting study, monensin was used as a tool to isolate those cisternae that contained accumulations of viral capsids because they sedimented at a higher density that normal cisternae ( ) . in the presence of monensin, terminal glycosylation is inhibited, i.e., addition of complex sugars such as galactose, sialic acid, fucose, etc., but the incorporation of amino acids into protein and the hydroxylation of proline were not affected. thus, in monensin-treated cells, newly synthesized proteins accumulate in intracellular vesicles that appear to be derived from the golgi apparatus, and some of the posttranslational modifications are blocked. apparantly there exists an acidic compartment in the golgi that is necessary for the secretory process ( ) . a recently discovered and vegry useful antibiotic, called brefeldin a, also inhibits the secretion of glycoproteins in hepatocytes but does not affect protein synthesis. brefeldin inhibited the terminal glycosylation of c^-protease inhibitor and haptoglobulin, as well as the conversion of proalbumin to albumin. both of these modifications appear to occur in the trans-golgi ( ) . on the other hand, the proteolytic processing of haptoglobulin occurs in the endoplasmic reticulum and this modifi cation was not affected by brefeldin ( ) . the effect of brefeldin on the golgi complex and endoplasmic reticululm of retrovirus-transformed murine erythroleukemia (mel) cells was studied. within min of addition of this compound, most stacked cisternae were converted to vesicles that were scattered throughout the centrosphere region, and by min, the golgi complexes were completely disassembled. over the next few hours, the number of vesi cles in the golgi area decreased concomitantly with an expansion of a predominantly smooth endoplasmic reticulum that consisted of a net work of dilated tubules in continuity with regular rer cisternae, annulate lamellae, and the nuclear envelope. by electron microscopy, viral glycoproteins appeared to accumulate on these membranes and immature virions budded preferentially into the cisternal space. these findings suggest that brefeldin blocks the intracellular transport of newly synthesized cellular and viral proteins immediately distal to the distinct compartment of the er. the effects of brefeldin were completely reversible and, within a short time after removal of the drug, golgi cisternae bergan to reappear ( ) . in a cell free system, brefeldin a prevented the assembly of nonclathrin-coated vesicles from golgi cisternae. furthermore, when coated vesicle assembly is blocked, extensive tubule networks form. these connect previously separate cisternae and stacks into a single topological unit that allows the mixing of the contents of golgi cisternae ( ) . these studies are consistent with in vivo studies that showed that the earliest detectable effect of brefeldin was the displacement from the golgi stacks of a -kda protein ( , ) , and this protein was found to be identical to one of the major coat protein subunits (cops) of golgi non-clathrin-coated vesicles, β-cop ( ) . thus, a simple explanation for the effect of this interesting and very useful compound is that it acts by blocking the budding of coated transport vesicles. brefeldin a is now being widely used in cell biology and biochemistry and its use will undoubtedly provide important insight into mechanisms of protein transport and golgi assembly and disas sembly. as indicated earlier, many membrane proteins are anchored to the cell surface by a glycosylphosphatidylinositol (gpi) anchor in which the protein is linked from its carboxyl-terminal region to the glycan via a phosphoethanolamine bridge. since the glycan portion of the molecule is essential to this anchoring mechanism, inhibitors of glycan formation should be of considerable interest for determining the role of this anchoring mechanism, as well as for understanding the function of the anchored proteins. as discussed in a previous section, the manno syl residues of the anchor are donated via dolichyl-p-mannose, so inhibi tors that block the formation of this glycolipid should affect anchor formation. thus, amphomycin was effective in cell-free experiments to demonstrate that the mannosyl donor for the three mannose units of the glycan portion of the anchor was indeed dolichyl-p-mannose ( ) . the sugar analog, -fluoro- -deoxyglucose, which was previously shown to inhibit dolichyl-p-mannose formation and lipid-linked oligosaccha rides ( ) , also altered the synthesis of the gpi-anchored protein alka line phosphatase in jeg- cells and caused the accumulation of a proform of the enzyme. this proform was gradually processed into a secretory form by proteolytic removal of the carboxy-terminal hy drophobic peptide ( ) . thus, both of these studies indicated that inhibition of the synthesis of dolichyl-p-mannose can block proteinanchor assembly. another recently reported inhibitor of protein-anchor formation is mannosamine. mannosamine was previously shown to be an inhibitor of lipid-linked saccharide formation and to cause the production of truncated lipid-linked oligosaccharides in mdck cells incubated with micromolar concentrations of this compound ( ) . when mannosamine was included in the growth medium of mdck cells, it blocked the incorporation of glycosylphosphatidylinositol into gpi-anchored pro teins. thus, in polarized mdck cell cultures, this amino-sugar markedely reduced the appearance at the cell surface of a recombinant gpi-anchored protein and converted this apically oriented membrane protein to an unpolarized secretory protein. it also blocked the surface appearance of other endogenous gpi-anchored proteins. in this system, mannosamine also inhibited the incorporation of [ h]ethanolamine into gpi-anchored proteins and gpi intermediates, suggesting that this amino-sugar inhibits the assembly of the glycan portion of the anchor ( ) . the effect of mannosamine on the biosynthesis of intermediates in volved in anchor formation was also examined in mdck cell cultures by incubating these cells with [ - h]mannose in the presence of various amounts of mannosamine. this amino-sugar inhibited the incorpora tion of mannose into gpi intermediates in a dose-dependent and revers ible manner. mannosamine was shown to be incorporated into the glycan as mannosamine and probably mostly in the second mannose position. thus, it appears to block further addition of mannose because the next mannose is attached to the -hydroxyl of this second mannose. various smaller sized intermediates were detected in cells incubated in the presence of this drug. as a result, ethanolamine-p could not be added, nor could the protein be transferred to this anchor. this inhibi tion was specific for mannosamine, and other amino-sugars such as galactosamine and trehalosamine were not inhibitory ( ) . further studies with mannosamine or with other inhibitors as they become known should help us to understand the physiological significance of this anchoring mechanism. inhibitors are certainly not a panacea for all biochemical studies, and as indicated earlier there are potential problems in their utiliza tion, such as lack of specificity or interference with other key reactions. nevertheless, they can provide valuable insights into understanding specific steps in a biochemical pathway as well as in determining the physiological function of various biochemical molecules. for example, in terms of the biosynthesis of the n-linked oligosaccharides, inhibi tors of the lipid-linked oligosaccharide pathway, such as amphomycin, have helped to establish that the mannosyl donor for the last four mannose residues of the glc man (glcnac) -pp-dolichol was dolichyl-p-mannose, rather than gdp-mannose. other inhibitors of this path way, such as tunicamycin, have provided evidence to suggest that the carbohydrate portion of the asparagine-linked glycoproteins may play a number of different roles, including enhancing the stability of the glycoprotein, affecting the conformation of the protein, providing a recognition signal for targeting the protein to specific sites in the cell or to uptake from the circulation, providing a receptor molecule for cell-cell adhesion or interactions and so on. the actual role of the carbohydrate depends on the amino acid sequence of the protein in question. the second group of important inhibitors are those that affect glycosidases. these types of enzymes are widespread in nature and are in volved in many natural processes such as cell division and cell separa tion, turnover of macromolecules, n-linked glycoprotein processing, and so on. thus, inhibitors of these enzymes have great potential for use as biochemical tools or even as therapeutic agents, or they may also be lethal as shown for the glucosidase and mannosidase inhibitors castanospermine and swainsonine. again these latter compounds have gained widespread use for studies on the role of carbohydrate in nlinked glycoprotein function. however, all of these studies point out the need for additional inhibi tors as well as for structurally different inhibitors. that is, the studies with glycosidase inhibitors have shown that even the best of these compounds is still somewhat nonspecific and inhibits more than one enzyme. for example, castanospermine inhibits the processing enzymes glucosidase i and glucosidase ii, as well as lysosomal a-glucosidase, and other glucosidases. swainsonine, which is somewhat more specific, still inhibits mannosidase ii and lysosomal α-mannosidase. can we find, or synthesize, more specific inhibitors that act only on one enzyme? of course, the answer to that question is not known at this time since the mechanism of catalysis has not been demonstrated for most of these enzymes. however, even if some of these enzymes share a common catalytic mechanism, it should still be possible to narrow the site of action of these inhibitors to groups of similarly acting enzymes. since there are now several inhibitors that appear to distinguish between glucosidase i and glucosidase ii, these two enzymes must differ in their catalytic mechanism or in their active site. thus, such studies provide hope that more specificity can be obtained with the right inhibitors. how can we find better inhibitors? it seems that there are a number of approaches that could be used. one of the most logical, of course, would be to work out and understand the catalytic mechanism of each of the enzymes to be inhibited and then design inhibitors based on this knowledge. while this approach is certainly possible, it is not likely to be feasible in the immediate future. most of the enzymes involved in the biosynthesis of n-linked oligosaccharides or membrane anchors appear to be present in cells in relatively low abundance, and therefore even the purification of these enzymes to homogeneity is difficult. actually, several of these proteins have now been purified to homogeneity, and perhaps with modern approaches of molecular biol ogy, they can be cloned and produced in sufficient amounts for crystalli zation. a second approach is to design new inhibitors based on the structures of already known prototypes. in this regard, various laboratories are currently conducting modeling studies with the known inhibitors to provide the basis for developing analogs of already-known compounds. while this is a viable and logical way to develop new inhibitors, it does have one major shortcoming, which is that this approach cannot provide completely new and novel structures that could lead to even better inhibitors than those that we already have available. in that respect, perhaps we need to take a lesson from nature since evolution has had millions of years to develop all sorts of unusual chemical structures. it is unlikely that penicillin, streptomycin, or bleomycin, to name a few, could have been chemically synthesized de novo without first hav ing been isolated and characterized from natural sources. thus, the third approach to better inhibitors that should not be ignored is to look for new inhibitory compounds from natural sources, because this seems like the most likely way to find new and previously unknown structures. early events in mammalian fertilization the biogenesis of lysosomes carbohydrate specific receptors of the liver a molecular model for cell interactions lymphocyte homing: progress and prospects carbohydrate specificity of the surface lectins of e. coli, k. pneumoniae, and s. typhimurium carbohydrates as antigenic determinants of glycoproteins glycosylation engineering 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homologues by reversed high performance liquid chromatography mechanism of action of tunicamycin on the udp-glcnac:dolichyl-p glcnac-l-p transferase tunicamycin inhibits glcnac lipid formation in plants evidence for the participation of saccharide-lipids in the synthesis of the oligosaccharide chain of ovalbumin inhibition of glycoconjugate biosynthesis by tunicamycin tunicamycin, an inhibitor of bacillus peptidoglycan synthesis, a new site of inhibition an intermediate in teichoic acid biosynthesis purification and properties of the udp-glcnac:dolichyl-pyrophosphoryl-glcnac glcnac transferase from mung bean seedlings udp-af-acetylglucosamine:glycoprotein n-acetylglucosamine- -phosphotransferase: proposed enzyme for the phosphoryla tion of the high mannose oligosaccharide units of lysosomal enzyme glycosylation in the nucleus and cytoplasm the effect of tunicamycin on wallpolymer synthesis in bacilli inhibition of lipid-linked saccharide synthesis: comparison of tunicamycin, streptovirudin, and antibiotic the tunicamycins-useful tools for studies on glycoproteins amplification and molecular cloning of the hamster tunicamycin sensitive glcnac-l-p transferase gene: the hamster and yeast enzymes share a common peptide sequence effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes inhibitors of the addition, modification or processing of the oligosaccharide chains of the n-linked glycoproteins effect of tunicamycin and cycloheximide on the secretion of acid hydrolases from i-cell cultured fibroblasts studies of the mechanism of tunicamycin inhibition of iga and ige secretion by plasma cells differing requirement for glycosylation in the secretion of related glycoproteins is determined neither by the producing cell nor by the relative number of oligosaccharide units carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in cul ture post-transtional processing of the epidermal growth factor receptor effects of tuni camycin on the binding and degradation of low density lipoproteins and glycoprotein synthesis in cultured human fibroblasts effects of tunicamycin on ngf binding and neurite outgrowth in pc cells post-translational glycosylation induced activation of aglycoinsulin receptor accumulated during tunicamycin treatment intracellular movement of cell surface recep tors after endocytosis: resialylation of asialo-transferase receptor in human erythroleukemia cells glycosylation of thyroidstimulating hormone in pituitary tumor cells: influence of high mannose oligosac charides units on subunit aggregation suppression of glycoprotein formation of semliki forest, influenza, and avian sarcoma virus by tunicamycin the nonglycosylated glycopro tein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellu lar aggregation at elevated temperatures streptovirudins of series i and ii: chemical and biological properties comparison of the biological and chemical properties of tunicamycin and mycospocidin anomeric configuration of ΑΓ-acetylglucosaminyl phosphorylundecaprenols formed in bacillus cereus mem branes holomycin and an antibiotic (mm ) related to tunicamycin glycolipid toxins from parasitised annual ryegrass: a comparison with tunicamycin studies on bacterial cell wall inhibitors enzymatic activities in cultured human lymphocytes that dephosphorylate dolichyl pyrophosphate and dolichyl phosphate structure of the peptide antibi otic amphomycin stimulation of lipid-linked oligosaccharide assembly during oviduct differentiation biosynthesis of lipid-linked oligosaccha rides in embryonic liver: formation of mannose containing derivatives amphomycin: effect of the lipopeptide antibiotic and the glycosylation and extraction of dolichyl monophos phate in calf brain membranes stimulation by dolichol phosphate mannose and phospholipids of the biosynthesis of glcnac-pp-dolichol dolichyl phosphate-mediated mannosyl transfer through liposomal membranes the effect of tsushimycin on the synthesis of lipid-linked saccharides in aorta inhibition of lipid-linked saccharide synthesis by bacitracin inhibition of lipid-linked mannose and mannoprotein synthe sis in yeast by diumycin in vitro properties of a soluble polyprenyl phos phate: udp-d-af-acetylglucosamine iv-acetylglucosamine-l-phosphate transferase the effect of flavomycin on the synthesis and transfer of lipid-linked saccharides in pig brain the effect of showdomycin on glycolipid formation: inhibition of glucosyl-phosphoryl-dolichol in aorta and simulation of glycosylceramide in yeast glycolipid formation in volvox carteri f nagariensis: efects of tunicamycin and showdomycin effect of -deoxy-dglucose on the multiplication of semliki forest virus and the reversal of the block by mannose carbohy drates of influenza virus. i. glycopeptides derived from viral glycoproteins after labeling of its radioactive sugars the lipid pathway of protein glycosylation and its inhibitors the formation of lipid-linked oligosaccharides in madin-darby canine kidney cells: changes in oligosaccharide profiles induced by glucosamine formation of unusual mannosamine-containing lipid-linked oligosaccharides in mdck cells formation of -deoxyglucose-containing lipid-linked oligosaccharides: interference with glycosylation of glycoproteins mechanism of inhibition of protein glycosylation by the antiviral sugar analogue -deoxy- -fluoro-d-mannose: inhibition of synthesis of man (glcnac) -pp-dol by the guanosine diphosphate ester glucose starvation alters lipid-linked oligosaccharide biosynthesis in chinese hamster ovary cells transitory effects of glucose starvation on the synthesis of dolichol-linked oligosaccharides in mammalian cells modification of oligosaccharide-lipid synthesis and protein glyco sylation in glucose-deprived cells altered g-protein glycosylation in vesicular stomatitis virus-infected glucose-deprived baby hamster kidney cells effect of the threonine analog β-hydroxynorvaline on the glycosylation and secretion of α-acid glycoprotein by rat hepatocytes effect of energy depletion on the glycosyla tion of viral glycoproteins studies on the regulation of the biosynthesis of glucose-containing oligosaccharide-lipids: effects of energy deprivation the chemistry and biochemistry of simple indolizidine and related polyhydroxy alkaloids castanospermine, a , , , -tetrahydroxy-octahydroindolizidine alka loid from the seeds of castonospermum australe castano spermine, a tetrahydroxylated alkaloid that inhibits β-glucosidase and β-glucocerebrosidase castanospermine inhibits the processing of the oligosaccharide portion of the influenza viral hemagglutinin the effects of castanospermine and swainsonine on the activity and synthesis of intestinal sucrase new enzyme inhibitors from microorganisms glucosidaseinhibition aus bacillus inhibition of n-linked complex oligosaccharide formation by -deoxynojirimycin, an inhibitor of processing glucosidases the pyrrolidine alkaloid, , -dihydroxymethyl- , -dihydroxypyrrolidine, inhibits glycoprotein processing glucose removal from n-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from rough er to golgi complex castanospermine inhibits the function of the low density lipoprotein receptor effects of castano spermine and -deoxynojirimycin on insulin receptor biosynthesis the effects of processing inhibitors of n-linked oligosaccharides on the intracellular migration of glycoprotein e of mouse hepatitis virus and the muturation of coronavirus particles palmitylation, sulfation, and glycosyla tion of the a subunit of the sodium channel: role of post-translational modification in channel assembly inhibition of human immunodeficiency virus syncytium formation and virus replication by castano spermine interference with hiv induced syncytium formation and viral inefectivity by inhibitors of trimming glycosidases role of protein n-glycosylation in pathogenesis of human immunodeficiency virus type i australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing total synthesis of , -dideoxy- , -imino- - -/ -d-glucopyranosy-dglycero-l-gulo-heptitol hydrochloride: a potent inhibitor of α-glucosidases selective inhibition of glycoprotein processing enzymes: differential inhibition of glucosidases i and ii in cell culture swainsonine, an inhibitor of glycoprotein processing inhibition of lysosomal α-mannosidase by swainsonine, an indolizidine alkaloid isolated from swainsona canescens swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of golgi mannosidase ii synthesis of -amino- -deoxy-d-mannopyranose and l, -dideoxy-l, -imino-d-mannitol, and inhibition of α and β-d-mannosidases novel mannosidase inhibitor blocking conversion of high-mannose to complex oligosaccharides the effect of deoxymannojirimycin on the processing of influenza viral glycoproteins secretion of high-mannose-type a r proteinase inhibitor and a r acid glycoprotein by primary cultures of rat hepatocytes in the presence of the mannosidase inhibitor membrane traffic in animal cells: cellular glycoproteins return to the site of golgi mannosidase i the use of -deoxymannojirimycin to evaluate the role of various α-mannosidases in oligosaccharide processing in intact cells design, synthesis and preliminary evaluation of a potent α-mannosidase inhibitor, , -dideoxy-l, -imino-d-mannitol l, -dideoxy-l, -imino-d-mannitol inhibits glycoprotein processing structure of kifunensine, a new immunomodulator isolated from an actinomycete kifunensine, a potent inhibitor of the glycoprotein processing mannosidase i mimicking the glucosidase transition state: shape/charge considerations a spectrophotometric investigation of swainsonine, an α-mannosidase inhibitor isolated from swainsona canescens loco intoxication: indolizidine alkaloids of spotted locoweed (astragalus lentiginosus) isolation and characterization of swainsonine from texas locoweed (astragalus mollisimus) the indolizidine alkaloids, slaframine and swainsonine: con taminants in animal forages lysosomal storage in swainsona spp. toxicosis: an induced mannosidosis the similar effects of swainsonine and locoweed on tissue glycosidases and oligosaccharides of the pig indicate the alkaloid is the principle toxin responsible for the induction of locoism induced mannosidosis-excretion of oligosaccharides by locoweed-intoxicated sheep swainsonine affects the processing of glycoproteins in vivo swainsonine prevents the processing of the oligosaccharide chains of the influenza viral hemag glutinin alteration in oligosaccharide structure of vsv glycoproteins by swainsonine abnormal glycosylation of human cellular fibronectin in the presence of swainsonine properties of bhk cells treated with swainsonine, an inhibitor of glycoprotein synthesis inhibition of nlinked oligosaccharide processing does not prevent the secretion of thyroglobulin glycosylation and secretion of surfactant associated glycoprotein a n-linked oligosaccharides of the h- d k histocom patibility protein heavy chain influence its transport and cellular distribution inhibition of disulfide bonding of von willebrand protein by monensin results in small, functionally defective multimers complete glycosylation of the insulin and insulin-like growth factor i receptors is not necessary for their biosynthesis and function glycosylation of the epidermal growth factor receptor in a- cells influence of the n-linked oligosaccharides on the biosynthesis, intracellular routing and function of the hu man asialglycoprotein receptor swainsonine and castanosper mine blockade of mannose glycoprotein uptake by macrophages the effect of glycoprotein processing inhibi tors on fucosylation of glycoproteins effects of swainsonine and casta nospermine upon processing of influenza virus sulfated glycoproteins glucocorticoids modulate macrophage surface oligosaccharides and their binding activity the effect of swainsonine on the associa tion of trypanosoma cruzi with host cells oligosaccha ride modification by swainsonine treatment inhibits pulmonary colonization by b f murine melanoma cells studies of immunomodulator inhibition by swainsonine of the degradation of endocytosed glycoproteins in isolated rat liver parenchymal cells mannostatins a & b: new inhibitors of a-d-mannosidase, produced by streptoverticillium verticillus var, quintum mannostatin a, a new glycoprotein-processing inhibitor amino-sugar glycosidase inhibitors: versitile tools for glycobiologists design of potential anti-hiv agents: mannosi dase inhibitors lipid-saccharide intermediates in glycoprotein biosynthesis. i. formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation control of n-linked oligosaccharide synthesis: cellular levels of dolichyl-p are not the only regulatory factor synthesis of the n-linked oligosaccha rides of glycoproteins: assembly of the lipid-linked precursor oligosaccharide and its relation to protein synthesis in vivo relationship between oligosaccharide-lipid synthesis and protein synthesis in mouse lm cells inhibition of asparagine-linked glycosylation by incorporation of a threonine analog into nascent peptide chains enhanced degradation of cathep sin d synthesized in the presence of the threonine analog, / -hydroxynorvaline effect of the threonine analog βhydroxynorvaline on the glycosylation and secretion of a- -acid glycoprotein by rat hepatocytes incorporation of betafluoroasparagine into peptides prevents n-linked glycosylation: in vitro studies with synthetic fluoropeptides impairment of dolichyl saccharide synthesis and dolichol-mediated glycoprotein assembly in aortic smooth muscle cells in cul ture by inhibitors of cholesterol biosynthesis inter-relationships between dolichol and sterol synthesis in mammalian cell cultures studies on the regulation of glycoprotein biosynthesis: an investigation of the rate-limiting steps of dolichyl phosphate biosynthesis regulation of dolichol and of cholesterol biosynthesis in cholesterolfed rabbits receptor-mediated endocytosis: concepts emerging from the ldl receptor system inhibition of dolichyl phosphate biosynthesis by compactin in cultured rat hepatocytes inhibition of polyisoprenoid and glycopro tein biosynthesis causes abnormal embryonic development mevalonate reverses the developmental arrest of preimplantation mouse embryos by compactin, an inhibitor of hmg coa reductase the biogenesis of lysosomes plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex monovalent ionophores inhibit secretion of procollagen and fibronectin from cultued human fibroblasts perturbation of vesicular traffic with the carboxylic ionophore monesin vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores dissection of the golgi complex. ii. density separation of specific golgi functions in virally infected cells treated with monensin vesicles and cisternae in the trans golgi apparatus of human fibroblasts are acidic compartments novel blockade by brefeldin a of interacellular transport of secretory proteins in cultured rat hepatocytes the effects of brefeldin-a on the high mannose oligosaccharides of mouse thyrotropin, free α-subunits and total glycoproteins effects of brefeldin a on the golgi complex endoplasmic reticulum and viral envelope glycoproteins in murine erythroleukemia cells brefeldin a, a drug that blocks secretion, prevents the assembly of non-clathrin coated buds on golgi cisternae a microtubule binding protein associated with membranes of the golgi apparatus dissection of a kda peripheral membrane protein from the golgi apparatus is an early event in brefeldin a action a coat subunit of golgi-derived non-clathrin coated vesicles with homology to the clathrin-coated vesicle coat protein β-adaptin biosynthesis of the glycolipid membrane anchor of trypanosoma brucei variant surface glycopro teins: involvement of dol-p-man aberrant processing of alkaline phos phatase precursor caused by blocking the synthesis of gly cosy lphosphatidylinositol mannosamine, a novel inhibitor of glycosylphosphatidylinositol incorpora tion into proteins inhibition of glycosylphosphatidylinositol anchor formation by mannosamine key: cord- -so gp authors: nieto-torres, jose l.; verdiá-báguena, carmina; jimenez-guardeño, jose m.; regla-nava, jose a.; castaño-rodriguez, carlos; fernandez-delgado, raul; torres, jaume; aguilella, vicente m.; enjuanes, luis title: severe acute respiratory syndrome coronavirus e protein transports calcium ions and activates the nlrp inflammasome date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: so gp abstract severe acute respiratory syndrome coronavirus (sars-cov) envelope (e) protein is a viroporin involved in virulence. e protein ion channel (ic) activity is specifically correlated with enhanced pulmonary damage, edema accumulation and death. il- β driven proinflammation is associated with those pathological signatures, however its link to ic activity remains unknown. in this report, we demonstrate that sars-cov e protein forms protein–lipid channels in ergic/golgi membranes that are permeable to calcium ions, a highly relevant feature never reported before. calcium ions together with ph modulated e protein pore charge and selectivity. interestingly, e protein ic activity boosted the activation of the nlrp inflammasome, leading to il- β overproduction. calcium transport through the e protein ic was the main trigger of this process. these findings strikingly link sars-cov e protein ic induced ionic disturbances at the cell level to immunopathological consequences and disease worsening in the infected organism. coronaviruses (covs) cause respiratory diseases in humans ranging from common colds to fatal pneumonias perlman and netland, ) . at the end of , the etiological agent of severe acute respiratory syndrome (sars-cov) emerged in guandong province, southeast china, initiating a global epidemic. approximately people were infected by the virus, whose severe disease resulted in a % mortality rate rota et al., ) . the sars-cov epidemic was controlled by the summer of , and the virus has not naturally reemerged since (http://www.who.int). however, in a closely related cov appeared in saudi arabia causing the middle east respiratory syndrome (mers-cov) (zaki et al., ) . mers-cov induces acute pneumonia similar to that caused by sars-cov, and is sometimes accompanied with renal failure (danielsson and catchpole, ; zaki et al., ) . mers-cov is now circulating and spreading throughout the human population, and to date has infected at least people leading to death in cases. initial cases were confined to countries of the arabian peninsula; however, new extensive infection clusters have been recently reported in other regions such as south korea (http:// www.who.int). furthermore, covs similar to those causing sars, mers, and many other human and animal diseases have been detected in bat species circulating all over the globe (annan et al., ; chu et al., ; drexler et al., ; falcon et al., ; muller et al., ; quan et al., ) . bats are now considered the natural host for most covs, providing a perfect scenario for recombination events among different viral species leading to the emergence of new viruses, able to cross species barriers and cause devastating illness in other animals and humans . for this reason, development and implementation of broad spectrum treatments and general therapeutic strategies against covs are a high priority. covs are enveloped viruses containing the largest positive-sense rna genomes known, around kb, which encode the viral replicase and a set of structural proteins: spike (s), envelope (e) and membrane (m), present in the viral envelope, and nucleocapsid (n) located inside the viral particle . the s protein interacts with the cellular receptor to trigger viral entry into the host cell wong et al., ) . the e and m proteins actively contents lists available at sciencedirect journal homepage: www.elsevier.com/locate/yviro participate in viral morphogenesis (de haan et al., ; lim and liu, ; nguyen and hogue, ; ruch and machamer, ) , and the n protein coats the viral genome to form a helicoidal nucleocapsid that remains protected within the viral envelope (narayanan et al., ) . depending on the cov species, there are other accessory proteins, some structural, that are also encoded by the genome. sars-cov encodes the highest number of accessory genes ( a, , a b, a, b and b), which play diverse roles eventually related to pathogenesis . to analyze the mechanisms leading to the high virulence of sars-cov and mers-cov, several mutants affecting their different genes have been generated (almazan et al., ; dediego et al., ; dediego et al., a; scobey et al., ; yount et al., ) . remarkably, deletion of sars-cov e gene produced a virus that was attenuated in at least three different animal models and conferred protection against sars-cov challenge (dediego et al., fett et al., ; lamirande et al., ; netland et al., ) . small deletions in different domains of the e protein caused similarly attenuated viruses, and are promising vaccine candidates (regla-nava et al., ) . in a similar approach, a mers-cov missing the e gene was generated, resulting in a replicationcompetent, propagation-defective virus which may also constitute the basis of a safe attenuated vaccine (almazan et al., ) . the cov e gene encodes a small transmembrane protein highly synthesized during infection (maeda et al., ; nieto-torres et al., ; raamsman et al., ) that mainly localizes to the golgi apparatus and the endoplasmic reticulum golgi apparatus intermediate compartment (ergic), where it facilitates virus production and morphogenesis (cohen et al., ; corse and machamer, ; maeda et al., ; nal et al., ; venkatagopalan et al., ) . notably, when the e protein is present, sars-cov overstimulates the nf-κb inflammatory pathway (dediego et al., b) and, through its pdz-binding motif, interacts with the cellular protein syntenin, triggering p mapk activation . these signaling cascades result in exacerbated inflammation and immunopathology. the connection between the e protein and virulence has further encouraged the search for other e protein functions that may trigger disease worsening. one of the most striking functions displayed by the cov e protein is ion channel (ic) activity. the cov e protein self assembles in membranes forming pentameric protein-lipid pores that allow ion transport (pervushin et al., ; torres et al., ; verdia-baguena et al., ; wilson et al., wilson et al., , . interestingly, lipid head-groups are integral components of the pore and regulate ion conductance and selectivity (verdia-baguena et al., ) . the sars-cov e protein showed a mild preference for cations (na þ , k þ ) over anions (cl À ) when reconstituted in membranes mimicking the charge and composition of those of the ergic/golgi. in this scenario, the e protein ic showed no selectivity for any particular cation (verdia-baguena et al., ) , though the relevance and consequences of these ic properties in a cellular context remains unknown. alteration of ion, and specially, ca þ homeostasis in favor of infection has been already demonstrated in several viral systems. to that end a wide range of viruses encode ion conductive proteins similar to e protein, named viroporins (nieva et al., ) . highly pathogenic rna viruses such as human immunodeficiency virus- (hiv- ), hepatitis c virus (hcv), influenza a virus (iav), picornaviruses and covs, encode one or more of these proteins (nieva et al., ) . viroporins participate in several steps of the life cycle and are usually linked with pathogenesis. whether ic properties could promote pathways leading to disease worsening has been unknown for a long time. recently, it was described that point mutations that specifically inhibited sars-cov e protein ic activity caused attenuation (nieto- . mice infected with e protein ic proficient viruses presented extensive disruption of the pulmonary epithelia and edema accumulation . edema is the major determinant of acute respiratory distress syndrome (ards), the pathology induced by sars-cov, leading to death hollenhorst et al., ; matthay and zemans, ) . edema and an il- β mediated proinflammatory response was increased in the lung parenchyma when e protein ic activity was present . il- β is a potent proinflammatory cytokine crucial in resolving infectious processes; however, its overproduction has been correlated with diverse severe inflammatory diseases such as asthma, ards, gout, atherosclerosis and parkinson's (dinarello, ; dos santos et al., ; martinon et al., ; pugin et al., ; strowig et al., ) . organisms tightly control il- β production through macromolecular complexes called inflammasomes, which are mainly expressed in macrophages and dendritic cells although other cell types, such as those of the bronchiolar epithelium, synthesize their components (ichinohe et al., ; triantafilou and triantafilou, ) . one highly studied inflammasome is the nucleotide-binding oligomerization domain (nod)-like receptor pyrin domain-containing protein (nlrp ) inflammasome, relevant in the pulmonary tissue. this complex is composed of the sensing protein nlrp , the adapter component apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (asc) and the catalytically inactive procaspase- (elliott and sutterwala, ; latz et al., ) . the inflammasome components are synthesized under precise danger stimuli, such as molecular patterns associated to infections. however, a second signal is sequentially needed to induce their assembly, which triggers the inflammasome. this leads to the processing of procaspase- into active caspase- , which cleaves inactive pro-il- β into its mature form il- β that is released to the extracellular media to stimulate proinflammation (elliott and sutterwala, ; latz et al., ) . the presence of ionic imbalances within cells is a main trigger of the nlrp inflammasome assembly and activation . interestingly, several viroporins stimulate this pathway through alteration of cell ion homeostasis, frequently involving ca þ imbalances (ichinohe et al., ; ito et al., ; triantafilou et al., a; triantafilou and triantafilou, ) . there is clear evidence correlating nlrp inflammasomes and il- β driven proinflammatory cascades with worsening of several respiratory diseases, including those caused by viruses (dos santos et al., ; mcauley et al., ; pugin et al., ; triantafilou and triantafilou, ) . however, the specific role that viral proteins with ic activity may play in this process has not been fully determined. overproduction of il- β in the airways of the lungs of mice infected with e protein ic proficient sars-covs, strongly suggested that ion conductivity may be stimulating the inflammasome (nieto- . here we demonstrate that sars-cov e protein forms a ca þ permeable channel in ergic/golgi membranes. e protein ic activity alters ca þ homeostasis within cells boosting the activation of the nlrp inflammasome, which leads to the overproduction of il- β. this data supports that sars-cov e protein ca þ channel activity may play a role in disease, through overstimulation of inflammasomes leading to immunopathology. pharmacological inhibition of this pathway may constitute the basis for combined therapeutics applicable for sars-cov and other viruses. the sars-cov e protein forms ca þ permeable ion channels previously, we reported that the sars-cov e protein forms channels that are moderately selective for cations in membranes mimicking the ergic/golgi (verdia-baguena et al., , , ). within cells, the movement of different cations through the e protein pore should be dictated by their respective gradients. ca þ possesses the highest asymmetrical distribution between the er-golgi lumen (hundred μm) and the cytoplasm (around nm) (zhou et al., ) , which should allow the flow of this cation through the e protein ic channel if it is permeable. to test whether this is the case, e protein ics were first reconstituted in artificial neutral lipid membranes in order to exclude any effect coming from the lipid charge, using mm cacl solutions. current jumps corresponding to the assembly of channels in the lipid membrane were detected (fig. a) . the most frequent current jump, representing the insertion of a single e protein ic in the lipid membrane, displayed an intensity of . . pa. interestingly, values multiple of . pa were also detected, corresponding to the insertion of two ( . pa) or three channels ( . pa), reinforcing the previous observation ( fig. a and b). because lipid charge can affect channel conductance (verdia-baguena et al., , single channel conductance was analyzed in ergic/golgi-like membranes that contained approximately % negatively charged lipids. interestingly, the e protein ic showed a slightly higher unitary current jump of pa under these conditions, and multiples of this value, evidencing the insertion of additional channels, were also detected (figs. c and d). the presence of the negatively charged lipid head-groups in the channel pore may facilitate ca þ flow through the channel enhancing current intensity under these conditions. this effect was already reported for e protein and monovalent cations (verdia-baguena et al., ) . additional measurements were performed in aqueous solutions with decreasing cacl concentrations, to approach ca þ amounts found in the ergic/golgi lumen. ic conductance (g), which is the ratio between the current intensity and the applied voltage, was calculated. single channel conductance scaled almost linearly with cacl concentration in neutral membranes (fig. e ). this reflects that ion conduction inside the e protein ic is similar to the conduction of the solution, which increases as the electrolyte concentration rises. these data indicate that the interaction between the channel and the permeating ions is weak, and are in accordance with e protein forming a neutral pore in these conditions, as previously reported for monovalent cation salts (verdia-baguena et al., ) . in ergic/ golgi membranes two different regimes depending on the range of cacl concentrations were observed. a linear relation between conductance and cacl concentration was again detected in high concentrated solutions. however, ic conductance in ergic/golgi membranes was independent of cacl concentration in low concentration solutions (below . m), and higher than in neutral membranes (fig. e ). this result suggests that e protein acts as a charged protein-lipid pore in ergic/golgi membranes. in this scenario, the conductance is regulated by the balance between the ions that flow into the channel to neutralize the excess of negative charges and the bulk electrolyte concentration. collectively, these data indicate that the e protein forms channels that are highly conductive in cacl . furthermore, e protein ics worked very efficiently when reconstituted in ergic/golgi membranes under ca þ concentrations approaching those found in the lumen of the organelles (hundred μm). to specifically test e protein selectivity for ca þ , i.e. the ability of the ic to select ca þ either by its charge or by its intrinsic properties, reversal potential (erev) experiments were performed. the rational of these experiments is provided next. ics were reconstituted in lipid membranes that separated two solutions, one with high and the other with low cacl concentration. this concentration gradient induces the movement of ions through the channel to equilibrate their asymmetric distribution. the potential applied across the channel, which is required to counteract this ion movement leading to zero electric current is the erev. the erev can be transformed into channel permeability for ca þ (pca þ ) and cl À (pcl À ) by using the goldman-hodgkin-katz equation (hodgkin and katz, ) . to test whether lipid charge may have an effect on e protein, selectivity measurements were performed in neutral, ergic/golgi similar charged membranes, and fully negative-charged membranes. the e protein ic showed pca þ /pcl À values of . ( / ) in neutral membranes. the value of this permeability ratio is barely the same expected for a non-selective neutral pore just reflecting the different diffusivities of ca þ and cl À . therefore, e protein channel is equally selective for cl À and ca þ in neutral membranes. interestingly, the ratio pca þ /pcl À increased when the e protein ic was assembled in membranes containing negatively charged lipids, being . in ergic/ golgi membranes and . in negative membranes (fig. ) . these data indicated that lipid charge largely influenced the channel preference for ca þ showing that under conditions mimicking the ergic/golgi environment, e protein displayed a mild selectivity for ca þ . multivalent ions, such as ca þ , can modulate ion transport across ics by interacting with the internal charges of their pores (alcaraz et al., ; garcia-gimenez et al., ; queralt-martin et al., ) . therefore we tested whether e protein ion selectivity could be modified by the presence of a range of small cacl concentrations. ion selectivity was measured using a ten-fold concentration gradient of kcl at ph upon symmetrical addition of very small cacl concentrations (mm range), at both sides of the membrane. by applying the erev, the flow of k þ and cl À ions ca þ selectivity of sars-cov e protein channel. permeability ratios pca þ / pcl À in neutral dphpc (magenta column), ergic/golgi (blue column) or negativelycharged dphps membranes (green column). dotted line represents the permeability ratio value for a hypothetical neutral pore. values above the line represent cation selectivity, and those below correspond to anion selectivity. error bars show standard deviations. down their electrochemical potential gradient is prevented. therefore, erev measures the relative preference of the channel for k þ cations over cl À anions, but not for ca þ , because calcium concentrations are the same at both sides of the pore. to analyze the relevance of ca þ on ic selectivity, this type of experiments were performed in neutral and in negatively-charged membranes (fig. a) . in charged membranes the addition of small amounts of ca þ induced significant changes in the channel erev, whereas in neutral membranes the effect was very small, although still measurable. these results indicate that ca þ interaction with e protein channel, modifying the effective pore charge, mainly occur with the lipid charges that line the pore, rather than with acidic residues of e protein tm domain. previously we reported that ph also modulates e protein channel selectivity by protonation and deprotonation of the titratable residues present in the pore (verdia-baguena et al., ) . the effect of ca þ and ph on e protein selectivity was simultaneously tested. erev titration was studied in the presence or absence of mm cacl under different ph conditions (fig. b) . in negatively-charged lipid membranes, rising ph induced sequential deprotonation of lipid head-groups and e protein glutamic acid residues as their respective pk a ( . for lipid head-groups and . for glutamic acid) were overtaken, conferring an excess of negative charges to the channel and making it cation selective. the addition of ca þ when e protein is reconstituted in charged membranes reduces the cationic selectivity or increases the anionic selectivity (i.e., it shifts the reversal potential towards less negative values), and reversal potential values become closer to those obtained in neutral membranes. this indicates that ca þ ions interact with the acidic protein residues and the negative lipid head-groups and decrease the effective negative charge of the e protein pore. this effect was especially patent at physiological values of ph (ph - . ). in addition, this interaction of ca þ ions with the protein acidic residues shifts their effective pk a towards lower values. these results indicate that both ca þ and h þ ions change e channel transport properties by modifying the charge of the pore, and further support that ca þ enters within e protein ic. in order to assess the biological impact of sars-cov e protein with and without ca þ channel activity, we evaluated previously constructed mutants containing amino acids substitutions known to affect the e protein ic. previously we demonstrated that mutations n a and v f in the transmembrane domain of sars-cov e protein abolished ion conductance in kcl and nacl solutions (verdia-baguena et al., ) . new conductance measurements were performed in ergic/golgi membranes in mm cacl solutions (fig. ) . wildtype e protein transmembrane peptides showed conductance values in the range of ps, whereas no conductance was observed for n a or v f mutants, indicating that these mutations also inhibited ca þ transport, and probably prevent all ion passage as it was previously demonstrated that they also failed to transport k þ , na þ and cl À (verdia-baguena et al., ). ca þ transport through sars-cov e protein channel activates the nlrp inflammasome alteration of cellular ion homeostasis by sars-cov e protein ic could have several implications. previously, we linked e protein ic with il- β triggered proinflammation in the lungs of mice, leading to epithelial cell damage and death . higher levels of mature il- β were detected in the airways of infected animals when e protein ic activity was present, suggesting that ion conductance could stimulate the inflammasome. to determine whether this was the case, the inflammasome complex was reconstituted in vero e cells by transient transfection of its components (nlrp , asc and procaspase- ) and the inactive pro-il- β (lo et al., ) , in the absence or presence of e protein, with or without ic activity. all the components of the inflammasome, besides the different versions of e protein were efficiently expressed within cells (fig. a) . when pro-il- β was transfected as a control, no significant level of active il- β was detected in the supernatant. however, when all the components were supplied to the cells, the inflammasome was stimulated, and active il- β was detected in the media in the range of pg/ml (fig. b) . interestingly, the production of il- β was significantly enhanced in the presence of e protein with ic activity (ic þ ) ( pg/ml). this stimulation was ic activity dependent, as the e protein mutants lacking ion conductance (n a and v f), represented in the figure as eic À and eic À , respectively, did not boost il- β levels (fig. b ). stimulation levels found with wildtype e protein were in the range of those previously reported for other inflammasome activating proteins, using this system (lo et al., ) . imbalances in ca þ within cells have been described as an inflammasome inducer (ito et al., ; murakami et al., ; triantafilou et al., b) . to test the specific contribution of sars-cov e protein ca þ transport to inflammasome activation, the complex was reconstituted in cells in the presence or absence of e protein. cells were subsequently treated with the cell permeant calcium chelator bapta-am (fig. a ). this compound enters cells and binds ca þ preventing inflammasome activation (ito et al., ) . increasing amounts of bapta-am markedly decreased the levels of secreted il- β in the presence of e protein (fig. a , infl eic þ ), reaching levels close to those obtained when the inflammasome was assembled alone (fig. a, infl) . the ca þ ionophore ionomycin stimulated active il- β accumulation in the cell supernatant at levels similar to those seen with e protein, further confirming that ca þ imbalances boost il- β production (fig. b) . no significant differences in cell viability were found for any of the treatments (fig. c) . collectively, these results indicate that sars-cov e protein activates the nlrp inflammasome through its ca þ transport ability. ion conductive proteins are widely distributed among viruses, being especially common in rna viruses (nieva et al., ) . conductance of ions facilitate diverse processes of the viral life cycle such as entry, takeover of organelles to serve as platforms for viral replication, protection of viral proteins from acidic cell compartments, and trafficking of nascent virions (nieva et al., ) . in addition, viroporins are often linked to pathogenesis, and in general, mutant viruses lacking them are attenuated, in many cases serving as effective vaccines (dediego et al., ; netland et al., ; watanabe et al., ; whitehead et al., ) . viroporin removal is frequently accompanied by a defect in viral production, which by itself can explain virulence attenuation. however, recent studies have indicated that ic activity may specifically trigger pathways leading to pathology. previously we demonstrated that sars-covs proficient in e protein ic activity caused increased damage of the pulmonary epithelia and edema accumulation . these disease symptoms correlated with an immunopathological response mediated by proinflammatory cytokines such as il- , tnf and il- β, the latter being a crucial mediator of this cascade. il- β overproduction is linked to a wide range of inflammatory pathologies including those caused by respiratory viruses (dos santos et al., ; mcauley et al., ; pugin et al., ; triantafilou and triantafilou, ) . here, we report for the first time that cov e protein formed an ic that transported ca þ in ergic/golgi membranes, where this protein locates, which may have important consequences on cell physiology. in fact, ca þ leakage through e protein ic induced the activation of the nlrp inflammasome resulting in overproduction of il- β. this finding together with previous in vivo observations indicates that ic activity correlates with proinflammation and pathology. generally, viroporins form poorly selective ion channels (nieva et al., ) . therefore, the subcellular location where viroporins assemble and the conditions of that particular environment are crucial determinants of their impact on cellular ionic homeostasis. previously, we reported that sars-cov e protein showed mild selectivity for cations (na þ and k þ ) when reconstituted in ergic/ golgi membranes, mostly conferred by the negative charges of the lipids (verdia-baguena et al., . high concentration gradients are found for na þ and k þ between the cell interior and the extracellular media (dubyak, ) . however, there is no known asymmetric distribution for either of these ion species between the lumen of ergic/golgi and the cell cytoplasm (chandra et al., ; schapiro and grinstein, ) . accordingly, minimal net transport of na þ and k þ through e protein pore should be expected, and therefore the biological relevance of these processes may be also limited. in contrast, the er and golgi apparatus store high amounts of calcium ions by the action of pumps such as the sarcoendoplasmic reticulum ca þ atpase (serca) and the secretory pathway ca þ atpase (spca) (wuytack et al., ) . this creates an enormous gradient of around -fold between the lumen of these organelles and the cytoplasm (zhou et al., ) . the gradient allows controlled eventual and temporal leakages of ca þ into the cytoplasm that trigger several processes relevant to cell physiology. interestingly, we have shown above that sars-cov e protein ic was also permeable to ca þ in ergic/golgi membranes. furthermore, ca þ interacted with the negative charges of the protein-lipid pore, modulating its properties. several other viroporins are known to transport ca þ , resulting in leakage of this cation from its intracellular stores. rotavirus nsp protein as well as coxsackievirus, encephalomyocarditis virus, and poliovirus b proteins deplete er and/or golgi ca þ concentrations in favor of viral proliferation (campanella et al., ; crawford et al., ; de jong et al., ) . alteration of protein trafficking, manipulation of apoptosis, and control of autophagy are some of the processes controlled by these ca þ effluxes. whether some of these aspects are influenced in a similar manner during sars-cov infection will be explored in future experiments. protons (h þ ) are also actively confined to the lumen of the golgi apparatus and those of the organelles of the secretory pathway in a process that acidifies their interior and creates a gradient with the cytoplasm (paroutis et al., ) . considering that the e protein ic weakly interacts with circulating ions and that ph can modulate its net charge, it is highly likely that protons will also flow through the ic within cells. this is not an isolated case, as several viroporins such as hcv p and iav m are known to transport protons, and others such as the b protein of the picornaviridae family transport both h þ and ca þ (de jong et al., ; wang et al., ; wozniak et al., ) . alkalinization of the golgi lumen is crucial to protect acid-sensitive viral progeny and prevent premature activation of viral proteins involved in entry processes (sakaguchi et al., ; wozniak et al., ) . sars-cov e protein ic mutants did not show profound growth defects, although they were outgrown in competition assays by ic proficient viruses, which suggests better proliferation when e protein ion conductance was present . whether alkalization of intracellular compartments by e protein ic may assist in sars-cov production remains to be explored. besides these considerations, it cannot be excluded that ic activity may have a greater impact in sars-cov production. inhibition of e protein ic activity may be compensated by the action of two other viroporins encoded by sars-cov, the a and a (chen et al., ; lu et al., ) ; further experiments are being performed to answer this question. disruption of ion homeostasis can have profound deleterious effects for the cells; consequently, they have evolved mechanisms to sense and control these disturbances. ion imbalances associated with infecting pathogens are recognized by inflammasomes, which are components of the innate immune system. inflammasomes orchestrate proinflammatory responses to fight viral infections, il- β being one of the major players (strowig et al., ) . however, increasing evidence indicates that overstimulation of this pathway can lead to undesirable effects for the organism. in fact, immunopathology rather than direct viral destruction of infected cells is the main cause of severe disease in many viral illnesses (mcauley et al., ; meduri et al., ; triantafilou and triantafilou, ) . sars-covs that lack e protein ic activity induced less proinflammation and active il- β, suggesting that ic activity could be a trigger of this pathway . indeed, wildtype e protein but not its ic À mutants boosted the production of mature il- β through the mediation of a reconstituted inflammasome. ca þ was the main trigger of this process, as chelation of this ion abolished enhanced il- β production, and a ca þ ionophore produced a similar simulation. e proteins from other human respiratory covs, such as mers-cov and hcov- e, also display ion channel properties (surya et al., ; verdia-baguena et al., ; wilson et al., ) . investigating whether these proteins may favor ca þ transport and activation of the inflammasome represents a relevant issue. furthermore, the identification of a possible correlation between the extent of inflammasome activation and the disease symptoms caused by these viruses, highly deleterious in the case of sars-cov and mers-cov, and mild in the case of hcov- e, could provide key information on the impact of this pathway on the pathological outcome induced by different covs, and will be analyzed in future experiments. this study provides new insights into the molecular mechanisms governing cov viroporin activity and the consequences of this function in viral pathogenesis. what is more, il- β overproduction has been related with the pathology induced by sars-cov and other respiratory viruses (triantafilou and triantafilou, ) . our results suggest that development of specific ic inhibitors and implementation of novel compounds decreasing inflammasome driven immunopathology (coll et al., ) may be a valuable complement to other antiviral approaches for the treatment of these infectious diseases. the african green monkey kidney-derived vero e cells were kindly provided by eric snijder (medical center, university of leiden, the netherlands). cells were grown at c with an atmosphere of % humidity and % co , in dulbecco's modified eagle medium (dmem, gibco) supplemented with mm hepes, mm l-glutamine (sigma), % non-essential amino acids (sigma) and % fetal bovine serum (fbs, biowhittaker). synthetic peptides representing the full-length sars-cov e protein, or its transmembrane domain (amino acids - ) containing point mutations that inhibited ion channel activity (n a and v f), were generated by standard phase synthesis and purified by hplc, as previously described (verdia-baguena et al., ) . ion channels were reconstituted in lipid membranes composed of diphytanoyl phosphatidylcholine (dphpc), diphytanoyl phosphatidylserine (dphps), or a mixture of % dioleoyl phosphatidylcholine (dopc), % dioleoyl phosphatidylethanolamine (dope), and % dioleoyl phosphatidylserine (dops) (avanti polar lipids, inc., alabaster, al). the membranes separated two chambers filled with electrolytic solutions of cacl or kcl. ion channel insertion was achieved by adding . - μl of a μg/ml solution of synthetic protein or peptides in a acetonitrile:isopropanol ( : ) buffer to one of the chambers (cis). all measurements were performed at room temperature ( c). the single-channel conductance was obtained from current measurements under an applied potential of þ mv in symmetrical salt solutions of m cacl buffered with mm hepes at ph . the conductance values were evaluated using the gaussian fit tool of sigma plot . (systat software, inc). for the selectivity measurements the reversal potential (erev) was obtained under a ten-fold salt concentration gradient ( mm / mm). one or several channels were inserted into the bilayer causing a net ionic current due to the concentration gradient. then the ionic current was manually set to zero by adjusting the applied potential. the potential needed to achieve zero current represents the erev; conversion of erev into the channel permeability (p þ /p À ) was by done using the goldman-hodgkin-katz (ghk) equation (hodgkin and katz, ) . the effect of ca þ on e protein ion selectivity was defined by measuring erev in a ten-fold kcl concentration gradient ( mm/ mm) buffered with mm hepes at ph , in the presence of increasing concentrations of cacl . the functional interaction of ca þ with e protein-lipid pore was evaluated measuring erev in dphpc and dphps membranes under different ph values, with or without mm cacl . sub-confluent monolayers of vero e cells seeded onto -well plates were transfected using lipofectamine (invitrogen) and a set of plasmids encoding the components of the nlrp inflammasome (lo et al., ; wu et al., ) : ng of pcdna -proil- β, ng of pcdna -nlrp -ha, ng pcdna -asc-mcherry, and ng of pcdna -procaspase- -myc, with ng of empty pcdna , as a control, or ng of either pcdna -e, pcdna -en a or pcdna -ev f. cell lysates and culture supernatants were collected h after transfection. the secreted active il- β was measured by elisa according to the manufacturer's specifications (ebioscience). cells were lysed in a buffer containing tris/hcl mm, edta mm, nacl mm, igepal %, and complete protease inhibitor (roche) at ph . inflammasome proteins were detected by western blot using a mouse anti-ha antibody (sigma) to detect nrlp -ha, mouse anti-mcherry (abcam) to bind asc-mcherry, rabbit anti-myc (abcam) to label procaspase- -myc, and rabbit anti-human il- β (cell signaling). sars-cov e protein was detected using a polyclonal antiserum produced in rabbits . as a loading control, beta-actin was labeled using a mouse monoclonal antibody (abcam). bound antibodies were detected using hrp-conjugated goat anti-rabbit igg and rabbit anti-mouse igg and the immobilon western chemiluminicesce substrate (millipore), following manufacturer's specifications. fresh supplemented dmem containing the indicated concentration of the cell permeant calcium chelator bapta-am (life technologies) was added to cells h after transfection of the inflammasome components and incubated min at c. then, media was removed and a second treatment with fresh media containing bapta-am was performed. supernatants were collected h post-bapta-am treatment. ionomycin (life technologies) was added at μm h after transfection and incubated for h. the mtt colorimetric method was used to measure cell metabolic activity. cell media of transfected cells was replaced by fresh dmem supplemented with % fcs and μg/ml of mtt. cells were incubated h at c. 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technical assistance. key: cord- -che iwu authors: hart, kristen c; donoghue, daniel j title: derivatives of activated h-ras lacking c-terminal lipid modifications retain transforming ability if targeted to the correct subcellular location date: - - journal: oncogene doi: . /sj.onc. sha: doc_id: cord_uid: che iwu to examine the ability of ras to activate signal transduction pathways in the absence of lipid modifications, fusion proteins were constructed that target ras(wt) or activated ras( l) to cellular membranes as integral membrane proteins, using the first transmembrane domain of the e protein of avian infectious bronchitis virus (ibv), which contains a cis-golgi targeting signal. golgi-targeted derivatives of activated ras were completely inactive in transformation assays. however, when examined in focus formation assays, transformation of nih t cells were seen with derivatives of ras( l) containing a mutated e targeting sequence that results in plasma membrane localization. removal of the lipid modification sites in and upstream of the caax motif did not abrogate the transforming activity of plasma membrane-localized ras( l) derivatives, indicating that these lipid modifications are not essential for ras activity, as long as the protein is correctly localized to the plasma membrane. interestingly, the activity of integral membrane versions of ras( l) was strictly dependent on a minimum distance between the transmembrane domain anchor region and the coding sequence of ras. derivatives with only a -amino acid linker were inactive, while linkers of either - or -amino acids were sufficient to restore transforming activity. these results demonstrate that: ( ) activated ras targeted to golgi membranes is unable to cause transformation; ( ) lipid modifications at the c-terminus are not required for the transforming activity of plasma membrane-anchored ras( l) derivatives, and serve primarily a targeting function; ( ) a transmembrane domain can effectively substitute for c-terminal modifications that would normally target ras to the inner surface of the plasma membrane, indicating that ras( l) does not need to reversibly dissociate from the membrane as might be allowed by the normal lipidation; and ( ) in order to function properly, there exists a critical distance that the ras protein must reside from the plasma membrane. introduction ras proteins are critical modulators of signaling pathways involved in cell growth and dierentiation. p ras is a member of a large family of small proteins that act as molecular`switches', cycling between an active gtp-bound state and an inactive gdp-bound state . ras is activated in response to a wide variety of stimuli, including platelet-derived growth factor (pdgf). binding of pdgf to its receptors induces their dimerization and transphosphorylation, creating binding sites for eector molecules (reviewed in hart et al., ) , some of which regulate or activate ras. activation of ras by growth factors recruits raf- to the plasma membrane (stokoe et al., ; leevers et al., ) , leading to raf- activation and subsequent initiation of the mapk cascade. ras also has been shown to activate rac and rho proteins, which control signaling pathways that lead to cytoskeletal modi®cations khosravi-far et al., ; qiu et al., a,b) . activation of this pathway has recently been shown to be crucial for full transformation by ras (prendergast et al., ; khosravi-far et al., ; qiu et al., a,b) . thus, combined activation of the rac/rho and mapk pathways may constitute the entire intracellular response necessary for transformation by activated ras. there are multiple p ras proteins, including n-ras, k-ras, and h-ras (reviewed in santos and nebreda, ; lowy and willumsen, ) , which are localized to the inner surface of the plasma membrane (willingham et al., ; sefton et al., ) through speci®c sequences in the c-terminus (willumsen et al., ) . the mechanism by which ras proteins are targeted to the plasma membrane has been the subject of intense research. since ras is frequently mutated in tumors, interference with ras localization could become an important means of treating some forms of cancer. post-translational modi®cations of ras proteins occur at the c-terminus, which includes a domain known as the`caax' box, where a is an aliphatic amino acid, and x represents any amino acid. processing of this region consists of farnesylation of the conserved cys residue four amino acids from the c-terminus (cys in h-ras and n-ras), cleavage of the last three amino acids of the protein, and carboxymethylation of the new c-terminal cys jackson et al., ; casey et al., ; gutierrez et al., ; clarke et al., ; lowy and willumsen, ; shih and weeks, ; willumsen et al., ; sefton et al., ) . n-ras and hras have cysteines n-terminal of the caax motif, which become palmitoylated after the caax processing (buss and sefton, ) . in the case of k-ras- b, the c-terminus contains a polybasic domain in addition to the caax motif which contributes to plasma membrane localization (hancock et al., (hancock et al., , jackson et al., ) . the palmitoylation reactions are reversible, allowing for potentially dynamic localization of ras proteins (magee et al., ) . although truncation of the c-terminus does not aect the inherent structure or biochemical properties of ras , mutations in these motifs render ras proteins cytosolic and inactive, implying that lipid modi®cations are important for ras function. there is recent evidence suggesting that the lipid modi®cations of ras are also necessary for activation of raf- and b-raf (okada et al., ; kikuchi and williams, ) . however, since these lipid modifications are also responsible for targeting ras to the plasma membrane, it is unclear from these recent reports whether the lipid modi®cations per se are a necessity, or just localization of ras to the plasma membrane. the palmitoylation reactions that occur in the c-terminus of ras proteins have been shown to be reversible (magee et al., ) . there is also some speculation that ras may require reversible association with membranes, and the lipid modi®cations in the cterminus provide the potential for this dynamic localization. however, the experimental methods used to date are unable to conclusively distinguish the precise role of lipid modi®cations in the membrane anchoring and activation of ras. in order to examine these questions, we have used a novel method of targeting ras to the plasma membrane by creating fusion proteins using the transmembrane targeting signal from the e protein of avian ibv placed at the n-terminus (swift and machamer, ) . in its wild-type form, this transmembrane domain is capable of targeting heterologous proteins to cis-golgi membranes. when the point mutation gln ?ile is present, proteins are instead targeted to the plasma membrane. these fusions allow us to address the question of whether ras must be able to reversibly associate with the plasma membrane for biological function as assayed by transformation and gtpase activity. to examine whether c-terminal modi®cations of ras are required for functions other than targeting, we created similar fusion proteins that contain mutations which abolish processing of the caax box. results presented here demonstrate that ras l can transform ®broblasts if targeted to the plasma membrane by an n-terminal transmembrane domain, yet is not active when localized to golgi membranes. we also show that integral membrane versions of ras l require a minimum distance from the membrane in order to activate signal transduction pathways leading to transformation. this work describes the ®rst derivatives of ras l that lack any lipid modi®cations, and demonstrate that cterminal lipid modi®cations are not required for transformation by ras l . to examine the possibility that activated h-ras can transform if targeted to membranes by an n-terminal transmembrane anchor, a variety of fusion proteins were designed. as illustrated in the left side of figure , ras wt and ras l proteins are localized at the inner surface of the plasma membrane by means of lipid modi®cations at the c-terminus. the right side of figure depicts the orientation and design of the transmembrane-anchored ras derivatives. for derivatives containing the e cis-golgi targeting signal, the transmembrane domain anchors the fusion proteins at golgi membranes as type i integral membrane proteins, with the ras-derived portion of the protein still in the cytosol. these derivatives allowed us to examine whether ras could function if localized to any membranous environment, thereby assessing the importance of the interaction between ras and the plasma membrane. fusion proteins were also designed with a mutated e (qi) targeting signal and are expected to be transported to the plasma membrane, with the nterminus outside the cell, and the ras portion of the proteins on the inner surface of the plasma membrane. figure represents all of the fusion proteins designed for this study. the feasibility of targeting ras to the plasma membrane by the n-terminus was previously demonstrated in work by buss et al. ( ) and lacal et al. ( ) , which examined the eect of nterminal myristylation on ras l activity. buss et al. ( ) described a naturally-occurring myristylated derivative of ras, p gag-ras, containing a -amino linker region between the myristylated gly and the ras coding sequence. lacal and colleagues generated nterminally anchored ras derivatives by attaching the amino acid myristylation signal from pp c-src. our derivatives dier in that ras is anchored permanently to membranes as an integral membrane protein. the myristylation targeting signal used in previous studies has the potential to allow reversible association of ras with the plasma membrane. this is an important distinction, which allows us to examine whether ras needs to be able to come o of the membrane in order to function. by exploiting the e localization signal, we were also able to target ras speci®cally to dierent membranes in the cell. we were also able to examine in more detail the minimum distance from the membrane that ras requires for activity, by designing constructs with linker regions of either , or amino acids. additional fusion proteins were designed to examine the general requirement for lipid modi®cations for ras activity. ras derivatives with -or -amino acid linkers figure schematic of ras fusion proteins. all fusion proteins have either the normal c-terminus (amino acids ± ) of ras wt or ras l (cmsckcvls), or contain saax (cmscksvls) or s (smssksvls) mutations, which alter or abolish the normal post-translational processing and lipid modi®cations of the proteins. the n-terminus of each e derivative is oriented either into the lumen of the golgi, or is extracellular if the point mutation q?i is present. the whole of the ras protein is always cytosolic. the transmembrane domain (designated by the shaded box) is either the cis-golgi targeting signal contained in the ®rst transmembrane domain of the ibv e protein, or a mutated version (q ?i) that targets the protein to the plasma membrane. the linker region between the transmembrane domain and the ras protein denoted by the black box is either -, -, or -amino acids in length ras anchored as an integral membrane protein kc hart and dj donoghue were constructed in the context of the unmutated (caax) c-terminus, as well as the saax and s cterminal mutations.`caax' refers to the wild-type cterminal sequences of ras.`saax' indicates a mutation of cys in the caax motif to ser, which abolishes the farnesylation, cleavage, and carboxymethylation reactions.` s' refers to mutation of cys , , and to ser, which in addition destroys the palmitoylation sites . the -amino acid linker derivatives were only made in the context of the saax mutant c-terminus. since mutation of all three c-terminal cys residues results in a ras protein completely unable to be modi®ed by lipid moieties, these mutants allowed us to assay whether these lipid modi®cations are necessary for activity, or just utilized in a membrane targeting capacity. ras derivatives are unable to cause transformation if targeted to golgi membranes when tested in focus forming assays, golgi-targeted derivatives of ras wt with -, -, or -amino acid figure localization and transforming activity of ras derivatives. bars depict the ras wt and ras l constructs tested in this paper. the following abbreviations are used to describe the dierent derivatives.` l' refers to mutation of codon of ras from gln to leu, which oncogenically activates the protein (sekiya et al., ) .`e ' refers to the ®rst transmembrane domain (amino acids ± ) of the e protein of avian ibv, which constitutes a cis-golgi targeting signal that can target heterologous proteins to the golgi (swift and machamer, ) .`e (qi)' indicates a point mutation in the transmembrane domain (gln ?ile) that abrogates its golgi-targeting function, targeting proteins instead to the plasma membrane (swift and machamer, ) . shaded boxes indicate the e or e (qi) transmembrane domain; black boxes indicate the linker region; striped boxes indicate the c-termini of the clones which is either wild-type (caax) or mutant (saax or s). localization was determined by indirect immuno¯uorescence, as described in the legend for figure . transforming activity of the clones was analyzed in a focus forming assay, where nih t cells were transfected with the plasmids encoding the fusion proteins, and examined for foci after approximately ± days. each experiment was repeated at least three times for transforming derivatives, and at least two times for non-transforming constructs. results are expressed as an average of all experiments performed with each given construct. = ± % of ras l ; ++= ± % of ras l ; +++= ± % of ras l ; ++++= ± % of ras l transforming activity linker regions were all inactive. this was expected, since ras wt did not induce focus formation in our assays. the golgi-targeted derivatives of ras l were also unable to cause transformation. this was irrespective of whether the linker region was -, -, or -amino acids in length, or whether the c-terminal post-translational modi®cation signals were intact or not. figure e and i demonstrate localization of e - ras l -saax and e - -ras l -saax, respectively, to a perinuclear region typical of golgi staining. panel k shows the localization of e - -ras l -caax, and figure l shows that this fusion protein colocalizes with mab e (see arrows), which speci®cally reacts with a cis-golgi epitope, and has been used as a marker for the early golgi (hart et al., ; wood et al., ) . this con®rms that the golgi-targeted fusion proteins are localized correctly, and therefore we conclude that ras l cannot initiate transforming signal transduction pathways from golgi membranes, indicating that attachment to any lipid-®lled environment is not sucient for ras to be active. ras l derivatives with -or -amino acid linkers can transform if anchored to the plasma membrane, irrespective of c-terminal lipid modi®cations the plasma membrane-targeted derivatives of ras l with the -amino acid linkers (e (qi)- -ras l -caax, e (qi)- -ras l -saax, and e (qi)- -ras l - s) were all inactive in focus formation assays. however, derivatives with -or -amino acid linker regions were very active in transformation of nih t cells. the results of these assays are summarized in figure , which indicates the focus forming activity of each ras derivative as an average of at least three independent experiments. e (qi)- -ras l -caax, e (qi)- -ras l -saax, e (qi)- -ras l - s and e (qi)- -ras l - focus forming assays of ras derivatives targeted to the plasma membrane as integral membrane proteins. plasmids encoding ras fusion proteins were transfected into nih t cells, and foci were counted after ± days. cells were ®xed with methanol, stained with giemsa, and photographed. ras anchored as an integral membrane protein kc hart and dj donoghue saax exhibited signi®cant transforming activity in comparison with ras l (see figure e ± i). it is of interest to note that mutations in the c-terminus of ras l that abolish the lipidation of the proteins do not aect the ability of activated ras to transform nih t cells. indeed, the derivative with an intact c-terminus was consistently three-to four-fold less ecient at transformation than derivatives with mutated ctermini (see figure ; also compare figure e , f and g). this could re¯ect conformational strains conferred on the protein due to anchoring both termini to the membrane. plasma membrane-targeted derivatives containing -, -, or -amino acid linkers were also examined by immuno¯uorescence. as seen in figure , derivatives that were positive in transformation assays localize to the plasma membrane (f, g, h, and j), and exhibit staining patterns comparable to that of ras l (b). also, derivatives with -amino acid linkers were still targeted to the plasma membrane (d), although unable to transform cells. thus, derivatives of ras l targeted to the plasma membrane via an n-terminal transmembrane domain are able to cause transformation, provided that there is a¯exible linker region of sucient length between the transmembrane anchor and the n-terminus of ras. these results also clearly demonstrate that ras l does not require lipid modi®cations in order to transform cells, and suggests that the c-terminal processing of ras proteins primarily functions in a membraneanchoring capacity. in order to verify that the fusion proteins being expressed were of the correct size, proteins were immunoprecipitated from transiently transfected cells ( figure ) . ras proteins typically run as a doublet on sds ± page gels, one band representing the unprocessed form of ras, the other indicating the mature, processed form of the protein (see arrows) (shih et al., , . the saax and s mutations prevent the c-terminal cleavage step and subsequent lipid mod-i®cations from occurring, leaving only a single band (lanes and , arrows) . the subtleties of these processing steps are dicult to detect in the remaining fusion proteins shown in figure , due to their increased molecular weight. however, it is quite apparent that all fusion proteins do express the additional n-terminal transmembrane domain sequences (lanes ± ), and these targeting domains are not cleaved o during maturation of the proteins. the fusion proteins migrate slower than figure indirect immuno¯uorescence con®rms localization of ras derivatives. nih t cells were transiently transfected with various ras constructs, ®xed, permeabilized, and incubated with rat mab ab- (clone y - ) and¯uorescein-conjugated goat arat secondary antibody. for (l) mab e was used to detect the cis-golgi, and visualized with rhodamine-conjugated goat amouse secondary antibody. (a) mock; (b) ras l -caax; (c) e - -ras wt - s; (d) e (qi)- -ras l - s; (e) e - -ras l -saax; (f) e (qi)- -ras l -caax; (g) e (qi)- -ras l -saax; (h) e (qi)- -ras l - s; (i) e - -ras l -saax; (j) e (qi)- -ras l -saax; (k) e - -ras l -caax -a-ras antibody (¯uorescein); (l) e - -ras l -caax -mab e a-cis-golgi antibody (rhodamine). arrows indicate caax -mab in k and l expected, which may be due to the presence of an nlinked glycosylation site in the extreme n-terminus of the e -derived sequence (machamer et al., ) . the transforming ability of some of the plasma membrane-targeted derivatives of ras l indicates that their function was not adversely aected by addition of an n-terminal transmembrane domain. to analyse whether fusion proteins that were non-transforming were still able to function in some manner, gtpase activity was examined. ras l remains locked in the gtp-bound state longer due to a decreased rate of gtp hydrolysis (temeles et al., ) , despite a -fold increased binding anity for rasgap (krengel et al., ) . if the n-terminal transmembrane domain does not interfere with normal protein function, one would expect to see a higher gtpase activity exhibited by ras wt fusion proteins, as compared to ras l derivatives. using thin-layer chromatography, this dierence in gtpase activities is apparent, as shown by a higher proportion of labeled gdp versus gtp in ras wt samples as compared to ras l samples ( figure , lanes and ) . interestingly, ras l with the s mutation in the cterminus, which renders the protein cytosolic and inactive in transformation, does not aect the gtpase activity (lane ). derivatives of ras wt with the -amino acid linker hydrolyze gtp, irrespective of whether they are targeted to the plasma membrane (lane ) or golgi membranes (lane ). ras l derivatives with transmembrane anchors and -amino acid linkers exhibit impaired gtpase activity as expected (data not shown). finally, the -and -amino acid derivatives of ras wt (lanes and ) or ras l (lanes and ) exhibit the expected gtpase activity pro®les, regardless of the transmembrane domain attached at the n-terminus or the modi®cations present at the c-terminus. these results demonstrate that ras proteins can be targeted to the plasma membrane via a transmembrane anchor without aecting the expected gtpase activity of the proteins. also, we ®nd that c-terminal lipid modifications do not play a role in maintaining the intrinsic gtpase activity of wild-type or activated ras. the experiments described above demonstrate that ras l can activate transforming signal transduction pathways when targeted by the n-terminus to the plasma membrane as an integral membrane protein. this activity was completely independent of the normal post-translational processing that occurs at the c-terminus of ras proteins. since ras l derivatives (e (qi)- -ras l -saax, e (qi)- -ras l - s and e (qi)- -ras l -saax) lacking the c-terminal sequences required for normal processing of ras are still able to cause transformation, it is clear that lipid modi®cations per se are not required for activity of oncogenic ras. this is the ®rst demonstration of nonlipidated transforming derivatives of ras. our data suggests that post-translational modification of h-ras serves primarily to target the protein to the plasma membrane. whether there are other, more subtle functions of these c-terminal lipid modi®cations note that all ras wt derivatives have a higher gtpase activity (lanes , , , and ) compared to ras l derivatives (lanes , , and ), as evidenced by increased amount of labeled gdp versus gtp in ras wt samples. this result is irrespective of localization to golgi or plasma membranes, and regardless of c-terminal modi®cations. lane : ras wt -caax; lane : ras l -caax; lane : ras l - s; lane : e - -ras wt - s; lane : e (qi)- -ras wt -saax; lane : e - -ras wt -saax; lane : e (qi)- -ras l - s; lane : e - -ras wt -saax; lane : e (qi)- -ras l -saax ras anchored as an integral membrane protein kc hart and dj donoghue distinct from their more obvious targeting function remains to be demonstrated. the observation that three of the transmembrane-anchored derivatives, e (qi)- -ras l -saax, e (qi)- -ras l - s, and e (qi)- -ras l -saax, retain transforming activity despite their inability to be lipid-modi®ed strongly implies that downstream signaling molecules such as raf- and the mapk cascade are activated in these cells. this contradicts recent reports that posttranslational modi®cations of ras are required for activation of raf- and b-raf (okada et al., ; kikuchi and williams, ). the precise activation state of eectors downstream from ras, and whether these molecules are activated in a similar fashion by integral membrane versions of ras, is currently under investigation. transforming activity of transmembrane derivatives of ras demonstrates that ras does not require transient or reversible association with the membrane if the lipid modi®cations on ras proteins serve only a targeting function, as indicated by our results, this raises the question of why ras proteins evolved to contain c-terminal lipid modi®cations instead of a more permanent transmembrane anchor. small gtpbinding proteins related to ras are now known to function in a variety of capacities within the cell, such as mediating vesicular transport in the process of endocytosis, sorting and tracking through the secretory pathway, and regulating the structure of the cytoskeleton (reviewed in hall, ; ferro-novick and novick, ; pfeer, ; zerial and stenmark, ) . some of these processes may require the regulatory gtp-binding proteins to be transiently or reversibly associated with lipid bilayers, and only lipid modi®cations would allow this dynamic association to occur. our results suggest, in the case of ras, that a reversible association with the plasma membrane is not required for activation of transforming signal transduction pathways, since integral membrane versions of ras l are transforming. it would be interesting to examine the eects of anchoring other small gtp-binding proteins more permanently to speci®c membranes or compartments using targeting sequences such as the transmembrane domain from the e protein. ras proteins require a minimum spacing from the plasma membrane in order to function transforming activity of ras l derivatives was dependent upon a minimum distance from the plasma membrane mediated by the length of the linker region. altering the distance between ras and the plasma membrane does aect the ability of ras to activate signal transduction pathways, as demonstrated by the inactivity of -amino acid linker derivatives of ras l and activation of these derivatives in transformation assays by addition of -or -amino acid linker regions. the crystal structure of c-h-ras bound to gdp (residues ± ) or gtp (residues ± ) indicates that the extreme c-terminus forms an alpha-helical region that juts out from the globular catalytic region of ras (de vos et al., ; pai et al., ) , suggesting that the catalytic domain is well-removed from the membrane in normal ras proteins. one can imagine that the native c-terminus of ras constitutes a natural`linker' region, not unlike the arti®cial linker regions incorporated into our ras fusion proteins. this would explain the failure of the short amino acid linker at the n-terminus to allow for a functional fusion protein, whereas linkers of -or amino acids, corresponding more closely in length to the c-terminus of normal ras, do result in a transforming ras l derivative. conformational energy analysis performed on the c-terminal amino acids excluded from the crystal structure of the gdp-bound form of ras indicates that this region likely forms a helix ± turn ± helix or helical hairpin' motif, allowing the n-terminus and c-terminus of ras to be in close proximity (brandt-rauf et al., ) . perhaps these two regions of the protein interact in some manner. however, it appears from our results that interactions between the nterminus and lipid modi®cations at the c-terminus are not important for ras function. there could be one or more eectors of ras that bind to a site dierent from the identi®ed eector domain, and access to this site may be dependent upon orientation of the protein with respect to the membrane, or may require binding to sites at both the n-and cterminus of ras proteins. identi®cation of other membrane-associated factors involved in regulation or activation of ras proteins and their eectors will provide clues as to why ras proteins evolved their unique structural elements. clearly the intrinsic gtpase activities of our fusion proteins are not altered when compared to normal versions of ras wt and ras l . gtp binding and hydrolysis are also apparently unaected by mutation of the caax box. thus, it appears that neither the n-nor the c-terminus are important for this function of ras. speci®c amino acids in the eector regions of ras (amino acids ± or switch i, and amino acids ± or switch ii) are involved in association with guanine nucleotides and also in binding to regulators of gtpase and nucleotide exchange activities. since gtpase activity is normal in the fusion proteins, the overall structure of the catalytic regions is not expected to be drastically altered. to our knowledge, this is the ®rst study examining the role of c-terminal lipid modi®cations in regulating gtpase activity of ras. one discrepancy between our results and those of buss et al. ( ) is that anchoring of wild-type ras to the plasma membrane by the e (qi) transmembrane domain does not result in transformation. buss et al. ( ) found signi®cant focus forming ability of wildtype ras after addition of n-terminal myristylation. they speculated that perhaps their protein was binding to inappropriate cellular targets, which may not be promoted by a transmembrane anchor. it is also possible that interaction with nucleotide exchange factors was enhanced in myristylated ras wt derivatives, but this does not occur in e (qi)-ras wt constructs. to further clarify the role of membrane association in regulating the inherent enzymatic activity of ras proteins, it would be interesting to see if there are any subtle eects on the association of integral membrane versions of ras proteins with regulators of gtpase and nucleotide exchange activities. the observation that golgi-targeted derivatives of ras are unable to transform ®broblasts, while perhaps not unexpected, provides some insight into the requirements for signal transduction through the ras pathway. it is clear that some mediators of the transforming pathway initiated by oncogenic ras are only available at the plasma membrane. precisely what these mediators are remains to be elucidated. however, one can conclude that simple juxtaposition of ras to a lipidrich environment is not sucient to allow it to signal. examination of these golgi-targeted ras derivatives and their ability to interact with or activate the normal cytosolic substrates such as raf- will provide future insights into the function of ras. recent evidence demonstrates that other important pathways regulating cytoskeletal structure and organization are also activated by ras. rac regulates membrane ruing in response to growth factor stimulation , while rhoa functions in the regulation of actin stress ®ber and focal adhesion assembly in growth factor-stimulated cells ). it appears that activation of both the mapk and rac/rho pathways is required for full transformation by ras (prendergast et al., ; khosravi-far et al., ; qiu et al., a,b) . in light of this recent evidence, we are currently investigating activation of the rac/rho pathway in response to our integral membrane versions of ras. full-length harvey p ras wt and p ras l cloned into pcdnai at the hindiii-ecori sites were generously provided by j buss. restriction sites in the coding sequences of ras wt and ras l used to make these clones are as follows: bsahi at nt and fspi at nt were used in generation of clones with e or e (qi) transmembrane anchors; a¯iii at nt was utilized to generate mutations in the c-terminus. note that the mutation resulting in the gln?leu at codon occurs at nt , and consists of a a?t transversion. all constructs containing n-terminal transmembrane domains and/or c-terminal mutations described in this paper were constructed using the following strategy. pairs of complementary oligonucleotides were designed and synthesized such that, when annealed, overhangs for restriction sites were formed. more details about the making of the various fusion constructs are available upon request. all synthetic oligonucleotides were gel puri®ed as previously described (xu et al., ) , and all dna sequences derived from oligonucleotides were con®rmed by dideoxy nucleotide sequencing before use. nih t cells were maintained as previously described (hart et al., ) . cells were split at a density of cells per mm plate and transfected the following day using the calcium phosphate precipitation protocol (chen and okayama, ) . cells from each mm plate were split : days later, and scored for foci ± days later. these assays were repeated at least for each construct. nih t cells were split onto mm plates containing glass coverslips and transfected the following day with mg of plasmid dna, as described above. two days after transfection, the cells were ®xed with % paraformaldehyde/pbs and permeabilized with . % triton x- /pbs. the intracellular location of ras fusion proteins was detected with rat monoclonal antiserum ab- (y - ) directed against v-h-ras (oncogene science) and uorescein-conjugated goat a-rat secondary antibody (boehringer mannheim). for double-labeling experiments, cells were ®xed and permeabilized as described above. ras fusion proteins were detected as described above, then the coverslips were treated with monoclonal ab e , which detects the cis-golgi of cells (kindly provided by wj brown and v malhotra) and rhodamine-conjugated goat a-mouse secondary antibody (boehringer mannheim). nih t cells were split onto mm plates and days after transfection, monolayers were washed with tris-saline, incubated min with dme minus cys and met, and then labeled for h with mci each of [ s]-cys and [ s]-met. cells were lysed in ripa ( % triton x- , . m nacl, mm tris-hcl ph . , . % sds, % doc, mg/ml aprotinin), precleared with protein a-sepharose, and incubated with ab- rat monoclonal antibody. immunoprecipitates were collected with protein a-sepharose beads coated with rabbit a-rat igg, washed with ripa, and resuspended in sample buer ( mm tris ph . , % sds, % -mercaptoethanol, % glycerol). proteins were separated by % sds ± page and detected by¯uorography. nih t cells were transfected as described for immunoprecipitation. two days after transfection, monolayers were lysed with lysis buer ( mm tris-hcl ph . , . m nacl, mm mgcl , mm cacl , % triton x- , mg/ml aprotinin) and precleared with protein a-sepharose. one third of the total lysate was then subjected to immunoprecipitation using ab- . immune complexes were collected on protein a-sepharose beads coated with rabbit a-rat igg, washed with lysis buer, with ripa, then incubated for min on ice with ml of . m a-[ p]gtp in ripa. beads were then washed with ripa, with lysis buer, and incubated at c for h in ml of lysis buer. ml of each sample was spotted onto pei-cellulose plates (jt baker, phillipsburg, new jersey) and chromatographed in . m kh po , ph . . abbreviations e , avian coronavirus e glycoprotein; er, endoplasmic reticulum; ibv, infectious bronchitis virus. melanie webster and patricia d'avis for critical reading of the manuscript, and laura castrejon for editorial assistance. kch would like to thank scott robertson, ryan dellinger, and vincent ollendor for advice and support. kch gratefully acknowledges support from the lucille p markey charitable trust. this work was supported by grant rt- from the u.c. tobacco related disease research program and by grant cb- from the american cancer society. proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa, proc. natl. acad. sci. usa proc. natl. acad. sci. usa, proc. natl. acad. sci. usa cancer investigation rab gtpases in vesicular transport we thank jan buss for providing the p ras wt and p ras l plasmids that served as the starting point for construction of all clones described in this paper, and for helpful advice on gtpase assays. we thank wj brown and v malhotra for the mab e used for co-localization studies. we also thank april meyer for technical support, ras anchored as an integral membrane protein kc hart and dj donoghue key: cord- -q rsfzgw authors: lavi, ehud; wang, qian; weiss, susan r.; gonatas, nicholas k. title: syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus date: - - journal: virology doi: . /viro. . sha: doc_id: cord_uid: q rsfzgw abstract coronavirus mouse hepatitis virus (mhv) possesses a membrane glycoprotein (m) which is targeted to the golgi apparatus (ga). we used immunocytochemistry with an organelle-specific antiserum to investigate the morphologic changes of the ga during infection of l murine fibroblasts with mhv-a . twenty-four hours after infection the ga was fragmented and translocated in the center of syncytia, while the microtubular network was also rearranged displaying radiating elements toward the center of syncytia. two fusion-defective mutants, which contain an identical amino acid substitution in the cleavage signal sequence of the spike glycoprotein (s), induced fragmentation of the ga. however, the ga migrated only partially to the centers of syncytia during infection with these mutants. revertant viruses, in which the above mutation was corrected, had fusion properties and ga staining similar to wtmhv-a . experiments with brefeldin a (bfa), which induces redistribution of the ga into the rough endoplasmic reticulum (rer), revealed that an intact ga for a period of – hr postinfection, is required for coronavirus replication and syncytia formation. thus, during mhv infection, syncytia formation is associated with fragmentation of the ga, followed by a previously undescribed phenomenon of migration of the organelle into the centers of syncytia. the fragmentation of the ga, however, may occur without the formation of syncytia. therefore, two distinct mechanisms may be responsible for the fragmentation of the ga and its subsequent migration to the center of syncytia. undergo endocytosis into the trans-golgi-network (tgn), while shiga toxin is internalized into the ga and the rer polypeptides synthesized in the rough endoplasmic (sandvig et al., ; . pretreatment of cells reticulum (rer) and destined for plasma membranes, with bfa protects them against the lethal effects of cerlysosomes, and secretion are transported through the tain toxins, implying that the toxin's entry into tgn and golgi apparatus for posttranslational modifications and the ga is necessary for translocation into their cytosolic targeting (farquhar and palade, ; mellman and sitargets (yoshida et al., ) . in the human disease mons, ; rothman and orci, ) . the ga is a dyamyotrophic lateral sclerosis (als), the ga of spinal cord namic organelle which undergoes morphologic and funcmotor neurons is fragmented into numerous small eletional modifications under physiologic and pathologic ments which resemble the dispersion of the organelle conditions. in mitotic hela cells the ga disperses in early induced by agents depolymerizing microtubules (moureprophase and reaggregates in telophase (robbins and latos et al., , ; gonatas et al., ) . a similar gonatas, ) . during mitosis, the golgi apparatus fragfragmentation of the ga has been observed in motor ments into numerous small groups of vesicles which neurons of transgenic mice expressing a mutant cu,zn have been referred to as the mitotic form of the organelle superoxide dismutase (mourelatos et al., ) . (lucocq et al., ) . in interphase cells treated with migration and rearrangements of the ga have been drugs which depolymerize microtubules, the ga fragreported during syncytia formation and fusion of vero ments into small randomly distributed elements (robbins cells infected with sindbis virus (ho et al., ) . within and gonatas, ; turner and tartakoff, ) . in many - hr after infection, individual elements of the ga, cells the secretion blocker brefeldin a (bfa) reversibly which are associated initially with separate microtubuleredistributes membranes and enzymes of the ga back organizing centers in perinuclear areas of fused cells, into the rer, but does not inhibit endocytosis (doms et congregate in the center of syncytia and form an exal., ; lippincott-schwartz et al., ; johnston et al., tended network of nondisrupted intact golgi complexes ). ricin, cholera toxin, and wheat germ agglutinin (ho et al., ) . in contrast to sindbis infections, the golgi apparatus is fragmented in cells infected with herpes simplex virus (campadelli et al., ) . is characterized by retention of the viral envelope glyco- ). the primary structure of mg- revealed significant homology with a chicken fibroblast growth factor protein m (previously known as e ) within the ga (tooze et al., ; klumperman et al., ; krijnse-locker et receptor and a ligand to e-selectin (gonatas et al., ; steegmaier et al., ) . in chicken, mg- appears al., ) and budding of virions from internal membranes (holmes and behnke, ; tooze et al., ) . the close early in development and the gene coding the protein, named glg , has been assigned to chromosome interaction between coronavirus particles with the ga provides an unusual opportunity to study morphologic (stieber et al., ; mourelatos et al., ) . and functional properties of the ga and various aspects of virus-cell interactions. in this study, we used organ-immunohistochemistry elle-specific antibodies, immunohistochemistry, and transmission electron microscopy to examine the fate of cells, grown on poly-d-lysine-treated coverslips, were the ga during cell fusion and syncytia formation in mouse fixed with % paraformaldehyde for min at room tem-l- cells infected with mhv-a and fusion defective perature, washed three times in pbs, then incubated for mutants. fragmentation of the ga into small immuno- min in . % saponin/ % goat serum (gs) in pbs stained elements occurred prior to their migration into and washed three times in % gs in pbs. cells were the centers of syncytia. experiments with bfa revealed then incubated with primary antibody ( : dilution in that the period of - hr postinfection is critical for pbs of immunoaffinity-purified rabbit anti-mg- anticoronavirus replication and syncytia formation. the rebodies or with a supernatant from the anti mg- hysults of studies with fusion defective mutants suggest bridoma ( a ) overnight at room temperature. cultures that different mechanisms are responsible for the initial were then washed, incubated with a biotinylated goat fragmentation of the ga and its subsequent migration to anti-rabbit igg antibody, incubated with the avidin-biotin the centers of syncytia. complex (abc), and stained with diaminobenzidine tetrahydrochloride (dab) ( mg dab/ ml tris-saline con-materials and methods taining mm imidazole and . % h o ), according to standard methods (graham and karnovsky, ; gues-cell cultures don et al., ) . l- cells (murine fibroblasts), originally obtained from american type culture collection (atcc; rockville, md), electron microscopy were used to maintain viral growth cultures, preparation of viral stocks, and viral plaque assays. in some experi-cells grown on thermanox plastic (em sciences, fort ments cl- murine fibroblasts were used. the cultures washington, pa), were fixed overnight at Њ with . % were maintained in dmem/heat-inactivated % fetal boglutaraldehyde / % paraformaldehyde in . % cacodylvine serum/ % pen/strep/ mg/liter d-glucose/l-gluate buffer, ph . , / . % cacl . subsequently cultures tamine. were postfixed in % osmium tetroxide / . % potassium ferrocyanide, dehydrated in ethanol, and embedded in viruses and infections araldite. sections ( - nm thick) were stained with lead and uranyl salts and viewed in a transmission electron three times plaque purified mhv-a stock virus was microscope (jeol cx) at kv (karnovsky, ; used as previously described (lavi and weiss, ) . stieber et al., ) . fusion defective mutants and revertants were prepared as previously described (gombold et al., ; hingley et al., ) . infections of cultures were done by incuba-viral infectivity assay tion of virus with cells for hr at a multiplicity of infection (m.o.i.) of , followed by washing of cells with fresh me-viral titers were determined by duplicate plaque dium three times. assays of several -fold dilution of samples in l- cell grown in six-well plates (lavi et al., ) . mg- is a conserved sialoglycoprotein of the medial brefeldin a (bfa) treatment cisternae of the ga. the preparations of the anti-mg- monoclonal antibody ( a ), and the immunoaffinity bfa (sigma) stock solution ( mg/ml in ethanol) was diluted in pbs and applied to cultures at a concentration purified anti-mg- polyclonal antibodies, were described in previous publications (gonatas et al., ; of mg/ml for periods of hr or longer, as specifically indicated in the text (fig. ) . in control experiments cul- croul et al., croul et al., , . the preparation of the monoclonal antibody h , a rer marker of a -to -kda tures were incubated for the same time periods with the same dilution of ethanol used to dissolve bfa. protein, has been previously described (chen et al., and cyncytia formation when infected with mhv-a . following infection, cells were fixed and examined by immu-changes of the ga during mhv infection and nohistochemistry with identical methods used for l formation of syncytia cells. the changes in the ga observed in these cells were identical to those seen in l -infected cells. morphological changes of the ga during coronavirus infection of l cells ( , , , , and hr) were examined by immunocytochemistry using organelle-specific changes of the ga following infection of l cells with antibodies (croul et al., (croul et al., , gonatas et al., ) . fusion-defective mutants and a fusion-negative strain typical infection of l cells with mhv-a caused syncyof mhv tia formation of the entire monolayer within hr and complete cytolysis within hr. at hr postinfection, to investigate whether the fragmentation of the ga is dependent on cell fusion, we examined the organelle in cell borders were indistinct and the nuclei were aggregated; by hr postinfection, the syncytia acquired their l cells infected with two fusion defective mutants of mhv-a . the c and b mutants, used in these ex-typical morphology consisting of a ring of nuclei surrounding a cytoplasmic center devoid of nuclei. between periments, were isolated from primary cultures of glial cells infected with mhv-a . these mutants are fusion- and hr postinfection, cells underwent pyknosis, karyorhexis, and then died and detached from the culture delayed and defective but not fusion-negative and produce reduced number of syncytia formations. thus at dish. the immunostained ga of uninfected cells formed con-any given time point postinfection with the mutant viruses, only a small percentage of l cells form syncytia, tiguous coarsely granular focal or ring-like perinuclear profiles (figs. a and d). following infection of l cells as compared to cells infected with mhv-a (gombold et al., ) . these fusion-defective mutants have a histi-with mhv-a , the coarsely granular stain of the ga became fine and smaller individual elements of the ga dine to aspartic acid mutation (h d) within the cleavage signal of the spike (s) glycoprotein. cleavage of s were discernible. at hr postinfection, the majority of the syncytia consisted of clustered nuclei within a cyto-is necessary for efficient cell fusion during mhv-a infection (gombold et al., ) . infection with these mutant plasmic mass, lacking distinguishable cell borders; in those syncytia, the immunostained ga appeared as viruses does not affect the efficiency of viral replication or titers of infectious virus (gombold et al., ) . immu-strands of finely granular elements forming a honeycomb-like network with interspersed nuclei (figs. b and nostaining of the ga in l cultures infected with the fusion defective mutants b and c showed fragmen- e). however, before full syncytia formation, in cells surrounding the forming syncytia the distribution of the ga tation of the ga similar to that found in cells infected with mhv-a . however, the translocation and rear-was perinuclear, similar to that of controls but with finer granular ga elements (compare figs. e and d) . this rangement of the ga during infection with these two mutant viruses was distinctly different from the changes phenomenon probably represented an early stage of fragmentation and rearrangement of the ga. at hr of the organelle seen during infections with the wild-type mhv-a . specifically, cells infected with either b or postinfection, the process of fragmentation of the ga and its relocation in the centers of syncytia, as well as the c mutants displayed both central and peripheral aggregates of finely granular ga (figs. h and i). the rearrangement of the nuclei in a ring formation within the syncytia, was complete (figs. c and f) . the honey-granular stain of the ga at the periphery of syncytia, and specifically around the peripherally located nuclei, was comb morphology due to the interspersed nuclei was replaced by a typical central aggregate of finely granular observed only in cultures infected with these two mutant viruses. this pattern of ga distribution was not seen in or fragmented ga, surrounded by a rim of nuclei (compare figs. f with e). furthermore, the nuclei in the any of the stages of mhv-a viral replication (compare figs. i and f ). in order to rule out the possibility that syncytium were not surrounded by any residual ga. thus, in the fully developed syncytium, all elements of the frag-the fusion-defective variants showed only delay in translocation of the ga into the center of cyncytia, immunocy-mented ga had moved into the center, while the more peripherally arranged nuclei formed a ring devoid of adja-tochemical analysis was performed at and hr after viral infection. these experiments showed that the typical cent elements of the ga (fig. f) . staining of syncytia with rabbit anti-mhv polyclonal antibodies revealed an complete central translocation of the ga, similar to that seen in a -infected cultures, never occurred in cultures abundance of viral proteins in the center of syncytia (not shown). infected with the two fusion defective mutants, even prior to or at the stage of complete cell death. in order to determine whether the fragmentation and rearrangement of the ga was cell-type dependent, an-to further explore the link between fragmentation of the ga and fusion we then infected l cells with mhv-other cell line, cl- murine fibroblasts, was infected with mhv-a . this cell line supports both cell fusion , a fusion-negative strain of mhv. l cells are suscepti- the network of microtubules is rearranged during mhv-a infection pletely destroyed after hr with titers similar to mhv-a . however, there was no cell fusion or syncytia forma-since the ga is associated with microtubules of tion in these cultures. infection with mhv- produced interphase cells (robbins and gonatas, ; turner dispersion and fragmentation of the ga in individual and tartakoff, ) , we investigated whether microtucells, some of which appeared to be balooned after bules are affected during coronavirus infection. spehr (fig. g) . these observations are consistent with the cifically, we investigated whether the fragmentation of conclusion that the fragmentation of the ga during infecthe ga within the centers of the syncytia is associated tion with mhv is independent of cell fusion. however, with a similar change of the microtubules. the immuthe translocation of the ga in the center of the syncytia nocytochemical staining of the ga at , , , , and is probably linked to cell fusion since fusion-defective hr after mhv-a infection was compared with the mutants were also defective in their ability to induce immunostaining of microtubules with antibodies translocation of the ga to the center of the syncytia. against alpha and beta tubulin. while the ga appeared fragmented early during infection and syncytia forma-ultrastructural changes of the ga in mhv-infected tion, fragmentation and disintegration of microtubules cells occurred late ( hr), when cells die. the kinetics of to further investigate the morphologic aspects of the the microtubule changes after infection with mhv-a fragmented ga in syncytia of l cells infected with mhvwas depicted by immunofluorescence on l- cells a , an electron microscopic examination was perafter infection with mhv-a (m.o.i. Å pfu/cell) and formed in cells hr after infection and at a multiplicity after staining with anti-tubulin antibodies and fitcof infection (m.o.i.) of plaque forming unit per cell. areas conjugated secondary antibodies (fig. ) . in uninof syncytia with a typical peripheral rim of nuclei were fected cells and at hr postinfection the microtubules selected from semithin ( . - mm) sections. in uninwere distributed throughout the entire cytoplasm of fected cells the ga was seen in a perinuclear location the individual cells. at hr postinfection the syncytia and consisted of several groups of stacked cisternae were beginning to form and the microtubules were surrounded by numerous coated and uncoated vesicles still distributed within the entire cytoplasm. at hr (fig. b) . in contrast to this typical morphology of the ga, postinfection, when the ga was fragmented and transin infected cells the stacks of the cisternae were marklocated to the centers of syncytia, the microtubules edly diminished in size and were replaced by numerous were rearranged in a characteristic pattern (fig. ) . tubulovesicular structures, some of which containing vi-specifically, at the periphery of the syncytia, an inrus particles ( fig. a) . furthermore, in infected cells the tense stain for tubulins suggested that the nuclei were region containing remnants of ga cisternae and the surrounded by a rich network of microtubules. at the abundant tubulovesicular structures was rich in intermediate zone, between the periphery and the centransversing microtubules ( fig. a) . ter of syncytia, the microtubules formed a radiating network. at the center of the syncytia, the immunostain the distribution of the rer is not affected by mhvfor alpha and beta tubulin was less intense and amor-a infection phous (fig. ) . these changes suggest that during coronavirus infection the microtubules undergo rear-the distribution of the rer in mhv-a -infected and rangement and perhaps provide guidance for the control l cells was investigated by immunocytochemtranslocation of the fragmented elements of the ga istry with the organelle-specific monoclonal antibody into the center of the syncytia. h . there was no detectable difference between the infected and uninfected cells in the the effect of brefeldin a on coronavirus infection immunostaining of the rer. in both cases the fine granular staining of the rer was evenly distributed since coronavirus infection is associated with the processing of viral proteins through the ga, including within the entire cytoplasm including the centers of syncytia (not shown). within the organelle, we investigated the effect of bfa hr later. uninfected cells, however, had completely recovered from the bfa effect - hr later. these on the morphology and kinetics of coronavirus infection. as summarized in fig. , bfa was introduced at findings suggest that virus infection accentuates and prolongs the bfa effect (fig. ) . various time points during infection of l cell with mhv-a . in uninfected l cells incubated for hr when cells were treated continuously with bfa during infection there was dispersion of the ga, lack of syncytia with mg/ml of bfa, immunostaining with anti-mg- showed a diffuse cytoplasmic pattern consistent formation, and no detectable viral titers (fig. ) . if bfa treatment began at or hr after viral inoculation, the with the known redistribution of mg- and other golgi markers within the rer (doms et al., ; lip- effect of this treatment on infection was similar to the effect observed after continuous bfa treatment (i.e., re-pincott- schwartz et al., ; johnston et al., ) . following hr of bfa treatment at the beginning of duced syncytia formation and no viral titers). however, when the bfa treatment began hr or more after viral infection cells had diffuse staining of the ga when observed hr postinfection as did uninfected cells inoculation, there were relatively minimal effects on syncytia formation, and viral titers were - logs lower as following a similar treatment with bfa. however, at hr postinfection (and hr of treatment with bfa at the compared to viral titers without bfa treatment (which usually fluctuate about - . logs in various experi-beginning of infection) there was incomplete recovery from the bfa effect. in uninfected cells treated in a ments). when bfa was introduced min or an hour before similar fashion with bfa, the recovery was complete. therefore, when cells were infected with mhv-a inoculation and continued through the entire infection or , , or hr after inoculation, there was no significant prior to or at the same time of treatment with bfa, the effect of the drug on the ga was not abolished or effect on the ability of the virus to form syncytia hr by cisternae derived from the intermediate compartment between the er and the golgi stacks, thus acquiring two membranes in one step (sodeik et al., (sodeik et al., , . the second wrapping cisternae in vaccinia virus assembly is derived from the trans golgi network (schmelz et al., ) . in cmv, ultrastructural as well as biochemical studies suggested that short-term exposure of infected cultures to bfa during the late infectious cycle primarily prevented golgi-dependent processes, e.g., envelopment of naked cytoplasmic nucleocapsids in the trans-golgi network (tgn) and normal processing of glycoprotein b (eggers et al., ) . in uukuniemi virus, a member of the bunyaviridae, immunofluorescent staining indicated that g glycoprotein expressed alone localized to the ga. g expressed alone was associated with the rer (melin et al., ) . coronavirus mhv m glycoprotein (previously known as e ) is targeted to the ga and contains a retention signal for the ga (machamer et al., ; swift and machamer, ; armstrong and patel, ) . previous reports have shown that the ga undergoes rearrangement (ho et al., ) and fragmentation (campadelli et al., ) in viral infections. the data presented here detail the changes in the morphology of the ga in cells infected by a virus which induces the formation of syncytia. in coronavirus infection, the virus displays cultures were incubated with virus (") for hr and exposed to bfa (ٖ) for various periods. at the end of -hr incubation the cultured cells complex and close interactions with the ga. in coronaviwere stained for mg- by immuohistochemistry and the supernatants rus-induced syncytia formation there is a unique translowere titered for virus by plaque assay. titers labeled indicate levels cation and aggregation of the ga into the center of the below pfu/ml, which is the lowest level of detection in this assay. syncytia which is not accompanied by similar changes of the rer and cytoskeleton (fig. f) . the observed fragmentation of the ga is not related to the formation of postinfection and viral titers at that time were only - . syncytia as demonstrated by the experiments using a logs lower than without treatment. thus bfa did not fusion-negative strain of mhv and fusion-defective mublock the endocytosis and the initial processing of the tants in which fragmentation of the ga also occurs. fragvirus into cells. bfa reduced viral replication only by mentation and translocation of the ga may be important - logs when introduced at - or - hr after in the life cycle of coronavirus replication. further studies infection. the results of the exposure to bfa at the beginare necessary to determine whether there are viruses ning of infection for hr and for various periods at the that replicate within cells without causing alteration of end of infection (fig. ) indicated that the bfa effect on ga morphology. the ga during - hr postinoculation was associated central translocation and aggregation of the ga may with reduced viral replication. if bfa was introduced after be unique to coronaviruses as it was not associated with or before this stage ( - hr), there was no effect on other fusion and syncytia forming viruses such as sindbis viral replication, assembly, and maturation. these results virus infection (ho et al., ) or herpes simplex virus suggest that the interval of - hr postinfection is the infection (campadelli et al., ) . since the aggregation most significant period requiring an intact ga for viral of the fragmented ga in the centers of syncytia has not replication and syncytia formation. this conclusion is been previously reported, we investigated the ga in cells based on infection at an m.o.i. of pfu/cell which may infected with lacrosse bunyavirus, which is known to be an asynchronous infection. in a more synchronous induce the formation of syncytia (gonzalez-scarano et infection the period of bfa effect may terminate earlier al., ) . immunostaining of the ga in bhk cells infected than hr postinfection. with lacrosse virus for hr showed network formation and fragmentation of the ga, without the aggregation of discussion the ga into centralized zones and without the formation of amorphous centers of syncytia which is characteristic the ga plays an important role in the life cycle of many viruses such as vaccinia, cmv, bunyaviruses, and of mhv infection (unpublished observations). thus the translocation of the ga to the center of the syncytia may coronaviruses. vaccinia virus dna becomes enwrapped evolution of a coronavirus during persistent infection in vitro use of ferrocyanide-reduced osmium tetroxide cessed through the golgi apparatus after infection with herpes simin electron microscopy coronavirus m proteins accumulate in the - kd membrane polypeptide of the rough endoplasmic reticulum golgi complex beyond the site of virion budding an anti-organelle antibody in pathology. the chromatolytic reaction studied with a characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex rothman experimental demyelination produced by the a strain of ture and toxic effect assembly of vaccinia virus: the second lar aspects of neurotropic viral infections assembly of vaccinia virus: incorporation of p treated with brefeldin a: evidence for membrane cycling from golgi and p into the membrane of the intracellular mature virus form of the golgi apparatus in hela cells assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the golgi stacks proteolytic cleavage golgi complex ronnholm, r., and fusion activity virus contains a signal for localization to the golgi complex the golgi complex: in vitro veritas? fragmentation of the golgi apparatus of motor anti-organelle antibody neurons in amyotrophic lateral sclerosis revealed by organelle-spe-stieber mg- , a membrane protein of the golgi apparatus assignment of the glg gene for mg- , a fibroblast ligand for e-selectin, is found only in the golgi apparatus and apgrowth factor and e-selectin binding membrane sialoglycoprotein of pears early in chicken embryo development - . membrane-spanning domain of coronavirus e protein fusion formation by the uncleaved spike protein of in transgenic mice expressing mutant cu,zn superoxide dismutase murine coronavirus jhmv variant cl- acetyl-galactosamine to the e glycoprotein of mouse hepatitis virus-mourelatos histochemical and ultrastrucand membrane traffic in golgi complex organization the ultrastructure of a mammaof the golgi apparatus by brefeldin a inhibits the cytotoxicity of ricin, modeccin, and pseudomonas toxin monoclonal antibody against the golgi apparatus. am. j. pathol. , be related to a unique interaction between coronavirus - . proteins with the membranes of the ga. croul, s. e., mezitis, s. g. e., stieber, a., chen, y., gonatas, j. o., goud, since mutant viruses that are defective in their ability b., and gonatas, n. k. ( ) . immunohistochemical visualization of to cause efficient fusion are also inefficient in translocatthe golgi apparatus in several species, including human, and tissue ing the ga to the center of the syncytia, the translocation with antiserum against mg- , a sialoglycoprotein of rat golgi apparatus. j. histochem. cytochem. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] of the ga appears to be linked to the ability of the virus doms, r. w., russ, g., and yewdell, j. w. ( ) . brefeldin a redistribto cause fusion. although the fusion property has been utes resident and itinerant golgi proteins to the endoplasmic reticuassociated with the s gene and the site encoding cleav- lum. j. cell biol. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] age of the s protein (gombold et al., ) , other reports eggers, m., bogner, e., agricola, b., kern, h. f., and radsak, k. ( ). showed that cleavage of the s gene in coronaviruses is inhibition of human cytomegalovirus maturation by brefeldin a. j. gen. virol. , - not an absolute requirement for fusion (stauber et al., farquhar, m. g., and palade, g. e. ( ) . the golgi apparatus (com- ; taguchi et al., ) . however, fragmentation of plex)- useful in future studies of aspects of both viral replication gonzalez-scarano, f., pobjecky, n., and nathanson, n. ( ) . lacross and the biology of the ga.bunyavirus can mediate ph-dependent fusion from without. virology , - . key: cord- -e qq ngq authors: jackson, catherine l. title: arf proteins and their regulators: at the interface between membrane lipids and the protein trafficking machinery date: - - journal: ras superfamily small g proteins: biology and mechanisms doi: . / - - - - _ sha: doc_id: cord_uid: e qq ngq the arf small gtp-binding (g) proteins regulate membrane traffic and organelle structure in eukaryotic cells through a regulated cycle of gtp binding and hydrolysis. the first function identified for arf proteins was recruitment of cytosolic coat complexes to membranes to mediate vesicle formation. however, subsequent studies have uncovered additional functions, including roles in plasma membrane signalling pathways, cytoskeleton regulation, lipid droplet function, and non-vesicular lipid transport. in contrast to other families of g proteins, there are only a few arf proteins in each organism, yet they function specifically at many different cellular locations. part of this specificity is achieved by formation of complexes with their guanine nucleotide-exchange factors (gefs) and gtpase activating proteins (gaps) that catalyse gtp binding and hydrolysis, respectively. because these regulators outnumber their arf substrates by at least -to- , an important aspect of understanding arf function is elucidating the mechanisms by which a single arf protein is incorporated into different gef, gap, and effector complexes. new insights into these mechanisms have come from recent studies showing gef–effector interactions, arf activation cascades, and positive feedback loops. a unifying theme in the function of arf proteins, carried out in conjunction with their regulators and effectors, is sensing and modulating the properties of the lipids that make up cellular membranes. arf proteins are low molecular weight gtp-binding (g) proteins that are regulated through a cycle of gtp binding and hydrolysis, in which binding of gtp activates and gtp hydrolysis inactivates the g protein (donaldson and jackson ; gillingham and munro b) (fig. . ). in their active gtp-bound form, arf proteins are tightly associated with the membrane surface. hence they bring their effectors, proteins that bind specifically to the gtp-bound form, into close contact with the lipid bilayer. these arf effector proteins include coat complexes that deform membranes and promote cargo sorting, enzymes such as the phosphatidylinositol kinases that alter membrane lipid composition, and actin cytoskeletal components (table . ). arf is the founding member of the family, and was originally identified as a protein factor required for the adp-ribosylation of the adenylate cyclase activator gsα by cholera toxin (schleifer et al. ) . although subsequent studies led to the discovery that the major cellular function of arf is regulation of membrane trafficking, its name comes from this initial finding. the original discovery that arf has an essential function in the secretory pathway at the level of the golgi came from studies in yeast (stearns et al. a, b) . soon thereafter, the role of arf in recruiting the copi coat complex to membranes of the early secretory pathway to mediate copi vesicle budding was demonstrated both in vitro and in cells. the reconstitution in vitro of copi vesicle budding provided important mechanistic insights (rothman and wieland ) , and the function of arf in cells was greatly aided by use of the specific inhibitor of arf activation, brefeldin a (klausner et al. ) . mammalian arf proteins can be divided into three classes based on sequence homology: class i (arfs - ), class ii (arfs - ), and class iii (arf ). class i arfs are highly conserved and are present in all eukaryotes, whereas the class ii arfs arose during animal cell evolution, diverging from the class i arfs in the animal lineage after fungi separated, but before choanoflagellates did (manolea et al. ; schlacht et al. ) . consequently, in all invertebrates, such as d. melanogaster and c. elegans, there is one member of each of the three arf classes. in certain lineages, some arf classes have undergone expansion. for example, vertebrates have multiple members of class i and ii arf proteins (li et al. ) . although yeast lack class ii arfs, they have two highly similar class i arf proteins and a single class iii member. plants have numerous class i arfs (six in arabidopsis) that are all more closely related to each other than to class i arfs of other eukaryotic supergroups, as well as highly diverged arfs with some characteristics of mammalian class iii arf (gebbie et al. ) . the arf proteins are part of a larger family that also includes the arf-like (arl) proteins. there are more than arl proteins, with a wide range of functions including membrane trafficking, targeting of proteins to cilia, microtubule regulation, and lysosome function (donaldson and jackson ; gillingham and munro b) . the most divergent arl protein is sar , an evolutionarily ancient small g protein that shares a highly related function eukaryotic cells (dacks and field ) . intriguingly, the last eukaryotic common ancestor (leca) likely possessed only one arf family member, in contrast to having already nearly rab proteins (koumandou et al. ) . however, multiple arf gefs and gaps existed in this ancient eukaryotic ancestor, supporting the idea that a key feature of arf function is a single arf protein participating in multiple gef and gap regulatory complexes (koumandou et al. ; schlacht et al. ). this feature is conserved in modern organisms, for example, in humans, which have only arf proteins, yet at least gefs and gaps. perhaps a clue as to the arf tgn vxpx targeting motif ap adaptor protein, bar bin/amphiphysin/rvs, cc coiled-coil, cop coatomer protein, er endoplasmic reticulum, ergic er-golgi intermediate compartment, gat gga (golgi-localized, γ-adaptin homologous, adp-ribosylation factor-binding protein) and tom homologous, grab grip (golgin- /rabbp α/imh p/p )-related arf binding, pm plasma membrane, tgn trans-golgi network; nd, not determined nature of the primordial arf protein comes from the protozoan parasite trypanosoma brucei, which expresses a single arf protein that has characteristics of both class i and class iii mammalian arfs. tbarf is a basic protein with a high pi similar to that of human arf , but contains the golgi-targeting motif mxxe (price et al. ) , found in human arf and arf (honda et al. ) . tbarf localizes to the golgi, and functional studies indicate that it has roles in both endocytosis and in golgi-lysosome trafficking (price et al. ) . the spatiotemporal control of arf protein function is mediated by regulators of arf gtp binding and gtp hydrolysis (fig. . d ). the arf guanine nucleotideexchange factors (gefs) catalyse gdp release from their substrate arf, allowing gtp, which is more abundant in cells, to bind. this nucleotide-exchange activity is carried out by the sec domain, a highly evolutionarily conserved domain first identified as a homology domain in the yeast sec p protein, and whose function was first identified in the yeast gea p protein (peyroche et al. ) . a sec domain is present in all arf gefs identified to date. the arf gtpase activating proteins (gaps) catalyse the hydrolysis of gtp on their substrate arf, a function carried out by a conserved gap domain, characterized by the presence of a zinc finger. the arf gaps are essential because arf proteins have negligible intrinsic gtp hydrolysis activity (kahn and gilman ) . the primary sequence homology of the catalytic domains of the gefs and gaps facilitated their identification, but their arf substrate specificity has not been fully elucidated. a recent study of the arf gaps has revealed that they have likely co-evolved with their arf substrates (schlacht et al. ) . these results support the conclusion that the arfs function in a tightly coordinated manner with their regulators. there are seven subfamilies of arf gefs in eukaryotic cells (cox et al. ) (table . , fig. . ). the gbf/gea and big/sec gefs are localized to the golgi, and use arf as a substrate (donaldson and jackson ) . in animal cells and yeast, these gefs act sequentially, with gbf/gea proteins functioning at the early golgi, and big/sec proteins at the trans-golgi and trans-golgi network (tgn) (franzusoff et al. ; peyroche et al. ; zhao et al. ) . the cytohesin/ arno, efa , and iqsec/brag subfamilies function primarily in endosomal-pm trafficking pathways at the cell periphery, and primarily act on arf (casanova ; cox et al. ; gillingham and munro b) . the efa gefs regulate endocytosis, endosomal membrane recycling, and actin cytoskeleton remodelling (casanova ; franco et al. ) . yel p is the orthologue of efa in budding yeast, saccharomyces cerevisiae, exhibiting exchange activity in vitro on arf p (the yeast class iii member) (gillingham and munro a) , which is involved in endocytosis (smaczynska-de rooij et al. ). yel p has a homology domain in the c-terminus resembling that of efa family members (gillingham and munro a) . syt p has a sec domain that most closely resembles that of the iqsec/ brag family in mammalian cells, although it lacks an iq motif (cox et al. ) , and functions with arl p and arl p at the trans-golgi in yeast . the fbxo arf gefs contain an f-box in addition to the sec domain, and are present in vertebrates, and at least one invertebrate (gillingham and munro b) , but are not present in yeast, worms, or flies. little is known of its function, although --recently a form of bilateral cleft lip has been associated with a deletion of a chromosomal region including the fbxo gene (calcia et al. ) . ralf is the founding member of a family of bacterial arf gefs, first identified in legionella pneumophila and rickettsia prowazekii (amor et al. ; cox et al. ). these gefs do not have a prokaryotic origin, but rather were incorporated into bacterial genomes through horizontal transfer from their eukaryotic hosts (nagai et al. ) . the sec domain of ralf is autoinhibited by the capping domain of the protein, which is relieved by interaction with a specific lipid environment (alix et al. ; folly-klan et al. ). there are subfamilies of arf gaps, of which are found in humans (schlacht et al. ) (table . ). a recent phylogenetic study has indicated that six arf gap families (arfgap , arfgap / , smap, acap, agfg, and the newly identified arfgapc family) are ancient, probably existing in the leca prior to separation of the eukaryotic supergroups. on the other hand, asap, arap, and git families arose more recently in evolution, being found only in animals. through the course of evolution, the arf gaps have undergone extensive domain shuffling, losing and gaining different domains in a complex pattern to give rise to the proteins existing today (schlacht et al. ) . the ph, bar, rhogap, ankyrin repeat, and c domains of arf gap proteins are conserved across eukaryotes, and therefore were probably present in the primordial arf gaps, whereas other domains such as gold, calm, and sh domains are restricted to specific lineages (schlacht et al. ). the first arf gap identified was arfgap (cukierman et al. ) , which has two alps motifs in its c-terminal region that mediate specific binding to the highly curved membranes of copi vesicles (bigay et al. (bigay et al. , mesmin et al. ) . in an elegant self-organizing mechanism, copi vesicles are generated through activation of arf at the golgi, which recruits copi to deform the membrane into a highly curved vesicle, which then recruits arfgap through its alps motif to hydrolyse the gtp on arf , releasing the coat and allowing fusion of the vesicle with its target membrane (bigay et al. ) . arfgap and also bind to copi vesicles, but do so through direct interaction with the copi coat, and hence can also bind to copi-coated regions that are not highly curved (kliouchnikov et al. ; weimer et al. ). arfgap , , and and their yeast orthologues gcs p and glo p function at the early golgi (spang et al. ) , whereas most of the other arf gaps function at the cell periphery in tgn-endosomal-plasma membrane trafficking and actin cytoskeleton remodelling (inoue and randazzo ; sabe et al. ). the following chapter in this volume will describe the latter class of arf gaps in depth. a major distinguishing feature of the arf proteins is the presence of a myristoylated amino-terminal amphipathic helix that is necessary for membrane binding ( fig. . a , b, c). myristoylation is a cotranslational modification required for the essential functions of arf in vivo (kahn et al. ) . in cells, myristoylation is required for correct localization of arf proteins, including golgi localization of arf and pm localization of arf (donaldson and jackson ) . in vitro, the myristoyl group of the gdp-bound form of arf is available for membrane insertion, permitting a weak association of the inactive form of arf with membranes (franco et al. ) . upon gtp binding, the n-terminal amphipathic helix of arf is released and inserts into the membrane, resulting in tight membrane association (antonny et al. ) (fig. . c) . structural studies revealed the details of this change in conformation, showing that the interswitch region changes position to occlude the hydrophobic pocket that harbours the amphipathic n-terminal helix in the gdp-bound form of arf (goldberg ). nmr studies of n-myristoylated arf -gtp further confirmed this mechanism (liu et al. ) . thus, in addition to changes in the effector binding regions upon exchange of gdp for gtp, arf proteins undergo a second change in conformation that brings them into very close contact with the membrane (fig. . c) (antonny et al. ; chavrier and menetrey ) . this property distinguishes them from other small g proteins of the ras superfamily, including the ras, rho, and rab families, which have a long c-terminal linker to which their lipid membrane anchor is attached (gillingham and munro b) . arf effectors are thus constrained to a position close to the membrane, in contrast to those of rab and rho, which can be located at a distance from the membrane surface (gillingham and munro b; khan and menetrey ) . another distinction between arf proteins and those of the rab and rho families is that no guanosine diphosphate dissociation inhibitors (gdi) have been identified. all arfs are tightly membrane bound in their active gtp-bound conformation because of nucleotide regulation of the position of the n-terminal amphipathic helix, and simply hydrolysing the gtp on arf proteins is sufficient to render them soluble in vitro. indeed, arf and arf appear to be released from membranes into the cytosol upon gtp hydrolysis in cells. however, arf remains bound to membranes in cells in its gdp-bound conformation. there is also evidence that arf -gdp and arf -gdp remain membrane bound in cells, in the latter case, to the er-golgi intermediate compartment (ergic) duijsings et al. ). this membrane association is likely due to interaction of the gdp-bound forms with membrane-associated proteins. for arf , members of the kalirin family of rho gefs have been shown to bind specifically to the gdp-bound form through their spectrin-like repeat domain (koo et al. ). arf -gdp recruits kalirin to the membrane where it subsequently activates rac and rhog to regulate actin dynamics (koo et al. ). arf -gdp binds several tbc (tre- /bub/cdc ) domaincontaining proteins, which have rab gap activity (haas et al. ) , including tbc d , a protein mutated in familial infantile myoclonic epilepsy (falace et al. ) , and the tre oncogene (martinu et al. ). hence interactions with the gdp-bound form of a g protein could provide a mechanism for a single arf protein to trigger alternative signalling pathways depending on the nucleotide bound, which could have important implications in human disease (donaldson and jackson ) . following activation on membranes, gtp-bound arfs recruit coat proteins, lipid-modifying enzymes, tethers, and other effector molecules that modulate the properties of membranes and mediate vesicle trafficking (table . ). the first function of the arf proteins to be identified was their ability to recruit cytosolic coat proteins to membranes. in the early secretory pathway, arf recruits coatomer complex i (copi), which sorts cargo proteins into copi-coated vesicles as it curves the membrane to form the vesicle (beck et al. ). arf at the trans-golgi network (tgn) also recruits the heterotetrameric clathrin adaptor proteins (ap), ap- , ap- , and ap- and the three monomeric golgi-localized γ-ear-containing, adp-ribosylation factor-binding proteins (ggas - ) (bonifacino and lippincott-schwartz ) . these various coat proteins specifically bind to cargo proteins and incorporate them into forming vesicles for sorting and transport to their correct destination. arf proteins can also recruit and activate enzymes that alter membrane lipid composition. the first of these enzymes to be identified was phospholipase d (pld), which hydrolyses phosphatidylcholine to generate phosphatidic acid (brown et al. ; cockcroft et al. ) . pld is activated by all arf proteins and also by arl (hong et al. ) . pld activation by arf is involved in a number of processes at the cell periphery, including regulated endocytosis and cell migration (d'souza-schorey and chavrier ) . another major function of arf proteins is regulation of phosphoinositide levels in cells. all arf proteins can both recruit to membranes and stimulate the activity of phosphatidylinositol -phosphate, -kinase (pip k), an enzyme that phosphorylates inositol -phosphate (pi p) at the -position to generate phosphatidylinositol , -bisphosphate (pi( , )p ) (honda et al. ) . in cells, it is primarily arf that colocalizes with pip k at the pm to generate pi( , )p , which in turn stimulates pm ruffling (honda et al. ) . at the golgi, arf recruits and stimulates the activity of phosphatidylinositol -kinase, forming pi p, an important membrane lipid for golgi function (de matteis and godi a). as described above, arf can recruit the copi coat to early golgi membranes, as well as the heterotetrameric clathrin adaptor complexes (ap- , ap- , ap- ) and the three monomeric adaptors (gga , gga , and gga ) to trans-golgi, tgn, and endosomal membranes. how a single arf protein can recruit multiple coats to different membrane sites in cells is still not fully understood, but one important contribution to specificity comes from the arf gefs. in both mammalian and yeast cells, gbf and gea / , respectively, interact directly with copi (deng et al. ), and knockdown of gbf inhibits copi recruitment to membranes in mammalian cells (deng et al. ; ishizaki et al. ; manolea et al. ; szul et al. ). in contrast, knockdown of big and big arf gefs inhibits ap- and gga recruitment to the trans-golgi (ishizaki et al. ; manolea et al. ) . in yeast, a single class i arf is responsible for recruiting copi, ap- , and gga coats to membranes in cells, and hence mechanisms such as arf gef-mediated specificity are required. in mammalian cells, arfs - could contribute an additional layer of specificity. it has been shown recently that a specific subset of arf family members (arf , arf , and arf , but not arf or arf ) are incorporated into copi vesicles reconstituted using cytosol (popoff et al. ) . a full understanding of the mechanisms determining the specificity of coat recruitment by arf family members and their regulators is an important open question in the field. important insights into the recruitment of coats by arf have come from recent structural studies (ren et al. ; yu et al. ) . the structure of arf -gtp bound to a subcomplex of the copi coat revealed a binding site on the γ subunit, which would position the entire copi complex on the membrane surface in a conformation very similar to the membrane-bound ap- /clathrin complex (yu et al. ) . copi, ap- /clathrin, and ap- /clathrin complexes share a remarkable level of structural similarity, although ap- is recruited to membranes by plasma membrane pi( , )p rather than an activated arf protein (jackson et al. ). all of these coats have two large subunits that are symmetrically located within the complex, which in the case of copi are the β and γ subunits. biochemical studies confirmed a second arf -gtp binding site on the β subunit of copi (yu et al. ) . the structure of arf in complex with the entire central trunk region of the ap- adaptor complex revealed only one of the two arf -gtp binding sites on ap- , on the β subunit (ren et al. ). this arf -ap- structure is almost identical to the active, cargo-bound ap- complex (jackson et al. ) , and reveals a second interaction, involving the back side of arf, opposite the switch regions, to a second site on the ap- γ subunit. biochemical and cell biological analyses showed that recruitment of the ap- adaptor to membranes requires two binding sites for arf -gtp (one on each of the symmetrical β and γ subunits), in a manner analogous to those found for copi. however, the conformation of the active arf -gtp-ap- complex docked to a membrane bilayer is not compatible with binding of arf molecules at both recruitment sites (ren et al. ) . hence the proposed model based on these results is recruitment of a closed conformation of ap- by two arf molecules, a change in conformation to the open cargo-bound form of the adaptor with one molecule of cargo engaged, and then a large conformational change mediated by binding of the trunk of the γ subunit to the back side of arf -gtp, concomitantly releasing the γ recruitment site (ren et al. ) . given the structural similarities, this model will likely apply to other arf -gtpcoat complexes, including copi. the majority of initial studies on the arf proteins focused mainly on arf at the golgi and arf at the cell periphery (d'souza-schorey and chavrier ; donaldson and jackson ) . this exclusivity was warranted, as arf is the most highly expressed of the arf proteins in cells, and is essential for viability both in mammalian cells (reiling et al. ) and in budding yeast, s. cerevisiae (stearns et al. a ). arf has functions at the cell periphery in cell adhesion and motility that are involved in numerous human pathologies such as cancer, and is the only member of the class iii arfs in mammalian cells. arf , arf , and arf , similarly to arf , all localize to internal membranes, including the golgi. as described above, phylogenetic studies support the conclusion that these other class i and ii arfs arose late in animal cell evolution, so may be involved in more specialized functions, or in increasing the spatial or temporal resolution of arf -mediated processes (manolea et al. ) . interestingly, partial depletion experiments show that under conditions where single knockdowns have little if any phenotypic effects, sirna depletion of pairs of arf -arf has effects on specific membrane trafficking steps (volpicelli-daley et al. ) . for example, the double knockdown of arf and arf affects transport in the early secretory pathway, inhibiting copi coat recruitment, but has little effect on several other trafficking pathways (volpicelli-daley et al. ) . consistent with this observation, arf localizes to the ergic and cis-golgi , and together with arf at the cis-golgi, it organizes trafficking between these compartments (ben-tekaya et al. ) . arf and arf differ only at seven amino acid positions in their amino-and carboxy-terminal regions and previously they were thought to function and localize identically in cells. however, a golgi-targeting sequence contained within the α- helix of arf and arf targets a chimaera between arf and arf to the early golgi (honda et al. ) . furthermore, arf localizes specifically to the trans-golgi and tgn and this localization depends on four arf -specific amino acids contained in the n-terminal amphipathic helix, which are conserved among arf homologues (manolea et al. ) (fig. . b) . arf , but not arf , becomes cytosolic at c, the temperature at which exit from the tgn is blocked (manolea et al. ) . these results support the conclusion that arf has a critical role during exit from the golgi. recently, class ii arfs have been shown to participate in novel functions at the tgn. d. deretic and colleagues have found that arf specifically recognizes the vxpx cytosolic targeting motif in retinal rhodopsin to facilitate its transport into the rod outer segment, a specialized cilium (deretic et al. ) . this ciliary targeting complex includes rab , fip (a dual arf and rab effector), and asap , an arf gap (mazelova et al. ), in addition to arf . the mechanism by which this complex facilitates the packaging of rhodopsin into post-golgi carriers has not yet been determined, but it is known that rhodopsin itself initiates complex formation by recruiting arf . the rhodopsin-binding site of arf has been mapped to the α- helix (deretic et al. ) , which corresponds to the region of arf that binds the snare protein membrin to mediate targeting to the early golgi (honda et al. ) . hence, the α- helix might generally allow arf protein binding to membrane receptors. arf and arf can also directly bind to the calcium-dependent activator protein for secretion (caps), which regulates exocytosis of dense core vesicles from nerve terminals (sadakata et al. ). arf and arf together have been shown to play a role in endosome-tgn trafficking; the double knockdown causes tubulation of the recycling endosome and an inhibition of tgn and mannose- -phosphate receptor trafficking from there back to the tgn (nakai et al. ) . how these roles of arf and at the tgn in cells can be reconciled with findings of arf localization to, and arf and functioning at, the early golgi a recent study has found a specific role for arf in mediating brefeldin a-induced apoptosis, a pathway used by human pathogens such as chlamydia trachomatis and shigella flexneri (reiling et al. ) . in response to brefeldin a and other golgi-disrupting agents, cells upregulate expression of arf at the transcriptional level, through proteolytic activation of the basic leucine zipper transcription factor creb at the golgi (reiling et al. ) . interestingly, a previous study found that knockdown of gbf caused an upregulation of the unfolded protein response and apoptosis (citterio et al. ) , possibly through this newly defined creb /arf pathway. these results suggest that golgi stress might trigger a response through arf aimed at re-establishing golgi function, and if this fails, mediate a switch to induction of apoptosis (reiling et al. ). a novel function for arf in lipid droplet (ld) metabolism has been uncovered recently (beller et al. ; guo et al. ; soni et al. ). lipid droplets are well known for their function in storage of energy in the form of triglycerides (ducharme and bickel ; londos et al. ) . more recently, their dynamic structure and integration with membrane trafficking pathways have revealed that they are in fact bona fide organelles (walther and farese ) . lds are distinct from other organelles in having a neutral lipid core surrounded by a phospholipid monolayer (tauchi-sato et al. ) , rather than a bilayer encompassing an aqueous interior. in addition to triglycerides, which are the major energy storage molecules of eukaryotic cells, the neutral lipid core of lds also contains esterified cholesterol. these two classes of neutral lipids serve as storage precursors of the two major lipid components of cellular membranes. arf , along with its gef gbf and effector copi, associates with lds ( fig. . ) and is required for recruitment of a subset of lipid droplet associated proteins to the ld surface (soni et al. ). in mammalian cells, these ld components include a triglyceride lipase (atgl) and a perilipin family member (plin ) (soni et al. ), and for atgl at least, this function is conserved in drosophila (beller et al. ). gbf itself is recruited to lds via hds , the domain just downstream of the catalytic sec domain, which binds both liposomes and artificial droplets directly in vitro (bouvet et al. ) . this domain and the downstream hds domain are both required for localization of gbf to the golgi. however, when expressed alone as gfp fusions in cells, hds and hds are targeted only to lds, not to the golgi (ellong et al. ). the sec domain regulates hds association with membranes, acting as an inhibitor of localization, and the n-terminal dcb and hus domains are required in addition to target gbf to the golgi (bouvet et al. ) . b. antonny and colleagues have proposed a novel mechanism of protein recruitment to the unique surface of lds. in a recent study, they demonstrated that the density of phospholipids in the monolayer surrounding the neutral lipid core of an artificial ld could be decreased, leading as expected to an increase in surface tension (thiam et al. ) . hence in contrast to a bilayer, which would tear with even a small decrease in phospholipid density in one leaflet, the phospholipids of the ld surface can be spread apart, albeit with the unfavourable effect of exposing the hydrophobic core to the aqueous environment of the cytosol. this increase in surface tension could favour the recruitment of cytosolic proteins to the ld, which would compensate for the lower density of phospholipids by helping to cover the ld surface. b. antonny and f. pincet further hypothesized that gbf , arf , and copi might function to recruit proteins to lds by decreasing phospholipid density, as a result of their removal by budding of small droplets from the surface of a large ld. indeed, arf -gtp and copi were sufficient to cause budding of - nm diameter droplets from an artificial droplet surface in vitro (thiam et al. ) . in support of their hypothesis, these authors then found that the binding of α-synuclein, known to associate with lds in cells (cole et al. ) , was promoted when the phospholipid density of artificial droplets decreased (thiam et al. ) . this result supports the hypothesis that lds can become more "reactive" when they experience a decrease in phospholipid density on their surface, because of the unfavourable increase in surface tension. together, these findings provide an elegant explanation for the function of gbf , arf , and copi in recruitment of proteins to the ld surface. in addition to its function in lipid metabolism of lds, arf has an important function in lipid trafficking at the golgi that is distinct from its role in vesicle formation. a number of lipid transfer proteins are recruited by arf to membranes at sites of close contact between the er and other organelles, known as membrane contact sites (mcs) (levine and rabouille ; stefan et al. ) . fapp , cert, and osbp transfer glucosylceramide, ceramide, and sterol, respectively, and all three possess a ph domain that requires both arf and pi p in order to bind to trans-golgi membranes (de matteis and godi b) (table . ). the first clue that these proteins might bridge two different organelles came from studies indicating that in addition to a late golgi-specific ph domain, they also carry a motif mediating binding to the er-localized protein vap (lev ; levine and loewen ) . a recent study showed that these two localization regions of osbp are sufficient to mediate contact between the er and golgi, and moreover, that osbp activity regulates this association (mesmin et al. ). fapp -mediated delivery of glucosylceramide to the trans-golgi has been shown to be dependent on arf and also to be required for vesicular trafficking from the golgi to the pm (d'angelo et al. ). hence arf , by recruiting proteins that mediate transfer of sphingolipid precursors and sterol, plays an essential role in establishing the characteristic lipid environment of the trans-golgi, pm, and endosomal system of the cell, an important feature of cellular organization (bigay and antonny ) . a key question regarding these transfer proteins is how they transport lipids such as sterols up their concentration gradient. for osbp, b. mesmin and colleagues showed that sterol is exchanged for pi p at the trans-golgi, where it is returned to the er and the phosphate hydrolysed by the er-resident pi p phosphatase sac p. the energy from hydrolysis of phosphate on pi p drives the transport of sterol up its concentration gradient (mesmin et al. ). arf is localized to the plasma membrane and regulates both the cortical actin cytoskeleton and endosomal membrane recycling. at the plasma membrane, arf modulates membrane lipid composition through activation of pi p -kinase (pip k) and pld, resulting in production of pi( , )p and pa. these phospholipids can influence the sorting of membrane proteins within the pm, and are important for the formation of clathrin-coated pits during endocytosis. they are also required for the recruitment and activation of rho family g proteins such as rac to induce actin polymerization. arf is associated with endosomal membranes derived from clathrin-independent forms of endocytosis, where it functions to recycle membrane components back to the plasma membrane (grant and donaldson ). recycling through endosomal compartments by arf is required for the polarized delivery of cdc , rac, and the par complex to the leading edge of migrating cells (osmani et al. ) , and for recycling of proteins involved in cell adhesion (balasubramanian et al. ). arf is also associated with clathrincoated vesicles, where it mediates the rapid recycling of transferrin receptor back to the plasma membrane through interaction with the microtubule motor adaptor protein jip after clathrin uncoating (montagnac et al. ) . arf can interact with adaptor protein (ap- ) (paleotti et al. ) and with ap- and clathrin during g-protein coupled receptor cell signalling (poupart et al. ) . hence arf has a general function in mediating recycling of components back to the pm after endocytic internalization via different routes. studies of the arf homologues in model organisms have illustrated the evolutionarily conserved nature of arf function. arf p, the yeast arf homologue, affects polarization events such as bud site selection in s. cerevisiae (huang et al. ) , and the switch in cellular growth from monopolar extension to bipolar extension in fission yeast (fujita ) . budding yeast arf p contributes to pm pi ( , )p levels like its mammalian homologue (smaczynska-de rooij et al. ) , and is involved in uncoating of clathrin-coated endocytic vesicles along with its specific gap gts p (toret et al. ). in the filamentous fungus aspergillus nidulans, the class iii arfb localizes to both the plasma membrane and endomembranes, and regulates endocytosis and polarity establishment during hyphal growth . arf in drosophila is also involved in endocytic recycling, which is required for cytokinesis in spermatocytes (dyer et al. ). in mammalian cells, arf is involved in cytokinesis through interaction with jip ). the crystal structure of arf in complex with jip shows that residues adjacent to the switch regions are structural determinants for the specific binding of jip to arf (isabet et al. ). in mammals, arf is not required for early embryonic development, as demonstrated by the fact that homozygous knockout mice can develop to mid-gestation or even to birth, although they die shortly thereafter (suzuki et al. ) . hence, the critical physiological roles of arf in cell adhesion and cell migration appear to be more important for functions that become crucial late in development of the organism, such as wound healing and metastasis, rather than functions necessary for early development. detailed descriptions of the functions of arf in these processes have been shown in a large number of studies, and the reader is referred to recent excellent reviews on the subject (d'souza-schorey and chavrier ; schweitzer et al. ). of the seven subfamilies of arf gefs in eukaryotic cells, probably the most intensively studied are the cytohesin/arno proteins. this subfamily was the first one to be identified in mammalian cells (chardin et al. ) , and in addition to a wide range of crucial physiological functions, its members have proved to be highly amenable to biochemical and structural characterization. the cytohesin/arno gefs function in plasma membrane-endosomal membrane trafficking routes, in cytoskeleton regulation, as well as in signal transduction pathways important for cell proliferation, immune response, and growth control (casanova ; kolanus ) . members of this gef family can catalyse exchange on both arf and arf in vitro and in cells, although in vitro they are more efficient gefs for arf (casanova ; macia et al. ). cytohesin activation is spatially regulated through relief of autoinhibition, positive feedback loops, and activation cascades. at the plasma membrane, the ph domains of cytohesin family members interact with pm-specific phosphoinositides and with the gtp-bound forms of arf (cohen et al. ) and arl (hofmann et al. ; li et al. ), leading to cytohesin recruitment and further activation of arf or arf at the plasma membrane. a crystal structure of the autoinhibited sec domain in tandem with the ph domain of cytohesin /grp revealed that the c-terminal helix that follows the ph domain and the linker between the sec and ph domains block the catalytic site (dinitto et al. ) . interaction of the ph domain with arf -gtp and phosphoinositides, either pi( , )p or pi( , , )p , as well as the interaction of the polybasic c-terminus of cytohesin/arno with acidic phospholipids, all contribute to relieving this autoinhibition (dinitto et al. ). reconstitution of the cytohesin/arno exchange assay on liposomes, in the presence of both activating arf -gtp and substrate arf , revealed that mutations in the ph domain of cytohesin/arno that abolished interaction with arf -gtp were completely inactive (stalder et al. ) . hence interaction of the ph domain with an activating arf protein is an absolute requirement for relief of cytohesin/arno autoinhibition on membranes. a recent structure of the sec and ph domains of arno in complex with arf -gtp, when compared to the autoinhibited structure, reveals a large conformational change upon arf -gtp binding. binding of arf -gtp creates grooves at the arf -ph domain interface into which the autoinhibitory elements bind, thus uncovering the binding site for the substrate arf (malaby et al. ) . together these studies demonstrate how precise spatial regulation of cytohesin/arno activation is achieved: a specific phosphoinositide (pip and/or pip ), additional acidic phospholipids, and an active arf localized to the plasma membrane must all coincide to relieve autoinhibition, thus restricting the membrane domain at which these gefs can become active. the activation of cytohesins by a gtp-bound arf family member raises the question of whether they can engage in a positive feedback loop whereby the product of the reaction can stimulate exchange. indeed, such a positive feedback loop has been demonstrated (stalder et al. ) . cytohesin/arno is one of the most efficient gefs in vitro on its myristoylated arf substrate (k cat /k m~ m À s À ), and this is due in part to the stimulation of exchange by interaction of arf -gtp with the ph domain (stalder et al. ) . given the high level of efficiency of this gef, it would be reasonable to expect a tight regulation of its activity. as described above, one such mechanism is found in the absolute requirement for an activating arf protein to relieve autoinhibition (stalder et al. ) . this study also showed that other arf effectors are able to compete with the cytohesin/arno ph domain for available arf-gtp, suggesting that the activating arf protein (arl -gtp or arf -gtp) must be present in excess of other effectors to reach a level sufficient to stimulate this gef (stalder et al. ) . hence, in order to activate cytohesin/arno, a burst of arl -or arf -gtp must be produced to overcome autoinhibition, but once in its active conformation, this gef has a high capacity to stimulate exchange on its arf substrates due to the effect of positive feedback. interestingly, stimulation of exchange activity by the product of the reaction has been demonstrated for the trans-golgi localized sec p arf gef (richardson et al. ) , as well as for the ras gef sos (boykevisch et al. ) . hence, this type of positive feedback regulation may be a general property of small g proteins. in the case of sec p, the hds domain downstream of the catalytic sec domain binds to membranes and to arf -gtp both in vitro and in cells, and like the ph domain in the cytohesin proteins, is responsible for mediating relief of autoinhibition and the positive feedback effect (richardson et al. ) . for sec , the hds -arf -gtp interaction is required to maintain golgi localization of sec p (richardson et al. ) . interestingly, reduction of arf levels in yeast cells is particularly detrimental to trans-golgi function (gall et al. ) , a result that could potentially be explained by the need for sec to have a high level of arf for its autocatalytic activity. one consequence of this mechanism could be to maintain directionality in trafficking through the golgi. as described above, the gbf/gea family of gefs act early in the secretory pathway, at the cis-golgi, and sec /big gefs act later at the trans-golgi. interestingly, the hds domain of gbf , also immediately downstream of the sec domain, is a direct lipid binding domain that does not require arf -gtp for membrane binding like sec p does (bouvet et al. ) . these results taken together suggest that one mechanism to drive trafficking forward through the golgi is initial recruitment of gbf /gea gefs to produce arf -gtp, which then can recruit the later-acting sec /big gefs to golgi membranes. whether arf , arf , or both are the primary substrates for the cytohesin gefs has been a long-standing controversy and is still an open question. however, arf -gtp is more efficient at relieving autoinhibition of cytohesins than arf -gtp, both in vitro and in cells (cohen et al. ; dinitto et al. ). this fact, combined with the requirement for acidic phospholipids in cytohesin membrane binding, would restrict activation of cytohesins to pm or endosomal membranes. these results illustrate the complexity of arno activation and shed light on this long-standing debate over the physiological substrate of cytohesins in cells. the fact that arf -gtp can activate cytoshesin/arno, that the capacity to activate this gef is highly dependent on relative levels of cytohesin/arno and effectors, and that both arf and arf positive feedback loops exist, all need to be taken into consideration in evaluating in vivo results. arf is required for specific processes at the pm such as recruitment of proteins to focal adhesions and in phagocytosis, and arf -gtp localizes to these sites (beemiller et al. ; furman et al. ; kruljac-letunic et al. ; norman et al. ). in the forming phagocytic cup, arf -gtp is recruited early, followed by arf -gtp, at a stage that requires rapid insertion of new membrane. these results support the idea that the arf -cytohesin-arf cascade may play an important role in processes that require a high level of arf protein. arf is less abundant than arf in cells, and since both arf and arf can recruit effectors such as pi p kinase and pld, processes requiring an acute activation of such effectors may rely on the more abundant arf to provide an adequate supply. another process in which this mechanism may operate is in the insulin signalling pathway, where both arf and arf were shown to contribute to activation of pi p kinase and pld by cytohesin- /arno (lim et al. ). the implication of arf proteins and their gefs and gaps in human pathologies is a rapidly expanding area. arfs and their regulators have been linked to neurodevelopmental disorders, neurodegenerative diseases, cancers, and both viral and bacterial infections. here, i will describe examples of their roles in human disorders, and the reader is referred to other sources for a more comprehensive description of specific topics in this growing field (dani et al. ; lovrecic et al. ; poincloux et al. ; sabe et al. sabe et al. , seixas et al. ; stafa et al. ; tan and evin ) . mutations in the big arf gef have been linked to autosomal recessive periventricular heterotopia (arph), a disorder of neuronal migration that leads to severe malformation of the cerebral cortex (microcephaly) and developmental delay (sheen et al. ) . two mutations in big have been identified in arph patients, one of which is a frame shift mutation that results in truncation of the majority of the protein (sheen et al. ). the disease symptoms are a result of the failure of a specific class of neurons to migrate from their point of origin in the lateral ventricular proliferative zone to the cerebral cortex (ferland et al. ; sheen et al. ). this defect arises from a defect in vesicular trafficking that alters the adhesion properties of these neurons (ferland et al. ). in addition to big , mutations in the gene encoding filamin a also cause arph. a recent study has found a mechanistic connection between these two proteins in a mouse model of the disease (zhang et al. ) . the iqsec/brag arf gefs are highly expressed in the postsynaptic density of the central nervous system (casanova ) , and play important roles in signalling during synaptic transmission (myers et al. ). this family of arf gefs use arf as a substrate, but can also act on arf (moravec et al. ) . brag /iqsec is mutated in x-linked nonsyndromic intellectual disability, a form of mental retardation. three point mutations isolated from patients map to the sec domain and result in proteins that cannot activate arf normally (shoubridge et al. a, b) . brag has been linked to alterations in synaptic content during long-term depression (ltd). signalling through ampa-type glutamate receptors facilitates ltd, and donwregulation of activated ampa receptors is normally regulated by ampa receptor-mediated recruitment of brag , which in turn activates arf and endocytosis (scholz et al. ) . the gefs and gaps for arf and arf at the cell periphery have roles which are becoming more clearly defined in the progression of numerous cancers (d' souza-schorey and chavrier ; muller et al. ; peng et al. ; sabe et al. sabe et al. , sangar et al. ; yoo et al. ) . cytohesin/arno gefs affect signalling through epidermal growth factor (egf)/erbb, promoting conformational changes that increase trans-phosphorylation of egfrs upon ligand-induced dimerization (bill et al. ) . importantly, these gefs are specifically inhibited by secinh , which holds promise as anti-cancer drug, since treatment of an egf receptor-dependent lung cancer cell line with secinh resulted in reduced proliferation (bill et al. ) . arf proteins and their regulators are hijacked by numerous bacterial and viral pathogens (dautry-varsat et al. ; goody and itzen ; hsu et al. ; humphreys et al. ; matto et al. ) . the arf gef gbf is required for the replication of numerous viruses, including enteroviruses, hepatitis c virus, and coronaviruses (belov et al. ; goueslain et al. ; lanke et al. ; winchester et al. ) . these viruses remodel er and early secretory pathway membranes to form replication complexes, for which they subvert the function of gbf . interestingly, a major requirement for replication of several of these viruses is gaining control of host lipid trafficking and metabolism pathways (alvisi et al. ; hsu et al. ; ilnytska et al. ). the activity of arf proteins is regulated in a spatiotemporal manner by their gefs and gaps, highlighting the importance of precise localization of these regulators. the mechanisms of this spatiotemporal control are now beginning to emerge. coincidence detection mechanisms involving binding to specific lipids and protein partners play an important role, and for the cytohesins, a fairly complete description of how relief of autoinhibition is coupled to precise spatial cues has been obtained. arf activation cascades have been demonstrated for the cytohesins, but may play an important role in the activation of other families of gefs as well. such cascades, similarly to those demonstrated for rab g proteins, could be involved in transforming one membrane domain into another during highly dynamic membrane trafficking maturation events. these transformations involve coordinate changes in both the lipid and protein composition of each membrane domain, a specific function of the arf family members, which recruit both lipid-modifying enzymes and protein effectors such as coats and tethers. the signature feature of arf family proteins, their n-terminal membrane-binding amphipathic helix, ensures that they are closely associated with the lipid bilayer in their gtp-bound form. future studies on how arf family proteins function will therefore continue to require in vitro reconstitution on model membranes. among the small g proteins, the arf family is perhaps the most enigmatic. why so few? what is the unifying principle in the broad range of functions defined for each one? the small number of arf proteins in a given eukaryotic cell is an ancient property of the arf proteins, since phylogenetic analyses indicate that the last common ancestor of the eukaryotes had only one arf protein, despite a significant level of complexity, and numerous distinct arf regulators and effectors (copi, trans-golgi clathrin-adaptor proteins, coiled-coil tether, and phospholipase d). one obvious consequence of having a single protein with different functions is that the various processes can be integrated in a simple manner. for example, the single pool of arf must be distributed among all of the different gef, gap, and effector complexes in the cell, which could provide a type of global regulation of all of the functions that these different complexes carry out. in particular, arf is involved in the recruitment of coat complexes and membrane tethers at the golgi that mediate vesicular trafficking, and also in lipid droplet metabolism and in recruitment of at least three lipid transfer proteins that mediate non-vesicular lipid trafficking in cells. hence a fundamental role of the arf proteins may be in coordinating both vesicular and non-vesicular lipid trafficking pathways in the cell. the capping domain in ralf regulates effector functions hepatitis c virus and host cell lipids: an intimate connection the structure of ralf, an adp-ribosylation factor guanine nucleotide exchange factor from legionella pneumophila, reveals the presence of a cap over the active site n-terminal hydrophobic residues of the g-protein adp-ribosylation factor- insert into membrane phospholipids upon gdp to gtp exchange arf and microtubules in adhesion-dependent trafficking of lipid rafts the copi system: molecular mechanisms and function a phosphatidylinositol- -kinase-dependent signal transition regulates arf and arf during fcgamma receptor-mediated phagocytosis copi complex is a regulator of lipid homeostasis a critical role of a cellular membrane traffic protein in poliovirus rna replication adp ribosylation factors and and group via phospholipase a( ) regulate morphology and intraorganellar traffic in the endoplasmic reticulum-golgi intermediate compartment curvature, lipid packing, and electrostatics of membrane organelles: defining cellular territories in determining specificity lipid packing sensed by arfgap couples copi coat disassembly to membrane bilayer curvature arfgap responds to membrane curvature through the folding of a lipid packing sensor motif cytohesins are cytoplasmic erbb receptor activators coat proteins: shaping membrane transport targeting of the arf-gef gbf to lipid droplets and golgi membranes regulation of ras signaling dynamics by sos-mediated positive feedback adp-ribosylation factor, a small gtp-dependent regulatory protein, stimulates phospholipase d activity bilaterally cleft lip and bilateral thumb polydactyly with triphalangeal component in a patient with two de novo deletions of hsa q and q involving pdgfc, gria , and fbxo genes regulation of arf activation: the sec family of guanine nucleotide exchange factors a human exchange factor for arf contains sec -and pleckstrin-homology domains toward a structural understanding of arf family:effector specificity syt p promotes activation of arl p at the late golgi to recruit imh p characterization of class i and ii adp-ribosylation factors (arfs) in live cells: gdp-bound class ii arfs associate with the er-golgi intermediate compartment independently of gbf unfolded protein response and cell death after depletion of brefeldin a-inhibited guanine nucleotideexchange protein gbf phospholipase d: a downstream effector of arf in granulocytes active arf recruits arno/cytohesin gefs to the pm by binding their ph domains lipid droplet binding and oligomerization properties of the parkinson's disease protein alpha-synuclein phylogenetic analysis of sec -domaincontaining arf nucleotide exchangers the arf gtpase-activating protein: zinc finger motif and golgi complex localization glycosphingolipid synthesis requires fapp transfer of glucosylceramide evolution of the eukaryotic membrane-trafficking system: origin, tempo and mode adp-ribosylated proteins as old and new drug targets for anticancer therapy: the example of arf recent insights into the mechanisms of chlamydia entry pi-loting membrane traffic protein-lipid interactions in membrane trafficking at the golgi complex a copi coat subunit interacts directly with an early-golgi localized arf exchange factor rhodopsin c terminus, the site of mutations causing retinal disease, regulates trafficking by binding to adp-ribosylation factor (arf ) structural basis and mechanism of autoregulation in -phosphoinositide-dependent grp family arf gtpase exchange factors arf family g proteins and their regulators: roles in membrane transport, development and disease arf proteins: roles in membrane traffic and beyond lipid droplets in lipogenesis and lipolysis differential membrane association properties and regulation of class i and class ii arfs spermatocyte cytokinesis requires rapid membrane addition mediated by arf on central spindle recycling endosomes interaction between the triglyceride lipase atgl and the arf activator gbf tbc d , an arf -interacting protein, is mutated in familial infantile myoclonic epilepsy disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia a novel membrane sensor controls the localization and arfgef activity of bacterial ralf myristoylation of adp-ribosylation factor facilitates nucleotide exchange at physiological mg + levels efa , a sec domaincontaining exchange factor for arf , coordinates membrane recycling and actin cytoskeleton organization localization of components involved in protein transport and processing through the yeast golgi apparatus adp-ribosylation factor arf p may function as a molecular switch of new end take off in fission yeast def- /asap is a gtpase-activating protein (gap) for arf that enhances cell motility through a gap-dependent mechanism the auxilin-like phosphoprotein swa p is required for clathrin function in yeast genes encoding adp-ribosylation factors in arabidopsis thaliana l. heyn.; genome analysis and antisense suppression identification of a guanine nucleotide exchange factor for arf , the yeast orthologue of mammalian arf the small g proteins of the arf family and their regulators structural basis for activation of arf gtpase: mechanisms of guanine nucleotide exchange and gtp-myristoyl switching modulation of small gtpases by legionella identification of gbf as a cellular factor required for hepatitis c virus rna replication pathways and mechanisms of endocytic recycling functional genomic screen reveals genes involved in lipid-droplet formation and utilization analysis of gtpaseactivating proteins: rab and rab are key rabs required to maintain a functional golgi complex in human cells the arl family of small g proteins can recruit the cytohesin arf exchange factors to the plasma membrane phosphatidylinositol -phosphate -kinase alpha is a downstream effector of the small g protein arf in membrane ruffle formation targeting of arf- to the early golgi by membrin, an er-golgi snare phospholipid-and gtp-dependent activation of cholera toxin and phospholipase d by human adp-ribosylation factor-like protein (harl ) viral reorganization of the secretory pathway generates distinct organelles for rna replication role for arf p in development of polarity, but not endocytosis, in saccharomyces cerevisiae arf coordinates actin assembly through the wave complex, a mechanism usurped by salmonella to invade host cells enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication arf gaps and their interacting proteins the structural basis of arf effector specificity: the crystal structure of arf in a complex with jip redundant roles of big and big , guanine-nucleotide exchange factors for adp-ribosylation factors in membrane traffic between the trans-golgi network and endosomes a large-scale conformational change couples membrane recruitment to cargo binding in the ap clathrin adaptor complex the protein cofactor necessary for adp-ribosylation of gs by cholera toxin is itself a gtp binding protein mutational analysis of saccharomyces cerevisiae arf structural biology of arf and rab gtpases' effector recruitment and specificity brefeldin a: insights into the control of membrane traffic and organelle structure discrete determinants in arfgap / conferring golgi localization and regulation by the copi coat guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction arf recruits the rac gef kalirin to the plasma membrane facilitating rac activation molecular paleontology and complexity in the last eukaryotic common ancestor the tyrosine kinase pyk regulates arf activity by phosphorylation and inhibition of the arf-gtpase-activating protein asap gbf , a guanine nucleotide exchange factor for arf, is crucial for coxsackievirus b rna replication bi-directional protein transport between the er and golgi aspergillus nidulans arfb plays a role in endocytosis and polarized growth non-vesicular lipid transport by lipid-transfer proteins and beyond inter-organelle membrane contact sites: through a glass, darkly endoplasmic reticulum: one continuous network compartmentalized by extrinsic cues functional genomic analysis of the adp-ribosylation factor family of gtpases: phylogeny among diverse eukaryotes and function in c. elegans arl d recruits cytohesin- /arno to modulate actin remodeling the cnk scaffold binds cytohesins and promotes insulin pathway signaling dynamic structure of membrane-anchored arf*gtp role of pat proteins in lipid metabolism adp-ribosylation factor guanine nucleotideexchange factor (arfgef ): a new potential biomarker in huntington's disease specificities for the small g proteins arf and arf of the guanine nucleotide exchange factors arno and efa structural basis for membrane recruitment and allosteric activation of cytohesin family arf gtpase exchange factors distinct functions for arf guanine nucleotide exchange factors at the golgi complex: gbf and bigs are required for assembly and maintenance of the golgi stack and trans-golgi network, respectively arf is activated uniquely at the trans-golgi network by brefeldin a-inhibited guanine nucleotide exchange factors the tbc (tre- /bub /cdc ) domain protein tre regulates plasma membrane-endosomal trafficking through activation of arf role for adp ribosylation factor in the regulation of hepatitis c virus replication ciliary targeting motif vxpx directs assembly of a trafficking module through arf two lipid-packing sensor motifs contribute to the sensitivity of arfgap to membrane curvature a four-step cycle driven by pi( )p hydrolysis directs sterol/pi( )p exchange by the er-golgi tether osbp arf interacts with jip to control a motor switch mechanism regulating endosome traffic in cytokinesis decoupling of activation and effector binding underlies arf priming of fast endocytic recycling brag /gep /iqsec interacts with clathrin and regulates alpha beta integrin endocytosis through activation of adp ribosylation factor (arf ) asap promotes tumor cell motility and invasiveness, stimulates metastasis formation in vivo, and correlates with poor survival in colorectal cancer patients arf -gef brag regulates jnk-mediated synaptic removal of glua -containing ampa receptors: a new mechanism for nonsyndromic x-linked mental disorder a bacterial guanine nucleotide exchange factor activates arf on legionella phagosomes arf and arf regulate recycling endosomal morphology and retrograde transport from endosomes to the golgi apparatus arf mediates paxillin recruitment to focal adhesions and potentiates rho-stimulated stress fiber formation in intact and permeabilized swiss t fibroblasts cdc localization and cell polarity depend on membrane traffic the small g-protein arf gtp recruits the ap- adaptor complex to membranes mat b-git interplay activates mek / erk and to induce growth in human liver and colon cancer nucleotide exchange on arf mediated by yeast gea protein the arf exchange factors gea p and gea p regulate golgi structure and function in yeast matrix invasion by tumour cells: a focus on mt -mmp trafficking to invadopodia several adp-ribosylation factor (arf) isoforms support copi vesicle formation arf regulates angiotensin ii type receptor endocytosis by controlling the recruitment of ap- and clathrin trypanosoma brucei arf plays a central role in endocytosis and golgi-lysosome trafficking a creb -arf signalling pathway mediates the response to golgi stress and susceptibility to pathogens structural basis for recruitment and activation of the ap- clathrin adaptor complex by arf the sec arf-gef is recruited to the trans-golgi network by positive feedback protein sorting by transport vesicles arfgap family proteins in cell adhesion, migration and tumor invasion the egfr-gep -arf -amap signaling pathway specific to breast cancer invasion and metastasis interaction of calciumdependent activator protein for secretion (caps ) with the class ii adp-ribosylation factor small gtpases is required for dense-core vesicle trafficking in the trans-golgi network involvement of small arfgap (smap ), a novel arf -specific gtpase-activating protein, in microsatellite instability oncogenesis ancient complexity, opisthokont plasticity, and discovery of the th subfamily of arf gap proteins requirements for cholera toxin-dependent adp-ribosylation of the purified regulatory component of adenylate cyclase ampa receptor signaling through brag and arf critical for long-term synaptic depression arf -mediated endocytic recycling impacts cell movement, cell division and lipid homeostasis rab and arf proteins in genetic diseases mutations in arfgef implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex mutations in the guanine nucleotide exchange factor gene iqsec cause nonsyndromic intellectual disability subtle functional defects in the arf-specific guanine nucleotide exchange factor iqsec cause non-syndromic x-linked intellectual disability yeast arf p modulates plasma membrane ptdins( , )p levels to facilitate endocytosis coatomerdependent protein delivery to lipid droplets arf gaps: gatekeepers of vesicle generation gtpase activity and neuronal toxicity of parkinson's disease-associated lrrk is regulated by arfgap kinetic studies of the arf activator arno on model membranes in the presence of arf effectors suggest control by a positive feedback loop adp ribosylation factor is an essential protein in saccharomyces cerevisiae and is encoded by two genes adp-ribosylation factor is functionally and physically associated with the golgi complex er-pm connections: sites of information transfer and inter-organelle communication crucial role of the small gtpase arf in hepatic cord formation during liver development dissecting the role of the arf guanine nucleotide exchange factor gbf in golgi biogenesis and protein trafficking beta-site app-cleaving enzyme trafficking and alzheimer's disease pathogenesis the surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition copi buds -nm lipid droplets from reconstituted water-phospholipid-triacylglyceride interfaces, suggesting a tension clamp function multiple pathways regulate endocytic coat disassembly in saccharomyces cerevisiae for optimal downstream trafficking isoform-selective effects of the depletion of adp-ribosylation factors - on membrane traffic lipid droplets and cellular lipid metabolism differential roles of arfgap , arfgap , and arfgap in copi trafficking methods for a prompt and reliable laboratory diagnosis of pompe disease: report from an international consensus meeting the adaptor protein and arf gtpase-activating protein cat- /git- is required for cellular transformation a structure-based mechanism for arf -dependent recruitment of coatomer to membranes filamin a regulates neuronal migration through brefeldin a-inhibited guanine exchange factor -dependent arf activation localization of large adp-ribosylation factor-guanine nucleotide exchange factors to different golgi compartments: evidence for distinct functions in protein traffic acknowledgements work in the author's laboratory is funded by grants from the agence nationale de la recherche (anr) and the fondation pour la recherche médicale (frm) and by the cnrs, france. key: cord- -yjobthaj authors: hirschberg, carlos b. title: transporters of nucleotide sugars, nucleotide sulfate and atp in the golgi apparatus membrane: where next? date: - - journal: glycobiology doi: . /glycob/ . . sha: doc_id: cord_uid: yjobthaj nan the complete sequencing of the human genome is underway while that of s.cerevisiae and c.elegans has been completed. this and the ongoing cloning of the above golgi membrane transporters raises the challenge to determine the relationship of structural homologs of the above transporters to specific functions. do many of the golgi membrane transporters have common structural features? biochemical experiments with golgi vesicles have suggested that nucleotide sugar transporters have a putative binding motif, determined by the nucleoside base (capasso and hirschberg, ) and a putative translocation motif, which is determined by the sugar (capasso and hirschberg, ) , that is, gmp is a competitive inhibitor of gdp-fucose transport in mammals (sommers and hirschberg, ) and gdp-mannose transport in yeast (abeijon et al., ) even though the former nucleotide sugar doesn't cross the yeast golgi membrane and the latter not the mammalian one. transport of all undine nucleotide sugars is inhibited com-petitively by ump while the sugars have no effect (capasso and hirschberg, ) . ump is the antiporter for all undine containing nucleotide sugars (hirschberg and snider, ; waldman and rudnick, ; milla et al., ) ; thus, one would expect all these uridine nucleotide sugar transporters to have common structural features facing the lumenal and cytosolic side of the membrane. by analogy to the atp/adp transporter of mitochondria (klingenberg, ) , one would expect the affinity for the corresponding nucleoside monophosphate of each transporter to be higher in its lumenal recognition domain than in its cytosolic one and the opposite for the nucleotide derivative. hydrophobicity plots and different algorithms for the putative orientation of membrane proteins provide only a beginning hypothesis for the topography of such proteins; thus, the number of transmembrane spanning domains and regions, including the amino and carboxy terminus, facing the cytosol or the lumen will need to be determined directly. the quaternary structure of these transporters is important to establish: to what extent is recent evidence showing that the golgi membrane paps transporter is a homodimer (mandon et al., b) , a general feature of these transporters. recent evidence in mammals and yeast suggests that golgi membrane transporters play a regulatory role in determining which macromolecules undergo specific posttranslational modifications in the lumen of the golgi apparatus (abeijon et al., ; toma et al., ) . the supply of nucleotides and nucleotide derivatives in the golgi lumen is limiting under physiological conditions thereby allowing those reactions with low kn, values to take preference over those with higher ones. an open question is whether overexpression of transporter proteins in the membrane of the golgi apparatus will affect transport activity. can intrinsic activities of these different transporters be modulated by different effectors including cytosolic and lumenal nucleotides that are known to be competitive inhibitors of nucleotide sugar transport (capasso and hirschberg, ) ? is the expression of the different transporter proteins subject to transcriptional or translational regulation during different physiological conditions and development? detailed functional studies of these transporters will require high microgram amounts for reconstitution into liposomes. these proteoliposomes should then be useful in electrophysiological studies analogous to recent ones with cftr (bear et al., ) to address what nucleotide and phosphate species cross the membrane, what their charges are, and to study the possible existence of cotransporters coupled to the antiporters. a combination of genetics and overexpression of wild-type and mutant transporter proteins followed by reconstitution into liposomes should allow determination of structural motifs required for membrane insertion, nucleotide recognition, and specific sugar translocation. studies of the yeast gdpase, which plays a pivotal role in the antiport mechanism for gdp-mannose entry into the golgi lumen (abeijon et al., ; berninsone et al., ) , have shown that the specificity of this enzyme can be altered depending on whether ca or mn + are added to the reaction (abeijon et al, ) . to what extent may this be another regulatory mechanism of nucleotide sugar transport in mammals which use uridine and guanosine nucleotide sugars? genetic diseases affect transport of sugars and amino acids in membranes of lysosomes (gahl et al., ; rosenblatt et al, ; mancini et al., ; tietze et al., ) . are there diseases related to the above golgi membrane transporters? one would expect mammalian homozygotes in some golgi transporter mutations to be lethal, as many transporters are highly specific and gene disruptions of glycosyltransferases acting downstream from these transporters were found to be so, that is, n-acetylglucosaminyltransferase i (ioffe and stanley, ; metzler et al., ) . nevertheless, the possibility exists that during pathological conditions, where some of these transporter activities are absent or diminished, other transporters may partially compensate for lost function, that is, the udp-glcnac transporter may have some affinity, although greatly diminished, for udp-galnac transport. a combination of genetics, gene disruption, and rna antisense technology should be applied to determine the possible role of these transporters during development and differentiation. what structural features determine that these transporters become localized in the golgi apparatus and/or the endoplasmic reticulum and not another organelle? to what extent will transporters for the same solute, which are localized in different organelles, differ in structure? atp transporters occur in mitochondria (klingenberg, ) , the endoplasmic reticulum (clairmont et al., ; mayinger and meyer, ; mayinger et al., ) , and the golgi apparatus (capasso et al, ): all of them should have recognition features for atp and the putative antiporters adp or amp, as well as specific organellar targeting features. elucidations of them will be of primary importance. the sub-golgi distribution of the different transporters relative to each other and to transferases that use the same nucleotide sugar as substrate will be of importance. will there be polarization in the golgi apparatus of these transporters in the same general manner as glycosyltransferases in some cells? will there be golgi apparatuses in which there is major overlapping of these different proteins? in the case of the yeast golgi apparatus, the possibility exists that these transporters are not polarized because in many instances, this organelle consists of only one cisternae (preuss et al., ) . nevertheless, different cisternae may be enriched in individual transporters. clearly, all transporters could colocalize and still allow oligosaccharide chain specificity to proceed normally as a result of substrate specificities of glycosyltransferases. within the golgi membrane, do the transporters and the transferases exist as structural or functional complexes? radiation inactivation studies suggest that the paps transporter is not in a functional complex with any corresponding sulfotransferases (mandon et al., a,b) and that galactosyl and sialyltransferases are not in a functional complex with the transporters (fleischer et al., ) . is this a general observation? undoubtedly, studies will address the golgi targeting features of these transporters. these approaches will consist of either mutagenizing different amino acids of the transmembrane and adjacent regions of these proteins or attaching such regions to plasma membrane proteins and studying their subcellular localization (machamer, ; gleeson et al, ; colley, in press ). these studies with multitransmembrane proteins of the golgi membrane (rudolph et al, ; swift and machamer, ; antebi and fink, ) and the above nucleotide sugar transporters, by analogy to glycosyltransferases, may not always yield interpretable results. the extent to which constructs expressed in one cell line or organism will localize to the same organelle in another cell line or organism must be determined. do transporters recycle between the golgi and the er? some, as in s.cerevisiae (abeijon et al., ) , have a kkxx signal (jackson et al., ; townsley and pelham, ; schroeder et al, ) , while some in k.lactis (abeijon et al, ) do not. is this of relevance? in summary, although much has been learned about golgi transport of nucleotide sugars, paps, and atp during the past years we are clearly at the dawn of a new era. this will, hopefully, bring more investigators into this field and thereby allow a faster pace in obtaining answers to the above important questions. topography of glycosylation in yeast. characterization of gdp-mannose transport and lumenal guanosine diphosphatase activities in golgi-like vesicles guanosine diphosphatase is required for protein and sphingolipid glycosylation in the golgi lumen of saccharomyces cerevisiae a mutant yeasl deficient in golgi transport of uridine diphosphate n-acetylglucosamine molecular cloning of the golgi apparatus uridine diphosphate-n-acetylglucosamine transporter from kluyveromyces laclis the yeast ca *-atpase homologue, pmr , is required for normal golgi function and localizes in novel golgi-like distribution purification and functional reconstirution of the cystic fibrosis transmembrane conductance regulator (cftr) the golgi guanosine diphosphatase is required for transport of gdp-mannose into the lumen of saccharomyces cerevisiae golgi vesicles effect of nuclcotides on translocation of sugar nucleotides and adenosine ' phosphate ' phosphosulfate into golgi apparatus vesicles mechanism of phosphorylation in the lumen of the golgi apparatus: translocation of adenosine ' triphosphate into golgi vesicles for rat liver and mammary gland translocation of atp golgi membrane transporters into the lumen of the rough endoplasmic rcticulum-derived vesicles and its binding to lumenal proteins including bip (grp ) and grp golgi localization of glycosyltransferases: more questions than answers expression cloning of the golgi cmp sialic acid transporter target sizes of galactosyltransferase, sialyltransferase and uridinediphosphatase in the golgi apparatus of rat bver cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis targeting of proteins to the golgi apparatus topography of glycosylanon in the rough endoplasmic reticulum and golgi apparatus mice lacking n-acetylglucosaminyltransferase i activity die at mid-gestation revealing an essential role for complex or hybrid n-lmked carbohydrates identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reuculum dialectics in carrier research: the adp/atp carrier and the uncoupling protein golgi gdpmannose requires leishmania lpg : a member of a eukaryotic family of putative nucleotide-sugar transporter targeting and retention of golgi membrane proteins characterization of a proton-driven carrier for sialic acid in the lysosomal membrane purification of tbe golgi adenosine ' phosphate '-phosphosulfate transporter, a homodimer within the membrane a monomeric protein in the golgi membrane catalyzes both n-deacetylation and n-sulfation of heparan sulfate an atp transporter it required for protein translocation into the yeast endoplasmic reticulum saclp mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation complex asparagine-linked oligosaccharides are required for morphogenic events during postimplantation development reconstitution of golgi vesicle cmpsialic acid and adenosine ' phosphate ' phosphosulfate transport into proteoliposomes characterization of the saccharomyces cerevisiae golgi complex through the cell cycle by immunoelectronmicroscopy defect in vitamin b metabolism the yeast secretory pathway is perturbed by mutations in pmr , a member of a ca + atpase family the golgi-localization of yeast emp p depends on its di-lysine motif but is not affected by the retl- mutation in a-cop transport of sugar nucleotides into rat liver golgi: a new golgi marker activity a golgi retention signal in a membrane-spanning domain of coronavirus el protein defective lysosomal egress of free sialic acid (nacetylneuraminic acid) in fibroblasts of patients with infantile free sialic acid storage disease transport of udp-galactose into the golgi lumen regulates the biosynthesis of proteoglycans the kkxx signal mediates retrieval of membrane proteins from the golgi to the er in yeast eur udp-glcnac transport across the golgi membrane: electroneutral exchange for dianionic ump acknowledgments i thank c. abeijon, j. baenziger, m. milla, and e. mandon for helpful comments, and annette stratton and karen welch for excellent secretarial assistance. the work in my laboratory was supported by grants nth and from the national institute of general medical sciences. key: cord- -g zhoyo authors: mukherjee, shruti; bhattacharyya, dipita; bhunia, anirban title: host-membrane interacting interface of the sars coronavirus envelope protein: immense functional potential of c-terminal domain date: - - journal: biophys chem doi: . /j.bpc. . sha: doc_id: cord_uid: g zhoyo the envelope (e) protein in sars coronavirus (cov) is a small structural protein, incorporated as part of the envelope. a major fraction of the protein has been known to be associated with the host membranes, particularly organelles related to intracellular trafficking, prompting cov packaging and propagation. studies have elucidated the central hydrophobic transmembrane domain of the e protein being responsible for much of the viroporin activity in favor of the virus. however, newer insights into the organizational principles at the membranous compartments within the host cells suggest further complexity of the system. the lesser hydrophobic carboxylic-terminal of the protein harbors interesting amino acid sequences- suggesting at the prevalence of membrane-directed amyloidogenic properties that remains mostly elusive. these highly conserved segments indicate at several potential membrane-associated functional roles that can redefine our comprehensive understanding of the protein. this should prompt further studies in designing and characterizing of effective targeted therapeutic measures. induce global metabolic changes on infected cells, leading to the rearrangement of the lipid dynamics to facilitate viral multiplication. ( ) in some cases, these alterations produce the reorganization of intracellular membranes of the host cell, building an adequate microenvironment for viral replication. all these findings highlight the intimate connections between viruses and host lipid interactions. typical of the genus, the initial steps of sars-covs entry include the attachment of the virus particle to a cellular receptor, angiotensin-converting enzyme (ace -specific for sars-cov) located on the host plasma membrane surface. ( ) the viral genome has to take entry into the host cell to reach the replication sites. different lipids found either on the plasma membrane and/or the endosomal membranes can contribute to these processes by enabling receptor clustering, virus internalization, or membrane fusion. the host spectrum/tropism of individual covs have been known to be primarily determined by the s protein. ( ) the functional aspects of the s protein have been known to mediate receptor binding and virus-cell fusion that occur at either the interface of the plasma membrane or within the endosomal vesicles. ( ) the mechanism of membrane fusion of sars-cov- mostly belongs to the type i fusion system, where s protein of sars-cov- comprises of a number of membrane binding regions in the s domain identified as fusogenic peptide or fps by several researchers. ( ) ( ) ( ) recently bhattacharjya et. al. have shown that one of these fusion peptides adopts β-sheet conformation either in the presence of phosphatidylcholine or phosphatidylcholine/cholesterol comprising membrane models with the help of circular dichroism spectroscopy. ( ) moreover, it has also been shown that the peptide undergoes oligomerization with increasing concentration of cholesterol in the membrane. in addition to receptor binding, the action of host cell-specific proteases can likely cleave the s protein, in turn, regulating the cov infection and host-or tissue-tropism. ( ) once inside the host cellular environment, positive-stranded rna viruses such as cov compartmentalize their replication machinery efficiently within the host membranous compartments to evade detection by host pattern recognition receptors. ( ) such compartments are formed for the benefit of the virus by taking advantage of cellular pathways and lipid-modifying enzymes. ( ) after transcription within the rough endoplasmic reticulum (er), the viral membrane proteins associate with the endoplasmic reticulum-golgi intermediate compartment (or the ergic) before being folded and promoting the budding off of the viral particle by hijacking the cellular secretory mechanism.( ) a major portion of the viral e protein has been known to remain localized at the site of intracellular trafficking, viz. the ergic system, participating in cov assembly and budding. ( , ) thus, much of the lipid components of the virus particles are comparable to these host organellar membranes being rich in phosphatidylcholines, phosphatidylethanolamines and a small fraction of phosphatidylinositols. ( ) this has provided direct evidence that the host membranes can serve as crucial functional domains that are indispensable for viral propagation. thus, mechanistic insight into the functional interface of host-membranes is particularly essential to obtain a comprehensive understanding of the target conformers for effective therapeutic strategies. e protein of cov is an integral membrane protein about - residues long and has been known to be most dynamic at the membranous functional interface. ( ) the primary structural elements of the protein make it particularly interesting that delve into its possible role in viral propagation. interestingly, despite the everevolving nature of the virion particles, the primary sequence of the protein remains mostly conserved. the sars-cov- e protein bears about % sequence similarity with that of the sars-cov. this alternatively directs into understanding the uniqueness of the sequence that is conserved to mediate some crucial functional roles of the protein (figure ). the primary amino acid sequence reveals a very short n-terminal hydrophilic sequence of about ten amino acids. ( , ) this is followed by a stretch of hydrophobic sequence that serves as the transmembrane domain (tmd). this twenty-eight residue long stretch has been known for the membrane-induced topologies of the protein that have been extensively defined and studied. ( ) the absence of a canonical cleaved signal sequence suggests equal likelihood for the protein to be a type ii (with its c-terminal targeted to the er lumen) or type iii (with its n-terminal targeted to the er lumen) membrane protein. therefore, the topology of the cov e protein is highly debated in the literature and remains largely inconclusive. table summarizes the contradictory topology predicted for some of the widely studied cov strains. the predicted topologies of the sars cov- e protein using prediction servers have been represented in table . however, further complicating matters, the topology models devised using prediction programs are mostly inconsistent with the previous experimental observations. two membrane-associated topologies have been demonstrated for e protein (figure )-hairpin or transmembrane. ( , ) in a report by arbely et al. ( ) , the protein was shown to adopt a highly unusual topology, comprising of a short transmembrane helical hairpin conformation that inverses about a previously unidentified pseudo-center of symmetry. ( ) this hairpin structure could deform lipid bilayers and cause fragmentation. ( , ) it has been suggested that post-translational modifications and several intermediate stages of maturation in the er and golgi environment can modulate the overall conformation of the molecule in response to the physiochemical properties of the membrane. ( ) ( ) ( ) the specific lipid composition of the particular lipid bilayers of the er-golgi protein synthesis-packing machinery of the cell defines divergent physicochemical properties at the membranous interface. the lipid packing density has been known to underlie significant features of the resulting membrane-inducing characteristic local curvatures, surface charge, phase behavior, elasticity, hydrophobicity, degree of hydration, etc.( ) membrane phase behavior in a cell has been known to permit the transient concentration of specific proteins and lipids into dynamic nanoscopic domains. ( ) the level of palmitoylation may moderate the relative proportion of the membrane-associated conformers of the protein that again, in turn, can modulate the local membrane curvatures, facilitating further favorable association of the protein conformers. ( ) substitutions of the hydrophobic amino acid residues in the tmd of the e protein with charged amino acids have been shown to alter the migrating properties of the e protein in sds-page. ( ) this, therefore, suggested changes in the overall conformation and membrane association of the mutant forms in comparison to the wild type e protein. the tmd is followed by a lesser hydrophobic c-terminus with a fraction incorporated as part of the envelope in the virion particle ( figure ). the c-terminus, in particular, has been known to be associated with the redirection to the host er and golgi complex membranes that underlies some of the major functional domains of the protein. ( , ) comprising of more than % of the entire protein sequence, this segment has been known to influence the topology in the host cell. there are several conflicting theories that either suggests a luminal localization of the c-terminal when associated with the ergic system or directed towards the cytoplasm. ( , ) the several experimental and in-silico prediction studies have been mostly inconclusive with respect to its localization and hence functional domain in the different strains of cov. nevertheless, the amino acid sequence of the c-terminal harbors some of the answers to the many known functional attribute of the protein sequence. interestingly, it comprises of the conserved "fyxy" peptide sequence (where x= any amino acid) that can be correlated to its high amyloidogenic propensity. ( , ) also, another highly conserved cysteine containing motif-"cxxc" can enable disulfide isomerization to prompt membrane-directed conformational changes. ( ) apart from these highly conserved sequences throughout the genus, there are distinct potent glycosylation sites along the stretch that can serve as chaperone interacting motifs to help in the protein folding and/or aid in j o u r n a l p r e -p r o o f journal pre-proof trafficking along with the cellular machinery.( ) glycosylation of particular asparagine residues (asn , asn , asn , and asn ) in the sars-cov has been shown to be crucial in maintaining the proteinoligomerization events associated with the host membranes. ( ) conversely, maintaining the glycosylation events have been suggested to be critical to maintaining the broad functional roles of the protein. ( , ) further terminally, there is a distinct phosphorylation domain comprising of the "s sr " motif. additionally, a pdzbinding domain (pbm) in the c-terminal plays an essential role in host cell modifications necessary for viral infection and pathogenesis (figure ). ( , ) mutation studies with the pbm have established its importance for viral infection, making it a significant factor of research for therapeutic and vaccine designing. ( , ) . the e protein as a functional viroporin. the primary structural features of the e protein have striking similarities with the viroporin class of proteins. ( ) ( ) ( ) viroporins ideally comprise of about - amino acids, with a distinct hydrophobic transmembrane domain. they are known for their interactions with membrane surfaces, often resulting in the expansion of the lipid bilayer. ( ) viroporins participate in several viral functions, including the prompting of viral particle release from host cells. ( ) although not essential for viral replication, viroporins modulate the host cellular machinery to make it favorable for viral propagation. they affect host cellular functions, including membrane vesicularization, glycoprotein tracking, and membrane permeability. the transmembrane domain could form hydrophilic pores in the membranes of virus-infected cells by oligomerization. ( , ) these hydrophilic channels would allow low molecular weight hydrophilic molecules to cross the membrane barrier, leading to the disruption of membrane potential, collapse of ionic gradients, and release of essential compounds from the cell several studies with sars cov e protein have demonstrated the structural and functional similarities with viroporins. it has been demonstrated that the sars-cov e protein could enhance the membrane permeability of bacterial cells to o-nitrophenyl-β-d-galactopyranoside and hygromycin b, suggesting that the protein may function as a viroporin. ( ) similar observations were reported on mouse hepatitis virus e protein. ( ) the e protein, as a viroporin, is present in low copy numbers in the virus particle, but has been implicated in membrane scission being mostly associated with the ergic and cis golgi-consistent with a predominant role as a mediator of virus assembly and release at this location. ( ) the lipid content at these locations may also enhance virus budding. ( ) compartments are formed by manipulating the protein-synthesis, packaging, and distribution system of the cell involving the er-ergic-golgi complex. this helps the viral replication machinery to evade detection by host pattern recognition receptors ( figure ). both viral and hijacked host proteins are used in this process, taking advantage of cellular pathways and lipid-modifying enzymes to the benefit of the virus, sequestering newly formed rnas away from host immune sensors. ( , ) this eventually allows the virus to manipulate the normal secretory pathway of the host cellular machinery to transport and deliver the virus protein cargo at the site of final packaging, and eventual budding. ( ) the virulence of the strain is, in fact, determined by the efficiency in their mechanism to concentrate the rna synthesis machinery and subsequent packaging within these specialized compartments that are, in turn, modulated by the associated structural proteins.( ) among the structural proteins, the e protein has been particularly intriguing either alone or in collaboration with the m protein. ( ) interestingly, studies have shown that overexpression of the e protein is sufficient to induce the packaging and assembly of the cov particles upon efficient manipulation of the host cellular machinery. ( ) rottier et al. suggested that the e protein has no genuine structural function in the virion envelope itself where it occupies frequent, regular positions within the lattice built by m protein. ( ) however, its primary role lies in the functional interface of the host packaging and assembling membranes ( figure ). the frequent but strategic positions within the lattice hint at a possible morphogenetic function to generate the required membrane curvature for the final viral particle formation ( figure , inset). studies have endowed the c-terminal to be associated with the redirection of the e protein to the er, and golgi complex for assembly, budding, and intracellular trafficking of infectious virions.( ) interestingly, sequence analyses of the c-terminal residues suggest the prevalence of some degenerate er export signal sequences. these are closely comparable to the most common fxxxfxxxf( ) and the [r/k](x)[r/k]( ) dibasic motifs that have been known to be responsible for the export of glycosyltransferases. the last -residue "r dklys " stretch from the ibv e protein terminal has been defined to serve as the er retention signal. ( ) the site-directed mutagenesis of this composite lysine residue (k ) to glutamine resulted in the accumulation of e in the golgi apparatus. [ ] maturing through the golgi complex, the e protein is expected to impart some very crucial functions with respect to vesicularization events, by inducing local curvatures at the site of membrane-directed accumulation ( figure ). two principle mechanisms have been described for moving and delivering cargo proteins through the secretory pathway of the golgi complex, the cisternal maturation, and the formation of mega-vesicles. ( , ) despite the extensive studies performed, experimental evidence for both the systems remains inadequate and far from being elusive. during cov infection, virions have been observed in large vesicle depots resembling megavesicles derived from golgi/ergic membranes, indicating that remodeling of the golgi complex may be crucial for virion trafficking. ( ) understanding the structural transitions that the e protein undergoes, from after its synthesis to its association with the ergic membrane complex, would greatly facilitate the characterization of potential targets. we expect the protein to undergo stepwise dynamic structural transitions with specific roles bound to the membranous component. this gradual transition is dependent equally on both the respective membrane composition along with the conformational changes in the protein as it is translated, modified, and matures along with the er-golgi network system. previous studies performed by knoops as membrane fusion and fission to prompt viral propagation by hijacking the entire cellular machinery. however, progress in our in-depth understanding of this whole machinery has been stymied by the inability to track the functional interface of the protein at the atomic-level resolution in real-time. identifying these intermediate functional conformers could broaden our chances of finding the best target motif for therapeutic design. the cov e protein has been known for its possible pro-apoptotic viroporin activity majorly by mobilizing calcium ions, prompting an overall change in the ionic environment of the homeostatic cell. ( , ) the virus particles, generally, hijack the cellular homeostatic machinery of the host cell by compromising the membrane integrity that facilitates its regulatory mechanisms within the environment of the host cells. it has been suggested that the e protein can directly disrupt membranes through the formation of ion channels upon membrane-directed oligomerization events mediated upon the insertion of the tmd. in this context, the tmd has received much interest owing to their membrane-spanning attribute. molecular dynamic simulation studies with synthetic peptides have revealed that the tmd could form ion channels by homo-oligomerization into stable dimers, trimers, and pentamers. ) additionally, in silico prediction studies based on high-resolution nmr spectroscopic studies provided useful insight into understanding the probable conformers that define the tmd induced ion channels. hence, these studies directed the defining of therapeutics targeting the resultant ion channel pores. ( ) the hollow structure of the oligomers allows the unregulated efflux of ions from the cellular compartments across the hydrophilic interior of the pore, modulating the membrane potential, altering the ionic environment of the lumen. alterations in ion concentration would promote translation of viral mrnas, given the translation of mrnas from many cytolytic animal viruses is reasonably resistant to high sodium concentrations. ( , ) since the secretory pathway of the host cellular machinery is highly sensitive to perturbations in ionic strength, viral propagation can trigger a detrimental apoptotic cascade. unregulated ion channel formation upon e protein oligomerization in the host membrane is believed to be one of the primary processes underlying the viral pathophysiology.( ) as discussed previously, much of the studies have been focusing on the tmd for the high molecular weight conformers (multimers) of the protein upon host membrane interactions. ( , ) however, interestingly, the immediate adjacent segment (towards the c-terminal) hint, at least in part, at a possible role in the membrane directed oligomerization propensity of the molecule. upon inspection into the c-terminal segment of the protein, we can see that it is marked by the presence of unique motifs that can prompt much of the membrane-directed conformational changes of the protein molecule. the primary sequence of the c-terminal region reveals possible functional characteristics of the protein that are lesser known in literature. interestingly, the sequence is distinctive of amyloidogenic proteins and peptides that have been studied in the literature for their membrane-directed indispensable functions. highresolution structural models of amyloid oligomers have been obtained in the context of membrane interactions in neurodegenerative diseases.( ) membrane-induced oligomerization prompts membrane compromise, often leading to vesicularization into smaller lipid aggregates. ( ) ( ) ( ) studies with amyloid proteins have hinted at the possibilities of a change in the local curvature being induced by the membrane-associated high molecular weight conformers of the proteins (figure , inset).( ) several reports have suggested specific lipid domains to serve as templates for protein anchorage. this is followed by rearrangement of the protein conformers resulting in aggregates that form "wedges" within the bilayer to facilitate curvature change. ( , ) such protein aggregates are driven mostly by the hydrophobic interactions that relocate within the acyl chain region of the lipid bilayer. these interactions result in a change in the lipid packing density and hence the overall membrane integrity. ( ) the structural similarity of the c-terminal segment of the e protein to amyloid proteomics indicates at analogous purposes that can be complementary to or function in parallel to its viroporin activities. the oligomerization properties of e protein have been defined previously.( ) e protein expressed in bacterial and mammalian cell systems under reducing conditions was shown to exist as monomers. however, upon change to non-reducing conditions, they formed homodimers and homotrimers. ( ) the existing literature has focused on the phenomenon of tmd multimerization associated with the ion channel formation that lacks mechanistic details on the initiation. however, site-directed mutagenesis studies revealed that the two-cysteine residues, immediately adjacent to the tmd in the c-terminal, were essential for the oligomerization, leading to the induction of membrane permeability. the tmd is immediately followed by a "cxxc" redox motif, which is highly conserved among the coronaviridae family of viruses. this motif is very crucial for several reasons. the oxidized cxxc motif can maintain the structural topology of the transmembrane region as well as that of its contiguous cytoplasmic domain, inclusive of glycosylation sites, involved in signaling and protein-protein interaction.( ) a probable activation of a thiol in this motif can trigger the participation of the other cys residues in the formation of the inter-subunit disulfide bond. the activated thiol will then attack the disulfide and cause its isomerization into a disulfide isomer within the motif. this may lead to the refolding of the transmembrane region and may activate its fusogenic potential.( , ) interestingly, a similar cxxc motif is present as a part of the c-terminal of s protein sequence. bioinformatic studies have suggested at the possibilities of forming inter-protein disulphide bridges that might indicate at a crossover or cooperation between these two structural proteins in the viral membrane interactions. ( ) the formation of a disulfide bond may also play a crucial role in the oligomerization of the e protein, forming stable dimers, trimers, and pentamers depending on its functional requirement.( ) thus even though the tmd spans the lipid bilayer, the cxxc motif could serve as an essential key to defining the membrane-associated oligomerization events-providing newer targets for preemptive therapeutic intervention. apart from the consequential involvement of this segment in the viroporin function, the amyloidogenic segment of the e proteins can function independently at the membranous interface. further downstream, the cterminal harbors the "fyxy" composite peptide sequence ( figure ) that has been known for a high propensity of amyloid formation. ( , ) interestingly, a self-assembling short peptide segment with the composite sequence has been studied to have a membrane-mediated function. bhunia and co-workers studied a -residue long peptide stretch (tk , t vyvysrvk ) in sars cov e protein (figure ) for its physical changes in the presence of both zwitterionic and negatively charged model membrane micelles.( ) the tk peptide segment was shown to adopt amyloid fibrillary structures in solution ( figure ). ( ) further studies with tk provided evidence for its aggregation propensity that closely resembles amyloid proteins. intriguingly, the composite -residue peptide motif-t vyv in tk was found to serve as the critical sequence motif that may be critical for the beta-sheet formation. ( ) however, in vitro measures demonstrated the differential orientation of the tk peptide with respect to the membranous environment: correlating well with the structure and function at the membrane-protein interactions interface. this atomic-resolution nmr based structural elucidation in the presence of membrane mimics have improved understanding of the molecular mechanism of cov infection under physiological conditions. studies demonstrated the helical propensity of this particular segment from the c-terminal region (figure ) of the protein to impart host specificity to the composite virus. the peptide's affinity was further j o u r n a l p r e -p r o o f journal pre-proof manifested by its pronounced membrane-integrity disruption ability towards the mammalian compared to the bacterial membrane mimic-implicated in the viral pathogenesis. tk penetrated deeper into the acyl region of the neutral micelles mimicking the mammalian membrane and its intracellular organelles as opposed to the superficial conformation when in association with the bacterial model membrane mimics (figure ). this provided evidence for the host membrane-associated conformational change in the protein segments that is specific to a particular lipid composition (species specificity). this study alternatively offered useful insight into the fact that the composite lipid molecules play particularly important roles in the interaction dynamics and conformational uptake. these segment-based studies highlighted the exclusive function of the amyloidogenic segment of the e protein that acts independently of the tmd-associated oligomerization events. similarly, another short -residue peptide, nk (n ivnvslvk ) further upstream, was identified and characterized, being marked by sheet breaker-hydrophobic residues, like ile, val, and leu. ( ) the experimental intervention into these segments can provide useful insight into the topology of the protein, its membrane-bound orientation, and eventual functional conformation uptake. these preliminary studies have provided useful links to the immense possibilities of the functional membrane-directed conformers of the e protein, which remains largely unexplored. nevertheless, epitope mapping for these interfaces can serve as critical targets for effective therapeutic intervention. preliminary sequence homology analyses performed to compare the e protein primary sequence of sars cov to that of the novel cov- , revealed a minimal difference with the "fyxy" motif remaining conserved (figure ). the first threonine and the second valine from the tk sequence have only been substituted by a serine and phenylalanine residues, respectively ( figure ). this might indicate at an unperturbed oligomerization potency of the segment. this conserved amyloidogenic sequence indicates a very crucial functional role of the segment that contributes significantly to the virulence. the fact that hydrophobic interactions can provide stability in localization within the acyl-chain layer of the lipid bilayers leaves us with some obvious open questions. does the c-terminal induce the initial docking of the protein to the host membranes? do these hydrophobic interactions prompt the downstream oligomerization events to the formation of the membrane-associated multimers? alternatively, does the lipid-composition specificity of the c-terminal determine the conformational uptake and the eventual functional topology? these questions are vital for a comprehensive understanding of viral propagation. interestingly, hydrophobic protection of these segments within the acyl-chain region can serve as open templates for downstream aggregation. this can result in membrane-associated aggregates that, in analogy to the amyloidogenic proteins, can modulate the overall membrane integrity. thus, it is essential to study the interface of interaction between the e protein and the host membrane, either as whole or directed to particular functional peptide segments. one of the major functional aspects of cov e protein has been associated with virus assembly and its subsequent release for propagation in the host physiology. indirect immunofluorescence microscopy showed that e protein is localized to the golgi complex in cells transiently expressing ibv e.( ) when co-expressed with ibv m, both from cdna and in ibv infection, the two proteins are colocalized in golgi membranes, near the cov budding site.( ) thus, even though ibv e is present at low levels in virions, it is apparently expressed at high levels in infected cells near the site of virus assembly. the subcellular localization of the sars cov e protein was analyzed using the sera from immunized mice by elisa and immunofluorescence using cells j o u r n a l p r e -p r o o f infected with recombinant (rsars-cov) and viruses lacking the e gene (rsars-cov-∆e) as a negative control. ( ) several parallel studies have endowed the c-terminal to be associated with the redirection of the e protein to the er, and golgi complex for the intracellular trafficking of infectious virions. ( ) experimental studies have shown that the c-terminal domain of the e protein, in fact, plays a crucial role in virus budding. ( , ) the deletion of the domain resulted in the free distribution of the mutant protein and a dysfunctional viral assembly. alternatively, mutations introduced into the cytoplasmic tail of mhv e protein by targeted rna recombination resulted in elongated virions, further indicating at the critical involvement of the domain in the budding mechanism. ( ) the difference in the lipid composition along the secretory pathway might, in fact, have a direct role on the e protein topology. moving from the er to the golgi and finally the plasma membrane, there is a definite gradient in the concentration of specific lipid components that induce a unique physical property of the lipidbilayers in terms of thickness and rigidity. ( ) the er presents a lower fraction of cholesterol or sphingolipids that allows maintenance of a relatively fluid lipid compartments for the remodeling associated with the protein association, sorting and accumulation. ( ) this fluidity can have a direct role in the gradual compartmentalization of the er-associated membranes prompting viral particle formations. the host membrane-mediated oligomerization of the e protein may be responsible for much of the membrane compromise in favor of the viral propagation. the membrane-associated aggregates can compromise the overall lipid bilayer integrity. ( , ) the hydrophobic interactions involving the lipid acyl regions can result in increased fluidity of the membrane surface, prompting phase separation of the lipid domains. biomolecular phase separation is suggested to drive the organization of cellular organelles involved in cellular packaging and compartmentalization.( ) differential distribution of the membrane proteins and their site of interaction with specific lipid rafts, help determine the local curvature and hence characterize propensity to bud off from the surface. ( , ) the membranous compartments of er and the golgi complex presents with a fair fraction of zwitterionic and neutral lipids-i.e., phosphatidylcholine and phosphatidylethanolamines. ( ) in vitro studies by khattari et al have demonstrated that the membrane-directed topology of the sars cov e protein imposes a direct constraint on the lipid-bilayer thickness and the acyl-chain ordering. ( ) their studies suggested that the increasing protein concentrations in the organellar membrane along the secretory pathway (greater protein/lipid concentrations), the tmd of the sars cov e protein induces bilayer lipid arrangements. specific inter-molecular interactions with the lipid molecules and intra-chain hydrogen bonds and salt-bridge interactions allow the uptake of a topology that traverses across the membrane bilayer. however, the exact transmembrane topology of the protein was highly debated and remains elusive.( ) nevertheless, these intra-and intermolecular interactions can be considered to have the ability to rearrange and induce direct morphological changes in the host membranes. the final viral packaging requires assembling of the replicated rna strands and the other viral proteins before the final membrane budding. the putative transmembrane domains of the e protein can also serve a 'catalytic' function in the membrane packaging. ( ) cross-talk between distant protein molecules can, in fact, serve as a "zipper" to close the 'neck' of the viral particle as it pinches off from the membrane in the terminal phase of budding. ( , ) despite the immense possibilities, much of the progress in our understanding of the actual mechanism of action is still hypothetical and far from being elusive. it is, therefore, essential to study the interface of interaction between the e protein and the replication complex. particularly, the membrane-mediated aggregation, that compromises the local curvature, inducing the final scission. cov gained particular notoriety when the sars cov outbreak shook the world around - . the aftermath leads to the identification of many newer family members. soon after that, studies were in full swing all around the globe to determine the mechanism of viral propagation. several parallel studies provided useful insight into the understanding of the structural and functional uniqueness of the viral proteins and rna in their search for finding the ideal target for therapeutic intervention. almost immediately after sars, the endemic spread of the mers-cov in reignited the urge to gain in-depth insights into the system all over the world for designing effective therapeutic measures. the recent outbreak of the novel covid- soon turned into a pandemic, threatening health standards globally. lu et al. first published the genomic characterization of covid- and also reported it to be significantly divergent from sars-cov, claiming it to be a new human-infecting βcoronavirus. ( ) a comparative analysis of small membrane/ envelope protein of different coronaviruses could provide an interpretation of the molecular events that could advance these viruses from developing acute infections to one producing a pandemic. an analysis of the sequence conservation can also help in identifying the regions required in the typical functions of membrane interaction, oligomerization, localization in the host cell, and viral infection. such information is of immediate significance and would contribute to vaccine designing and facilitate the evaluation of vaccine candidate immunogenicity. additionally, this would help in reflecting the potential effects of mutational events as the virus is transmitted through the human population. recent studies have revealed that the homology with a cov strain isolated from pangolin was ~ %. this indicated that while sars-cov- have been known to evolve from the bat cov, pangolins might have helped as an intermediate host. ( ) nevertheless, further research is required to justify these theories. we performed a blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) search for the e protein and selected the sequences from the different genus of the coronaviridae family. homology analyses and sequence alignment were conducted using the mega software. the muscle program of the mega software was used to perform multiple sequence alignment, and a phylogenetic tree was created by using a maximum likelihood approach ( figure ). ( ) predictive tools such as tmhmm ( ), memsat( ), phobius ( ) , and the hydrophobic moment plot method( ) of eisenberg and co-workers allowed the calculation of the transmembrane regions. much has been defined to correlate the structural conformation of the tmd with its ion channel formation. however, sequence analyses of the c-terminal have revealed crucial information that has been out of the limelight for several years. despite the fact that the c-terminal has shown the potential to underlie much of the protein's dynamic functionality associated with membrane budding and scission, it has received much lesser attention. several secondary structure prediction programs predicted two β-strands within the sars-cov e protein tail sequence, including the n ivnvslvk and the t vyvysrvk . the predicted β-strands fit the criteria for forming a β-hairpin, which is a simple structural motif with two β-strands linked by a short loop of two to five amino acid residues. interestingly, a highly conserved proline residue, pro , resides between the predicted βstrands-responsible for the β-coil-β motif. ( ) the antiparallel β-strands form hydrogen bonds to stabilize the hairpin structure. previous reports with the synthetic peptide fragment of e (i.e., i -s ), encompassing the predicted β-hairpin segment, was found to produce ∼ % β-structure and was completely resistant to hydrogen-deuterium exchange in fourier transform experiments. alternatively, the study showed that titration with the drug molecule, hma ( -(n, n-hexamethylene) amiloride) resulted in large perturbations of the val and leu residues, which are far apart in the sequence. the structural model proposed by jaume torres's group, therefore, suggested that these two residues were coming spatially close in the membrane-associated pentamer and might belong to different monomers. comparison with mutation-based data hypothesized this β structure to be in dynamic equilibrium with an α-helical intermediate form. in fact, a delicate balance between j o u r n a l p r e -p r o o f these two forms may alter the membrane-dynamics and subsequent processes in the infected cell (e.g., membrane scission, binding to protein partners, or e protein localization). disturbing this predicted β-strand region along with the substitution of the conserved proline residues had resulted in the disruption of the cytoplasmic golgi complex signal, consequently changing the subcellular localization of e protein. ( ) taken together, the results support the hypothesis that the three-dimensional structure of e protein and not the primary sequence dictates much of its function. the sum of amino acid substitutions per site from between sequences is presented in figure . analyses were performed using the poisson correction model. the estimated value of the shape parameter for the discrete gamma distribution is ~ . , suggesting that the occurrence of the conserved amino acid residues among the coronavirus family e protein is not entirely random. however, upon close inspection with other e protein sequences of different species of the same genus, the value decreased to ~ . . substitution patterns and rates were estimated under the jones-taylor-thornton ( ) model (+g). ( , ) the model evolutionary rate differences among sites were obtained using a discrete gamma distribution ( categories, [+g]). all the ambiguous positions were eliminated for each of the sequence pairs (pairwise deletion option). evolutionary analyses were conducted in mega x. ( ) collectively, the data reveals that sites observed to be constant among sequences might have crucial biological significance correlated to their function. besides, deleterious mutations are much more likely to be found in population-level data. this indicates that sites that would predictably be considered as 'invariable' at the phylogenetic level might have transient polymorphisms at the population level. in the analyses of the e protein sequences from the cornaviridae family, altering the number of gamma categories did not have any noticeable effect on the estimate of the substitution rate. cov e protein has received lesser attention when compared to the other structural proteins (like s and m) majorly owing to a very low copy number in the viral envelope. ( , , ) it is the smallest and yet has been the most puzzling of the major structural proteins. earlier deletion based studies had proven that the viral life cycle endures the absence of e protein, implying that other viral genes could counterbalance for its loss. ( ) however, recent evidence collected with recombinant covs missing e protein, exhibit considerably lowered viral titers, crippled viral development, or produce progeny incapable of further propagation. ( , ) several parallel studies have highlighted the crucial role played by the e and m proteins in the propagation of the viral genome into the host cell and virulence. remarkably, studies have reported that viruses produced by the deletion of the sars-cov e gene can be attenuated in at least three different animal models that conferred protection against sars-cov pathogenesis. ( , ) but, the lack of complete information and a limited number of findings have prevented an understanding of the exact mechanism underlying the definite functional role of e protein in viral infection. nevertheless, these have provided the basis of understanding that the cov e might be involved in several aspects of the viral replication cycle, including host cell responses such as apoptosis, inflammation, and even autophagy in association with other nonstructural viral proteins.( ) soon after the identification, studies have been in full swing all around the globe to determine the mechanism of the viroporin action. several parallel studies from all over provided useful insight into understanding the structural and functional uniqueness of the viral protein in their search for finding the absolute target for therapeutic intervention. extensive studies performed in prof. torres's laboratory provide useful insight into the structural conformation of the ion channel pores created by e protein that seems to be a very attractive target for therapeutic intervention. ( ) their studies have prompted the designing and characterization of specific inhibitors for these channels that would perturb the primary functional attribute of the protein. hexamethylene amiloride has been studied to have blocked this e protein-associated ion channel activity in the mammalian cells expressing sars-j o u r n a l p r e -p r o o f cov e protein. ( ) however, viroporin proteins, like the sars-cov e protein, can exhibit a multitude of diverse functions unrelated to their ion-channel properties that are yet to be defined. the dynamic functional interfaces of the protein have been underestimated for its functionality in the propagation. this study can be correlated with earlier docking screening by gupta et al., where more than phytochemicals were evaluated on the same protein, and three of those, i.e., belachinal, macaflavanone e, and vibsanol b were found to be particularly promising. ( ) based on the experimental evidences, it is worth mentioning that the two hits, i.e., glycyrrhizic acid and cepharanthine, are supporting their significant activity against the sars viruses. ( ) more recently, anatoly chernyshev has established the potential of the sars-cov- e protein as a pharmaceutical target. ( , ) these studies have defined approved drugs, which theoretically could block the e protein-induced ion channel and eventually hinder the virus's life cycle. ( ) however, these studies have been based on in silico approaches, and hence much of the drug candidates have to be subjected to further experimental investigations. considering the lengthy procedure of new drug development, the existing strategy of drug repurposing has turned into one of the preferred solutions for the immediate treatment of sars-cov- affected individuals. ( ) long-term drug development objectives for the pharmaceutical industry incorporate the identification of inhibitors targeted at the replication or infection routes associated with sars-cov- or other allied coronavirus infections. ( ) knowledge of the coronavirus structural proteins and their mechanisms of viral action can be crucial in defining alternative therapeutic targets. despite the extensive research efforts worldwide, a definite cure to target cov propagation is still elusive. effective therapeutic intervention into viral systems without compromising the host cell integrity requires an in-depth understanding of the specific functional interface. especially, with the ever-evolving viral components, as seen in the covid- specimen, immense multidimensional research is the need for the hour. among the structural proteins, the e protein have not been a convincing choice for therapeutic targeting, despite the evidence for a crucial viroporin activity. much of the knowledge has remained restricted to only its ion channel formation ability. although this might be serving as a significant functionality, it is definitely not the comprehensive representation of the immense potential that the protein is possibly capable of undertaking. we believe, in-depth understanding of its functional interface with the host-membranes can undermine newer possibilities and functional attributes that have not received the deserved attention so far. attempts to determine the crucial protein domains associated with the host membranes are innovative in terms of targeted therapeutic designing. several studies have been gradually exemplifying the protein's essential role in viral packaging and propagation. the conserved sequences in the c-terminal region directly prompt the understanding of evolutionarily crucial functional roles of this segment that is yet to be conclusively defined. the knowledge is incomplete, essential insight into the protein's functional interface should provide newer insight into understanding and structure-function correlation. this should enable the effective designing of targeted therapeutics. the entire portrayals of biologics with characters suitable for inhibiting several key cov proteins could serve as a basis for drug development. j o u r n a l p r e -p r o o f infected cell table : list of predicted topologies of sars cov- envelope protein using in silico prediction servers. alternatively, the segment has been studied for the differential membrane-directed functioning. the peptide undergoes a helical conformation when in association with membranous environments. comparison between bacterial and mammalian model membrane mimicking systems showed the different orientation of the peptide. this suggested the specific functional role of this peptide segment and its membrane-directed structural change. preliminary studies with the ty peptide segment (underlined) had also shown the significance of the "fyxy" sequence, characteristic of amyloid proteins, and peptides. ( , ) the figure was prepared with https://biorender.com. j o u r n a l p r e -p r o o f highlights:  the sars cov envelope protein is a small structural protein of the virus that has been suggested to have significant viroporin like activity.  majority of its function is mediated at the interface of host-membrane interactions j o u r n a l p r e -p r o o f  focus at the membrane-directed features of the protein provide useful insight into gaining mechanistic insight into its viroporin functions.  studies have elaborated the central hydrophobic transmembrane domain of e protein, known to affect ion-channel formation.  the c-terminal region of the protein show further potential host-membrane directed functional roles.  the highly conserved amyloidogenic amino acid stretches of the c-terminal suggest at significant contribution to cov propagation. sars-cov- : an emerging coronavirus that causes a global threat world health organization declares global emergency: a review of the 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atpase involved in membrane remodelling a hinge in the distal end of the pacsin f-bar domain may contribute to membrane-curvature sensing membrane curvature at a glance constitutively oxidized cxxc motifs within the cd heterodimeric ectodomains of the t cell receptor complex enforce the conformation of juxtaposed segments furin cleavage potentiates the membrane fusion-controlling intersubunit disulfide bond isomerization activity of leukemia virus env intersubunit disulfide isomerization controls membrane fusion of human t-cell leukemia virus env formation of transitory intrachain and interchain disulfide bonds accompanies the folding and oligomerization of simian virus vp in the cytoplasm assessing the role of aromatic residues in the amyloid aggregation of human muscle acylphosphatase a possible role for pi-stacking in the self-assembly of amyloid fibrils infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles physical interaction between the membrane (m) and envelope (e) proteins of the coronavirus avian infectious bronchitis virus (ibv) analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes membrane lipid composition: effect on membrane and organelle structure, function and compartmentalization and therapeutic avenues protein aggregation in a membrane environment protein aggregation and membrane lipid modifications under lactic acid stress in wild type and opi deleted saccharomyces cerevisiae strains biological phase separation: cell biology meets biophysics membrane lipids: where they are and how they behave sars coronavirus e protein in phospholipid bilayers: an x-ray study membrane topology and insertion of membrane proteins: search for topogenic signals another triple-spanning envelope protein among 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east respiratory syndrome coronavirus found in a patient with characteristic symptoms coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus coronavirus virulence genes with main focus on sars-cov envelope gene genome-wide analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication . approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel antiviral activity of cepharanthine against severe acute respiratory syndrome coronavirus in vitro drug repurposing strategies for covid- recent progress and challenges in drug development against covid- coronavirus (sars-cov- ) -an update on the status tgev corona virus orf encodes a membrane protein that is incorporated into virions key: cord- - esuj ye authors: mironov, alexander a.; pavelka, margit title: the golgi apparatus and main discoveries in the field of intracellular transport date: journal: the golgi apparatus doi: . / - - - - _ sha: doc_id: cord_uid: esuj ye in this chapter, we summarize important findings in the field of intracellular transport, which have considerably contributed to the understanding of the function and organization of the golgi apparatus (ga). it is not possible to mention all authors in this huge field. we apologize for gaps and incompleteness, and are thankful for suggestions and corrections. . the golgi apparatus and main discoveries in the field of physiology of intracellular transport discovery of the ga confirmation of the presence of the ga (dalton ) the regional distribution of the thiamine-pyrophosphatase activity within the ga (novikoff and goldfischer ) the trans er (novikoff ; novikoff et al. ) gerl concept (novikoff ) isolation of golgi membranes from cells (morr e and mollenhauer ) the process of sulphation in the ga (godman and lane ) the sugar-nucleotide transport from the cytosol to the golgi lumen across the golgi membranes, the role of the ga in glycosylation (neutra and leblond ) the origin of lysosomes and the function of clathrin-coated vesicles during protein absorption (bainton and farquhar ; friend and farquhar ) the intracellular transport (jamieson and palade a,b) galactosyltransferase as a golgi marker (whur et al. ; morr e et al. ) isolation of clathrin-coated vesicles (pearse (pearse ) the pm-to-golgi transport of the endogenously added marker (herzog and farquhar ) m p-mediated sorting of golgi enzymes at the ga (tabas and kornfeld ) clathrin-coated buds in the trans side of the ga (griffiths et al. ) immunocytochemical localization of galactosyltransferase berger ) topology of n-glycosylation (dunphy and rothman ) reconstitution of intra-golgi transport in vitro (balch et al. ) the c temperature block (saraste and kuismanen ) clathrin-independent endocytosis (moya et al. ; sandvig et al. sandvig et al. ) sandvig et al. - the mitotic form of the ga and mechanisms of mitotic golgi transformation in animal cells (featherstone et al. ; lucocq et al. lucocq et al. ) the copi-coated vesicles and characterization of molecular mechanisms involved into the function of copi coat (orci et al. ; serafini et al. serafini et al. ) the structure and function of the tgn and the c temperature block simons ) kdel-signal for the retention of luminally located proteins (munro and pelham ) bfa was applied for the study of intra-golgi transport (doms et al. ; lippincott-schwartz et al. ) snares (newman et al. ) the main genes involved in intracellular transport, the genetic evidence in favour of the vesicular model of the transport in yeast (kaiser and schekman ) a golgi retention signal in the membrane-spanning domain (swift and machamer ) the role of oligomerization for the retention of golgi enzymes (weisz et al. (weisz et al. ) the role of pm-derived signalling for intra -golgi transport (de matteis et al. golgi matrix (slusarewicz et al. ) and cis-golgin, gm (nakamura et al. ) copi-dependent retrieval sorting signals (cosson and letourneur ) gaps (all authors quoted in the consecutive chapters deserve to be listed here). the list is open for suggestions. the development of the research in the field of intracellular transport has been comprehensively discussed in at the conference in pavia devoted to the th anniversary of the golgi discovery. historically, the first mechanism that had been proposed for intracellular transport was the progression. the origin of the progression model (or the concept of cis-to-trans flow) links to grasses name ( ) who proposed that the continuous formation of cis golgi cisternae balances the conversion of trans one into secretory granules. however, the first experimental data in favour of the progression concept were obtained in (franke et al. ) . in , it has been demonstrated that proteins newly synthesized in the er appeared, after a few minutes, not only over golgi stacks but also over round profiles surrounding the ga and the conclusion that secretory proteins bypass the ga was made (jamieson and palade a,b, a,b) . then, in , the vesicular model replaced the progression model because the main support for the progression model, the cis-trans movement of scales in algae has been considered to be a rare formula connected with unusual geometry and size of the product (farquhar and palade ) . ironically, the major supporting data for the vesicular model at that time was based on the isolation of golgi-derived clathrin-coated vesicles (rothman et al. ). however, after the discovery of coat protein i (copi) (orci et al. ), the vesicular model was changed, and instead of clathrin-dependent vesicles, copi-dependent vesicles were proposed to serve as anterograde carriers. the strongest support for the vesicular model appeared from the experiments in yeast with the temperature sensitive sec genes (kaiser and schekman ). the in vitro isolation of functional (containing vsvg and able to fuse with acceptor golgi membranes) copi-coated vesicles (osterman et al. ) was interpreted as the second proof for the role of copi-coated vesicles in the anterograde intra-golgi transport. importantly, however, that the first author of this paper later stressed, that actually, these data support the cisterna maturation model (ostermann ) . on the other hand, it has also been demonstrated that min after fusion of two (or more) cells (one cell is vsv-infected, another is a non-infected cell) and formation of a heterokaryon, vsvg seems to move from the ga derived from the infected cell to the ga derived from non-infected cells . these results were interpreted as confirmation of the ability of vesicular carriers to diffuse through the cytosol of the heterokaryon from one ga to another. however, later, the rothman group (orci et al. ) laid less emphasis on the heterokaryon experiments, suggesting that those observations appeared as a result of the treatment of cells with an acidic medium. instead, the string theory was proposed, according to which a proteinaceous-like string links vesicles to cisternal elements and prevents budded vesicles from diffusing away, while still allowing them to diffuse laterally. with time, due to accumulation of contradictions, the current vesicular paradigm became less and less effective in the explanation of growing body of observations (mironov et al. ) . as a result, the original version of the vesicular paradigm began to be modified not only by the opponents of the vesicular model but also by its authors and proponents (orci et al. ) . in order to resolve accumulated contradictions within the field, almost simultaneously several groups (bannykh and balch ; mironov et al. ; glick et al. ; schekman and mellman ) have published the cisterna maturation-progression model based on the copi vesicles-mediated golgi enzyme recycling. the first experimental confirmation that large aggregated cargo, such as procollagen i, can be transported through the ga by maturation mechanism came in . previous stereological observations in p. scheffelii suggesting that their scales being much too large to be packaged into vesicles are transported by the progression of golgi cisternae towards the plasmalemma were published not in an original paper but in a review (becker et al. ) and were not confirmed later because glycoprotein and polysaccharide synthesis are uncoupled during flagella regeneration (perasso et al. ) . next, it has been demonstrated (mironov et al. ) that both diffusible and non-diffusible cargoes are transported in the same carriers through the golgi stacks. it has been proved that vesicles are not transport carriers for cargo in the intra-golgi transport not only in situ, but also in vitro, in cell-free assay (happe and weidman ) . after these publications, there was a short period when the cisterna maturation model became dominant. with time new contradictions not compatible with the cisterna maturation-progression model have accumulated (mironov et al. ). the attempts to use transport models based on combination of basic principles were not successful (see chapter . ). therefore now, there is no consensus on the models of intra-golgi transport. the existence of the maturation mechanism is almost finally established for the secretion of large polymeric structures incompatible in size with copi-dependent vesicles in many types of cells and under the infection of some viruses. jamieson jd, palade ge ( a) intracellular transport of secretory proteins in the application of gfp-technology for the study of the ga in living cells (cole et al. ) characterization of the er exit sites characterization of er-to-golgi transport carriers in living cells characterization of post-golgi transport carriers in living cells the role of endocytic tgn in the formation of the most-trans golgi cisterna tomographic reconstruction of the ga the concentration of regulatory secretory proteins within the golgi cisternae the understanding of the evolution of small gtpases had changed the model of the golgi evolution characterization of golgi-to-apical pm transport carriers in living cells characterization of the golgi-to-endosome carriers in living cells the role of gm in the maintenance of the golgi ribbon the role of er-to-golgi transport in the maintenance of the golgi ribbon origin of granules in polymorphonuclear leukocytes. two types derived from opposite faces of the golgi complex in developing granulocytes reconstitution of the transport of protein between successive compartments of the golgi measured by the coupled incorporation of n-acetylglucosamine the organization of endoplasmic reticulum export complexes membrane dynamics at the endoplasmic reticulum-golgi interface copii: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum anterograde transport of algal scales through the golgi complex is not mediated by vesicles the golgi apparatus: from discovery to contemporary studies procollagen traverses the golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation diffusional mobility of golgi proteins in membranes of living cells the golgi apparatus and main discoveries * coatomer interaction with di-lysine endoplasmic reticulum retention motif observations of the golgi substance with the electron microscope receptor and protein kinase c-mediated regulation of arf binding to the golgi complex ap- : an adaptor-like protein complex with ubiquitous expression ap- , a novel protein complex related to clathrin adaptors brefeldin a redistributes resident and itinerant golgi proteins to the endoplasmic reticulum camillo golgi and the discovery of the golgi apparatus compartmentation of asparagine-linked oligosaccharide processing in the golgi apparatus the golgi apparatus (complex)-( - )-from artifact to center stage conserved structural features of the synaptic fusion complex: snare proteins reclassified as q-and r-snares newly synthesized g protein of vesicular stomatitis virus is not transported to the golgi complex in mitotic cells synthesis and turnover of membrane proteins in rat liver: an examination of the membrane flow hypothesis functions of coated vesicles during protein absorption in the rat vas deferens a cisternal maturation mechanism can explain the asymmetry of the golgi stack on the site of sulfation in the chondrocyte golgi c ( a) intorno alla struttura della cellula nervosa sur la structure des cellules nerveuses des ganglions spinaux grasse pp ( ) ultrastructure, polarity and reproduction of golgi apparatus the role of clathrin-coated vesicles in acrosome formation the trans golgi network: sorting at the exit site of the golgi complex cell-free transport to distinct golgi cisternae is compartment specific and arf independent luminal membrane retrieved after exocytosis reaches most golgi cisternae in secretory cells kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport in living cells a c-terminal signal prevents secretion of luminal er proteins characterization of a cis-golgi matrix protein, gm radioautographic comparison of the uptake of galactose-h and glucose-h in the golgi region of various cells secreting glycoproteins or mucopolysaccharides bet , bos , and sec are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the golgi complex nucleosidediphosphatase activity in the golgi apparatus and its usefulness for cytological studies gerl, its form and function in neurons of rat spinal ganglia golgi apparatus and lysosomes the er to golgi interface is the major concentration site of secretory proteins in the exocrine pancreatic cell a new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the golgi stack vesicles on strings: morphological evidence for processive transport within the golgi stack stepwise assembly of functionally active transport vesicles stoichiometry and kinetics of transport vesicle fusion with golgi membranes endocytic routes to the golgi apparatus clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles the golgi apparatus of the scaly green flagellate scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between golgi apparatus and plasma membrane ultrastructure of long-range transport carriers moving from the trans golgi network to peripheral endosomes er-to-golgi transport visualized in living cells gm and grasp -dependent lateral cisternal fusion allows uniform golgi-enzyme distribution immunocytochemical localization of galactosyltransferase in hela cells: codistribution with thiamine pyrophosphatase in trans-golgi cisternae transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrincoated vesicles intercompartmental transport in the golgi complex is a dissociative process: facile transfer of membrane protein between two golgi populations effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin pre-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface visualization of er-to-golgi transport in living cells reveals a sequential mode of action for copii and copi does copi go both ways? adp-ribosylation factor is a subunit of the coat of golgi-derived cop-coated vesicles: a novel role for a gtp-binding protein isolation of a matrix that binds medial golgi enzymes a golgi retention signal in a membrane-spanning domain of coronavirus e protein biosynthetic intermediates of beta-glucuronidase contain high mannose oligosaccharides with blocked phosphate residues secretory traffic triggers the formation of tubular continuities across golgi sub-compartments microtubuledependent transport of secretory vesicles visualized in real time with a gfp-tagged secretory protein oligomerization of a membrane protein correlates with its retention in the golgi complex radioautographic visualization of the incorporation of galactose- h and mannose- h by rat thyroids in vitro in relation to the stages of thyroglobulin synthesis key: cord- -aqtceqqo authors: hunter, eric title: membrane insertion and transport of viral glycoproteins: a mutational analysis date: - - journal: protein transfer and organelle biogenesis doi: . /b - - - - . -x sha: doc_id: cord_uid: aqtceqqo nan the eukaryotic cell faces a continual problem of partitioning a variety of enzyme activities into different subcellular organelles where specific macromolecular reactions can occur. this subcellular organization requires specific intracellular targeting of macromolecules through the cytoplasm, by as yet ill-defined mechanisms, and through the secretory pathway via a more clearly defined vesicular transport mechanism (sabatini et al. y ) . as obligate intracellular parasites with only limited genetic complexity, viruses must utilize the existing cellular transport mechanisms to colocalize their virion components in a part of the cell where assembly can take place. this problem of intracellular targeting is compounded for the enveloped viruses because they must transport capsid polypeptides through the cytoplasm and envelope components through the secretory pathway to a common point of assembly. since these viruses depend on the preexisting host cell processes and possess lipid envelopes that are biochemically similar to cellular membranes, they can provide ideal systems for probing the cellular mechanisms involved in glycoprotein biosynthesis and transport. the relatively simple structure of virions, the high level of expression of viral genes, the ease of molecularly cloning and manipulating those genes, together with the availability of conditional lethal mutants with defects in viral protein transport, confer several additional advantages for such studies. we have chosen an enveloped, rna-containing virus, rous sarcoma virus (rsv), for an analysis of viral glycoprotein biosynthesis and transport, because in addition to the aspects delineated above, the availability of molecularly cloned, infectious dna copies of the genome of this retrovirus has also allowed us to pose questions about the role of the glycoproteins in virus assembly and virus infectivity. like most simple enveloped viruses, the retroviruses consist of a host membrane-derived, lipid bilayer that surrounds (envelopes) a protein capsid structure (fig. ) . the icosahedral capsid of rsv assembles as the virus particle buds from the plasma membrane of the cell, and so these two events are linked both temporally and spacially. glycoprotein knobbed spikes extend from the surface of the virion, and it is generally thought that during virus assembly a specific interaction between the glycoproteins and capsid (and/or "matrix" protein) is required, since cellderived polypeptides are for the most part excluded from the budding structure. the envelope glycoproteins span the lipid bilayer and are thereby divided into three distinct domains: an external, hydrophilic receptor-bind- ing domain that functions in virus-cell attachment; a hydrophobic membrane-spanning domain; and a hydrophilic cytoplasmic domain. in rsv two polypeptides, gp and gp , make up this structure (fig. ) ; the external domain is primarily made up of the amino acid long gp which contains regions that define the host-range and neutralization properties of the virus. the amino acid long gp polypeptide, on the other hand, is a bitopic protein that anchors the envelope glycoprotein complex into the virion via disulfide linkages to gp . it is less heavily glycosylated than gp (having only versus potential glycosylation sites) and contains two apolar regions in addition to the hydrophilic cytoplasmic domain. one apolar region is located near the amino terminus of gp and may be analogous to the fusion peptide of the hemagglutinin ha polypeptide of influenza virus that mediates viral entry into the cell. the hatched regions represent the highly hydrophobic signal and anchor sequences found at the n and c terminus, respectively. the location of a nonpolar region that may be analogous to the fusion peptide of ortho-and paramyxoviruses is shown by the stippled box. the aug at the start of the env orf was used to initiate translation of transcripts expressed in an sv vector (wills et ai, ) , but during a virus infection the genomic length transcript is spliced such that the aug and first codons from gag (black box) are spliced into the env orf . in both cases the signal peptide is removed during translation, and cleavage of the polyprotein precursor to gp and gp occurs in the golgi at the basic tetrapeptide, -arg-arg-lys-arg-. branched structures denote potential n-linked oligosaccharide addition sites (asn-x-ser or asn-x-thr). (b) orientations of gp and pg in the viral membrane are depicted schematically. in the electron microscope this structure is seen as a spiked knob, where gp is the spike and gp is the knob. second apolar region in gp consists of a amino acid long stretch of hydrophobic residues near the carboxy terminus that functions during translation to stop the movement of the protein into the lumen of the rough endoplasmic reticulum (rer) and to anchor the complex in the membrane. the general orientation and structure of the rsv glycoprotein is thus similar to that of the influenza virus hemagglutinin (ha) (porter et al, ; gething et al, ) , the vesicular stomatitis virus (vsv) g protein (gallione and rose, ) , and several membrane-spanning cellencoded glycoproteins. the two viral glycoproteins of rsv are encoded by a single viral gene, env, and are translated in the form of a heavily glycosylated precursor polypeptide, pr e/ii; . since several processing and maturation events occur during the transport of the env gene products to the plasma membrane, they provide excellent markers for the subcellular compartments of the cell. a long ( amino acid) amino-terminal signal peptide, which mediates translocation of the env gene product across the rough endoplasmic reticulum (rer), is removed cotranslationally from the precursor protein, and in the lumen of the rer - high-mannose core glycosylation units are added to the nascent pr . the marked increase in molecular weight (mw) (approximately k) that results from the addition of this endo-ß-tv-acetylglucosaminidase h (endo h) sensitive carbohydrate provides a clear indicator for the translocation event. removal of glucose residues and some mannose moities appears to occur prior to transport of the protein to the golgi, where further trimming of the mannose residues and addition of glucosamine, galactose, and fucose are observed. cleavage of pr to gp and gp takes place after galactose and prior to fucose addition; it thus provides an excellent marker for trans-golgi locations. while the transit time of pr from the rer to golgi is quite long (t = min) compared to other viral glycoproteins, movement through the golgi appears rapid and precludes any dissection of individual compartments within this organelle. these major biochemical modifications to the env glycoprotein coupled with sensitive immunological probes allow a fairly accurate mapping of the cell's secretory pathway. several viruses assemble at points within the secretory pathway, and since in many cases the location of the viral glycoproteins defines the virus maturation point (roth et al., b; jones et al., ; gottlieb et al., ; gahmberg, ; kabcenell and atkinson, ) these systems are proving useful in investigating the signals that target proteins to specific subcellular locations. figure is a schematic summary of the assembly points for the major groups of enveloped viruses. herpes simplex virus (hsv), a complex enveloped dna virus that encodes several glycoproteins (gb, gc, gd, ge) , buds into the nuclear envelope (spear, ) . virion glycoproteins synthesized on the rer appear to be transported to the nuclear membrane in an endo h-sensitive form where they are incorporated into nascent virions (compton and courtney, ) . it has been postulated that intact hsv virions traverse the secretory pathway thereby exposing the glycoproteins to the entire array of carbohydrate-modifying enzymes such that the mature virion contains glycoproteins with complex carbohydrate side chains (compton and courtney, ; spear, ) . a majority of the hsv glycoproteins can be found on the surface of infected cells, suggesting that targeting to the nuclear membrane is not absolute. expression of the cloned hsv gd gene in the absence of other viral components resulted in more rapid alphaviruses ortho-, paramyxoviruses transport to the plasma membrane and reduced accumulation on the nuclear membrane, indicating that interactions with other viral-encoded proteins may be required for normal nuclear membrane localization (johnson and smiley, ) . the corona-, flavi-, and rotaviruses have been reported to undergo assembly at the rer (dubois-dalcq et al, ) , but the rotaviruses appear to target this organelle most specifically. while the mature rotavirus is not enveloped, it contains a glycosylated capsid protein in its outer shell that is derived from a transient membrane during the assembly process. the inner and outer protein shells of these viruses form sequentially and by very different mechanisms. the inner capsids, containing the genomic rna segments, are the equivalent of nucleocapsids of other viruses and assemble in the cytoplasm at the edge of electron-dense inclusions called "viroplasm" (petrie et al, ) . they acquire a transient envelope, or pseudoenvelope, by budding at rer membranes adjacent to the viroplasm. further maturation of rotaviruses occurs within the cisternae of the rer where enveloped particles are converted to mature double-shelled virions and the lipid bilayer is removed by a process that remains to be elucidated (dubois-dalcq et al, ) . vp , the glycosylated protein found in the outer capsid, has been shown to have carbohydrate structures consistent with its rer location and to target specifically to this organelle when expressed in the absence of other viral proteins from a recombinant expression vector (kabcenell and atkinson, ; poruchynsky et al, ) . the golgi body is the site of assembly for several viruses. coronaviruses, for example, mature by budding into the lumina of rer or golgi cisternae; virions form as the intracytoplasmic, helical neucleocapsids align under regions of intracellular membranes containing viral proteins. two glycoproteins, el and e , comprise these membrane-associated polypeptides. e forms the large peplomers or spikes characteristic of coronaviruses and is a multifunctional molecule, being responsible for virus-induced cell fusion, binding of the virion to receptors on the plasma membrane of susceptible cells, and for inducing neutralizing antibody (dubois-dalcq et al, ) . el, in contrast, is an unusual polypeptide; it has only a short amino-terminal domain, which contains the glycosylation sites of the protein, and two long stretches of hydrophobic amino acids, suggesting that it may traverse the membrane more than once (dubois-dalcq et al, ; boursnell et al, ) . during infection, e can be transported through the secretory pathway to the plasma membrane, whereas el is transported only as far as the golgi apparatus, where it accumulates during the infection cycle (sturman and holmes, ) ; it has been suggested that this restricted intracellular movement of el ac-counts for the intracellular budding site of coronaviruses (sturman and holmes, ) . members of the bunyavirus family also mature intracellularly, by budding at the golgi complex (bishop and shope, ; dubois-dalcq et al., ) . by immunofluorescent microscopy, kuismanen and colleagues showed that in cells infected with uukuniemi virus the golgi region underwent an expansion and became vacuolized . both glycoproteins, gl and g , accumulated in the golgi region during virus infection; neither polypeptide could be chased out of the golgi even after a -hr treatment with cycloheximide (gahmberg et al., ) , conditions that would allow complete transport of the semliki forest virus membrane proteins from the golgi (green et al., a) . furthermore, the glycoproteins of a temperature-sensitive strain of uukuniemi virus were retained in the golgi even under conditions where no virus maturation took place and no nucleocapsids accumulated in the golgi region (gahmberg et al., ) . thus intracellular targeting of these viral components appears to be independent of other viral components and of the assembly process itself. moreover, it supports the concept that it is the glycoproteins themselves that dictate the cellular site of virus maturation. for several virus groups, virion assembly does not occur until the envelope components have traversed the entire secretory pathway. thus the ortho-and paramyxoviruses, rhabdoviruses, alphaviruses, and retroviruses mature at the plasma membrane. even these proteins, however, possess additional membrane-targeting information such that in polarized epithelial cells, where different cell proteins are inserted in the apical and basolateral membranes, the different viruses assemble from distinct membranes; for example, the ortho-and paramyxoviruses bud from apical membranes, rhabdo-and retroviruses from basolateral membranes (rodriguez-boulan and sabatini, ; herrler et al., ; roth et al., a; rindler et al., ) . as with those viruses that mature at points within the secretory pathway, it is the glycoproteins themselves that appear to specify the specific plasma membrane location for virus assembly, since glycoproteins expressed from recombinant expression vectors are transported in a polarized fashion (roth et al., b; jones et al., ; gottlieb et al., ; . the problem of polarized expression will be dealt with in more detail later in this chapter (see section ii, b, ) . from the brief outline presented above it is clear that viral envelope components provide a plethora of systems for studying intracellular protein targeting. recombinant dna approaches, described below, are already providing information on the role of the different glycoprotein domains in this important aspect of viral and cell biology. in addition to utilizing the secretory pathway of vertebrate cells for transporting viral components to the point of virus maturation, several enveloped viruses take advantage of a second vesicle-mediated transport system, the endocytic pathway, to gain entry into susceptible cells. these viruses, such as orthomyxoviruses, rhabdoviruses, and togaviruses, in contrast to paramyxoviruses, such as sendai virus, which bind and fuse with the plasma membrane of the host cell, bind to the host cell surface and are subsequently internalized by endocytosis. this latter process serves an important role in the normal uptake of nutrients and in the internalization of receptor-bound ligands such as hormones, growth factors, lipoproteins, and antibodies (mellman et al., ; hopkins, ) . bound virions are carried into clathrin-coated pits, which form continually on the surface of the cell, and which fold inward and pinch off into the cytoplasm to form "coated vesicles." as the coated vesicle moves into the cytoplasm, it loses its clathrin and fuses with an endosome, a large acidic vacuole with a smooth outer surface. for viruses entering by this pathway, membrane fusion occurs in the endosomal compartment (marsh, ; yoshimura and ohnishi, ) . fusion is triggered by the mildly acidic endosomal ph and is catalyzed by the virally encoded glycoproteins which undergo a low ph-dependent configurational change (skehel et al., ; kielian and helenius, ) . the ph dependence of fusion varies among virus types, with the optimal ph for fusion generally falling within the range of ph . - . for endocytosed viruses (white et al., ) . in order to obtain an understanding of the molecular mechanisms involved in these low ph-induced fusion reactions, virus mutants have been isolated which fuse with ph optima different from those of their respective parents. through the use of an elegant selection scheme, in which mutagenized virus was allowed to fuse with nuclease-filled liposomes at a ph below . , kielian et al. ( ) isolated the first such fusion mutant of semliki forest virus. this virus, fus- , fused at a ph optimum . ph units lower than that of the wild type (ph . versus . ). the mutant was, nevertheless, fully capable of infecting cells under standard infection conditions and even under conditions that prevent fusion of endosomes with lysosomes. on the other hand, the fus- mutant showed increased sensitivity to lysosomatropic agents that increase the ph in acidic vacuoles of the endocytic pathway. in addition to proving that alterations within viral structural components can significantly affect the ph at which virus-induced fusion can occur, these results showed that a ph below . exists within the endosomal compartment and thereby demonstrated the usefulness of mutant viruses as biological ph probes of this pathway. in parallel studies, rott and co-workers ( ) have shown that variants of the x strain of influenza virus, selected for their ability to undergo activation cleavage and growth in madin-darby canine kidney (mdck) cells, have an elevated fusion ph threshold (approximately . ph units higher than the wild type). similar virus variants have been selected by growth of influenza virus in the presence of amantadine, a compound that raises endosomal ph (daniels et al., ) . in this latter study, viruses were obtained that fused at ph values . - . units higher than the parental strain. analogous mutants have been reported to occur naturally within stocks of the x strain of influenza virus (doms et al., ) . such mutants should provide useful probes for elucidating the endocytic pathway. the mechanisms by which cells send membrane-bound and secreted proteins to their proper subcellular locations remain a central problem in cell biology. it has been postulated to involve the specific interaction of "sorting signals," located within the structure of the newly synthesized proteins, with membrane-bound receptors in the rer and golgi apparatus of the cell (for review, see sabatini et al., ; silhavy et al., ) . this concept is supported by the facts that cell, as well as viral, glycoproteins can be retained at or targeted to specific points in the secretory pathway and that cells can transport and secrete a variety of glycosylated and nonglycosylated proteins at distinctly different rates (strous and lodish, ; fitting and kabat, ; gumbiner and kelly, ; ledford and davis, ; , kelly, . very little is known about the composition(s) or indeed the exact role of sorting signals, but it is generally thought that they are composed of protein. clearly, the initial step that introduces polypeptides into the secretory pathway is mediated by the interaction of a sequence of amino acids (the signal sequence) within the polypeptide and the signal recognition particle (srp)/docking protein (dp) complex (blobel and dobberstein, ; blobel, , a,b; walter et al., ; meyer and dobberstein, ; meyer et al., ; gilmore et al., a,b; . mutants of secreted proteins that are defective in later stages of transport have been identified that differ from the wild-type forms by one (mosmann and williamson, ; wu et al., ; shida and matsumoto, ) or two (yoshida et al., ; hercz et al., ) amino acid substitutions, supporting the concept that sorting signals are composed of protein. also, several conditional transport-defective mutants of membrane-bound viral glycoproteins have been identified (for example, knipe et al, b; zilberstein et al, ; pesonen et al, ) . studies using tunicamycin, an inhibitor of glycosylation, suggest that carbohydrate moieties are not recognized directly by the sorting machinery but may be important for maintaining the proper secondary or tertiary structures of (protein-composed) sorting signals (struck et al., ; gibson et al, gibson et al, , leavitt et al, ; hickman et al, ; roth et al, ; strous et al, ; green etal, b) . the lack of a direct role for carbohydrate moieties in the sorting process is perhaps to be expected in view of the fact that many secreted proteins are not glycosylated at all (for example, strous and lodish, ; underdown et al, ) . nevertheless, addition of carbohydrate to molecules that are unable to be transported through the secretory pathway can release the block to their transport (guan et al, ; machamer et al, ) . in addition, the transport of certain hydrolases to the lysosome (and away from the secretory pathway) does appear to require the addition of a carbohydrate moiety (mannose -phosphate) (hasilik and neufeld, ; sly and fisher, ; creek and sly, ) , but these additions in turn must require the recognition of signals within the polypeptide chains. even less is known about the intramolecular location(s) of sorting signals. in the case of the membrane-spanning glycoproteins, three protein domains exist which together or separately may harbor sorting signals: ( ) the internal or cytoplasmic domain, ( ) the hydrophobic or transmembrane domain, and ( ) the extracytoplasmic or external domain. since most secreted proteins (which may be cotransported with membranebound glycoproteins, strous et al, ) possess only external domains, it might be reasonable to expect the transmembrane and cytoplasmic domains to be unimportant to the sorting process. we have tested this hypothesis by introducing genetic lesions into the gene encoding the envelope glycoproteins of rsv (wills et al, (wills et al, , hardwick et al, ; , as have others for the vsv g protein bergmann, , ; rose et al, ; adams and rose, a,b) , the influenza virus hemagglutinin protein (sveda et al, gething and sambrook, ; doyle et al, doyle et al, , gething et al, ) , and the glycoproteins of semliki forest virus (garoff et al, ; garoff, ; . genetic analyses of protein transport in prokaryotic systems have provided both support for the role of the signal peptide in protein translocation and valuable insights into the polypeptide interactions that are required for the intracellular targeting of bacterial secreted and membrane proteins (reviewed by michaelis and beckwith, ; silhavy et al, ; benson et al, ; oliver, ) . while similar experiments are more difficult to perform in eukaryotic cells with the enveloped virus systems described here, both the classic and molecular genetic approaches outlined below are providing information on the role of different protein domains in the transport process. during the genetic analysis of enveloped virus replication through the isolation and biochemical characterization of spontaneous and mutageninduced variants, complementation groups were established for several viruses that contained mutants defective in normal transport of the viral glycoproteins (knipe et al., a,b; zilberstein et al., ; pesonen et al, ; gahmberg, ; ueda and kilbourne, ) . the existence of conditional lethal mutants that were blocked at different stages of virus glycoprotein maturation suggested that the viral polypeptides themselves might contain the signals necessary for normal sorting by the cells' transport machinery and raised the possibility that such mutants could be used tq dissect the maturation pathway of a glycoprotein. since it is impossible in this chapter to provide a detailed review of the characterization of mutants in each of these systems, and since the transport of the influenza virus glycoproteins has recently been discussed in detail by , this section will concentrate primarily on mutants of the vsv g protein gene as an example of these approaches. temperature-sensitive (ts) mutants in complementation group v of this vsv have defects in the structural gene for the viral glycoprotein, g, and cells infected at the nonpermissive temperature with such mutants produce markedly reduced yields of virus like particles which are noninfectious and specifically deficient in g protein. at the nonpermissive temperature the mutant g polypeptide is synthesized normally; however, it does not accumulate on the cell surface, nor is it incorporated into virions (knipe et al., b; zilberstein et al., ) . the ts(v) mutants can be subdivided into two subclasses with respect to the stage of posttranslational processing at which the block occurs (zilberstein et al., ) . three mutants, tsl \ , tsm l, and ts , encode g proteins that at the nonpermissive temperature are blocked at an early, pre-golgi step of the secretory pathway. while insertion into the er membrane, removal of the amino-terminal hydrophobic signal sequence, and addition of the two n-linked high-mannose core oligosaccharides occur in a way that is indistinguishable from the wild type, all subsequent golgi-mediated carbohydrate processing reactions are blocked (zilberstein et al., ) . these results are consistent with subcellular fractionation and immunoelectron microscopy studies which indicated that the g protein in tam or ta -infected cells was arrested in its transport from the rer to the golgi complex at the nonpermissive temperature (zilberstein et al., ; bergmann et al., ; bergmann and singer, ) . the defect in transport in these mutants is a reversible phenomenon, thereby excluding irreversible denaturation as the basis for lack of movement; proteins synthesized at the nonpermissive temperature rapidly move by stages to the plasma membrane upon shift to the permissive temperature bergmann and singer, ) . within min after shift to °c, g protein of tsoa could be seen by immunoelectron microscopy at high density in saccules at one face of the golgi complex and by min later was uniformly distributed through the complex (bergmann and singer, ) . movement of the mutant proteins to the cell surface occurred rapidly and was accompanied by incorporation into virions . the second class of ts mutants of vsv is represented by tal . g protein encoded by this mutant is transported normally through most of the golgi-mediated functions involved in the processing of carbohydrate side chains, including addition of the terminal sialic acid residues. however, this molecule does not undergo two posttranslation modification reactions that take place with the wild-type g protein. the first is the addition of fucose to the tel oligosaccharide chains, which is reduced at both permissive and nonpermissive temperatures (zilberstein et al., ) . in the second, a molecule of palmitic acid (a -carbon fatty acid) is covalently attached to the wild-type g polypeptide near the membranespanning region rose et al., ) . attachment occurs at a late stage of maturation, just before oligosaccharide processing is completed (schmidt and schlesinger, ) , probably in the eis compartment of the golgi complex (dunphy et al., ) . this modification of the g protein does not occur at the nonpermissive temperature in cells infected with the tsl ll mutant. taken together with the almost complete processing of the mutant's oligosaccharide side chains, this suggests that at the nonpermissive temperature the asl glycoprotein accumulates at a specific region within the golgi complex. the mutants of vsv, together with equivalent mutants in other viral systems that were blocked at different stages of the secretory pathway (pesonen et al., ; gahmberg, ; kuismanen et al., ) , raised the possibility of identifying and understanding the nature of sorting signals in secreted polypeptides. with the advent of recombinant dna and rapid nucleotide sequence techniques, it has been possible to determine at the amino acid level the basis for these defects (gallione and rose, ; arias et al., ) , but the interpretation of this information with regard to protein transport has been less than straightforward. gallione and rose ( ) determined the nucleotide sequence of the ts mutant of vsv and compared it to that of the parent and a wildtype revertant. the mutant and revertant differed in three amino acid residues, and through the construction and expression of hybrid genes it was possible for these investigators to demonstrate that the basis of the temperature-sensitive phenotype was a single amino acid change of phenylalanine to serine. since this polar substitution occurred within a very hydrophobic region of the g protein, it was suggested that it might significantly affect protein folding in this region such that reversible denaturation of the protein might occur at the nonpermissive temperature. this denaturation could prevent further transport to the golgi apparatus and the cell surface. alternatively, gallione and rose ( ) pointed out that the conformational change at the nonpermissive temperature might be more subtle, perhaps preventing recognition by a component of the protein transport machinery. however, since hydrophobic residues are generally buried within a protein (kyte and doolittle, ) , it is unlikely that the mutated sequence itself would play a direct role in such an interaction. nothing is known about the -dimensional structure of the g protein or whether accessory proteins are involved at this stage of protein transport, thus both suggestions remains viable possibilities. a similar analysis has been carried out by arias et al. ( ) , who sequenced the genes encoding the viral glycoproteins of tslo and ts , mutants of sindbis virus defective in the intracellular transport of their glycoproteins, and of revertants of these mutants. these investigators found ts to have a double mutation in glycoprotein el, while ts was a single mutant in the same glycoprotein. in each case reversion to temperature insensitivity occurred by changes at the same site as the mutation, in two cases restoring the original amino acid and in the third case substituting a homologous amino acid (arginine in place of lysine). since the three mutations were far apart from each other in the protein, these authors concluded that the -dimensional conformation of el was very important for the correct migration of the glycoproteins from the er to the plasma membrane. similarly, two ts mutants in the ha gene of influenza virus that result in ha protein transport being arrested in the rer are also caused by single point mutations that probably disrupt the tertiary structure of the molecule (nakajima et al., ) . in summary, several conditional mutants have been isolated by classic genetic approaches that at the nonpermissive temperature disrupt the normal transport of viral glycoproteins through the secretory pathway. while these mutants carried the promise of defining specific protein domains that might interact with components of the transport machinery, the evidence from the nucleotide sequencing experiments outlined above suggests that many or all of the mutations may exert their phenotype through distortion of the -dimensional shape of the molecule. while this mechanism does not preclude a role for specific protein-protein interactions in the secretory pathway, it provides no direct evidence for it at this time. the recent development of cdna cloning, gene sequence manipulation, and gene expression technologies has opened up new approaches for localizing and characterizing those structural features of a protein that act as sorting signals. these new methodologies have allowed investigators to delete or modify potentially important structural regions of a protein at the nucleotide level and then to determine the effect of such changes by expressing modified genes in suitable eukaryotic expression vectors. furthermore, in some instances it has been possible to test directly the functional role of a particular peptide region by fusing it to another protein and analyzing the behavior of the chimera. since these general approaches to protein sorting have been reviewed recently (garoff, ; gething, ) , this chapter will describe our recent recombinant dna analyses of the biosynthesis and transport of the rsv envelope glycoproteins, within the context of similar analyses in other viral systems. as we have discussed earlier, the rsv env gene encodes two viral glycoproteins, gp and gp , that mediate recognition of, attachment to, and penetration of the susceptible target cell. these proteins are synthesized as a glycosylated precursor protein, pr , that is proteolytically cleaved in the golgi complex. the coding sequences for gp and gp have been placed in an open reading frame that extends from nucleotide to nucleotide , and predict sizes of amino acids ( , mw) for gp and amino acids ( , mw) for gp ( fig. ) . carbohydrate makes up a significant contribution to the observed molecular weights of these polypeptides-the predicted amino acid sequence contains potential glycosylation sites (asn-x-ser/thr) in gp and in gp . experiments aimed at determining the number of carbohydrate side chains yielded results consistent with most or all of the sites being occupied . although an initiation codon is located early (codon ) in the open reading frame, during a viral infection splicing yields an mrna on which translation initiates at the same aug as the gag gene to produce a nascent polypeptide in which gp is preceded by a amino acid long leader (signal) peptide (fig. ) . this peptide contains a hydrophobic sequence that we have shown (see below) is necessary for translocation across the rer and is completely removed from the env gene product during translation . it represented one of the longest signal peptides described to date, and we were therefore interested in determining the signal peptide requirements for normal biosynthesis of gp and gp . for these studies the env open reading frame was excised from the rsv genome and inserted into an sv expression vector under the control of the late-region promoter (wills et al., ; . in this construction translation is initiated at the aug present at the start of the open reading frame (at nucleotide of the rsv genome) and results in the synthesis of an even longer ( amino acid) signal peptide; nevertheless, biosynthesis of pr and signal peptide cleavage occur normally. furthermore, expression of the rsv env gene in african green monkey (cv- ) cells parallels that seen in a normal virus infection in avian cells (wills et al., ; hardwick et al., ) , making it an excellent system for the analysis of mutant env genes. a. deletion/substitution of the signal peptide. in order to examine the role of the signal peptide in rsv glycoprotein biosynthesis we constructed a series of deletion mutations within the ' coding region of the env gene using the double-stranded exonuclease babl. oligonucleotide linkers of the sequence catcgatg were ligated to the ends of the truncated molecules to introduce a unique restriction endonuclease cleavage site and to replace the deleted in-frame aug. the mutants were then sized and their nucleotide sequence determined to find those with a suitable deletion and an in-frame aug. one such mutant, al, contained a deletion of nucleotides within the env coding sequences and encoded an env product that completely lacked an aminoterminal hydrophobic sequence (fig. ) . expression of this gene from the sv vector resulted in the synthesis of a nonglycosylated, kda cytoplasmic protein that was similar in size to the nonglycosylated wild-type env gene product produced in the presence of the glycosylation inhibitor, tunicamycin. in contrast to the tunicamycin product, however, the al protein was not associated with membrane vesicles and was rapidly degraded (half-life < min; e. hunter, k. shaw, and j. wills, unpublished) . thus the signals for initiating translocation of the rsv env gene product must reside within the cotranslationally removed amino-terminal sequence, and in their absence the molecule is synthesized as an unstable cytoplasmic protein. this result is similar to those obtained by gething and sambrook ( ) and by sekikawa and lai ( ) with the influenza virus ha gene product. influenza hemagglutinin sequences are depicted by italicized text. underlined text denotes amino acid residues encoded by oligonucleotide linkers. arrows depict the signal peptidase cleavage site at which the signal is removed cotranslationally from each of the constructs. plus symbols indicate that translocation, glycosylation, or transport to a plasma membrane location is observed; minus symbols mean that the properties above are not observed. since the signal peptide of the rsv env gene product is exceptionally long, it was of interest to determine whether another signal peptide could substitute for it. for these experiments we have utilized the signal sequences of the influenza virus ha gene (a/jap/ / ; gething et al., ) . two constructions were made: in the first of these the al deletion mutant coding sequence was fused in-frame to the ha signal coding sequence at the signal peptidase cleavage site of the latter (fig. ) and in the second, we made use of a sail restriction enzyme site in the ha gene and an xhol site in the rsv env gene, so that the signal sequence and amino acids of ha were fused with env amino acids into gp (fig. ) . expression of these hybrid genes in cv- cells resulted in the biosynthesis of a glycosylated pr protein that was transported to the golgi complex, cleaved to gp and gp , and displayed on the cell surface (e. hunter, k. shaw, and j. wills, unpublished) . to demonstrate that the pr molecules expressed from the ha-a fusion gene had undergone signal peptide cleavage, pr was immunoprecipitated from [ h]leucine, pulse-labeled cells and analyzed by sequential edman degradation, in order to determine the amino-terminal sequence. to our surprise the signal peptidase had cleaved at the ha cleavage site, despite the fact that according to the analyses of von heijne ( ) only the rsv cleavage sequence should have been recognized. thus the h a -al fusion protein contains two potential signal peptidase cleavage sites (that from the ha and that remaining in the env sequences), but only the first of these is utilized. both gene fusions, therefore, result in the synthesis of aberrant gp proteins-that from the ha-a fusion having an amino acid amino-terminal extension, and that from the sallxho fusion having lost amino-terminal amino acids and gained from ha which nevertheless can be transported to the plasma membrane (wills et al., unpublished data) . reciprocal gene fusions, in which the env gene signal peptide was fused to the structural sequences of ha (fig. ) , also resulted in translocation of the ha molecule across the rer membrane, supporting the concept that this transient sorting sequence is not polypeptide specific. however, only in the construction where the signal sequence of env was precisely fused to the amino terminus of ha was transport beyond the rer observed (wills et al., unpublished data) . in constructions where the amino-terminal sequence of ha was perturbed, the recombinant protein was apparently prevented from assembling into trimers and its transport was blocked in the rer . thus, while signal peptides may be capable of mediating the translocation of foreign polypeptides across the rer, other sorting "signals" must be active for transport of the molecule to continue. site. the experiments described above have been extended to determine the following: (a) whether the hydrophobic region of the signal peptide carries all the information required for transfer of the env gene product into the rer; (b) what the structural specificities of the signal peptide are; and (c) where the specificity for signal peptidase cleavage is located. more than prokaryotic and eukaryotic signal peptides have been sequenced (watson, ) . comparison shows that most extensions comprise - amino acid residues; one of the longest being that of the rsv envelope glycoprotein. there is no homology between sequences, but a characteristic distribution of amino acid chains is observed. three structurally distinct regions have been observed so far: a positively charged aminoterminal region, a central region of or more hydrophobic residues, and a more polar carboxy-terminal region that appears to define the cleavage site (von heijne, (von heijne, , (von heijne, , perlman and halvorson, ) . the importance of these general features has been supported by the genetic studies in prokaryotic systems (reviewed by silhavy et al., ; benson et al, ) . to investigate these questions we initially constructed a series of internal deletion mutants that initiated within the amino-terminus of gp and extended into the signal peptide. the deletion mutations were introduced into the coding region for the envelope glycoprotein by digestion of a plasmid containing the env gene at a unique xhol site located base pairs (bp) from the ' end of the coding sequence for gp , followed by digestion with the double-stranded exonuclease bal l. potential mutants were identified by restriction enzyme analysis and dna sequencing, and those of interest were engineered into the sv expression vector. these are depicted in figs. and . mutants x -a -b, and -c were derived from a single out-of-frame parent, and they represent a nested set of mutants in which the hydrophobic sequence varies from the wild-type length of to only amino acids. expression of these mutant genes in cv- cells gave the results summarized in fig. . mutant polypeptides with the shortest hydrophobic domain (x -c) resembled the al mutant polypeptides in that they had a cytoplasmic location, were nonglycosylated, and were rapidly degraded. they differed from the al mutant in length ( versus kda) confirming that the mutated signal was not removed. mutant x -a polypeptides, on the other hand, were translocated and glycosylated with an efficiency equivalent to wild type, despite the substitution of serine and isoleucine for leucine and cysteine residues within the hydrophobic domain. mutant x -b expressed a phenotype intermediate between that of x -a and x -c, approximately % of the polypeptides being translocated and glycosylated. none of the mutants contain the sequences that specify the signal peptidase cleavage site, and molecules of x -a/x -b that were translocated across the rer retained an uncleaved signal peptide. the data from these mutations suggest the following: ( ) that the length, rather than the amino acid composition, of the hydrophobic domain of the env signal peptide is critical for translocation across the rer and ( ) that signal peptide cleavage is not a requirement for translocation. the first of these conclusions is supported by genetic experiments in prokaryotic systems, where a requirement for secondary structure in the signal peptide was suggested bankaitis et al., ) . the second is consistent with the presence of permanent insertion sequences in secreted and membrane-spanning proteins that are translocated across the rer membrane without removal of an amino-terminal signal peptide (palmiter et al., ; bos et al., ; markoff et al., ; zerial et al., ; spiess and lodish, ) . several membrane-spanning proteins are anchored in the membrane by an amino-terminal anchor/signal domain and display what is termed group ii protein topology (garoff, ; wickner and lodish, ) , where the amino terminus of the protein is cytoplasmic and the carboxy terminus is luminal. after translocation of a nascent chain across the endoplasmic reticulum has been initiated, the signal peptide is removed. this cleavage is carried out by signal peptidase, a cellular gene product. two classes of signal peptidases have been described. a signal peptidase of escherichia coli (spase i) has been cloned into pbr (date and wickner, ) , and has been shown to accurately cleave eukaryotic precursor proteins as well as bacterial protein precursors (talmadge et al., ) . conversely, the eukaryotic signal peptidase will accurately cleave prokaryotic proteins (watts et al., ) . the latter enzyme has been studied, using detergentsolubilized dog pancreas signal peptidase (jackson and white, ) and hen oviduct signal peptidase (lively and walsh, ) , and been demonstrated to be an integral membrane protein that can be solubilized only when the lipid bilayer is dissolved. a second prokaryotic signal peptidase (e. coli spase ii) has been described that is specific for prolipoproteins (hussain et al., ; tokunaga et al., ) and membrane-bound penicillinases (nielsen and lampen, ) . this enzyme maps to a different locus on the e. coli genome and requires a glyceride-modified cysteine for cleavage. perlman and halvorson ( ) and von heijne ( ) have examined sequences of a number of membrane proteins and have described amino acid sequence patterns that allow prediction of signal peptidase cleavage sites with greater than % accuracy. the most striking feature of signal peptidase cleavage sites is the presence of an amino acid with a small, uncharged side chain at the carboxy terminus of the signal peptide. the most common amino acids found at this position are alanine and glycine. from statistical analyses, the peptidase cleavage site appears to be determined by sequences within the signal peptide and not by sequences beyond the cleavage site. this is in contrast to the observations that mutations within the structural protein itself prevent signal peptidase cleavage of the lamb gene product and the m coat protein (emr and bassford, ; benson and silhavy, ; rüssel and model, ) . to investigate this question with regard to the rsv env gene product, the deletion mutants shown in fig. were constructed as described above. mutant xi has a amino acid deletion encompassing residues - of gp , which results in the loss of one potential glycosylation site. this deletion resulted in the synthesis of a slightly smaller precursor polypeptide that lacked one carbohydrate side chain but was otherwise glycosylated normally. based on size estimations in pulse-labeling experiments, in the presence and absence of tunicamycin, the precursor polypeptide lacked the long, signal-containing leader peptide. thus, although the mutation in xi significantly alters the sequences near the signal peptidase site, the signal peptidase still recognized and removed the signal peptide. in mutants x and x the amino-terminal nine and six amino acids, respectively, of gp are deleted. they therefore encode gp poly- xhol site in dna r\- | °| asp val his leu leu glu gin pro gly asn leu trp ile thr trp ala asn arg . fig. . amino acid sequence deduced from dna sequence of mutants xi, x , and x . the rsv glycoprotein is schematically represented: the location of the hydrophobic signal sequence within the long ( amino acid) leader peptide is denoted by a stippled bar, and the mature gp glycoprotein by a hatched bar. the signal peptidase cleavage site in both the cartoon and the numbered amino acid sequence is denoted by a long arrow. the potential glycosylation site in the amino terminus of gp is shown as a cho. the amino acid sequence of the last amino acids of the signal peptide and the amino-terminal amino acids of gp are shown for the wild-type gene product. the solid black bars show the lengths and positions of the deletions in mutants xi, x , and x . peptides with novel amino termini that alter the signal peptidase cleavage site from ala/asp-val-his to ala/asn-leu-trp and ala/gln-pro-gly, respectively. thus both the charge and secondary structure of the cleavage site would be predicted to be altered by the loss of asp and his (x ) and by the relocation of proline near the cutting site (x ). nevertheless, the signal peptidase efficiently cleaved the leader peptide from the nascent polypeptide, and its specificity of cleavage was unaffected by these alterations (hardwick et al, ) . these experiments then support the generalized conclusion from statistical analysis, that the sequence to the right of the signal peptidase site is not critical for signal peptidase cleavage. none of the deletions generated in the rsv glycoprotein gene resulted in a loss of recognition and cleavage by the signal peptidase. this result contrasts with previously described prokaryotic mutants. a amino acid deletion starting at the fifth residue beyond the signal peptidase site of the lamb gene product blocked cleavage of the signal (emr and bassford, ; emr et al, ) , and a deletion of amino acids beginning amino acids downstream from the signal abolished signal cleavage although the shortened protein was localized correctly . in addition, the substitution of a leucine, in place of the glutamic acid, at residue of the mature m coat protein also inhibited signal peptidase cleavage; however, in this latter instance the procoat protein was transported inefficiently across the inner membrane (boeke et al., ; rüssel and model, ) . although more mutants will be required to properly define these systems, the prokaryotic cleavage site appears to be more sensitive to manipulation than that of eukaryotes. there is accumulating evidence that transported prokaryotic proteins, unlike those of eukaryotes, may not be transferred across membranes in a strictly cotranslational manner (randall and hardy, ) . thus, altered regions within the structural protein portion of a molecule would have the opportunity to interact and interfere with signal peptidase cleavage; such as interaction would not be possible in the cotranslational system described for eukaryotes. although the mutant xi polypeptides were translocated across the rer membrane in a normal fashion, immunofluorescence experiments and posttranslational modification probes indicated that the transport and maturation of the xi glycoprotein was halted shortly after exiting the rer, perhaps within pre-or cis-golgi vesicles. cells synthesizing the mutant protein showed no surface immunofluorescence, no cleavage of the pr to gp /gp , and no terminal sugar additions (hardwich et al., ) . the basis for this block appears to be the altered amino acid sequence rather than the loss of the carbohydrate side chain since by using a mutagenic oligonucleotide we have modified the amino terminus of the xi gp from asp-val-his-arg-thr-to asp-val-asn-arg-thr-, thereby reinserting the glycosylation site missing from this mutant. the derivative mutant, x a, is glycosylated at this site but remains blocked at the same intracellular point in the secretory pathway (k. shaw, k. kervin, and e. hunter, unpublished) . a second deletion mutant of the rsv env gene is also blocked in intracellular transport. this mutant, c , has an engineered deletion at the carboxy terminus of gp that removed the cytoplasmic tail and transmembrane region (see below and wills et al., ) . its transport is clearly blocked at an earlier stage than that of the xi mutant since it was localized to the er and never reached the golgi apparatus, whereas by immunofluorescent staining of fixed cells the xi protein appeared to colocalize with the golgi complex (see fig. ; hardwick et al., ) . although the xi and c mutants contain alterations at opposite ends of the env gene product, they both appear to lack an element that normally signals their transport to and beyond the cis-golgi. while there may be a specific amino acid sequence (analogous to the amino-terminal signal sequence) that is required for these transport steps, it is as likely that a correctly aligned tertiary structure is the critical factor. just as small changes as the amino terminus of ha can disrupt assembly and transport un rhodamine fig. . intracellular immunofluorescence of cells expressing wild-type and mutant polypeptides. fixed infected cv- cells were stained to detect the intracellular localization of wild-type and mutant glycoproteins. rabbit anti-glycoprotein antibodies were tagged with fluorescein-conjugated goat anti-rabbit antibodies which in wild-type and xi-infected cells could be localized on the nuclear membrane (nm), endoplasmic reticulum (er), and golgi apparatus (g). the golgi was localized by staining the same cells with rhodamine-conjugated wheat germ agglutinin. in mutant c -infected cells neither the nuclear membrane nor the golgi apparatus stained with the antiglycoprotein antiserum. magnification is . of the ha trimer, the deleted amino acids unique to the xi mutation may similarly play a critical role in the tertiary structure of the env complex, such that sorting signals required for transport through the golgi complex are lost. mutants x and x have deletions that begin at the amino terminus of gp and extend nine and six amino acids into this structural protein; thus these mutations overlap with the deletion in xi. nevertheless, both mutant proteins were transported to the cell surface and were indistinguishable from the wild type. mutants x and x thus indicate that the terminal nine amino acids of pr are not required for normal intracellular transport and define the critical region in gp as the seven amino acids that are uniquely deleted in the xi mutant. a. truncation of the cytoplasmic sequences. dna and protein sequence studies demonstrated the presence of a amino acid long hydrophobic (and presumably membrane-spanning) domain and a amino acid long cytoplasmic domain at the carboxy terminus of gp ; see fig. ). comparison of these domains with those of other exogenous and endogenous strains of rsv has revealed that the sequence within the hydrophobic domain is highly conserved and that within the cytoplasmic domain the sequence of the first amino acids (adjacent to the hydrophobic domain) is also highly conserved while those at the carboxy termini diverge greatly (hughes, ; hunter et al., ) . these results raised the possibility that the conserved region of the cytoplasmic domain might play a functional role in either transport of the env gene product through the secretory pathway or in virus assembly. to investigate this question we initially altered the cytoplasmic domain by introducing deletion mutations into the molecularly cloned sequences of the proviral env gene and examined the effects of the mutations on transport and subcellular localization in cv- cells. we found that replacement of the nonconserved region of the cytoplasmic domain with a longer unrelated sequence of amino acids from sv vector sequences (mutant cl) did not alter the rate of transport to the golgi apparatus nor the appearance of the glycoprotein on the cell surface. larger deletions, extending into the conserved region of the cytoplasmic domain (mutant c ), however, resulted in a -fold slower rate of transport to the golgi complex, but did not prevent transport to the cell surface (wills et al., (wills et al., , . these results were thus consistent with the cytoplasmic domain of the rsv env gene product playing some role in transport to the golgi complex. similar results were obtained by rose and bergmann ( ) who introduced into the cdna clone encoding the vsv g protein a series of deletions that affected the cytoplasmic domain. these mutants fell into two classes; the first was completely arrested in their transport at a stage prior to the addition of complex oligosaccharides (presumably the rer) and the second showed severely reduced rates of transport to the golgi complex although the proteins were ultimately transported to and expressed on the cell surface. the method by which these mutants were constructed (as with the rsv env mutants) meant that the truncated g proteins terminated in sv sequences, and in at least one case the block to transport could be alleviated by substitution of a termination codon for these "poison" sequences. even in these constructions, however, three foreign amino acids were translated prior to termination (rose and bergmann, ) . the concept that the cytoplasmic domain might influence or govern the rate at which membrane-spanning proteins were transported to the golgi complex was supported by similar studies of doyle et al. ( ) on the ha polypeptide. while the ha cytoplasmic domain could be replaced by the equivalent region from the rsv env gene product without affecting the rate of transport of the hybrid ha from the rer to the golgi complex, truncation of the ha cytoplasmic domain or addition of the amino acid long cytoplasmic domain from gp slowed transport significantly, and addition of amino acids encoded by pbr sequences blocked transport of the ha from the rer. on the other hand, studies on the class i histocompatibility antigens (zuniga et al., ; murre et al., ) , the p of semliki forest virus (garoff et al., ) , and additional studies on the ha of influenza indicated that the cytoplasmic domains of these proteins could be truncated without affecting transport to the cell surface, although the kinetics of transport were not determined in every case. b. substitution mutations. it should be noted that in most of the mutant constructions described above, the carboxy-terminal region contained one or several aberrant amino acids as a result of the recombinant dna approach. thus in order to determine more directly the role of the cytoplasmic domain of gp , we have used oligonucleotide-directed mutagenesis to introduce an early termination codon in the coding sequences of gp such that the arginine residue that represents the first amino acid of the cytoplasmic domain is changed to an opal terminator. this mutation creates a truncated viral glycoprotein lacking specifically the cytoplasmic domain of gp . the biosynthesis and transport of the products of this mutant viral glycoprotein gene were analyzed by expression from an sv late-region replacement vector, and its ability to be active in viral assembly was investigated by substitution of the mutated gene for the wild-type gene in an infectious avian retrovirus vector. in contrast to our previous results, deletion of the entire cytoplasmic domain alone had no effect on the biosynthesis or rate of intracellular transport of the env glycoprotein. thus it seems unlikely that the conserved amino acids present in this region play a role in intracellular transport. although the cytoplasmic domain contains several charged, hydrophilic residues, it does not appear, by itself, to be required for anchoring the complex in the membrane, since molecules lacking the cytoplasmic domain were expressed stably on the plasma membrane and were not shed into the cell culture medium . a recent study by gething et al. ( ) has demonstrated that mutations within the cytoplasmic domain of the influenza virus ha can affect the conformation of the extracellular domain by preventing assembly and trimerization of the ha molecule, thereby resulting in a failure of those mutants to be efficiently transported. a similar requirement for the assembly of oligomeric forms of the vs v g protein prior to its transport to the golgi complex has also been reported (kreis and lodish, ) . the inconsistency of our previous results with the ones obtained with the opal mutant could be explained in a similar way. we cannot rule out the possibility that in our earlier experiments the extra amino acids, added as a consequence of the loss of the env termination codon, created a conformational change in the extracellular domain of pr and slowed its transport from the rer. our present results indicate that the cytoplasmic domain of gp is neither a recognition signal for transport to the plasma membrane nor a requirement for anchoring the molecule to it. these findings also support the idea that the charged amino acids present in most of the cytoplasmic domains of many transmembrane proteins (garoff et al., ; sabatini et al., ) are dispensable for anchor function (davis etal., ) . this latter question has also been addressed by cutler and co-workers , who mutated the cytoplasmic domain of the p polypeptide of semliki forest virus. this region, which normally contains a charge cluster (arg-ser-lys) flanking the hydrophobic domain, was changed to a neutral (met-ser-gly) or an acidic (met-ser-glu) one using oligonucleotide mutagenesis. expression analyses of these mutant proteins confirmed that the basic amino acids were not required for cell surface transport since they reached the surface in a biologically active form. nevertheless, both mutant polypeptides showed reduced stability when membranes containing them were extracted with high-ph buffer . charged residues within the cytoplasmic domain may thus provide an additional measure of stability to the membrane-bound complex. since the conserved residues in the cytoplasmic domain of gp were not required for protein transport it seemed possible that this region might play a role in the process of infectious virus assembly. the fact that the mutant protein was efficiently transported to the cell surface allowed us to analyze this potential role for the cytoplasmic domain in the process of virus budding. chemical cross-linking experiments have demonstrated an interaction between gp and pl , one of the gag gene products that structure the viral core of rsv (gebhardt et al, ) . while it is clear that virus assembly can occur in the absence of glycoproteins, it was suggested that the pl /gp interaction may be part of the driving force for the process of viral assembly and budding. furthermore, since host membrane glycoproteins are excluded from the viral membrane there must be some positive signal for inclusion of the viral env gene products in the budding virion. to determine whether the cytoplasmic domain is involved in this interaction and required for infectious virus assembly, we reconstructed a retrovirus genome carrying the "tail(-)" env gene mutation. surprisingly, such mutant viruses were infectious on avian cells and spread through the culture with similar efficiency to those containing a native env glycoprotein complex. furthermore, this truncated env gene complex was incorporated as efficiently into virus particles as the wildtype complex . this fact suggests that if an interaction between gp and pl is required to mediate the incorporation of the glycoproteins into the envelope of the budding viral particle, it must occur within the lipid bilayer, presumably with the hydrophobic anchor domain. it is thus unlikely that interactions between viral capsid proteins and the cytoplasmic domain of the env complex constitute a driving force for preferential incorporation of the viral glycoproteins in the avian retroviral envelope. what then is the function of the cytoplasmic domain of the env glycoprotein? since this segment of the viral polypeptide does show a region of conserved sequence, it is possible that it has evolved to facilitate transport to the plasma membrane without being a requirement for it; clearly, randomly inserted alterations within this domain can exert a negative effect on the transport process. while we have observed normal assembly and infection by virus encoding a "tail(-)" env product, it will be of interest to determine whether continued growth of the virus results in the dominant appearance of revertants that encode a functional cytoplasmic domain. many cell surface and membrane proteins of animal viruses are bound to the lipid bilayer by a membrane-spanning hydrophobic peptide close to the carboxy terminus of the polypeptide (reviewed by warren, ; armstrong et al, ) . experimental evidence for this first came from deletion mutants of the influenza virus ha (gething and sambrook, ; sveda et al, ) , the vsv g protein (rose and bergmann, ) , and the minor coat protein of phage fl (boeke and model, ) in which removal of sequences that encoded the cytoplasmic and membrane-spanning domains resulted in secretion of the protein. the hydrophobic membrane-spanning peptide of these polypeptides is thought to be an essential component of the cotranslational signal that results in the arrest of chain transfer across the rer membrane during synthesis. these stop-translocation sequences have been proposed to be a region of the nascent protein molecule which halts insertion through the membrane by disassembling the translocation apparatus and thereby creates proteins with three topological domains (blobel, ) . they appear to be inseparable from the anchor sequences (yost et al, ; rettenmier et al., ) since the transfer of intact transmembrane domains to normally secreted proteins has caused translocation of the constructed hybrid molecules to stop at the added sequences (yost et al., ; guan and rose, ) . however, the precise structural and physical properties of the stop-translocation sequences have not been defined. wold et al. ( ) have suggested that the cytoplasmic domain of membrane-spanning proteins might act to interrupt translocation; however, this seems unlikely since deletion mutants lacking this domain are found to be associated with the membrane in a normal manner (see above; garoff et al., ; zuniga et al, ; murre et al, ; doyle et al, ; . while length and sequence vary widely among regions described as transmembrane anchors, they do have characteristics in common. most often, they are long stretches ( - residues) of predominantly nonpolar and hydrophobic amino acids, bounded by charged residues, at the carboxy terminus of membrane proteins. membrane-spanning sequences have also been described, however, at the amino terminus of some viral proteins (blok etal.,a s ; palmiter et al, ; bos et al, ; markoff et al, ; zerial et al., ; spiess and lodish, ) and in the middle of other proteins (rettenmier et al, ; kopito and lodish, ; finer-moore and stroud, ) . we have investigated the structural requirements for a functional anchor/stop-translocation sequence in the rsv env system by constructing both deletion and point mutations in this region. a. deletion of the anchor domain. during our studies on the role of the cytoplasmic domain in env product transport, we characterized a mutant (c ) in which the entire cytoplasmic and transmembrane domains were deleted. this mutant, in contrast to those described for the influenza virus ha and the vsv g protein, was arrested in its transport at the rer and thus was not secreted from the cell (wills et al., ) . pulse-chase experiments coupled with oligosaccharide precursor labeling experiments showed that the c polypeptide was not transported to the golgi complex, even though it accumulated in a soluble, nonanchored form in the lumen of the rer; the mutant thus appeared to lack a functional sorting signal. surprisingly, immunofluorescent labeling studies showed that the c protein (unlike the wild type) did not accumulate on the nuclear membrane but rather in vesicles distributed throughout the cytoplasm (fig. ) , suggesting that movement to the nuclear membrane, blocked in c , may require a specific transport event, even though the rer and nuclear membranes appear to be continuous. this hypothesis is supported by studies on the vsv g protein that indicate that transitional vesicles (for the transport of glycoproteins to the golgi apparatus) may be derived from "blebs" in the nuclear membrane (bergmann and singer, ) . although these studies raised the possibility that sorting signals might exist within the deleted region of c , the amino acid deletion in this mutant, which extends into the external domain of gp , would be expected to prevent normal folding of the glycoprotein. to determine whether the transmembrane domain was required for intracellular transport, we have modified the env gene by oligonucleotide-directed mutagenesis, changing the lysine (aaa) codon, which precedes the hydrophobic domain of gp , to an ochre nonsense codon (taa). this modified gene thus encodes a protein consisting of the entire external domain of pr and lacking precisely the hydrophobic membrane-spanning and hydrophilic cytoplasmic domain. the biosynthesis and intracellular transport of the truncated protein in cv- cells was not significantly different from that of the wild-type glycoprotein, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. in contrast to the case of the c mutant, this complex lacking just the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the culture medium . since the glycoprotein complex lacking only the cytoplasmic domain of gp is stably expressed on the cell surface, in a manner similar to the wild-type complex, it can be concluded that the transmembrane domain alone is required for anchoring the rsv env complex in the cell membrane. b. requirements for a functional stop-translocation/anchor sequence. we have approached the question of the compositional requirements for membrane anchoring and orientation (stop-translocation) of a membrane-spanning protein by substituting an arginine for a centrally positioned leucine in the hydrophobic anchor region of the rsv env gene product (fig. ) . the arginine substitution is one of the most drastic ^ membrane-spanning mutant « domain * wt his leu leu lysjgly leu leu leu gly leu val val ile leu leu leu leu val cys leu pro cys leu leu gin phe val ser ser ser ile|arg lys met μΆ^ his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val cys|arg|pro cys leu leu gin phe val ser ser ser ile) arg lys met t his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val cysj ;;;-::;;;: : :;|i|leu leu gin phe val ser ser ser ile|arg lys met t his leu leu lys|gly leu leu leu gly leu val val ile leu leu leu leu val| |ser ser se Üe|arg lys met t his leu leu lys|gly leu leu leu gly leu val val ile leu leu[ jval ser ser ser ile| arg lys met t his leu leu lys|gly leu leu leu gly leu val| |ser ser ser ile|arg lys met t his leu leu lys|gj leu||| |ser ser ile|arg lys met compositional point mutations that could be made since it is only rarely found buried in hydrophobic environments (kyte and doolittle, ) and has a high predicted potential for terminating membrane-buried helices (rao and argos, ) . the substitutions we have made fall within the conserved leucine-rich " i c " region proposed by patarca and haseltine ( ) and near the two cysteine residues where palmitate may be covalently added (gebhardt et al, ; kaufman et al., ) . by changing the anchor's hydrophobic integrity through the insertion of point mutations we hoped to define better what constituted a functional anchor sequence. placing a highly charged basic side chain into the hydrophobic core of the membrane might be expected to either ( ) terminate the membrane-spanning helix, thereby partitioning the charged residue to one side or the other of the membrane, or ( ) destroy the stop-translocation signal, causing the protein to be secreted. the results of these experiments showed that a single amino acid substitution in the transmembrane anchor did not affect membrane association or its orientation in the membrane; unexpectedly, however, it affected targeting of the protein at a stage late in the transport pathway, such that the mutant protein was rapidly degraded in lysosmes . the early translation products of both the arginine-mutant and wild-type genes behaved normally: they were synthesized with equal efficiency, had normal bitopic symmetry, and were glycosylated. the kinetics for the turnover of these precursors were nearly identical to those previously reported in infected chicken embryo fibroblasts (bosch and schwarz, ) and in sv expression vectors (wills et al., ) . furthermore, in the golgi, palmitate was added to the precursors, they were cleaved to gp -gp , and they received terminal sugars. only after this last stage did the presence of the charged side chain of the substituted arginine alter expression. at the level of the trans golgi, a post-golgi compartment (saraste and kuismanen, ) , or at the cell surface, the gp -gp complex was rapidly shunted to lysosomes and degraded-as shown by the protection afforded the terminally glycosylated env proteins by the lysosomatropic agent, chloroquine . the exact pathway that the molecules take to the lysosome is not known. they may be transported directly from the trans golgi or first to the surface where they are rapidly endocytosed. discriminating between the alternate pathways has not been possible from current data. why the insertion of an arginine into the anchor should result in targeting to lysosomes is not obvious. a possible explanation is that we have introduced a specific sorting signal into the molecule; however, this is unlikely since other polypeptides with charged residues in the membranespanning domain are not so targeted (kabcenell and atkinson, ; saito et al., ; hayday et al., ) . the arginine's charge is incompatible with the hydrophobic environment of the lipid bilayer; to achieve stability, the charged guanidinium group needs to be neutralized, and how this is done inside the bilayer is not clear. parsegian ( ) has postulated that a lone charge sequestered in a membrane must form a pore or tunnel along with localized membrane thinning to acheive the lowest energy state. if the charged residue in the gp anchor causes the mutant molecules to aggregrate and form channels in the membrane in an analogous manner, it would likely kill the cell unless there was a mechanism to remove it rapidly. alternatively, since the env protein is not an isolated entity in the membrane, it is conceivable that it aggregates with other components of the membrane to reduce net charge cooperatively, and thereby triggers the endocytotic machinery (mellman and plutner, ) . charged residues are found in several proposed membrane-spanning helices (kabcenell and atkinson, ; saito et al., ; hayday et al., ; reviewed by rao and argos, ) . the charged residues in bacteriorhodopsin membrane-spanning a helices have been suggested to be neutralized by forming ion pairs (engelman et al., ) . this is likely a special case, however, since the energy required to bury an ion pair in the membrane is not much different from that required to bury the free charged group itself (parsegian, ) . neutralization of strong charges, particularly of lysine and arginine, may occur through the formation of strong hydrogen bonds with tyrosine (kyte and doolittle, ) ; however, no tyrosine residues are present in the anchor domain of the env gene product which could participate with the arginine. the t cell a, ß, and y gene products and the rotavirus vp protein have a putative structure similar to that of the arginine mutant, with a lysine centered within the transmembrane anchor; however, unlike the molecule we created, they invariably have tyrosine residues adjacent to the lysine (saito et al., ; kabcenell and atkinson, ; hayday et al., ) which could stabilize the charge through hydrogen bonds (kyte and doolittle, ) . adams and rose ( a) have described the similar insertion of an arginine (and glutamine) residue at the center of the transmembrane domain of the vsv g protein. their mutant protein, like the env mutant we have characterized, was bitopic, could be seen localizing in the golgi, and did not accumulate on the cell surface. since these investigators observed a "lower level of protein expression" with their mutant g protein, it is possible that it also was rapidly degraded in lysosomes following terminal glycosylation. in contrast, observed no alteration in the biosynthesis and transport of a mutant p polypeptide of semliki forest virus in which the hydrophobic domain was interrupted by insertion of a glutamic acid residue. in this protein, however, the outer boundary of the hydrophobic domain is not delineated by a charged residue and so it is possible that additional uncharged residues from the external domain were pulled into the membrane. since the insertion of a charged polar residue into the transmembrane region of the rsv env gene product did not interfere with its anchor/stoptranslocation function, we have investigated the requirement for the long ( amino acid) hydrophobic domain in arresting translocation and anchoring the env complex. a series of deletion mutations was generated by progressively removing base pairs to either side of a unique sph\ restriction site that had been previously engineered into the center of the anchor coding region. this produced env proteins with truncated transmembrane anchors that ranged in length from (t ) to a single apolar amino acid (tl) summarized in fig. ; . while the effects of the deletions on the transport and subcellular localization of the env gene product appeared to be a complex function of the length and composition of the remaining anchor, the mutants appeared to fall into three broad phenotypic classes (summarized in table i ). even the smallest deletion (t ), which removed only three amino acids, greatly reduced the surface expression of the mature env proteins. t and mutants t , t , and t had a normal bitopic orientation in the membrane but appeared to be cleared from the cell surface and degraded in lysosomes, since they accumulated only in the presence of choroquine, an inhibitor of lysosomal degradation. the reduced surface expression of the as determined by the distance between charged residues. b hydrophobicity score as determined using the hydropathicity values of kyte and doolittle ( ) . mean hydrophobicity is hydrophobicity score divided by the apparent anchor length. c symbols: + denotes either a wild-type or positive response; ± denotes an intermediate response; -denotes a negative response. d sec, secreted. e nd/na, not determined/not applicable. f with chloroquine treatment, pr appears in the medium with gp and gp . « anchorless and tailess deletion mutant . largest deletion mutant (t ) was surprising since the effective hydrophobic peptide remaining in this construct was as long or longer than functional anchors reportedly present in other integral membrane proteins [e.g., amino acids: m protein of influenza (lamb, ) ; amino acids: vsv g protein (rose et al., ) and adenovirus e protein (wold et al., ) ]. deletions which reduced the size of the transmembrane anchor to seven amino acids (t ) or less resulted in the secretion of mature glycoproteins into the medium. a third class of mutants with hydrophobic regions of (t ) and (til) amino acids, respectively, while remaining membrane associated, no longer appeared to span the rer as bitopic proteins. neither mutant could be found at the surface of cells, nor could their degradation be arrested by chloroquine treatment. from the sum of the data obtained with these mutants, it would appear that bitopic insertion of the env gene product is possible with effective anchor domains of at least amino acids; if additional amino acids are removed from the domain, the protein can no longer exist bitopically, and it either partitions monotopically to the luminal side of the rer membrane or withdraws amino acids from the cytoplasmic side of the membrane into the bilayer. nevertheless, such sequences from the cytoplasmic domain are not able to stabilize the shortest anchors (tl, t , and t ) since these are secreted from the cell. davis and model ( ) have investigated the requirements of a functional anchor domain by inserting artificial hydrophobic peptides of varying length into the membrane-associated pill protein of the bacteriophage fl. their results show that hydrophobic amino acids are sufficient to maintain the protein in a bitopic configuration; however, the amino acid anchor was "deleterious to the cell" presumably because it was too short to assume a stable conformation compatible with existence in the bilayer and thereby destabilized the membrane. a construct with an anchor of hydrophobic amino acids was membrane associated but showed an intermediate phenotype in its sensitivity to solubilization by alkali. in contrast, a construct with only hydrophobic amino acids-also membrane associated-was completely released into the supernatant at high ph. adams and rose ( b) reduced the anchor domain of vsv g by precisely deleting amino acids from within the hydrophobic core. when the length of the anchor was reduced from amino acids to as few as , the protein was normally membrane associated and expressed in a bitopic fashion on the cell surface. on the other hand, proteins with an anchor domain of or amino acids, while spanning the membrane, appeared to be transported only as far as the golgi where they accumulated; the surface expression of these proteins was greatly reduced ( amino acid anchor) or undetectable ( amino acids). doyle et al. ( ) have characterized a series of carboxy-terminal deletion mutants of the ha polypeptide in which the amino acid anchor domain was truncated to , , and amino acids, respectively. in this case, molecules with a amino acid long transmembrane domain were stably anchored but were transported less efficiently to the plasma membrane. truncation of the hydrophobic anchor to or residues resulted in ha proteins that were unstable and whose transport appeared to be blocked in the rer or in a pre-golgi compartment-resembling the til mutant of the rsv env gene described above. from the results of these different systems and approaches it would seem that in strictly physical terms, anchors may be significantly reduced in their length without any consequence to the membrane association. the limits for this length ap-pear to be about - amino acids, but it must be appreciated that the mere presence of a stretch of hydrophobic amino acids within a protein does not serve to constitute an anchor. several mammalian virus envelope proteins contain in their external domain long hydrophobic amino acid regions that are equivalent to the truncated bitopic anchors have described here (gething et al, ; white et al, ) . indeed, gp contains a strongly hydrophobic amino acid region that clearly does not act as a stop-translocation sequence or play a role in membrane association . the work of davis and model ( ) , on the other hand, implies that the length of a hydrophobic region is the major determinant as to whether or not it will confer membrane association properties to a protein, although they point out that the position of such sequences within the molecule may play a role. most eukaryotic membrane-spanning polypeptides have a complex tertiary structure that is stabilized by multiple disulfide linkages, and it is possible that the entropy of a correctly folded molecule is sufficient to pull potential stop-transfer regions through the membrane. a corollary of this hypothesis, therefore, is that, once folding is complete, short hydrophobic regions that can potentially span the membrane as an a helix would stop translocation. the length requirements for such a region could be shorter than the residues predicted from a-helix dimensions if the region were flanked by arginines or lysines. since the latter have long side chains, equivalent in length to a single turn of an a helix, a stretch of hydrophobic amino acids - amino acids long might be sufficient. such a prediction fits well with the data we have obtained and with those of adams and rose ( b) and davis and model ( ) . it will be of interest to determine what effect inserting the truncated anchors of mutants t and t into the middle of the env precursor has on translocation; if the above speculations are correct they should be extruded into the external domain. finally, it should be reemphasized that merely providing a bitopic membrane anchor/stop-translocation is not sufficient to confer wild-type biological activity on a polypeptide. mutant t of the rsv env gene has a hydrophobic domain which might be expected to be sufficiently long and hydrophobic in character to span the membrane stably; it is modified normally by palmitic acid and can clearly be transported to its targeted cellular location. nevertheless, it is degraded rapidly by the cell. these results imply that hydrophobic transmembrane domains contain additional (and perhaps subtle) signals that remain to be deciphered, a conclusion that is supported by the finding that deletion of the anchor domain of the rotavirus vp protein abolishes its specific targeting and retention in the rer (poruchynsky et al, ) . polarized epithelial cells exhibit apical and basolateral membrane domains that are separated by well-defined tight junctions. each membrane domain has a unique protein composition (louvard, ; reggio et al., ) , indicating that mechanisms must exist to specifically target membrane proteins to different surfaces. the sorting process occurs during or shortly after passage of the glycoproteins through the golgi complex pfeiffer et al., ; rindler et al., rindler et al., , . however, the mechanisms determining this directed transport to either the apical or basolateral membranes are not understood. their study has been facilitated by the use of cultured epithelial cell lines, such as the mdck cell line, and by the observation that certain rna viruses bud exclusively from apical or basolateral domains of these polarized cells in culture (rodriguez-boulan and sabatini, ; herrler et al., ; roth et al., a; rindler et al., ) . avian and mammalian retroviruses together with rhabdoviruses such as vsv mature from the basolateral surface, while ortho-and paramyxoviruses bud from the apical surface. the carbohydrate residues present on the different proteins do not appear to play a role in this sorting process, since tunicamycin does not interfere with the polarized release of the viruses (roth et al., ; green et al., b) . as with the maturation of viruses that assemble at intracellular locations within the secretory pathway, polarized budding of enveloped viruses is dependent on the site to which viral glycoproteins are transported. expression of cloned viral glycoprotein genes from both sv -based and vaccinia expression vectors in polarized cells has demonstrated that the ha and neuraminidase polypeptides of influenza virus are targeted to the apical surface (roth et al., b; jones et al., ; gottlieb et al., ) while the g protein of vsv and the gp /pl e complex of murine leukemia virus (mulv) are transported exclusively to the basolateral membranes . in an attempt to locate the signals which direct these glycoproteins to the apical or basolateral domains, recombinant dna techniques have been employed to construct chimeric proteins and express these in polarized cells in culture. in experiments where sequences encoding the external domain of ha were fused to those encoding the transmembrane and cytoplasmic domains of the vsv g protein, the hybrid glycoprotein behaved in the same manner as wild-type ha and was transported to the apical domain of polarized cells (mcqueen et al., ; roth et al., ) . conversely, fusing the external domain of g protein to the anchor/cytoplasmic domain of ha results in basolateral transport (mcqueen et al., ) . these experiments thus suggest that the ectodomains of ha and g protein contain signals for apical and basolateral transport, respectively. while expression of a secreted form of the ha glycoprotein in an apical polarized manner supports this conclusion (roth et al. y ) , the unanchored ectodomain of the mulv gp /pl e complex, which is normally targeted to the basolateral membrane, is secreted in a nonpolarized fashion . it is possible that this soluble protein is improperly folded and thus is unable to interact with the sorting machinery, alternatively it also raises the possibility that targeting signals may be located in more than one domain of these molecules. further analyses should shed light on this problem. the studies described in this chapter demonstrate the breadth of information that has been and can be obtained from studies on enveloped virus glycoprotein biosynthesis. many of the studies were performed at a time when the cloned genes and molecular probes for cellular glycoproteins were unavailable and thus provided valuable insights into the manner in which cells compartmentalized and transported membrane proteins. the exciting possibility of utilizing viral glycoprotein genes for genetic analyses of the transport pathway, in a way analogous to that pursued in prokaryotic systems (michaelis and beckwith, ; silhavy et al. y ; oliver, ) , has led to a plethora of studies that utilized both classic and recombinant dna genetic approaches. these investigations have resulted in great progress in our understanding of the general processes involved in intracellular transport of proteins through the secretory pathway but at the same time have raised difficult questions about the molecular interactions required for protein sorting. the initial observation that proteins destined for secretion contain an amino-terminal sorting sequence provided a precedent on which to build models for protein targeting based on topogenic sequences (blobel, ) . to a large extent the identification of signal sequences was facilitated by their transient nature, not by a conserved primary sequence. indeed, while some common characteristics of signal peptides can be recognized (von heijne, ) , the identification of signal peptides in proteins where they are not removed has proved difficult and has required the use of sophisticated recombinant dna technology [e.g., ovalbumin (tabe et ah, ) ]. the requirement for a signal sequence to initiate translocation across the er membrane has been clearly confirmed through the isolation and construction of mutants which lack this functional region (as discussed above) and by fusion of this sequence to proteins that are not normally translocated (lingappa et al., ) . these approaches have also been facilitated by the transient, nonstructural nature of many aminoterminal, translocation signals. recent experiments by friedlander and blobel ( ) and kaiser et al. ( ) , however, raise questions about the informational content of signal sequences. in particular kaiser and colleagues showed that several random amino acid sequences derived from human genomic dna fragments could act as signal sequences for translocation of the yeast invertase enzyme. thus even in this well-defined situation, where an amino acid sequence is known to play a functional role in the sorting process, it can be impossible to predict with confidence its location in the protein; how then might we expect to identify additional sorting sequences that may or may not exist within the structural domain of a transported protein? the possibility that additional sorting sequences might be involved in steering the transport of a membrane-spanning or secreted protein through the vesicular maze of the secretory pathway remains open. at the present time the necessity for a native tertiary structure cannot be separated from the possibility of such additional sorting sequences. it is clear that disruption of a poly peptide's normal folding can completely prevent its transport from the er kreis and lodish, ) , and the simplest explanation for the phenotypes of a variety of conditional and nonconditional transport mutants would be that they alter the tertiary structure of the mature protein (see section ii, a, above). a -dimensional structure has been determined for only a few molecules that traverse the secretory pathway, and even with these proteins the current, predictive algorithms are insufficiently accurate to model potential changes in molecular shape in response to mutations. the question of a direct role for tertiary structure in protein transport thus represents a major challenge to molecular biologists. furthermore, one might argue that a change in protein shape could also mask or distort a necessary (peptide) sorting sequence. this possibility is supported by the observation that for a majority of eukaryotic proteins the amino-terminal signal peptide is unable to initiate translocation across the er if translation is allowed to proceed to completion, presumably because the tertiary structure of the nascent polypeptide precludes the interaction of the signal peptide with the translocation machinery. it is quite feasible that sorting signals and targeting signals could be represented by different entities within a single polypeptide, particularly if the latter were required to fix the intracellular location of a protein. for example, the rotavirus vp polypeptide accumulates within the er un-less its amino-terminal hydrophobic anchor region is deleted, whereupon it is transported to the cell surface and secreted (poruchynsky et al., ) ; in this instance the deleted region presumably contains a sequence that can fix the intracellular location of the protein despite the fact that the molecule has the potential to be exported from the cell. since most translocated polypeptides appear to follow a common pathway to a late compartment of the golgi (kelly, ) , it might be argued that a native conformation is the sole requirement for transport to this organelle and that the observed differences in the transport rates of proteins to the golgi merely reflect the time necessary for completion of the folding process. nevertheless, proteins leaving the golgi appear to be sorted into specific pathways; for example, in secretory cells proteins may follow either the constitutive pathway or be sequestered in secretory granules (moore and kelly, ; reviewed by kelly, ) , and in epithelial cells specific proteins appear to be transported directly to either the apical or basolateral membranes (see above). thus, it would seem likely that some form of sorting signal must be present in the polypeptide at this point in the secretory pathway in order to correctly direct its transport; initial results from viral glycoprotein expression studies indicate that at least in polarized cells the ectodomain of the sorted protein plays a dominant role (mcqueen et al., ; . additional studies should provide a clearer picture of this complex process. in summary, studies on the biosynthesis and transport of enveloped virus glycoproteins have provided important insights into the general processes involved in the intracellular movement of these membrane-associated molecules. the specific questions that remain to be answered are many and difficult, but it is likely that these viral systems will continue to play a vital role by providing clues and direction in this important area of cell biology. incorporation of a charged amino acid into the membrane spanning domain blocks cell surface transport but not membrane anchoring of a viral protein structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport sequence analysis of two mutants of sindbis virus defective in the intracellular transport of their glycoproteins domain structure of bacteriophage fed adsorption protein intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide information within the mature lamb protein necessary for localization to the outer membrane of eseherichia coli k- genetic analysis of protein export in escherichia coli k- immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (g) of vesicular stomatitis virus in infected chinese hamster ovary cells passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the golgi apparatus en route to the plasma membrane bunyaviridae. in "comprehensive virology intracellular protein topogenesis transfer of proteins across membranes. i. presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma studies on the size, chemical composition and partial sequence of the neuraminidase (na) from type a influenza viruses show the nterminal region of na is not processed and serves to anchor na in the viral membrane a prokaryotic membrane anchor sequence: carboxyl terminus of bacteriophage fl gene iii protein retains it in the membrane processing of filamentous phage precoat protein: effect of sequence variations near the signal peptidase cleavage site nh -terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation processing of gpr env, the precursor to the glycoproteins of rous sarcoma virus: use of inhibitors of oligosaccharide trimming and glycoprotein transport sequence of the membrane protein gene from avian coronavirus ibv virus-specific glycoproteins associated with the nuclear fraction of herpes simplex virus type -infected cells mutants of the membrane-binding region of semliki forest virus e protein. i. cell surface transport and fusgenic activity mutants of the membrane-binding region of semliki forest virus e protein. ii. topology and membrane binding fusion mutants of the influenza virus hemagglutinin glycoprotein isolation of the escherichia coli leader peptidase gene and effects of leader peptidase overproduction in vivo a charged amino acid substitution within the transmembrane anchor of the rous sarcoma virus envelope glycoprotein affects surface expression but not intracellular transport altered surface expression, membrane association and intracellular transport result from deletions within the transmembrane anchor of the rous sarcoma virus envelope glycoprotein an artificial anchor domain: hydrophobicity suffices to stop transfer fine structure of a membrane anchor domain variant influenza virus hemagglutinin that induces fusion at elevated ph mutations in the cytoplasmic domain of influenza virus hemagglutinin affect different stages of intracellular transport analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin assembly of enveloped rna viruses early and late functions associated with the golgi apparatus reside in distinct compartments localization and processing of outer membrane and periplasmic proteins in escherichia coli strains harboring export-specific suppressor mutations suppressor mutations that restore export of a protein with a defective signal sequence importance of secondary structure in the signal sequence for protein secretion path of the polypeptide in bacteriohodopsin amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor evidence for a glycoprotein "signal" involved in transport between subcellular organelles bovine opsin has more than one signal sequence characterization of two recombinant-complementation groups of uukuniemi virus temperature-sensitive mutants uukuniemi virus glycoproteins accumulate in and cause morphological changes of the golgi complex in the absence of virus maturation nucleotide sequence of a cdna clone encoding the entire glycoprotein from the new jersey serotype of vesicular stomatitis virus a single amino acid substitution in a hydrophobic domain causes temperature-sensitive cell-surface transport of a mutant viral glycoprotein using recombinant dna techniques to study protein targeting in the eucaryotic cell expression of semliki forest virus proteins from cloned complementary dna. ii. the membrane-spanning glycoprotein e is transported to the cell surface without its normal cytoplasmic domain rous sarcoma virus pl and gp can be chemically crosslinked to high molecular weight complexes. an insight into viral association cold spring harbor laboratory, cold spring habor construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein purification of the fusion protein of sendai virus: analysis of the nh -terminal sequence generated during precursor activation cloning and dna sequence of double-stranded copies of haemagglutinin genes from h and h strains elucidates antigenic shift and drift in human influenza virus expression of wild-type and mutant forms of influenza hamagglutinin: the role of folding in intracellular transport synthesis and infectivity of vesicular stomatitis viruses containing nonglycosylated g protein the nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures protein translocation across the endoplasmic reticulum. i. detection in the microsomal membrane of a receptor for the signal recognition particle protein translocation across the endoplasmic reticulum. ii. isolation and characterization of the signal recognition particle receptor the mechanism of protein translocation across the endoplasmic reticulum membrane sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells passage of viral membrane proteins through the golgi complex glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells conversion of a secretory protein into a transmembrane protein results in its transport to the golgi complex but not to the cell surface glycosylation allows cell-surface transport of an anchored secretory protein two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells amino-terminal deletion mutants of the rous sarcoma virus glycoprotein do not block signal peptide cleavage but block intracellular transport biosynthesis of lysosomal enzymes in fibroblasts: phosphorylation of mannose residues structure, organization, and somatic rearrangement of t cell a genes antitrypsin: the presence of excess mannose in the z variant isolated from liver isolation and structural analysis of influenza virus c virion glycoproteins studies on the mechanisms of tunicamycin inhibition of iga and ige secretion by plasma cells the importance of the endosome in intracellular traffic sequence of the long terminal repeat and adjacent segments of the endogenous avian virus rous-associated virus complete sequence of the rous sarcoma virus env gene: identification of structural and functional regions of its product mechanism of signal peptide cleavage in the biosynthesis of the major lipoprotein of the escherichia coli outer membrane phospholipid is required for the processing of presecretory proteins by detergent-solubilized canine pancreatic signal peptidase intracellular transport of herpes simplex virus gd occurs more rapidly in uninfected cells than in infected cells surface expression of influenza virus neuraminidase an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus sah many random sequences functionally replace the secretion signal sequence of yeast invertase cysteines in the transmembrane region of major histocompatability complex antigens are fatty acylated via thioester bonds pathway of protein secretion in eukaryotes ph-induced alterations in the fusogenic spike protein of semlilci forest virus membrane fusion mutants of semliki forest virus separate pathways of maturation of the major structural proteins of vesicular stomatitis virus maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus primary structure and transmembrane orientation of the murine anion exchange protein oligomerization is essential for transport of the vesicular stomatitis virus glycoprotein to the cell surface uukuniemi virus maturation: an immune fluorescence microscopy study using monoclonal glycoprotein-specific antibodies a simple method for displaying the hydropathic character of a protein influenza m protein is an integral membrane protein expressed on the infected-cell surface impaired intracelluiar migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and sindbis virus kinetics of serum protein secretion by cultured hepatoma cells: evidence for multiple secretory pathways determinants for protein localization: / -lactamase signal sequence directs globin across microsomal membranes hen oviduct signal peptidase is an integral membrane protein reversible block in intracellular transport and budding of mutant vesicular stomatitis virus glycoprotein hepatoma secretory proteins migrate from rough endoplasmic reticulum to golgi at characteristic rates apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells l ). a single n-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface polarized expression of a chimeric protein in which the transmembrane and cytoplasmic domains of the influenza virus hemagglutinin have been replaced by those of the vesicular stomatitis virus g protein basolateral expression of a chimeric protein in which the transmembrane and cytoplasmic domains of vesicular stomatitis virus g protein have been replaced by those of the influenza virus hemagglutinin glycosylation and surface expression of the influenza virus neuraminidase requires the n-terminal hydrophobic region the entry of enveloped viruses into cells by endocytosis internalization and degradation of macrophage fc receptors bound to polyvalent immune complexes acidification of the endocytic and exocytic pathways identification and characterization of a membrane component essential for the translocation of nascent proteins across the membrane of the endoplasmic reticulum secretory protein translocation across membranes-the role of the "docking protein mechanism of incorporation of cell envelope proteins in escherichia coli secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways structural mutations in a mouse immunoglobulin light chain resulting in failure to be secreted construction, expression and recognition of an h- molecule lacking its carboxyl terminus identification of the defects in the hemagglutinin gene of two temperature-sensitive mutants of a/wsn/ influenza virus membrane-bound penicillinases in grampositive bacteria carbohydrate moieties of glycoproteins, a reevaluation of their function protein secretion in escherichia coli ovalbumin: a secreted protein without a transient hydrophobic leader sequence energy of an ion crossing a low dielectric membrane: solutions to four relevant problems similarities among retro virus proteins mutations within the proteolytic cleavage site of the rous sarcoma virus glycoprotein precursor block processing to gp and gp mutants of the rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and/or cytoplasmic domains: analysis of intracellular transport and assembly into virions a putative signal peptidase recognition site and sequence in eucaryotic and procaryotic signal peptides reversible defect in the glycosylation of the membrane proteins of semliki forest virus tsl mutant localization of rotavirus antigens in infected cells by ultrastructural immunocytochemistry intracellular sorting and basolateral appearance of the g protein of vesicular stomatitis virus in mdck cells complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned dna deletions into an nh -terminal hydrophobic domain result in secretion of rotavirus vp , a resident endoplasmic reticulum glycoprotein export of protein in bacteria a conformation preference parameter to predict helices in integral membrane proteins surface and cytoplasmic domains in polarized epithelial cells transmembrane orientation of glycoproteins encoded by the w-fms oncogene viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellular transport in doubly infected madin-darby canine kidney cells polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of madin-darby canine kidney cells infected with temperature-sensitive viruses asymmetric budding of viruses in epithelial monolayers: a model system for study of epithelial polarity intracellular transport of influenza virus hemagglutinin to the apical surface of madin-darby canine kidney cells expression from cloned cdna of cell-surface secreted forms of the glycoprotein of vesicular stomatitis virus in eukaryotic cells altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein vesicular stomatitis virus is anchored in the viral membrane by a hydrophobic domain near the cooh terminus the presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition polarity of influenza and vesicular stomatitis virus in mdck cells; lack of a requirement for glycosylation of viral glycoproteins influenza virus hemagglutinin expression is polarized in cells infected with recombinant sv viruses carrying cloned hemagglutinin dna basolateral maturation of retroviruses in polarized epithelial cells heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway membrane insertion and intracellular transport of influenza virus glycoproteins the large external domain is sufficient for the correct sorting of secreted or chimeric influenza virus hemagglutinins in polarized monkey kidney cells studies on the adaption of influenza viruses to mdck cells a mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein mechanisms for the incorporation of proteins in membranes and organelles complete primary structure of a heterodimeric t-cell receptor deduced from cdna sequences pre-and post-golgi vacuoles operate in the transport of semliki forest virsus membrane glycoproteins to the cell surface fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of posttranslational modification of the viral glycoprotein relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and sindbis virus membrane glycoproteins evidence for covalent attachment of fatty acids to sindbis virus glycoproteins defects in functional expression of an influenza virus hemagglutinin lacking the signal peptide sequences analysis of the hemagglutinin glycoprotein from mutants of vaccinia virus that accumulates on the nuclear envelope mechanisms of protein localization changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion the phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes glycoproteins specified by herpes simplex viruses an internal signal sequence: the asialoglycoprotein receptor membrane anchor nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells polarized transport of the vsv g surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors intracellular transport of secretory and membrane proteins in hepatoma cells infected by vesicular stomatitis virus vesicular stomatis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same golgi vesicles effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chicken hepatocytes the molecular biology of coronaviruses cell surface expression of the influenza virus hemagglutinin requires the hydrophobic carboxy-terminal sequences influenza virus hemagglutinin containing an altered hydrophobic carboxy terminus accumulates intracellularly segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in xenopus oocytes: identification of an ovalbumin signal sequence eukaryotic signal sequence transports insulin antigen in escherichia coli prolipoprotein signal peptidase in escherichia coli is distinct from the m procoat protein signal peptidase temperature-sensitive mutants of influenza virus: a mutation in the hemagglutinin gene structural studies of iga myeloma proteins having anti-dnp antibody activity patterns of amino acids near signal-sequence cleavage sites how signal sequences maintain cleavage specificity signal sequences, the limits of variation purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum signal recognition protein (srp) mediates the selective binding to microsomal membranes of m-i>//ro-assembled poly somes synthesizing secretory protein translocation of proteins across the endoplasmic reticulum iii. signal recognition protein (srp) causes signal sequence-dependent and sitespecific arrest of chain elongation that is released by microsomal membranes translocation of proteins across the endoplasmic reticulum i. signal recognition protein (srp) binds to m-wvro-assembled polysomes synthesizing secretory protein membrane proteins: structure and assembly compilation of published signal sequences m procoat and a preimmunoglobulin share processing specificity but use different membrane receptor mechanisms cell fusion by semliki forest, influenza, and vesicular stomatitis viruses membrane fusion proteins of enveloped animal viruses multiple mechanisms of protein insertion into and across membranes alterations in the transport and processing of rous sarcoma virus envelope glycoproteins mutuated in the signal and anchor regions mutations of the rous sarcoma virus env gene that affect the transport and subcellular location of the glycoprotein products the -kda glycoprotein precursor coded by region e of adenovirus secretion of a x immunoglobulin chain is prevented by a single amino acid substitution in its variable region molecular abnormality of human a r antitrypsin variant (pi-zz) associated with plasma activity deficiency uncoating of influenza virus in endosomes a stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems the transmembrane segment of the human transferrin receptor functions as a signal peptide mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein expression and function of transplantation antigens with altered or deleted cytoplasmic domains key: cord- -cqvnxsbo authors: bryant, nia j.; stevens, tom h. title: two separate signals act independently to localize a yeast late golgi membrane protein through a combination of retrieval and retention date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: cqvnxsbo the localization of proteins to late-golgi membranes (tgn) of saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. these signals could act by directing retrieval from a post-golgi compartment or by preventing exit from the tgn. to investigate the mechanism of localization of yeast tgn proteins, we used the heterologous protein a-alp (consisting of the cytosolic domain of dipeptidyl aminopeptidase a [dpap a] fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [alp]), which localizes to the yeast tgn. insertion of the aromatic residue–based tgn localization motif (fxfxd) of dpap a into the cytosolic domain of alp results in a protein that resides in the tgn. we demonstrate that the fxfxd motif confers golgi localization through retrieval from a post-golgi compartment by detecting a post-golgi processed form of this protein in the tgn. we present an assay that uncouples retrieval-mediated golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast tgn. we also demonstrate that the cytosolic domain of dpap a contains additional information, separate from the retrieval motif, that slows exit from the tgn. we propose a model for dpap a localization that involves two distinct mechanisms: one in which the fxfxd motif directs retrieval from a post-golgi compartment, and a second that slows the rate at which dpap a exits the tgn. membranes (tgn) of saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. these signals could act by directing retrieval from a post-golgi compartment or by preventing exit from the tgn. to investigate the mechanism of localization of yeast tgn proteins, we used the heterologous protein a-alp (consisting of the cytosolic domain of dipeptidyl aminopeptidase a [dpap a] fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [alp] ), which localizes to the yeast tgn. insertion of the aromatic residue-based tgn localization motif (fxfxd) of dpap a into the cytosolic domain of alp results in a protein that resides in the tgn. we demonstrate that the fxfxd motif confers golgi localization through retrieval from a post-golgi compartment by detecting a post-golgi processed form of this protein in the tgn. we present an assay that uncouples retrieval-mediated golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast tgn. we also demonstrate that the cytosolic domain of dpap a contains additional information, separate from the retrieval motif, that slows exit from the tgn. we propose a model for dpap a localization that involves two distinct mechanisms: one in which the fxfxd motif directs retrieval from a post-golgi compartment, and a second that slows the rate at which dpap a exits the tgn. t he secretory pathway of eukaryotic cells is composed of a series of membrane-bound organelles, each with its own unique complement of components. transport of components between these organelles is achieved by means of vesicular transport, which results in a flow of membrane traffic throughout the pathway (rothman, ) . to maintain its identity, an organelle must ensure that its resident proteins do not get swept with those proteins passing through en route to other destinations in the cell. to achieve this localization, resident proteins can either be retrieved once they have exited the organelle, or they may be prevented from ever leaving that organelle in the first place . these two mechanisms need not be mutually exclusive, since efficient localization of a protein to an organelle could arise through a combination of both mechanisms. both luminal and membrane proteins are localized to the er through continuous retrieval from a post-er compartment after recognition of specific localization signals (lewis and pelham, ; townsley et al., ) . in addition, it seems that the selection of cargo for entry into vesicles budding from the er also plays an important role in the retention of proteins in this organelle (schekman and orci, ) . the mechanism for localization of proteins to the golgi apparatus is less clearly understood, but it is likely that both modes of localization are at work. studies on glycosyltransferases have shown that the transmembrane domains of these molecules are essential for golgi localization in both yeast and mammalian cells (munro, ; nilsson et al., ; machamer et al., ; chapman and munro, ; graham and krasnov, ; lussier et al., ) . current theories favor models in which the transmembrane domains prevent entry of resident proteins into transport vesicles. this may be achieved either by the formation of aggregates too large to enter transport vesicles (swift and machamer, ; nilsson et al., ) or by exclusion of proteins from vesicles because of the length of their membrane spanning domain (bretscher and munro, ) . however, localization of the yeast glycosyltransferase, och p, which resides in the cis -golgi, requires retrieval from a more distal golgi compartment (harris and waters, ) . other golgi membrane proteins residing in the tgn achieve their localization through more dynamic means. tgn , furin, and the mannose- -phosphate receptor are localized to the tgn of mammalian cells by virtue of aromatic residue containing signals in their cytosolic tails (bos et al., ; wong and hong, ; humphrey et al., ; canfield et al., ; takahashi et al., ; voor-hees et al., ; schafer et al., ) . these proteins are localized through a similar mechanism to that in operation at the er: they continuously exit the tgn and are later retrieved from endosomal compartments (kornfeld, ) . although the morphology of the last definable subcompartment of the golgi of saccharomyces cerevisiae has not been described at the ultrastructural level, it is taken as being functionally equivalent to the tgn of mammalian cells (graham and emr, ; wilcox and fuller, ; wilsbach and payne, ; nothwehr et al., ) . the yeast tgn is defined as the compartment where proteins destined for the cell surface are sorted from those destined for delivery to the vacuole, and contains the three processing proteinases involved in the maturation of the mating pheromone ␣ -factor (kex p, kex p, and ste p; also called dipeptidyl aminopeptidase a [dpap a] ; bryant and boyd, ; nothwehr et al., ) , as well as the vacuolar protein sorting receptor (vps p; marcusson et al., ; cereghino et al., ; cooper and stevens, ) . the cytosolic tail of each of these four integral membrane proteins is required for localization in the yeast tgn wilcox et al., ; cooper and bussey, ; cereghino et al., ; cooper and stevens, ) , and more specifically aromatic residues have been shown to be essential for the proper localization of dpap a, kex p, and vps p (wilcox et al., ; nothwehr et al., ; cereghino et al., ; cooper and stevens, ) . the cytosolic tail of dpap a is sufficient to localize the transmembrane and luminal domains of the vacuolar membrane protein alkaline phosphatase (alp) in golgi membranes (nothwehr et al., ) . the resulting fusion protein, a-alp, has served as a model tgn marker protein and has been used to show that a -residue segment (containing the motif fxfxd) within the cytosolic domain of dpap a is critical for the protein's localization to golgi membranes (nothwehr et al., ) . insertion of this motif into the cytosolic domain of alp (rs-alp; retention sequence alp) results in a protein that colocalizes with kex p in the yeast tgn (nothwehr et al., ) . mutagenesis of the fxfxd motif leads to delivery of the protein to the vacuole (nothwehr et al., ) , an observation that contributed to the formulation of a model of how proteins are retained in the s. cerevisiae tgn (nothwehr and stevens, ; nothwehr et al., ) . in this model, proteins continually leave the golgi and enter a late endosomal/prevacuolar compartment (pvc), from which they are retrieved back to the golgi apparatus after recognition of the fxfxd motif. evidence in support of the above model comes from the observation that the exaggerated pvc that accumulates in class e vps mutants contains proteins normally resident to the tgn (vps p, kex p, dpap a, and a-alp), as well as vacuolar proteins and endocytosed proteins (raymond et al., ; piper et al., ) . vps p has been shown to be involved in controlling traffic through the pvc, and it functions to allow vps p to return to the tgn from the pvc (piper et al., ) . these data support a model in which proteins resident to the yeast tgn enter the pvc, from where they are efficiently retrieved back to the golgi apparatus. to date, little direct evidence exists to discriminate between this sort of retrieval mechanism and a static retention mechanism where proteins would be excluded from vesicles leaving golgi membranes and, consequently, have a longer residency period in the tgn. the mechanistic role of the aromatic localization signals identified in tgn proteins of s. cerevisiae is not known. to determine the mechanism by which information within the cytosolic tail of dpap a confers golgi localization, we devised an assay to uncouple "retrieval"-mediated golgi localization from "static retention"-mediated golgi localization by analyzing the trafficking of a series of a-alp mutants in vps ⌬ cells. here, we report that the aromatic residue based motif fxfxd of the dpapa tail mediates retrieval from the pvc. we also report the discovery of a second signal near the nh terminus of the dpap a tail that acts independently of the retrieval mechanism to slow the rate of departure of this protein from the tgn. the yeast strains used in this study are listed in table i . strains were constructed by standard genetic techniques and grown in rich media ( % yeast extract, % peptone, % dextrose; yepd) or standard minimal medium (sd) with appropriate supplements as described by sherman et al. ( ) . strain nby was constructed by transforming sny - a with the vps ⌬ ::leu disruption cassette (bamhi/ecori fragment) from pkjh , and by screening leu ϩ transformants first for the secretion of carboxypeptidase y (cpy) and subsequently for loss of production of vps p, as determined by immunoblot analysis of cell lysates. the pho gene of my was disrupted to give rise to nby by transforming psn linearized with sali, selecting for ura ϩ colonies, and then selecting for ura Ϫ loop outs on media containing -fluoroorotic acid. immunoblot analysis was used to identify ura Ϫ loop outs lacking pho p. yeast strains nby and nby were constructed by integrating the vps - * allele into the vps locus of sny - a and nby , respectively, as described by cooper and stevens ( ) . this plasmids used in this study are listed in table ii . deletions within the cytosolic domain of a-alp were constructed by oligonucleotide-directed mutagenesis of pcjr according to the method of kunkel et al. ( ) . to incorporate these deletions into the a-alp fusion protein, eagi-bglii fragments from derivatives of pcjr carrying the appropriate deletions were subcloned into the eagi-bglii sites of psn . the following dele- . abbreviations used in this paper : alp, alkaline phosphatase; cpy, carboxypeptidase y; dpap a, dipeptidyl aminopeptidase a; e, extracellular; i, intracellular; procpy, cpy precursor; pm, plasma membrane; pvc, prevacuolar compartment; rs-alp, retention sequence alp. tions were made: pnb ( ⌬ - ), pnb ( ⌬ - ), pnb ( ⌬ - ), and pnb ( ⌬ - ), where the deletion endpoints are indicated (e.g., ⌬ - is missing amino acid residues - ). met labeling and immunoprecipitation of alp fusion proteins, vps prelated proteins, and cpy were performed as described previously (piper et al., ; nothwehr et al., ; cooper and stevens, ) . briefly, yeast cultures were grown overnight in selective synthetic media without methionine to od ϭ . cells were harvested and resuspended in fresh media to the same od . cells were pulse labeled for min with mci s-express label/ . od , followed by the addition of unlabeled methionine and cysteine, both to g/ml. at specified times, samples were removed and treated by the addition of sodium azide to mm at Њ c. vps p, cpy, alp, and related proteins were immunoprecipitated from protein extracts of these cells using polyclonal antibodies specific for these proteins, as described previously (piper et al., ; nothwehr et al., ; cooper and stevens, ) . half-times of processing of alp-and vps p-related proteins were determined as described previously (nothwehr et al., ; cooper and stevens, ) using a radioanalytic imaging system (ambis, inc., san diego, ca), and linear regression analysis plotting percentage total protein was processed as a function of time. fractionation studies were carried out essentially as described using the differential sedimentation of subcellular membranes followed by equilibrium gradient analysis (becherer et al., ) . in brief, cells were grown in ml sd medium lacking uracil to an od ϭ . . sodium azide was added to a final concentration of mm before the cells were harvested and resuspended in ml mm tris-hcl, ph . , mm edta, % ␤ -mercaptoethanol. after incubation at Њ c for min, cells were harvested and resuspended in . m sorbitol, mm kpo , ph . , mm mgcl (spheroplast buffer) containing g/ml zymolyase. cells were incubated for h at Њ c and washed twice with spheroplast buffer. spheroplasts were lysed in ml mm tris-hcl, ph . , . m sorbitol, mm edta (lysis buffer) containing added proteinase inhibitors (pmsf [ mm], leupeptin [ g/ml], antipain [ g/ml], chymostatin [ g/ml], and pepstatin [ g/ml]) using strokes in a dounce homogenizer (this and subsequent steps were all performed at Њ c). cell debris and unlysed cells were removed by centrifugation at g for min. centrifugation at , g for min yielded pellet (p ) and supernatant (s ) fractions. the s fraction was further separated by centrifugation at , g for min to yield a membrane pellet fraction. the , g membrane pellet was resuspended in ml cold lysis buffer and loaded on the top of a - % sucrose step gradient. after centrifugation at , g for h, ϫ -ml fractions were collected from the top of the gradient, and the proteins from these fractions were precipitated using tca. equal percentages of each fraction were subjected to immunoblot analysis after sds-page. the preparation of fixed spheroplasted yeast cells, attachment to microscope slides, and costaining of alp fusion proteins using mab d -a (molecular probes) and vph p using affinity-purified polyclonal antibodies were carried out as described previously (nothwehr et al., ) . essentially, fixed spheroplasts attached to slides were incubated with the following solutions, followed by extensive washing with mg/ml bsa in pbs after each step: ( a ) pbs-bsa containing a : dilution of mouse anti-alp mab d -a (molecular probes) and a : dilution of affinity-purified rabbit anti-vph p polyclonal antibody; ( b ) : dilution of a biotin-conjugated goat anti-mouse igg (heavy and light chains [h ϩ l]); and ( c ) : dilution of both fitc-conjugated streptavidin and rhodamine-conjugated goat anti-rabbit igg (h ϩ l). staining using affinity-purified antibodies specific for vps p was also carried out as described previously (cooper and stevens, ) , using a : dilution of the affinity-purified polyclonal antibody, followed by a : dilution of a biotin-conjugated goat antimouse igg (h ϩ l) and, subsequently, streptavidin-conjugated fitc. to assess the morphological redistribution of alp fusion proteins and vps p in cells harboring a temperature-sensitive allele of vps , cells were grown in yepd at Њ c to od ϭ , at which time cycloheximide was added to a final concentration of g/ml, and an aliquot of these cells was fixed and prepared for immunofluorescence microscopy. to follow the fate of these proteins upon loss of vps p function, the remainder of the cells were warmed rapidly to Њ c, and the culture was moved to a to observe the fate of various alp fusion proteins after their accumulation in the pvc as a result of a loss of vps p function, vps mutant cells harboring vps under control of the gal promoter (ϩ) or empty plasmid (Ϫ) were grown to od ϭ in synthetic media containing % raffinose. galactose was added to a final concentration of %, and the cultures were split in half. one half was treated immediately (Ϫ galactose) and the other half was returned to Њc for min (ϩ galactose). at both time points, samples were prepared for immunofluorescence microscopy, and whole-cell extracts were prepared to monitor the induction of vps p production by immunoblot analysis. . od units were analyzed for cpy secretion after pulse-chase labeling with [ s]methionine. the cytosolic domain of dpap a is necessary and sufficient to retain the transmembrane and luminal domains of the vacuolar protein alp in the last definable golgi subcompartment in yeast (nothwehr et al., ) , which is functionally equivalent to the tgn of mammalian cells (franzusoff et al., ; redding et al., ; graham and emr, ) . mutational analysis identified the aromatic residue-containing motif, fxfxd, as being essential for the localization of the fusion protein a-alp (consisting of the cytosolic domain of dpap a fused to the transmembrane and luminal domains of alp) to the yeast tgn. forms of the a-alp protein lacking this signal (such as (f/a)a-alp, in which the two phenylalanine residues have been mutated to alanines) fail to be retained in the golgi complex and are delivered to the vacuole wilcox et al., ; cooper and bussey, ) . delivery of proteins containing the luminal domain of alp to the vacuole can be monitored by detecting a vacuolar protease-dependent (pep -dependent) cleavage, which results in the production of a lower molecular weight form of the protein (klionsky and emr, ; nothwehr et al., ) . whereas alp is delivered to the vacuole and processed with a half-time of Ͻ min (klionsky and emr, ) , a-alp is localized to the tgn and does not undergo any significant processing (nothwehr et al., ) . insertion of the fxfxd motif into the cytosolic domain of alp causes the protein rs-alp to be retained in the tgn, as determined by indirect immunofluorescence (nothwehr et al., ) . like a-alp, rs-alp has been shown to colocalize with the tgn protein kex p (nothwehr et al., ) , but rs-alp undergoes pep -dependent proteolytic cleavage with a half-time of min in wild-type cells, whereas a-alp does not undergo proteolysis, even after chase times of up to h (fig. b) . these data indicate that even though rs-alp localizes to the tgn in the steady state, it becomes exposed to vacuolar proteases in a post-golgi compartment. if rs-alp is being retrieved back to the tgn from a vacuolar protease-containing, post-golgi compartment (i.e., the pvc), then proteolytically processed rs-alp should fractionate with tgn membranes. to test this possibility, we separated yeast membranes by sucrose density gradient centrifugation. membranes from cell lysates were sedi-mented at , g to obtain a membrane pellet containing er, vacuolar, mitochondrial, and plasma membranes (paravicini et al., ; piper et al., ) . the resulting supernatant was subjected to centrifugation at , g, and the resulting membrane pellet (containing ‫% ف‬ of the total cellular vph p and - % of the total cellular pep p, kex p, and a-alp or rs-alp) was resuspended and loaded on a - % sucrose gradient. in this gradient, the residual vacuolar membranes, defined by vph p (manolson et al., ; piper et al., ) , and prevacuolar membranes, defined by pep p (becherer et al., ; piper, r.c., and t.h. stevens, unpublished results) , were found at the top of the gradient (fractions - , fig. a) . by con- ]methionine for min and chased by adding unlabeled methionine and cysteine each to a final concentration of g/ml. at the indicated times, the cells were spheroplasted and extracts were immunoprecipitated with a polyclonal antibody against alp, followed by sds-page and fluorography. the product of the initial pep -dependent proteolysis is indicated by a single asterisk, with a double asterisk indicating a further breakdown product of this primary product. this breakdown pattern differs from that seen for a-alp (which has only one pep -dependent breakdown product, e.g., fig. ), but clearly both of the lower bands shown here are produced in a pep -dependent manner. trast, tgn membrane proteins (a-alp and kex p) fractionated near the bottom of the gradient. both the processed and unprocessed forms of rs-alp also cofractionated with kex p in the tgn membrane fractions (fig. b) . these data indicate that rs-alp has reached a post-golgi compartment containing activated vacuolar proteases, and has been retrieved back to the tgn. to investigate retrieval from the pvc in greater detail, we turned to yeast cells harboring mutations in the vps gene. vps is required for the movement of traffic out of the pvc, both back to the tgn and onto the vacuole (raymond et al., ; piper et al., ) . vps mutant cells accumulate an exaggerated form of the pvc (the class e compartment) containing endocytosed proteins, as well as tgn membrane proteins and proteins en route to the vacuole, such as activated vacuolar proteases ( fig. ; raymond et al., ; piper et al., ) . we have followed the fate of a-alp, (f/a)a-alp, and rs-alp in a vps mutant before and after induction of vps under control of the gal promoter. immunoblot analysis (fig. a) revealed that vps mutant cells carrying the gal -vps plasmid (phy ) produced vps p only after the addition of galactose. pulse-chase immunoprecipitation of the vacuolar protease cpy demonstrated that secretion of cpy was suppressed in vps mutant cells now producing vps p (fig. b) , indicating that the cpy sorting receptor (vps p) had regained the ability to cycle between the pvc and the tgn. we performed indirect immunofluorescence to determine the fate of a-alp, rs-alp, and the retrieval-defective form of a-alp, (f/a)a-alp in vps mutant cells after restoration of vps p function. all three proteins localized to the exaggerated pvc in vps mutant cells (fig. a) , indicating that they are transported to the pvc, but either fail to be retrieved to the tgn (a-alp and rs-alp) or fail to be transported to the vacuole ((f/a)a-alp). approximately min after induction of vps p synthesis by addition of galactose (fig. b) , a-alp and rs-alp redistributed to a punctate pattern as commonly observed for golgi proteins in wild-type cells (franzusoff et al., ; redding et al., ; roberts et al., ; wilcox et al., ; nothwehr et al., ) . in the same cells in which a-alp and rs-alp redistributed to the tgn, the vacuolar membrane protein vph p redistributed from the class e compartment to the vacuole (fig. b) . by contrast to a-alp and rs-alp, min after induction of vps p synthesis, (f/a)a-alp colocalized to the vacuole membrane with vph p (fig. b) . finally, in vps cells that did not contain vps under gal control, a-alp and vph p remained in the class e compartment after the addition of galactose (data not shown). taken together, these data show that the fxfxd motif found in the dpap a cytosolic domain is both necessary and sufficient for retrieval from the pvc to the tgn. we were interested to further investigate the observation that whereas both a-alp and rs-alp localize to the tgn, only rs-alp undergoes measurable proteolytic processing in wild-type yeast cells. one possibility, suggested by the observation that (f/a)a-alp is processed with a half-time of min while alp is processed with a half-time of Ͻ min (nothwehr et al., ; klionsky and emr, ) , is that the cytosolic tail of dpap a contains a second signal, not present in rs-alp, that slows its exit from the tgn. to test this hypothesis, we designed an assay to estimate the rate of exit of membrane proteins from the tgn. the assay is based on measuring the rate of proteolytic processing of tgn proteins in vps mutant cells, since these proteins accumulate in the class e compartment and this compartment contains activated vacuolar proteases (raymond et al., ; piper et al., ) . therefore, we predict that proteins that exit the tgn slowly will exhibit long half-times for proteolytic processing in vps figure . sucrose gradient fractionation of a-alp/rs-alp containing membranes. high speed pellet membrane fractions ( , g) were prepared from od units of sny - a cells harboring either psn (a-alp) or psn (rs-alp). these membranes were fractionated by a - % sucrose density step gradient. after centrifugation, ϫ -ml fractions were collected from the top of the gradient, and the proteins were precipitated by the addition of tca. equal percentages of each fraction were subjected to immunoblot analysis after sds-page to detect the following proteins: a-alp/rs-alp using an anti-alp mab ( d -a ); kex p was used as a marker protein for tgn membranes and detected using affinity-purified polyclonal antibodies; anti-vph p mab d -a -b was used to detect vph p as a marker protein for vacuolar membranes; and pep p, a marker protein for membranes of the prevacuolar compartment, was detected using affinity-purified polyclonal antibodies. mutant cells whether or not they are competent to be retrieved to the tgn. by contrast, proteins that exit rapidly from the tgn would be rapidly proteolytically processed in vps mutant cells independently of their potential to be retrieved. pulse-chase immunoprecipitations ( fig. and table iii) revealed that whereas a-alp was very stable in wild-type cells, this protein became proteolytically processed with a half-time of min in vps mutant cells. by contrast, retrieval-defective a-alp, (f/a)a-alp, was proteolytically processed with a half-time of ‫ ف‬ min in both wildtype and vps mutant cells. rs-alp was proteolytically processed with half-times of min in wild-type cells and min in vps cells, suggesting that rs-alp exits rapidly from the tgn and relies solely on retrieval for golgi localization. these data indicate that the dpap a cytosolic domain indeed slows exit from the tgn, and that a-alp may avoid proteolytic processing in the pvc of wild-type cells by only rarely exiting the tgn. a slow rate of tgn exit would be consistent with the function of dpap a, which is to proteolytically process the ␣-factor polyprotein in the tgn (sprague and thorner, ) . to extend the analysis of the exit rates of tgn membrane proteins, we compared the rates of proteolytic processing of the cpy sorting receptor, vps p, in wild-type and vps mutant cells. to gain further insight into vps p, we also determined processing rates for the recycling-defective form of vps p (vps p- *), which lacks the cytosolic domain but otherwise binds cpy precursor (procpy) normally (cereghino et al., ; cooper and stevens, ) . whereas vps p was very stable in wildtype cells, vps p- * was cleaved by vacuolar proteases with a half-time of ‫ ف‬ min ( fig. and table iii) . interestingly, both vps p and vps p- * were proteolyzed with a half-time of ‫ ف‬ min in vps mutant cells, suggesting that vps p exits the tgn rapidly, independently of the presence of the recycling/retrieval signal in the cytosolic domain. rapid exit from the tgn by vps p is consistent with its function as the cpy sorting receptor, binding procpy in the tgn and releasing it in the pvc before recycling back for more rounds of vacuolar hydrolase sorting. to further address the rate of protein exit from the tgn membrane proteins such as vps p, a-alp, and rs-alp continuously leave golgi membranes and enter the pvc, from which they are retrieved via a mechanism that involves the recognition of an aromatic residuecontaining motif. this retrieval requires vps p, and in vps ⌬ cells, tgn membrane proteins accumulate in an exaggerated form of the pvc, the class e compartment (raymond et al., ; piper et al., ) . the class e compartment is proteolytically active, and the rate at which proteolytically sensitive proteins are processed in vps ⌬ cells can be used to measure the rate at which they enter this compartment. this is the basis for the assay to measure the leaving rates of a-alp-and vps p-related proteins from the tgn. wt, wild type. , and phy (vps under gal control) (ϩ) or empty vector (Ϫ) were grown in synthetic media containing % raffinose. at time t ϭ , galactose was added to a final concentration of %, and cells were allowed to grow for an additional min. at times t ϭ (Ϫ galactose) and t ϭ min (ϩ galactose), samples were removed and treated in the following ways (also see fig. ): (a) whole-cell extracts were prepared from od units of nby cells harboring psn and phy or empty vector to detect the presence of vps p using immunoblot analysis (similar results were obtained using the same cells harboring either psn or psn in conjunction with phy ; data not shown); and (b) . od units of cells were radiolabeled to determine whether the cells were secreting cpy. tgn, we followed the transfer of three membrane proteins from the tgn to the pvc (class e compartment) in vps mutant cells by indirect immunofluorescence. yeast cells carrying a temperature-sensitive allele of vps accumulate vps p rapidly into the pvc upon shift to Њc, and this protein redistributes to golgi membranes once cells are returned to the permissive temperature (piper et al., ) . the rapid onset of the transport defect in vps -ts cells allowed us to monitor the rate at which a-alp, rs-alp, and vps p enter the exaggerated pvc. when vps -ts cells were maintained at Њc, indirect immunofluorescence revealed (fig. ) that a-alp, rs-alp, and vps p exhibited staining patterns typical of golgi membrane proteins ( - dispersed dots) in a vast majority of the cells (Ͼ %). min after shifting vps -ts cells to Њc, both rs-alp and vps p had redistributed to the exaggerated pvc, yet a-alp staining appeared golgilike as long as - min after the temperature shift. these immunofluorescence data support the conclusion that exit from the tgn is the rate-limiting step for a-alp processing in vps mutant cells. therefore, by both pulsechase and indirect immunofluorescence analyses, a-alp has been shown to exit the tgn more slowly than rs-alp and vps p. the differences in the rate of exit from the tgn between a-alp and rs-alp/vps p could either arise through information contained within the tails of alp and vps p, specifying fast exit of the latter two proteins, or it could arise through information contained within the tail of dpap a that acts to slow the exit of a-alp. to determine whether tail sequences are responsible for slowing the exit of a-alp from the tgn, a series of deletions was made in the -amino acid cytosolic tail of ste p in the context of a-alp (fig. a) . the proteolytic processing rates of a number of mutated a-alp fusion proteins were determined in both wild-type and vps mutant cells (table iii and fig. ) . the a-alp protein lacking the first amino acids of the dpap a cytosolic domain ((⌬ - )a-alp), which localizes to the tgn (nothwehr et al., ; our unpublished results) , was found to behave like rs-alp. (⌬ - )a-alp was processed with a half-time of min in wild-type cells and ‫ ف‬ min in vps cells (table iii) , indicating that it exits the golgi rapidly and achieves its golgi localization through retrieval alone. by contrast, (⌬ - )a-alp behaved just like (f/a)a-alp in that it localized to vacuolar membranes (nothwehr et al., ; our unpublished results) and was processed with halftimes of ‫ - ف‬ min in both wild-type and vps mutant cells ( fig. and table iii) presumably since it contains information to slow exit from the tgn. these data indicate that the signal to slow tgn exit resides in the first amino acids of the dpap a cytosolic domain. in an experiment similar to that described above, vps -ts cells producing a version of dpap a lacking residues - accumulated the protein in the class e compartment after - min at the restrictive temperature, whereas those producing the full-length protein took longer ‫ ف(‬ min) to accumulate ste p in the class e compartment (data not , or psn (rs-alp) and phy (vps under gal control) (ϩ) or empty vector (Ϫ) were grown in synthetic media containing % raffinose. at time t ϭ , galactose was added to %, and cells were allowed to grow for an additional min. at times (a) t ϭ (Ϫ galactose) and (b) t ϭ min (ϩ galactose) (also see fig. ), cells were fixed and prepared for immunofluorescence using anti-alp antibodies in combination with biotin-linked secondary antibody and streptavidin-conjugated fitc, as well as anti-vph p antibodies in combination with rhodamine-conjugated goat anti-rabbit igg (h ϩ l). shown). these data indicate that amino acid residues - are important for slowing the exit of full-length, wild-type dpap a from the tgn. analysis of a set of deletions lacking various portions of the cytosolic tail of dpap a revealed that a-alp missing only amino acid residues - exited the tgn with a rate of - min (fig. ) . these data indicate that the dpap a tgn retention signal can be mutationally separated into two components, an nh -terminal signal for slowing exit from the tgn, and the fxfxd motif (residues - of the cytosolic domain), which directs retrieval back to the tgn from the pvc. eukaryotic cells face the challenge of maintaining the integrity of secretory organelles despite a continuous and dynamic flow of membranes and proteins through the pathway. the data presented in this paper demonstrate that the yeast s. cerevisiae uses two different modes of retention to ensure localization of dpap a to the tgn. there are two separable signals for golgi localization of dpap a found in the cytosolic domain. the first is the well-characterized motif containing aromatic amino acid residues (fxfxd in dpap a), which functions in retrieval from the prevacuolar compartment (pvc) back to the tgn. a novel assay for measuring the rate of membrane protein exit from the tgn revealed the second signal, which serves to slow exit from the tgn. these two retention signals serve to efficiently localize dpap a to the yeast tgn. while kex p, dpap a, and vps p have been shown to contain phe and/or tyr residues in their cytosolic domains that are required for golgi localization (wilcox et al., ; nothwehr et al., ; cereghino et al., ; cooper and stevens, ) , the evidence that these localization motifs direct retrieval from a post-golgi compartment has remained indirect. however, subcellular fractionation of membranes from wild-type yeast cells expressing rs-alp (alp containing fxfxd) revealed that the vacuolar protease-activated form of rs-alp cofractionated with the tgn membrane protein kex p. these data demonstrate that the fxfxd retention motif of dpap a functions as a retrieval signal, and that rs-alp was exposed to active vacuolar proteases in a post-golgi compartment and then retrieved back to the tgn. further evidence for the involvement of the fxfxd motif in retrieval from the pvc comes from our data that this motif is required to specify redistribution of a-alp from the class e compartment (exaggerated pvc) to golgi membranes after induction of vps p synthesis, an experiment that also indicates that the golgi localization of a-alp involves retrieval from the pvc. having established that localization of a-alp directed through aromatic amino acid residues involves retrieval from the pvc, we used vps mutant cells (which are blocked in traffic from the pvc back to the golgi apparatus) to measure the rate at which proteins exit the tgn and enter the proteolytically active and exaggerated form of the pvc (raymond et al., ; piper et al., ) . as described below, this assay successfully uncouples retrieval-mediated localization from static retention-based golgi localization by blocking the retrieval mechanism. comparison of pro- at the indicated times, the cells were spheroplasted, and the extracts were immunoprecipitated with polyclonal antibodies against alp and/or vps p, followed by sds-page and fluorography. the products of pep -dependent proteolysis are indicated using asterisks, as described in fig. . Ͼ golgi vps p- * vacuolar ‡ the kinetics of processing of the proteins listed in table iii were determined in sny - a (wild-type) and nby (vps ⌬) cells harboring the appropriate plasmid or producing vps p- *. in brief, cells were labeled with [ s]methionine for min and chased for various times by adding methionine and cysteine each to a final concentration of g/ml. cells were spheroplasted and extracts were immunoprecipitated with a polyclonal antibody against alp and/or vps p, followed by sds-page and fluorography. half-times of processing were determined using linear regression analysis, as described in materials and methods. § the localization of each of the proteins was also determined in sny - a (wildtype) cells by indirect immunofluorescence (iif) using affiniity-purified polyclonal antibodies specific for alp. tein processing half-times in wild-type and vps mutant cells allows us to determine whether a particular protein is localized to golgi membranes through retrieval from the pvc, retarded exit from the tgn, or a combination of both mechanisms. for example, when the signal that directs retrieval from the pvc is defective, there is no difference between the half-time of processing in wild-type and vps mutant cells. in contrast to this, when a signal that acts to slow the exit of a protein from the tgn is defective, increased processing in vps mutant cells is observed. consistent with earlier work (nothwehr et al., ) , mutations in the fxfxd retrieval motif in the cytosolic domain of dpap a were found to result in vacuolar delivery of the a-alp fusion protein with a half-time of ‫ ف‬ min. to determine the molecular basis for the half-time of delivery of (f/a)a-alp to the vacuole ‫ ف(‬ min) being so much slower than for alp ‫ - ف(‬ min), we investigated whether there was additional localization information in the dpap a cytosolic domain. vps mutant cells, in which tgn membrane proteins accumulate in a proteolytically active class e compartment (exaggerated pvc; piper et al., ) , were used to estimate whether the various a-alp fusion proteins were all delivered to the pvc at the same rate. based on proteolytic processing of the lumenal (cooh-terminal) propeptide of alp, these studies revealed that a-alp and (f/a)a-alp were delivered to the pvc with a half-time of ‫ ف‬ min, while rs-alp and (⌬ - )a-alp exhibited half-times of ‫ - ف‬ min. these data indicate that there is information in the nh -terminal amino acids of the dpap a cytosolic domain that confers the slower rate of delivery of the a-alp fusion proteins to the pvc, and that this rate is independent of the presence or absence of the aromatic amino acid-based retrieval motif located elsewhere (fxfxd, amino acids - ) in the cytosolic domain. since all our data indicate that the a-alp fusion proteins are delivered rapidly to the golgi (a-alp receives golgi modifications with similar kinetics to alp; bryant, n.j., and t.h. stevens, unpublished results), the slow rate of transport of a-alp (and (f/a) a-alp) to the pvc reflects a slow rate of exit from the tgn. vps p, the cpy sorting receptor, is a yeast tgn membrane protein that was found to be delivered rapidly to the pvc (half-time of ‫ - ف‬ min). as with a-alp, the rate of vps p delivery to the pvc was independent of the presence or absence (vps p- *) of the tyr-based retrieval motif. therefore, vps p exits the golgi at a rapid rate and achieves golgi localization through retrieval, whereas dpap a contains both retrieval and static retention signals. such a result is consistent with the biological functions of the two proteins. dpap a is involved in the processing of the mating pheromone ␣-factor precursor as this polyprotein passes through the golgi apparatus before its packaging into secretory vesicles (sprague and thorner, ) . by contrast, vps p must continuously cycle between the golgi and the pvc, binding procpy in the golgi and releasing it in the pvc before recycling back to the golgi to bind more ligand (cereghino et al., ; cooper and stevens, ) . it is thus easy to rationalize the rapid tgn exit rate of vps p and the rather slow rate of dpap a. consistent with this interpretation are the observations that overexpression of vps p leads to processing of a-alp in wild-type cells, yet the rate of processing of a-alp in vps ⌬ cells is unaffected by overexpression of vps p (bryant, n.j., and t.h. stevens, unpublished results) . these observations suggest that vps p and a-alp compete for the retrieval machinery but not for entry into tgn-derived vesicles bound for the pvc. deletion of the first amino acids of the dpap a cytosolic domain did not affect the localization of the resulting (⌬ - )a-alp fusion protein (nothwehr et al., ) . aanalysis of the rate of delivery of (⌬ - )a-alp to the pvc in vps mutant cells, however, revealed that this protein rapidly exited the tgn just like rs-alp, containing only the fxfxd retrieval motif. conversely, deletion of residues - of the dpap a cytosolic domain produced a retention-defective form of a-alp, (⌬ - )a-alp (localized to the vacuole membrane), which was delivered to the pvc slowly (half-time of ‫ ف‬ min) in vps mutant cells. these results indicate that the first amino acid residues of the dpap a cytosolic domain are necessary for slowing the tgn exit rate, and that the first are sufficient to slow the rate. the region important to slow the rate of a-alp from the tgn was further narrowed by construction and analysis of a set of deletions, each smaller by amino acids. the smallest deletion, removing only residues - of the dpap a cytosolic domain, still eliminated the static mode of retention. thus, the first amino acid residues of dpap a are required for static retention. deletion of these same amino acid residues from wild-type dpap a caused the protein to accumulate rapidly in the class e compartment of vps -ts cells (data not shown), indicating that this static retention motif is important in the context of the full-length protein. additional studies will be required to test whether this region is sufficient for static retention. the presence of the static retention signal in the cytosolic domain of dpap a helps explain why both a-alp and rs-alp are localized to the tgn, yet only rs-alp gets proteolytically processed by vacuolar proteases in a post-golgi compartment. while we cannot as yet rule out the possibility that rs-alp is retrieved from the vacuole itself, it is most likely that both rs-alp and a-alp are transported to and retrieved from the same pvc, and that this compartment contains low levels of activated vacuolar proteases en route to the vacuole. evidence for low levels of activated vacuolar proteases in the yeast late-endosomal/ prevacuolar compartment comes from the work of schimmoller and riezman, , indicating that ␣-factor endocytosed into late endosomes encounters proteases. there is abundant evidence from mammalian cells that hydrolases are distributed throughout the endocytic pathway (blum et al., ) . if rs-alp and a-alp are in fact retrieved from the same pvc, then the failure to detect proteolytic processing of a-alp must reflect its very slow exit rate from the tgn, thus reducing the time a-alp spends in the pvc compared to rs-alp. if rs-alp is being retrieved from the same pvc as vps p, then the differential rate of proteolysis between these two proteins in wild-type cells may reflect a differential susceptibility to the low levels of proteases in the pvc. the data presented in this paper indicate that dpap a requires both static retention and retrieval for efficient localization to the yeast tgn. it remains to be determined whether the two other tgn proteins that have been identified (kex p and kex p) also contain signals to slow their exit from the tgn. we propose that the nh terminus of dpap a serves to keep this protein anchored in the tgn membrane, and that this protein only occasionally exits the golgi apparatus (half-time of ‫ ف‬ min). molecules of dpap a that enter the pvc are efficiently recognized by virtue of the fxfxd motif and are recycled back to the tgn. some proteins, such as the cpy sorting receptor vps p, are only retained in the tgn by retrieval because they must continually cycle between the tgn and pvc to fulfill their function in the cell. the identification of separable signals for retention of dpap a in the tgn enables us to determine whether the recently identified grd mutants (golgi retention defective; nothwehr et al., ) are defective in the static retention of dpap a, or defective in its retrieval from a post-golgi compartment. we are currently investigating whether the grd proteins are involved in recognition of either the static or retrieval signals in the cytosolic domain of dpap a. figure . kinetics of processing of (⌬ - )a-alp and (⌬ - )a-alp in wild-type and vps ⌬ cells. sny - a (wild-type) and nby (vps ⌬) cells harboring either pnb ((⌬ - )a-alp) or psn ((⌬ - )a-alp) were labeled with [ s]methionine for min and chased by adding unlabeled methionine and cysteine, each to a final concentration of g/ml. at the indicated times, the cells were spheroplasted, and the extracts were immunoprecipitated with a polyclonal antibody against alp, followed by sds-page and fluorography. the products of pep -dependent proteolysis are indicated using asterisks, as described in fig. . novel syntaxin homologue, pep p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast localization of cathepsin d in endosomes: characterization and biological importance tgn is maintained in the trans-golgi network by a tyrosine-containing motif in the cytoplasmic domain cholesterol and the golgi apparatus immunoisolation of kex p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single golgi compartment localization of the signal for rapid internalization of the bovine cation-independent mannose -phosphate/insulin-like growth factor-ii receptor to amino acids - of the cytoplasmic tail the cytoplasmic tail domain of the vacuolar protein sorting receptor vps p and a subset of vps gene products regulate receptor stability, function, and localization the functioning of the yeast golgi apparatus requires an er protein encoded by anp , a member of a new family of genes affecting the secretory pathway yeast kex p is a golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane vps p cycles between the late-golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases localization of components involved in protein transport and processing through the yeast golgi apparatus compartmental organization of golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec (nsf) mutant sorting of yeast alpha , mannosyltransferase is mediated by a lumenal domain interaction, and a transmembrane domain signal that can confer clathrin-dependent golgi localization to a secreted protein localization of a yeast early golgi mannosyltransferase, och p, involves retrograde transport localization of tgn to the trans-golgi network: involvement of a cytoplasmic tyrosine-containing sequence membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase structure and function of the mannose- -phosphate/insulin growth factor-ii receptors rapid and efficient site-specific mutagenesis without phenotypic selection ligand-induced redistribution of a human kdel receptor from the golgi complex to the endoplasmic reticulum localization and targeting of the saccharomyces cerevisiae kre p/mnt p alpha , -mannosyltransferase to a medial-golgi compartment retention of a cis-golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain the vph gene encodes a -kda integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar h( ϩ )-atpase the sorting receptor for yeast carboxypeptidase y is encoded by the vps gene sequences within and adjacent to the transmembrane segment of alpha- , -sialyltransferase specify golgi retention. embo (eur the membrane spanning domain of beta- , -galactosyltransferase specifies trans golgi localization kin recognition. a model for the retention of golgi enzymes sorting of membrane proteins in the yeast secretory pathway membrane protein retention in the yeast golgi apparatus: dipeptidyl aminopeptidase a is retained by a cytoplasmic signal containing aromatic residues golgi and vacuolar membrane proteins reach the vacuole in vps mutant yeast cells via the plasma membrane the newly identified yeast grd genes are required for retention of late-golgi membrane proteins alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps null mutant missorts and secretes only a subset of vacuolar hydrolases sorting of membrane proteins in the secretory pathway vps controls vacuolar and endocytic traffic through a prevacuolar compartment in saccharomyces cerevisiae yeast vps p is a sec p-like protein required for the consumption of vacuole-targeted, post-golgi transport vesicles morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class e vps mutants immunolocalization of kex protease identifies a putative late golgi compartment in the yeast saccharomyces cerevisiae membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment mechanisms of intracellular protein transport two independent targeting signals in the cytoplasmic domain determine trans-golgi network localization and endosomal trafficking of the proprotein convertase furin coat proteins and vesicle budding involvement of ypt p, a small gtpase, in traffic from late endosome to the vacuole in yeast methods in yeast genetics pheromone response and signal transduction during the mating process of saccharomyces cerevisiae a golgi retention signal in a membrane-spanning domain of coronavirus e protein localization of furin to the trans-golgi network and recycling from the cell surface involves ser and tyr residues within the cytoplasmic domain mutational analysis of the human kdel receptor: distinct structural requirements for golgi retention, ligand binding and retrograde transport an acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-golgi network localization and internalization from the cell surface posttranslational processing of the prohormone-cleaving kex protease in the saccharomyces cerevisiae secretory pathway mutation of a tyrosine localization signal in the cytosolic tail of yeast kex protease disrupts golgi retention and results in default transport to the vacuole dynamic retention of tgn membrane proteins in saccharomyces cerevisiae the sxyqrl sequence in the cytoplasmic domain of tgn plays a major role in trans golgi network localization we thank rob piper and antony cooper for useful discussions about this work and for providing pep p and vps p antibodies. laurie graham is thanked for affinity-purified anti-vph p antibody and for critical reading of the manuscript. we also thank jason brickner and bob fuller for providing anti-kex p serum, as well as members of the stevens lab and greg flynn for reading the manuscript. this work was supported by a grant from the national institutes of health ( ) to t.h. stevens.received for publication september and in revised form november . key: cord- -alv uk authors: mellman, ira; simons, kai title: the golgi complex: in vitro veritas? date: - - journal: cell doi: . / - ( ) -a sha: doc_id: cord_uid: alv uk nan iplex has proved to be among the more challenging probllems in cell biology. the last several years have turned out ito be particularly exciting in this respect since they have iyielded new insights and ideas at an increasingly rapid ipace. this period of advance has largely been due to the idevelopment of powerful new biochemical, morphological, #and genetic approaches to unraveling the complexities of 'this organelle. while much remains to be discovered, the iproblem now is how to integrate this wealth of information. 'to see if this is possible, we will first summarize how the lslolgi is commonly believed to work and then evaluate the lstrength of the evidence that underlies these views. present view of the golgi 'the golgi complex is essentially a carbohydrate factory. in <animal cells, it is engaged in the biosynthesisof glycolipids (and of oligosaccharide portions of glycoproteins and proteoglycans. in plant cells, it has the additional task of producing a wide range of extracellular polysaccharides. the factory receives its substrates for glycosylation from the iendoplasmic reticulum (er), transports its raw materials (monosaccharide precursors) in from the cytosol, and dis-,tributes its completed protein, lipid, and polysaccharide iproducts to a variety of destinations. 'transport through the golgi is directional (one of the most compelling features of the golgi complex ,is its structure. in electron micrographs, the golgi is seen #as a set of flattened cisternae with dilated rims arranged llike a stack. the cisternae are also associated with an #array of small vesicles, as well as with a network of tubules (emanating from both sides of the stack (figure ). morphologically and functionally, the golgi complex has a distinct ,polarity (farquhar and palade, ) . newly synthesized membrane and secretory proteins coming from the er enter the golgi through the cis face, traverse across the stack, and leave via the trans face. a reticulum of tubules iemanating from the trans-most cisterna, collectively referred to as the trans-golgi network (tgn) or trans-golgi reticulum, probably reflects the actual sites of exit (griffiths ,and simons, ; geuze and morrb, ) . on route ,through the golgi, newly synthesized glycoproteins are subjected to a series of posttranslational modifications, most notably the ordered remodeling of their n-linked oli-gosaccharide side chains and biosynthesis of o-linked glycans. given the directionality of transport and the sequential nature of the glycosylation events, it is commonly thought that the golgi consists of a series of functionally distinct compartmentscorresponding to structurally distinct cisternae or sets of cisternae (dunphy et al., ; duden et al., a) . accordingly, enzymes involved in the early golgi events in terminal n-glycosylation-such as the trimming of the high mannose chain to its mans form by mannosidase l-have been placed in the cis golgi (kornfeld and kornfeld, ; pelham, ) . enzymes involved in intermediate steps, such as the transfer of n-acetylglucosamine to the mans sugars (glcnac transferase i), are thought to be restricted to medial cisternae (dunphy et al., ) . finally, enzymes that catalyze the addition of terminal galactose and sialic acid moieties (such as galactosyltransferase and sialyltransferase) are placed in trans cisternae and/or the tgn (roth and berger, ; roth et al., ; griffiths and simons, ; duncan and kornfeld, ; taatjes et al., ) . the scheme thus predicts that a series of sequential biochemical reactions occurs within separate compartments, through which glycoprotein substrates pass as if on an assembly line. a consequence of this multicompartment arrangement is that newly synthesized proteins must be transferred from one golgi compartment to the next. the most widely held view is that transport vesicles bud from the dilated rims of cisternae and then fuse with the next. such a mechanism is consistent with the existence of the numerous small vesicles found in the vicinity of the golgi (farquhar and palade, ) . since transport of proteins and small molecules through the secretory pathway does not appear to require any identifiable signals, it is also thought that transit through the golgi occurs by "bulk flow" (pfeffer and rothman, ) . in other words, the intercisternal transport vesicles may not selectively include components intended for transport. rather, resident proteins of any one cisterna must be selectively excluded from entering these transport vesicles, most likely reflecting the selective retention of resident proteins in their appropriate cisternae. a few cellular and viral glycoproteins are known that appear to have such retention signals, resulting in their accumulation in the golgi (colley et al., ; swift and machamer, ; nilsson et al., ; munro, ) . finally, at the level of the tgn, terminally glycosylated proteins are sorted to their destinations (griffiths and simons, ). proteins intended for transport to lysosomes electron micrograph is taken from rambourg and clermont ( ) with permission. the trans side is recognizable by maturing (pg) and mature (g) secretory granules. the discontinuous profile of the fenestrated network is seen on the cis side (ce). small vesicles are marked v and the endoplasmic reticulum is marked er. also notice areas where the stack is disrupted by discontinuities ( , x ). and endosomes are sequestered into clathrin-coated buds of the tgn, owing to specific recognition determinants (kornfeld and mellman, ; pearse and robinson, ) . proteins lacking these signals, including soluble proteins destined for secretion, are by default packaged into transport vesicles of the constitutive pathway that are targeted to the plasma membrane. in the tgn of polarized or secretory cells (endocrine and exocrine secretory cells, epithelial cells, and neurons), other routes of exit may occur, leading to inclusion either within storage granules (kelly, ; huttner and tooze, ) or within transport vesicles targeted specifically to the apical or basolateral plasma membranes (bennett et al., ; wandinger-ness et al., ) or to the dendritic and axonal cell surfaces (dotti and simons, ) . how good is the evidence for the current view of the golgi? as we have seen, there are three key elements underlying our general view of the golgi: that the golgi consists of several discrete compartments (cis, medial, trans, and tgn) corresponding to individual or groups of cisternae; that transit between these compartments occurs viavesicular carriers; and that the transport process is inherently nonselective with respect to the passenger proteins. let us examine the strength of the evidence in favor of each of these propositions and interpret the data accordingly. are there? although originally thought to be a "fixation artifact," the basic cisternal organization of the golgi complex is now accepted as a certainty. similarly, the cis to transdirectionality of transport is quite clear from early morphological and autoradiographic analysis in exocrine cells where the sidedness of the golgi is readily identifiable (farquhar and palade, ) as well as from immunoelectron microscopy studies using viral glycoproteins as markers (bergmann and singer, ) . what is less than clear, however, is the extent to which the golgi is divided into distinct compartments. as mentioned earlier, some of the enzymes involved in early, intermediate, and late steps in the terminal glycosylation of n-linked sugar chains have been localized to one to three individual cisternae by electron microscopy using immunoperoxidase or immunogold cytochemistry (roth and berger, ; slot and geuze, ; dunphy et al., ; roth et al., ) . however, a definitive demonstration that these enzymes exist in separate cisternae can only be provided by visualizing two or more enzymes simultaneously. the golgi complex in the cell is not the idealized stack that we are accustomed to recognize in diagrams, but it may be subject to twists that make individual sections difficult to interpret. to date, simultaneous localization of two or more enzymes has not been accomplished. in addition, the localization of key enzymes such as mannosidase i remains to be established; this is a critical unknown, since mannosidase i is thought to be the first glycosidase to modify n-linked oligosaccharides upon their arrival in the golgi. a further complication is introduced by the fact that there appears to be considerable variation between cell types. for example, in goblet cells of the intestine, galactose a , -sialyltransferase (the enzyme responsible for adding l.erminal sialic acid residues to n-linked sugars) has been localized to trans cisternae, as is the case in hepatocytes. in neighboring absorptive cells of the gut, however, almost lhe entire golgi stack is labeled (except forthe cis cisterna) (roth et al., ) . it has also recently become clear that golgi glycosyltransferases may have multiple isoforms with either golgi or plasma membrane distributions. for example, two isoforms of - galactosyltransferase have been found that differ by a amino acid addition at the cytoplasmic n-terminus (lopez et al., ) . the short galactosyltransferase remains in the golgi, while the long form is directed to the plasma membrane. cell fractionation experiments have also contributed to our view of the golgi as a multicompartment organelle, since it has been possible to partly separate membranes containing several of the enzymes involved in golgi processing events (goldberg and kornfeld, ; dunphy and hothman, ) . however, interpretation of these data is limited on two accounts. first, in the absence of definitive immunocytochemical localization, it is impossible to know whether the different fractions correspond to different sets of cisternae. second, the enzymes studied have been separated into only three fractions, the highest density fraclions containing enzymes acting early in glycosylation (glucosidase, enzymes involved in generating mannose -phosphates on lysosomal enzymes) and the lightest density fractions containing terminally acting glycosyltransferases (galactosyl and sialyltransferases). the five enzymes hat are involved in "intermediate" processing steps (manrnosidase i and ii, glcnac transferase i, ii, and iv, and fucosyltransferase) were all found to cosediment in a single peak of intermediate density. thus, although consistent with the existence of at least three physically and functionally distinct compartments, these observations also suggest that many of the carbohydrate processing steps may occur within a single compartment. since the enzymes involved in n-glycosylation act sequentially (and specifically) (that is, mannosidase i creates the substrate for glcnac transferase, which creates the substrate for mannosidase ii, which creates the substrate for galactosyltransferase, etc.), there is no a priori need for physical separation to ensure proper processing. indeed, having glcnac transferase and galactosyltransferase in a common compartment would explain the existence of lactosamine repeats in the terminal chains of many glycoproteins (cummings and kornfeld, ; howe et al., ) . a further reason for caution in interpreting existing imlmunocytochemistry and fractionation results is the growing appreciation of the fact that the individual golgi cister-inae-and perhaps even entire stacks-may not be as datic and structurally distinct as we have come to believe. iexamination of the golgi using high voltage electron milcroscopy has suggested that adjacent stacks of cisternae (are joined via extensive tubular interconnections (rambourg and clermont, ) . similarly, real-time imaging of iliving cells labeled with the fluorescent lipid analog nbd-iceramide has revealed that the golgi is capable of forming ,a large number of tubules that may serve to connect other-'wise "separate" stacks of cisternae (cooper et al., ) . whether the tubules form connections between cisternae in register (i.e., cis to cis, medial to medial) or out of register (cis to medial) is unknown. the possibility must also be considered that even within a single stack, adjacent cisternae might at least transiently possess similar tubular interconnections. anyway? a question that clearly needs discussion at this point is what exactly defines a cellular compartment. classically, membrane-bounded compartments have been viewed as comprising physically distinct entities each with its own distinct protein compositions and, presumably, unique functions. accordingly, all of any one type of organelle can be considered a compartment, but so can a distinct region (or "subcompartment") within an organelle. by this criterion, compartment boundaries should be easily delineated by immunoelectron microscopic localization of resident marker proteins. however, this simple view is complicated by several factors. for example, even the localization of two golgi enzymes to different cisternae by double label immunoelectron microscopy might not unequivocally demonstrate that they are present in physically distinct compartments. such a distribution of enzymes could reflect the existence of subdomains in an otherwise continuous membrane array, analogous to the lateral heterogeneity found between the rough and the smooth er. conversely, markers may be segregated into physically distinct membranes that nevertheless remain functionally continuous by repeated membrane fissions and fusions or transient tubular interconnections. the existence of such homotypic interactions-interactions among "like" elements-would play an important role in preserving continuity among equivalent but physically separated organelles. thus, the simple test of localizing different markers to distinct membranes cannot by itself serve to identify distinct compartments. the definition of a compartment is further confounded when considering organelles that are also involved in membrane traffic. given that there is likely to be continuous transport and recycling of membrane components between such compartments, compartment identities are not static and therefore may not always be entirely biochemically distinct. the object, then, is to identify only those components that are endogenous, i.e., not involved in recycling. conceivably, endogenous (or resident) components may be defined by their ability to interact with a compartment-specific structural framework, such as a cytosolic membrane coat or skeleton. it is clear that only by identifying the machinery responsible for entry into, retention within, or exit from a compartment will we be able to precisely and consistently determine compartment boundaries. in the absence of this information, compartments can only be tentatively defined as functionally equivalent and possibly physically continuous populations of membrane-bounded structures that are both functionally and physically distinct from all other compartments. taken from rambourg and clermont ( ) , with permission. the osmiophilic cis compartment, the saccules (cisternae) of the medial compartment, as well as the cisternae and tubules of the trans compartment are all indicated. scribe definitively the compartmentalization of the golgi complex, models for the organization of the golgi should be kept as simple as possible, adding complexity only as it is necessitated by additional functional requirements. as we have discussed, there are three minimal functions that the golgi must accomplish: first, receipt and sorting of membrane and soluble cargo arriving at the cis golgi from the er; second, glycosylation and processing of glycoproteins and glycolipids; and third, sorting of membrane and soluble cargo arriving and exiting at the trans face of the golgi. assuming that distinct compartments are required to accomplish these functions, at least three compartments are required, defined here as the cis-golgi network (cgn), the medial golgi, and the tgn, respectively. although the details of golgi morphology vary considerably between different cell types, such a three compartment subdivision is generally consistent with the overall structural organization of the mammalian golgi from threedimensional reconstructions (rambourg and clermont, ) (figures and ) . it is also consistent with the functional organization of the golgi in saccharomyces cerevisiae, which, on the basis of mutations affecting golgi function, has recently been proposed toconsist of at least three compartments (graham and emr, ) . the cgn at a minimum, the cis side of the golgi stack must function in the receipt of newly synthesized material from the er. structurally, however, the cis-most cisterna of the golgi is typically associated with a continuous array of tubules that are selectively stained after prolonged osmication (lindsey and ellisman, ; rambourg and clermont, ) ( figure ). given this distinctive morphology, and by analogy to the tgn, it seems appropriate to refer to this region as a distinct compartment called the cgn (huttner and tooze, ; hsu et al., ; pelham, ) . for several reasons, it also seems reasonable to suggest that the cgn also includes the poorly understood but possibly interconnected system of tubules between the er and the golgi, referred to as the "salvage compartment" (warren, ; pelham, ) (figure ). in addition to serving as a site of entry into the golgi, recent evidence suggests that the cgn functions in the recycling of protein and lipid components back to the er, while having a relatively limited role in glycosylation. using both animal and yeast cells, pelham and colleagues have found that soluble er proteins containing the kdel (or hdel) er retention marker are delivered to a post-er the ports of exit are the sites from which proteins leave the er. as discussed in the text, the "intermediate compartment" defined by the p and ~ antigens probably consists both of the cgn, the ports of exit from the er, and the carrier elements responsible for bidirectional transport between the er and the cgn. the lateral arrows would be sites where direct fusions would interconnect cgn-cgn, medialmedial, and tgn-tgn compartments of two adjacent stacks. compare these sites to the discontinuities in figure . site, in which little "golgi-like" oligosaccharide processing oiccurs and from which these proteins are returned to the er (pelham, (pelham, , dean and pelham, ) . in animal cells, the lysosomal enzyme cathepsin d was given a c-terminal kdel tag and found not to receive any terminal sugar modifications associated with golgi-linked glycosylation. however, mannose residues on the enzyme did receive a glcnac-phosphate. thus, although the kdel clonstruct reached a glcnac-phosphotransferase-containing compartment, it did not encounter mannosidase i, a glycosidase that would remove the phosphotransferase substrate. although it may yet turn out that some kdel proteins receive further oligosaccharide modifications, k.del receptors can retrieve these proteins before they reach the bulk of the oligosaccharide transferases; thus, recycling is a function associated with a distinct "early" golgi compartment. without immunocytochemical localization of these constructs (the kdel receptor or the phosphotransferase), it is impossible to be certain that these events occur in the cgn. however, this appears likely, given that our model assumes that the tubules and vesicles defining the cgn are also associated with antigens such as ~ , ~ , and mb (saraste et al., ; schweizer et al., ; chavrier et al., ) which have been used to define the so-called "intermediate compartment" . these antigens label structures that appear to serve as intermediates in transport from the er to the golgi as well a.s in the recycling of golgi components back to the er (i-ippincott- schwartz et al., ) . the markers also label presumed sites of exit from the er ("transitional elements," palade, ) as well as the cis-most golgi cisterna (saraste and svensson, ; lotti et al., ) , suggesting that they are not endogenous cgn components, but recycle between the er and cgn. thus, the intermediate compartment probably comprises the exit sites from both the e.r and the cgn as well as the dynamic "pool" of vesicles and tubules that reflects the continuous recycling between tlhe two sites ( figure ) . although incubation at low temperalture has also been used to delineate boundaries in this region, like all temperature blocks, forward transport is clependent on length of incubation, making precise interpretation even more difficult than usual. we thus view the cgn as a unique golgi compartment responsible for receiving and sorting components arriving from the er. structurally, we predict it will ordinarily (but perhaps not always) consist of the cis-most golgi cisterna and the associated vesicles and tubules that include the intermediate compartment of schweizer et al. ( ) . in addition to the transport of kdel-tagged proteins to and from the golgi and the limited post-er glycosylation events to which these proteins are subjected, the cgn rnight also be predicted to include a variety of other activities that have been relegated to the purgatory between the eir and the golgi. these would include the addition of the first n-acetylgalactosamine residue to o-linked sugars, the assembly of coronavirus particles (tooze et al., ; machamer et al., ) and fatty acylation of membrane protein cytoplasmic domains (rizzolo and kornfeld, ; e onatti et al., ) . experiments in yeast also give at least indirect support to the existence of lipid recycling to the er from the cgn as well (cleves et al., ) . the medial golgi would function primarily as a glycosylation device. structurally, it corresponds to the cisternae and tubules in the middle of the golgi stack (figure ) whose structures vary widely in different cells (rambourg and clermont, ) . while distinct from the cgn or tgn, we assume that the medial cisternae represent a single functionally continuous compartment, irrespective of the actual number of physically separate cisternal elements in any one golgi complex. nevertheless, owing to the sidedness of the golgi, the medial cisternae may appear structurally distinct. for example, since one face of a medial cisterna is apposed to the cgn while another face is apposed to the tgn, the two faces may have different compositions, owing to their interactions with structurally distinct compartments. most of the glycosylation reactions (n-linked, o-linked, as well as glycolipid synthesis), aswell as polysaccharide synthesis normally associated with the golgi, are viewed as occurring in the medial compartment. this arrangement would account for cell type heterogeneity in cisternal number and enzyme distribution by placing them all in asingle compartment. at present, there is insufficient data to know if this is really the case. if the medial golgi serves only to mediate glycosylation reactions, why the characteristic cisternal morphology? one possibility might be simply to enhance the efficiency of glycosylation. although precise measurements are difficult to make, transport systems for sugar nucleotide precursors are kinetically inefficient (deutscher et al., ) . moreover, while the terminal transferases also have relatively high k,s for substrate (high micromolar range), they may work at rates that may exceed the rate of sugar nucleotide transport (weinstein et al., ; c. hirschberg, personal communication) . thus, the cisternal stack by its flattened structure may serve to enhance the efficiency of glycosylation by minimizing lumenal volume and increasing membrane surface area. the tgn is viewed as the compartment that mediates the sorting and final exit of material from the golgi. structurally, it is likely to be defined by the sacculotubular network located on the trans side of the golgi that varies in structure in different cell types (rambourg and clermont, ) (figures and ) . the tgn can be visualized in the electron microscope after labeling cells with cs-nbd-ceramide (pagan et al., ) with cs-dmb-ceramide (pagan et al., ) or after accumulating vesicular stomatitis virus (vsv) g protein at % (griffiths et al., ) . the tgn may also have a lower ph than preceding compartments in the pathway (anderson and pathak, ) . the tgn is known to undergo dynamic changes in size, depending on the amount of protein traffic through the compartment (griffiths et al., ) . in glandular cells producing secretion granules, several cisternae on the trans side form condensing vacuoles, presuming that all these cisternae are part of the tgn (rambourg and clermont, ) . the extreme case is represented by the golgi complex of algae where flakes are assembled in the lumen of the golgi cisternae (melkonian et al., ) . since these large flakes are first observed several cisternae away from the cis side, it seems reasonable to assume that the flakes assemble in saccular portions of the tgn and that entire tgn cisternae peel off and are transported to the cell surface. the main function of the tgn is to sort proteins and lipids leaving for different post-golgi destinations (griffiths and simons, ; simons and van meer, ) . the tgn also receives membrane traffic from the endocytic pathway (farquhar and palade, ; kornfeld and mellman, ) and, unlike all other golgi subcompartments, its degree of continuity with the plasma membrane is increased following treatment with brefeldin a (bfa) (lippincott- . the tgn is also the only golgi compartment that fails to enter the er following bfa (chege and pfeffer, ; ulmer and palade, ; reaves and banting, ) . certain "late" protein modification events also occur in the tgn, such as galactose a , sialylation (duncan and kornfeld, ) tyrosine sulfation (huttner and baeuerle, ) and proteolytic cleavage at dibasic residues on viral glycoproteins and cellular precursor proteins (sossin et al., ) . we propose that the tgn also operates as a directional valve to control the flow of membrane proteins and lipids in biosynthetic transport (figure ) . such a function would contribute to the efficiency of forward transport by preventing passenger proteins and lipids from returning to the medial golgi after reaching the tgn. in practice, this would predict that there is no recycling from the tgn to the medial golgi. the need for this function is best illustrated by considering the transport and distribution of membrane lipids through the golgi. several lipids such as cholesterol, glycolipids, and sphingomyelin are present in very low concentrations in the er, although they are abundant in the golgi (van meer, ) . if there were lipid recycling from each golgi compartment, it would be difficult to account for these differences in lipid composition. placing a valve in the pathway makes the lipid sorting problem easier to solve. several studies have demonstrated directly that there is little backflow of glycoproteins from the tgn into proximal golgi compartments (duncan and kornfeld, ; neefjes et al., ; chege and pfeffer, ) . also, recycling of glycosphingolipids through the tgn (schwartzmann and sandhoff, ) would be easier to explain if these plasma membrane lipids would not encouter medial golgi enzymes; otherwise, with time, only complex glycolipids would be present on the cell surface. does transport through the golgi require vesicular carriers? the second key element underlying our current view of the golgi concerns the role of carrier vesicles in mediating transport through the golgi complex. while transport vesicles may represent the most likely mechanism to accomplish transfer between compartments and/or individual cisternae, how strong is the evidence that they actually perform this function? in intact cells, perhaps the best argument comes from the early cell-cell fusion experiments of rothman and colleagues (rothman et al., a (rothman et al., , b . here, cho cell glycosylation mutants were infected with vsv and then fused with uninfected cells. transfer of the vsv g protein from golgi membranes in the infected to the uninfected cells was monitored by a glycosylation event that could be carried out by only the uninfected recipient, which was chosen to complement the glycosylation defect of the infected cell. these observations indicated that efficient transfer of vsv g protein occurs between spatially distinct golgi stacks. this is most easily explained byamechanism involving vsvg proteincontaining transport vesicles that form from the infected cell golgi, diffuse through the heterokaryon's cytosol, and fuse with golgi membranes in the uninfected cell. conceivably, the same transport vesicles could mediate intercisternal transport within a single golgi stack. the major limitation of these data is that they are indirect. they do not identify the vesicles nor do they definitely establish their site of origin. one cannot exclude the possibility that the g protein moved by lateral diffusion through tubules connecting the heterologous golgi stacks, which otherwise remain separated during the assay. furthermore, even the existence of vesicles involved in inter-golgi transport does not demonstrate that they have a similar role in intercisternal transport within a single stack, as is assumed by our current view of the golgi. in vitro transport through the golgi and the role of carrier vesicles the most extensive series of considerations favoring the role of transport vesicles in intra-golgi transport comes from experiments designed to reconstitute transport in vitro. that such transport can be achieved has been demonstrated over the past several years by the pioneering work of rothman and coworkers. in their approach, golgi fractions prepared from vsv-infected glcnac transferase-deficient cells ("donor golgi") are incubated with similar fractions from uninfected wild-type cells ('acceptor golgi"). upon the addition of atp and cytosolderived factors, there is a progressive appearance of [ h]glcnac-labeled (balch et al., ) and endo h-resistant (fries and rothman, ) vsv g protein, reflecting its transfer from mutant "donor" to wild-type "acceptor" membranes. this transfer has also been dissected kinetically into two major stages: an early stage presumed to correlate with the budding of transport vesicles from the donor golgi, and a late stage reflecting the docking and subsequent fusion of the vesicles with the acceptor golgi. the early and late stages also differ in their requirements and inhibitor sensitivities (balch et al., ; rothman and orci, ) . given that vsv g protein in early golgi compartments was transported with the highest efficiency (fries and rothman, ) the transport event assayed was postulated to reflect the formation of vesicles from cis (early) cisternae and the fusion of these vesicles with medial cisternae (which contain the glcnac transferase) (rothman and orci, ) (figure ) . however, the identi-donor acceptor fllgure . two models to account for donor-acceptor connections in inter-golgi transport in vitro (a) vesicular transport, as predicted by the current view of the golgi alid by the work of rothman and colleagues (rothman and orci. ) . (es) vesicle-independent direct fusions between donor and acceptor compartments (cgn-cgn, medial-medial, and tgn-tgn), perhaps mediated by tubular extensions. while these tubules may be amplified by bfa. as described in the text, they may exist and function even in the absence of the drug. ties of the donor and acceptor cisternae have not yet been established, nor can kinetic analysis alone provide a unique interpretation, especially when transport is monitored indirectly by linkage to glycosylation, which itself is a kinetically complex series of reactions (see above). analysis of this golgi transport system has already led to the discovery of several discrete protein factors whose a.ctivities are required to mediate the assay (rothman and orci, ) . some of these represent the mammalian homologs of yeast genes known to be involved in intracellular transport in intact cells (wilson et al., ; clary et al., ) . one important factor, designated nsf (nem-.sensitive fusion protein), is homologous to the yeast sec gene product and is thought to act at a late stage in transport, at the time when transport vesicles fuse with the acceptor membranes orci et al., ; rexach and schekman, ) both in er to golgi and in golgi as well as in post-golgi transport (graham and emr, ) . the precise function of nsf, however, remains unknown. another factor, designated snap (for soluble nsf attachment protein), is homologous to the yeast sec gene product and is thought to be involved in nsf binding to membranes (clary et al., ) . the strongest evidence for the existence of a vesicular intermediate in the in vitro transport reaction has been provided by the identification and isolation of the presumed carrier vesicles themselves. during the early stagesof the reaction, isolated golgi membranes generate an array of coated tubules, buds, and vesicles (orci et al., ' ). the vesicles have been isolated (after elution from l:heir membrane-bound state by high salt), and their coats have been found to contain several unique components . one of these, b-cop, was previously identified by kreis and coworkers as a kd peripheral protein tightly associated with the golgi (allan and ikreis, ; duden et al., b; serafini et al., ) . these non-clathrin-coated vesicles are good candidates ,for carrier vesicles. first, they contain vsv g protein, the iprotein being transported (orci et al., serafini let al., ) . next, conditions that block the transport reac-tion in general result in the accumulation or lack of formation of vesicles. most interestingly, treatment of cells or isolated membranes with the nonhydrolyzable gtp analog, gtpys, both blocks glycosylation of the vsv g protein and results in the accumulation of vsv g protein-containing coated buds and vesicles . similar results were obtained using aif , an inhibitor of conventional gtp-binding "g proteins" kahn, ) . the stage at which these inhibitors act remains unknown (donaldson et al., b) . in spite of this large amount of correlative evidence, additional data will be required before it is certain that the coated vesicles serve as unique and obligatory transport intermediates either in vitro or in intact cells. perhaps the most important piece of missing information pertains to whether the vesicles have a composition consistent with their presumed transport function. for example, it will be important to confirm that the golgi vesicles do not contain resident proteins, e.g., glycosyltransferases, that are not intended for transport. the analogous point is well established for plasma membrane clathrin-coated vesicles. many examples are known where proteins destined for coated vesicle-mediated endocytosis accumulate at coated pits, while those that are not efficiently internalized do not enter coated pits (pearse and robinson, ) . another critical unknown is a demonstration that the golgi coated vesicles are functional in vitro, i.e., that vsv g protein-containing vesicle fractions can be used to reconstitute transport when added to acceptor golgi. the analogous experiment has recently been accomplished for a less well-characterized vesicle fraction that mediates transport from the er to the golgi in yeast (groesch et al., ; rexach and schekman, ) . thus, in principle, this approach should be possible. however, with all such experiments, it will be important to ensure that the coated vesicle fractions used are not contaminated with uncoated, fusion-competent membranes. bfa blocks vesicle formation without blocking transport a reason for examining the features of the golgi vesicles so carefully is indicated by a recent paper that raises the possibility that, at least under certain conditions, these vesicles may not be required for transport in vitro . in this work, the effect of bfa on the golgi transport assay was determined. bfa is a macrocyclic fungal antibiotic that blocks the transport of membrane and secretory proteins through the golgi (takatsuki and tamura, ; misumi et al., ) . in intact cells, the drug causes a dramatic and rapid retrograde transport of golgi components back to the er via a system of tubular extensions (lippincott- schwartz et al., ) . the mechanism of bfa action is unknown, but one intriguing property is its ability to rapidly, and reversibly, trigger the dissociation of b-cop (donaldson et al., a; klausner et al., ) . when isolated golgi was treated with bfa, p-cop was no longer associated with the membranes nor were any coated buds or vesicles detected . nevertheless, the transfer of vsv g protein from donor to acceptor golgi continued with similar kinetics and efficiency as in untreated controls. the only difference was that transfer in bfa-treated golgi lost its sensitivity to gtpys inhibition. there are two possible interpretations of these results. the first is that coated vesicles that form in the assay system are not obligatory intermediates in the transport event, even in the absence of the drug. alternatively, bfa may induce an aberrant form of transport that nevertheless utilizes enzymatic machinery required for vesicular transport in the absence of the drug. it is at present impossible to distinguish between these two possibilities. however, bfa-treated golgi in vitro appear to form tubular extensions that may serve to interconnect donor and acceptor golgi stacks, thus resulting in transfer to the acceptor across these tubular bridges . this possibility is appealing since it could reflect the bfa-induced tubulation of the golgi seen in intact cells. however, additional electron microscope immunocytochemistry will be required to establish continuous interconnections between antigenically distinct golgi elements. vesicular transport or golgi fusion? the fact that "transport" (i.e., g protein glycosylation) can occur in the absence of vesicle formation raises a number of important questions: what transport step(s) is actually being measured in vitro? what is the relationship of the events reconstituted in vitro to those found in intact cells? is transport in vitro ever completely dependent on vesicle formation, even in the absence of bfa? if vesicles are not involved, is transfer from donor to acceptor golgi mediated by direct fusion, perhaps via tubular extensions? as mentioned above, it is unclear whether the g protein is transferred across a predicted compartment boundary (donor cgn to acceptor medial) or within a single compartment (donor medial to acceptor medial). it is becoming increasingly clear that all membranebound organelles have a propensity to form tubules in intact cells and in vitro, with or without bfa treatment. for example, the er (lee and chen, ), mitochondria (johnson et al., ) peroxisomes (yamamoto and fahimi, ) endosomes (hopkins et al., ; tooze and hollinshead, ; hunziker et al., b; lippincott-schwartz et al., ) , and lysosomes (swanson et al., ; lippincott-schwartz et al., ) are all capable of forming dynamic tubular membrane networks. one wellstudied example is the early endosome, the structure of which varies in different cell types (marsh et al., ; tooze et al., ) . cell-free assays have documented that the early endosome elements can fuse avidly with each other (gruenberg and howell, ) . other examples of processes involving homotypic fusion in vitro are the assembly of the nuclear envelope (burke and gerace, ) and the formation of er (dabora and sheetz, ) . the golgi complex is also a dynamic structure that can be disassembled by microtubule depolymerization into fragments that remain functional despite being dispersed throughout the cytoplasm (thyberg and moskalewski, ) . upon microtubule reassembly, the golgi fragments rapidly reassemble and assume their characteristic cisternal organization (ho et al., ) . a similar series of events must occur during mitosis when the golgi fragments into populations of small vesicles and tubules (lucocq et al., ) . during telophase, these fragments reassemble. given that homotypic fusion occurs so commonly, it would be premature to dismiss the possibility that the intra-golgi transport activity observed in vitro actually reflects a process of regulated tubule formation and/or direct fusion of donor and acceptor golgi elements (figure ) . indeed, even in untreated golgi, both in vitro and in intact cells, tubules can be found that are similar to those observed in bfa-treated golgi (hermo et al., ; braell et al., [see figure b in this reference]; griffiths et al., ) . that a direct fusion event is possible is also suggested by the fact that the predicted diffusion coefficient of intact golgi stacks is ~ o-fold less than that of the much smaller coated vesicles (n. ktistakis, unpublished data). thus, one might expect a decrease in the kinetics of vsv g protein transfer if bfa were to switch the signal generated in vitro from a vesicle-dependent phenomenon (as is presumed to occur in the absence of the drug) to a direct, tubule-mediated fusion event. however, g protein is transported from donor to acceptor with the same kinetics in the presence or absence of bfa . finally, the fact that the two n-linked chains of vsv g protein are processed simultaneously in vitro as opposed to sequentially in permeabilized cells may also indicate that compartmental boundaries may not be strictly preserved, i.e., that direct fusion may occur, during the cell-free assay (schwaninger et al., ) . the minimal model that we discussed (see figure ) predicts only two functional and presumably physical discontinuities in thegolgi: from thecgn to the medial golgi, and from the medial golgi to the tgn. thus, according to this view, transport through the golgi involves two intercompartmental transfers, reflecting transfer from the site of entry to the site of glycosylation, and from the site of glycosylation to the site of exit. considering available data, we consider it likely that the intercompartmental transfers between cgnlmedial and medial/tgn boundaries occur in vivo via vesicular carriers while the putative transporters remain to be identified, the p-cop-containing vesicles serafini et al., ) are obvious but unconfirmed candidates, whether or not they are ultimately found to function as carrier vesicles in in vitro golgi assays. the tubular interconnections known to occur in the golgi may also play a role in intercompartmental transfer, but we view these as probably being more important for establishing and maintaining intracompartmental links that serve to interconnect functionally identical golgi compartments and to mix their contents. these tubular connections may break and fuse continuously, accounting for the dynamic behavior of the golgi complex after microtubule disassembly and reassembly. however, if their formation is tightly controlled by a regulated process of assembly and disassembly of the f&cop-containing golgi coat, even tubule-based intercompartmental transfer is not inconceivable. moreover, whether cell-free assays of golgi function reflect such an intercompartmental transfer, or intracompartmental transfer between wild-type and mutant golgi, remains to be solved. only further characterization of the machinery responsible for intra-golgi transport will decide between these two possibilities. nevertheless, based on the evidence both from the in vitro assays and from yeast genetics, it is clear that the few proteins identified thus far, such as nsf and snap, are generally important elements, irrespective of the precise pathways or whether transport occurs via vesicles or tubules. is transport through the golgi selective or nonselective? having considered the evidence for the compartmental organization of the golgi and for the role of carrier vesicles in transport, we come to the last of the three general elements underlying our view of golgi function, namely, that transport of passenger proteins through the golgi is inherently nonselective (pfeffer and rothman, ) . the evidence for this view is largely indirect or negative, in that efforts to identify discrete signals on proteins that are required for forward transport have thus far been unsuccessful. on the other hand, as discussed above, there is now considerable evidence in favor of several distinct "retenlion signals" that effectively prevent forward transport of membrane or lumenal proteins after reaching their prescribed destinations in the er or various golgi compartments (pelham, ) . if this view is correct, selectivity in i.ransport through the secretory pathway may occur by "default," i.e., transport of passenger proteins proceeds owing to the absence of a retention signal. this concept, however, is not necessarily incompatible with the existence of selective signals for forward transport. it is clear that such signals exist and play an important role in directing the traffic of proteins as they leave the tgn. examples include the mannose g-phosphate residues that specify transport of hydrolytic enzymes to lysosomes and mellman, ) and the cytoplasmic domain determiinants that target newly synthesized membrane proteins to ithe basolateral surface of polarized cells (hunziker et al., a; brewer and roth, ) . since the transport of ,fluorescent lipids from the golgi to the plasma membrane iin nonpolarized cells occurs very rapidly, it is thought that "constitutive" transport from the golgi to the cell surface may not require specific signals . ,analogous results were obtained in experiments in which a tripeptide containing a cognate site for n-linked glycosylation presumably also involved transport from the er (wieland et al., ; helms et al., ) . while these experiments suggest that signals are not necessary for transport, the fact that they are released with rapid kinetics does not alone establish the absence of such signals. experiments showing that different secretory and membrane proteins are transported with different kinetics may indicate that signals or receptors are involved in forward transport (lodish et al., ; lodish, ) . similarly, the suggestion that newly synthesized viral spike glycoproteins are present in golgi membranes at a density severalfold greater than in the er is also consistent with the existence of signal-driven forward transport (griffiths et al., ) . however, both of these observations might also be reconciled with a nonselective mechanism of transport. given recent evidence that exit from the er is linked to the folding of newly synthesized proteins, differential folding rates among proteins may indirectly affect their transport kinetics. moreover, if the intrinsic rate of transport of glycoproteins through the golgi is slow relative to the rate of er exit, then one might also expect a higher concentration of certain passenger proteins in the golgi. at present, most of the attention paid to the question of signals in transport concerns er to golgi or postqolgi transport. whether transport through the golgi complex itself is selective or nonselective is still an open question. after years of descriptive work, the golgi complex is slowly starting to reveal its secrets. we have now entered an exciting period of research, during which it will become possible to define the molecular mechanisms responsible for generating and maintaining golgi structure and function. the first phase is already well under way and has been characterized by a search for essential bits and pieces of the golgi machinery, a number of which have already been found (nsflsecl , asnaplsecl , (j-cop, arf[stearnsetal., , rab p[goudetal., ] ,sec p [achstetter et al., , and secl p [bankaitis et al., oj) . as we have seen, however, it is at present difficult to know precisely what steps are controlled by each of these components. nevertheless, the observed conservation of golgi proteins between s. cerevisiae and mammals is most encouraging for our ability to confirm in living cells the function of components identified in vitro. the combination of cell-free analysis and genetics has proven its worth. the next phase will have to deal with the questions that have arisen. how many golgi compartments are there? are compartment boundaries defined by specific protein frameworks? if so, how do they function and how are they regulated? does transport between golgi compartments require vesicular carriers? what is the role of tubules? how does the machinery responsible for forward traffic relate to the machinery controlling homotypic fusion? how is specificity of forward and backward traffic regulated? how does lipid composition and organization affect transport? what function does the stack structure have? how do microtubules interact with the golgi elements? the challenge will be to integrate the information we are now collecting in the context of how the golgi complex works as a whole. sec encodes an unusual, high molecular weight protein required for membrane traffic from the yeast golgi apparatus a microtubule-binding protein associated with membranes of the golgi apparatus vesicles and cisternae in the trans golgi apparatus of human fibroblasts are acidic compartments reconstitution of the transport of protein between successive compartments of the golgi measured by the coupled incorporation of n-acetylglucosamine an essential role for a phospholipid transfer protein in yeast golgi function release of putative exocytic transport vesicles from perforated mdck cells lmmunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (g) of vesicular stomatitis virus in infected chinese hamster ovary cells palmitylation of viral membrane glycoproteins takes place after exit from the endoplasmic reticulum the glycoprotein that is transported between successive compartments of the golgi in a cell-free system resides in stacks of cisternae a single amino acid change rn the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin a cell free system to study reassembly of the nuclear envelope at the end of mitosis localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments compartmentation of the golgi complex: brefeldin-a distinguishes bans-golgi cisternae from the trans-golgi network snaps, a family of nsf attachment proteins involved in intracellular membrane fusion in animals and yeast mutations in the cdp-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein conversion of a golgi apparatus sialyltransferase to a secretory protein by replacement of the nh?-terminal signal anchor with a signal peptide tubulovesicular processes emerge from tram-golgi cisternae, extend along microtubules, and interlink adjacent frans-golgi elements into a reticulum the distribution of repeating [gal , glcnachl, ] sequences in asparatine-linked oligosaccharides of the mouse lymphomacell lines bw and phak . the microtubule-dependent formation of a tubulovesicular network with characteristics of the er from cultured cell extracts recycling of proteins from the golgi compartment to the er in yeast translocation across golgi vesicle membranes: ach glycosylation mutant deficient in cmp-sialic acid transport guanine nucleotides modulate the effects of brefeldin a in semipermeable cells: regulation of the association of a -kd peripheral membrane protein with the golgi apparatus binding of arf and cop to golgi membranes: possible regulation by a trimeric g protein polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture involvement of .cop rn membrane traffic through the golgi complex kd protein associated with non-clathrin-coated vesicles and the golgi complex, shows homology to .adaptin intracellular movement of two mannose -phosphate receptors: return to the golgi apparatus compartmentation of asparagine-linked oligosaccharide processing in the golgi apparatus attachment of terminal n-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the golgi stack the golgl apparatus (complex)-( - )from artifact to center stage transrent activity of golgl-like membranes as donors of vesicular stomatitis viral glycoprotein in vitro small gtp-binding protem associated with golgi cisternae evidence for extensive subcellular organization of asparagine-linked oligosaccharide processing and lysosomal enzyme phosphorylation compartmental organization of golgi-specific protein modification and vacuolar protein sorting events defined in a yeast secl (nsf) mutant the trans golgi network: sorting at the exit site of the golgr complex density of newly synthesized plasma membrane proteins in intracellular membranes. i. stereological studies exit of newly synthesized membrane proteins from the trans cisterna of the golgi complex to the plasma membrane the dynamic nature of the golgi complex isolation of a functional vesicular intermediate that mediates er to golgi transport in yeast membrane traffic in endocytosis: insights from cell-free assays reconstitution of steps in the constitutive secretorypathwayin permeabilized cells three-dimensional architecture of the cortical region of the golgi-apparatus in rat spermatids reclustering of scattered golgi elementsoccurs along microtubules movement of internalized ligand-receptor complexes along a continuous endosomal reticulum derived protein sequence, oligosaccharides, and membrane insertion of the kd lysosomal membrane protein (igpl ): identification of a highly conserved family of lysosomal membrane glycoproteins a recycling pathway between the endoplasmic reticulum and the golgi apparatus for retention of unassembled mhc class i molecules basolateral sorting in mdckcells requiresadistinct cytoplasmicdomain determinant selective inhibition of transcytosis by brefeldin a in mdck cells protein sulfation on tyrosine biosynthetic protein transport in the secretory pathway localization of mitochondria in living cells with rhodamine fluoride is not an activator of the smaller ( - kda) gtp-binding proteins the rate of bulk flow from the golgi to the plasma membrane pathways of protein secretion in eukaryotes elrefeldin a: cytosolic coat protein assembly, organelle structure, and membrane traffic assembly of asparagine-linked a~ligosaccharides the biogenesis of lysosomes dynamic behavior of endoplasmic reticulum in living cells the neuronal endomembranesystem microtubule-dependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway brefeldin a's effects on endosomes, lysosomes, and the tgn suggest a general mechanism for regulating organelle structure and membrane traffic transport of secretory and membrane glycoproteins from the rough endoplasmic reticulum to the golgi hepatoma secretory proteins migrate from the rough endoplasmic reticulum to golgi at characteristic rates evidence for a molecular distinction between golgi and cell surface forms of immunocytochemical analysis of the transfer from the intermediate compartment to the golgi complex of vsv g protein the el glycoprotein of an avian coronavirus is targeted to the cis golgi complex purification of a novel class of coated vesicles mediating biosynthetic protein transport through the golgi stack three dimensional structure of endosomes in bhk- cells scale formation rn algae novel blockade by brefeldin a of intracellular transport of secretory transport of secretory proteins in cultured rat hepatocytes sequences within and adjacent to the transmembrane segment of a- , sialyltransferase specify golgi retention recycling glycoproteins do not return to the cis-golgi the membrane spanning domain of -l , -galactosyltransferase specifies rrans golgi localization a new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the golgi stack dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the golgi stack brefeldin a, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on golgi cisternae molecular trapping of a fluorescent ceramide analogue at the golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-golgi marker for both light and electron microscopy a novel fluorescent ceramide analog for studying membrane traffic in animal cells: accumulation at the golgi apparatus results in altered spectral properties of the sphingolipid precursor intracellular aspects of the process of protein secretion clathnn, adaptors, and sorting evidence that luminal er proteins are sorted from secreted proteins in a post-er compartment control of protein exit from the endoplasmic reticulum recycling of proterns between the endoplasmic reticulum and golgi complex brosyntheticprotetn transport and sorting by the endoplasmic reticulum and golgi three-dimensional electron microscopy: structureofthegolgr apparatus perturbation of the morphology of the trans-golgi network following brefeldin a treatment: redistribution of atgn-specific integral membrane protein distinct biochemical requirements for the budding, targeting, and fusion of er-derived transport vesicles post-translational protein modification in the endoplasmic reticulum. demonstration of fatty acylase and deoxymannoiirimycin-sensitive alpha-mannosidase activities lmmunocytochemical localization of galactosyltransferase in hela cells: codistribution with thiamine pyrophosphatase in the trans-golgi cisternae demonstration of an extensive trans-tubular network continuous with the golgi apparatus stack that may function in glycosylation differential subcompartmentation of terminal glycosylation in the golgi apparatus of intestinal absorptive and goblet cells movement of proteinsthrough the golgi stack: a molecular dissection of vesicular transport intercompartmental transport in the golgi complex is a dissociative process: facile transfer of membrane protein between two golgi populations transport of protein between cytoplasmic membranes of fused cells: correspondence to processes reconstituted in a cell-free system distribution of the intermediate elements operating in er to golgi transport antibodies to rat pancreas golgi subfractions: identification of a -kd cis-golgi protein sequential transport of protein between the endoplasmic reticulum and successive golgi compartments in semi-intact cells metabolism and intracellular transport of glycosphingolipids identification, by a monoclonal antibody, of a kd protein associated with a tubule-vesicular compartment at the c&side of the golgi apparatus identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to golgi apparatus a coat subunit of golgi-derived nonclathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein -adaptin lipid sorting in epithelial cells lmmunoelectron microscopic exploration of the golgi complex sorting within the regulated secretory pathway occurs in the rrans-golgi network adp-ribosylation factor is functionally and physically associated with the golgi complex tubular lysosome morphology and distribution within macrophagesdepend on the integrity of cytoplasmic microtubules a golgi retention signal in a membrane-spanning domain of coronavirus el protein post-golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody brefeldin a, a specific inhibitor of intracellular translocation of vesicular stomatitis virus g protein: intracellular accumulation of high mannose type g protein and inhibition of its cell surface expression microtubules and the organization of the golgi complex tubular endosomal networks in attpo and other cells replication ofcoronavirus mhv-a in sac(-) cells: determination of the first site of budding of progeny virions site of addition of n-acetyl-galactosamine to the el glycoprotein of mouse hepatitis virusa effects of brefeldin a on the processing of viral envelope glycoproteins in murine erythroleukemia cells lipid traffic in animal cells distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of mdck cells signals and salvage sequences purification of a gal - glcnac ~ - sialyltransferase and a galpl- glcnac a - sialyltransferase to homogeneity from rat liver the rate of bulk flow from the endoplasmic reticulum to the cell surface a fusion protein required for vesicle-mediated transport in both mammalian cells and yeast three-dimensional reconstruction of a peroxisomal reticulum in regenerating rat liver: evidence of interconnections between heterogeneous segments we are indebted to the many friends and colleagues who read, criticized, or otherwise contributed during the evolution of this review. in particular, wewould liketo thanksuzannepfeffer, jim rothman, pietro de camilli, marylin farquhar, rick klausner, ari helenius, regis kelly, and graham warren, each of whom devoted a considerable amount of time and effort. key: cord- - z xminb authors: nan title: oligomerization of a membrane protein correlates with its retention in the golgi complex date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: z xminb the first membrane-spanning domain (m ) of the m glycoprotein of avian coronavirus (formerly called e ) is sufficient to retain this protein in the cis-golgi. when the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (vsv g protein) is replaced with m , the resulting chimera (gm ) is retained in the golgi (swift, a. m., and c. e. machamer. . j. cell biol. : - ). when assayed in sucrose gradients, we observed that gm formed a large oligomer, and that much of this oligomer was sds resistant and stayed near the top of the stacking gel of an sds-polyacrylamide gel. the unusual stability of the oligomer allowed it to be detected easily. gm mutants with single amino acid substitutions in the m domain that were retained in the golgi complex formed sds-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. oligomerization was not detected immediately after synthesis of gm , but occurred gradually with a lag of approximately min, suggesting that it is not merely aggregation of misfolded proteins. furthermore, oligomerization did not occur under several conditions that block er to golgi transport. the lumenal domain was not required for oligomerization since another chimera (alpha m g), where the lumenal domain of gm was replaced by the alpha subunit of human chorionic gonadotropin, also formed an sds-resistant oligomer, and was able to form hetero-oligomers with gm as revealed by coprecipitation experiments. sds resistance was conferred by the cytoplasmic tail of vsv g, because proteolytic digestion of the tail in microsomes containing gm oligomers resulted in loss of sds resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. interestingly, treatment of cells with cytochalasin d blocked formation of sds-resistant (but not sds- sensitive) oligomers. our data suggest that sds-resistant oligomers form as newly synthesized molecules of gm arrive at the golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. the oligomerization of gm and other resident proteins could serve as a mechanism for their retention in the golgi complex. ization did not occur under several conditions that block er to golgi transport. the lumenal domain was not required for oligomerization since another chimera (otmlg), where the lumenal domain of gml was replaced by the o~ subunit of human chorionic gonadotropin, also formed an sds-resistant oligomer, and was able to form hetero-oligomers with gml as revealed by coprecipitation experiments. sds resistance was conferred by the cytoplasmic tail of vsv g, because proteolytic digestion of the tail in microsomes containing gml oligomers resulted in loss of sds resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. interestingly, treatment of cells with cytochalasin d blocked formation of sds-resistant (but not sds-sensitive) oligomers. our data suggest that sds-resistant oligomers form as newly synthesized molecules of gml arrive at the golgi complex and may interact (directly or indirectly) with an actinbased cytoskeletal matrix. the oligomerization of gml and other resident proteins could serve as a mechanism for their retention in the golgi complex. t he golgi complex is the site of oligosaccharide processing and sorting for proteins that are destined for delivery to secretory granules, lysosomes, the plasma membrane, and the extracellular space. the golgi-resident proteins that perform these functions must be retained in the golgi despite the large amount of lipid and protein traffic through this organelle. retention of these proteins is thought to involve recognition of positive signals that specify golgi localization. a useful model protein for studying golgi retention signals has been the m glycoprotein (formerly called el) of the avian coronavirus infectious bronchitis virus (ibv)k coronaviruses bud into the intermediate compartment or golgi complex of mammalian cells (griffiths and rottier, please address all correspondence to dr. carolyn machamer, department of cell biology and anatomy, the johns hopkins school of medicine, n. wolfe street, baltimore, md . dr. ann m. swifts current address is smithkline beecham pharmaceuticals, philadelphia, pa. ). when cdna encoding the m protein is expressed in animal cells, the protein is targeted to the cis-golgi (machamer et al., ) . structurally, the ibv m protein consists of a short glycosylated amino-terminal domain, three membrane-spanning domains, and a long carboxyterminal cytoplasmic domain. the first membrane-spanning domain (ml) of the m glycoprotein is sufficient to retain this protein in the golgi (machamer and rose, ) . furthermore, the ml domain can confer golgi localization to a wellcharacterized plasma membrane protein (the g protein of vesicular stomatitis virus, or vsv g). vsv g is a type i membrane protein which is cotranslationally inserted into the er, where it forms homotrimers. it is then rapidly transported through the golgi complex (as determined by the kinetics of n-linked oligosaccharide processing) and delivered to the plasma membrane (for review see doms et al., ) . when the membrane-spanning domain of vsv g is replaced with the ml domain of ibv m, the resulting chimera (gml, see fig. ) is retained in the golgi complex (swift and machamer, ) . extensive mutagenesis suggests that at least four specific amino acids in the ml domain are critical for golgi retention of gml: asparagine , threonines and , and glutamine (machamer et al., ) . interestingly, these residues form an uncharged polar face along one side of the alpha helix predicted for ml. the polar nature of this face of the helix suggests that protein-protein interactions along this face may mediate golgi retention of gml. there are two general ways to envision the specific retention of a protein by information contained within its transmembrane domain (for review see machamer, ) . the first involves the recognition of this domain by a specific receptor that either blocks further movement in the exocytic pathway or retrieves escaped proteins back to the appropriate compartment. a receptor-based model also requires a mechanism for retention or recycling of the receptor itself. the second mechanism invokes changes in the tertiary or quaternary structure of the protein that are induced via the membrane-spanning domain when the protein encounters a particular microenvironment. changes in protein structure could include oligomerization or aggregation (covalent or non-covalent) with other proteins or lipids, which would prevent movement of the protein into transport vesicles. the cis-golgi may differ from the er in lipid composition and divalent cation concentration. these changes in microenvironment could conceivably trigger a conformational shift in golgi resident proteins upon their arrival in the appropriate compartment. we have been investigating the mechanisms by which ml might mediate retention of gml in the golgi complex. we observed that gml formed a large oligomer soon after synthesis when assayed by sucrose gradient centrifugation. here, we show that this oligomer is unusually stable, and that its formation correlates with arrival and retention in the golgi complex. our findings point to a possible role for oligomerization in the retention of gml. aprotinin, colchicine, carbonyl cyanide m-chlorophenylhydrazone (cccp), cytochalasin d, tpck-trypsin, and soybean trypsin inhibitor were obtained from sigma chem. co. (st. louis, mo). brefeldin a was from epieentre technologies (madison, wi). stock solutions of reagents were stored at - °c. cos-'/and hela cells were maintained in dme with and % fcs, respectively. cos- cells plated in -mm dishes ( - % confluent) were transfected with an sv -based expression vector using deae-dextran (machamer et al., ) . expression was analyzed at -- h posttransfection. for expression using the vaccinia-t system, hela cells ( - % confluent) were infected with the recombinant vaccinia virus vtf - encoding t rna polymerase (fuerst et al., ) at a multiplicity of infection of - . after adsorption for min at °c, the inoculum was replaced with . ml of serum-free medium containing /~g of a vector (par ) encoding the appropriate gene behind the t promoter and - /~ of the cationic lipid ~transfectace" (gibco brl, gaithersburg, md; rose et al., ) . expression level was varied by changing the amount of dna added per well ( . - ~tg/dish). in coexpression experiments, cells were transfected with /~g of dna encoding each construct. expression was analyzed by metabolic labeling starting h after infection. gml and related mutants were generated using the kunkel method of oligonucleotide-direeted mutagenesis as described previously (swift and machamer, ; machamer et al., ) . point mutants were named by appending the original amino acid (in single letter code) followed by the new amino acid. ,m (guan et al., a) and tmr (doms et al., ) were obtained from jack rose (yale university). construction of ,~mlg was described previously (swift and machamer, ) . tgml was constructed from gml by inserting a stop codon at position using site-directed mutagenesis (kunkel et al., ) . indirect immunofluorescence staining of transiently transfected cos- ceils was performed as described (machamer and rose, ; machamer and rose, ) . gml and related mutants were detected using a monoclonal anti-vsv g antibody ( /~g/ml; lefrancois and lyles, ) followed by texas red-conjugated, affinity-purified goat anti-mouse igg ( /zg/ml; jackson immuno research laboratories inc., avondale, pa). cells expressing vsv g or mutant g proteins were rinsed once with pbs, starved for rain in methionine-free dme, and labeled for the indicated times with #ci/ml trans s-label (icn radiochemicals, irvine, ca) or l-[ s] in vitro cell labeling mix (amersham corp., arlington heights, il) in serum-free, methionine-free dme. cells were solubilized immediately or after the appropriate chase period in serum-free dme containing a threefold excess of unlabeled methionine. cells were lysed in detergent solution ( mm tris, ph . , % np- , . % deoxycholate, . mm edta, and . tiu/ml aprotinin). samples were immunoprecipitated using a polyclonal anti-vsv antibody (produced by immunization of rabbits with purified vsv) and fixed staphylococcus aureus (calbiochem-behring corp., san diego, ca). after heating to *c for rain in laemmli sample buffer containing % fl-mercaptoethanol, samples were electrophoresed on % sds-polyacrylamide gels as described (laemmli, ) . marker proteins were c-methylated standard molecular weight markers (amersham corp.). labeled proteins were detected by fluorography (bonner and laskey, ) . am chimeras were radiolabeled and solubilized as above but immunoprecipitated using anti-human chorionic gonadotropin (hcg) antibody (organon teknika-cappel, west chester, pa) and electrophoresed on % sds-pelyacrylamide gels. brefeldin a ( /tg/ml final concentration) was diluted into serum-free medium from a mg/ml stock in ethanol immediately before use. colehicine ( mm) was prepared in dmso, diluted : into serum-free, methionine-free medium, and added to cells ( ~tm final concentration) starting h before metabolic labeling. cytochalasin d ( . m in dmso) was added to cells ( /~m final concentration) h after infection and ineluded in subsequent changes of medium. an equal volume of dmso was added to control cells. in experiments involving cell treatment with cccp, ceils were starved in glucose-, methionine-, and cysteine-free medium for rain before metabolic labeling in the same medium for win. cccp ( . m stock in dmso) was added at the beginning of the chase in glucosefree medium to a final concentration of #m. hela cells were transfected, metabolically labeled for min, and chased for rain as described above. cells were rinsed three times in pbs supplemented with lnm mgci and . mm cac , and then biotinylated using sulfo-nhs-biotin (pierce chem. co., rockford, il) as described (lisanti et al., ) . cells were then solubilized as described above and gml and related proteins immunoprecipitated. samples were eluted from the s. aureus pellets by heating to °c in /~ mm "iris, mm nac , % sds, ph . , and divided into two equal aliquots. one aliquot was kept at - °c ("total") while the other was diluted tenfold with mm tris, mm naci, ph . , and incubated with streptavidin-coupled agarose beads (pierce chem. co.) for min at "c. the streptavidin beads were then pelleted and washed three times in ripa buffer. non-biotinylated proteins in the supernatant were precipitated using "i~a. samples were analyzed by sds-page followed by fluorography. oligomerization of the gml protein was analyzed by velocity gradient centrifugetion in sucrose performed essentially as described (doms et al., ) . continuous - % (wt/wt) sucrose gradients were poured over a % (wt/wt) sucrose cushion ( . nil) in sw . tubes. all solutions were in mnt ( mm naci, mm tris, mm mes, ph . ) containing . % triton x- . hela cells expressing either vsv g or gml were metabolically labeled for min and harvested (in mnt containing % triton x- ) either immediately or after min of chase. lysates were loaded on top of the gradients and spun at - , rpm for - h. fractions ( . ml) were collected from the top using a buchler auto densi-flow iic, immunoprecipitated with anti-vsv antibody, and electrophoresed to determine the location of the proteins in the gradient. for estimating the size of the oligomer, gradients ( - % sucrose [wt/wt] in mm nac , mm tris, ph . , . % triton x- ) were spun at rpm for h. the markers used were thyroglobulin ( . $ ,, ) and eatalase ( . $ ,, ). microsomes were prepared after metabolic labeling and the appropriate chase period from transfected hela cells plated in -cm dishes. cells were rinsed in pbs and swelled in . ml swelling buffer ( mm tris, mm naci, t mm mgci , ph . ) for rain on ice. after scraping with a rubbet policeman, . ml swelling buffer containing % sucrose (wt/vol) was added, and the cells were homogenized in a i ml dounce homogenizer with strokes of the a pestle. lysates were centrifuged briefly ( s , rpm) to remove nuclei and large debris. . ml swelling buffer containing % sucrose (wt/vol) was added, and the lysates were centrifuged for min at , rpm in a beckman tl- ultracentrifuge (t . rotor). pellets were resuspended gently in pbs and divided into two aiiquots. tpck-trypsin ( /~g) was added to one aliquot, and both were incubated at "c for min. proteolytic digestion was stopped by adding #g of soybean trypsin inhibitor to both tubes. samples were solubilized (in detergent solution containing . % sds or in mnt as noted) and processed further as described in the text and figure legends. previous studies from this laboratory showed that the first membrane span (ml) of the m glycoprotein of bv (previously called the e glycoprotein) causes the chimeric protein gml to be retained in the golgi. the sequence of ml is shown in fig. a. using site directed mutagenesis, four key residues in the ml domain were shown to be critical for golgi retention of gml: asparagine (n, ), two threonines (t~ and t, ), and glutamine (q~). some substitutions at q~ are tolerated (e.g., gmlqi'hs ) whereas other changes (e.g., gmlqi~o) result in transport of the protein to the cell surface (machamer et al., ) . another mutation in which two isoleucine residues were inserted near the center of the transmembrane domain (gmli,~) also disrupts retention in the golgi complex. these three chimeric proteins (gml~, gmlqlso, and gmlqh_~o) were expressed in transiently transfected cos-'/ cells and localized by indirect immunofluorescence ( fig. b) . vsv g and grnlqi~o were readily detected at the plasma membrane, while gml and gmlqi-hso were detected only in the golgi region and largely colocalized with lens culinaris lectin staining, even after treatment with nocodazole or brefeldin a (machamer et al., ) . gmlin~ staining was less prominent at the plasma membrane than gmlqi~ , and a considerable amount of fluorescence was detected in the golgi complex. the localizations of these proteins were confirmed in hela cells and bhk cells transfected using a vaccinia-virusmediated expression system (not shown). interestingly, when we analyzed expression of these constructs in hela cells and cos- cells by metabolic radiolabeling and immunoprecipitation, we noticed the appearance of a radiolabeled sds-resistant species that migrated at the top of the stacking gel, in addition to products migrating at the expected molecular masses. the material at the top of the stacking gel was more prevalent in samples immunoprecipitated from hela cells ( fig. c) than in those from cos- cells ( fig. d) . this species was present only in immunoprecipitates from cells that expressed mutants with golgi-resident populations (gml, gmli,, and gmlqh s ) and it appeared regardless of the level of expression (over a > -fold range). although ~ % of the total radioactivity immunoprecipitated from gml-expressing hela cells migrated at the top of the stacking gel, we have not quantitated this information routinely. one problem with quantitation is that this material could include radiolabeled components other than gml; furthermore, some of the oligomer may not enter the gel at all and be lost during subsequent processing. gmli,, was previously reported to move through the golgi complex rapidly (with a half time of rain in cos- cells; swift and machamer, ) . however, the kinetics of transport reflect only the population of protein that enters the separating gel. the sds-resistant protein may therefore represent a stable golgi-resident pool. the sds-resistant oligomerization did not depend on the type of cells used for expression, since the same results were found using transiently transfected bhk cells and stably transfected cho cells (not shown). because the sdsresistant material was easiest to detect in hela cells, we used these cells for subsequent experiments. the material at the top of the stacking gel could be detected after transfer to nitrocellulose and probing with anti-vsv g antibodies by immunoblotting, suggesting that it contained gml (or the related constructs; data not shown). furthermore, this large species was efficiently precipitated by several conformationsensitive anti-vsv g monoclonal antibodies that do not recognize grossly misfolded proteins (doms et ai., ) . we tried to solubilize the gml oligomer using a wide variety of lysis conditions and sample treatments. none of the modifications we tested, including changes in salt concentration, reducing agents, detergents, chaotropic agents (e.g., urea, guanidinium hc ), or temperature of solubilization or sample elution disrupted the sds-resistant oligomer (data not shown). a very small amount of sds-sensitive gml could be produced when the top of the stacking gel (containing the sds-resistant oligomer) was isolated after electrophoresis, placed in the well of a fresh gel, and reelectrophoresed (not shown). together, these observations suggest that sds-resistant oligomer formation is an intrinsic property of gml and mutant gml proteins that are retained in the golgi complex, but not of mutants that efficiently reach the plasma membrane. if the oligomerized protein represents a distinct intracellular pool of protein(s), it should not be susceptible to cell surface biotinylation. moreover, we predicted that an oligomer that formed nonspecifically after cell solubilization would be precipitated by streptavidin since incorporation of a few biotinylated molecules would allow precipitation of the entire oligomer. hela cells expressing gml, vsv g, or gmlin, were metabolically labeled for min, and then chased for min before cell-surface biotinylation with sulfo-nhsbiotin. after immunoprecipitation, an aliquot of each sample was treated with streptavidin-coupled agarose, and biotinylated and non-biotinylated proteins were recovered and analyzed by sds-page (fig. ) . under these conditions, ,o % of the total vsv g was biotinylated and could be recovered in the streptavidin pellet. only the mature (sialylated) form of vsv g was recovered in this fraction, suggesting that biotinylation was indeed restricted to cell surface proteins. in contrast, essentially none of the gml was precipitated by streptavidin, suggesting that this protein remained in an intracellular compartment. a small amount of sds-sensitive gmli~, was recovered in the streptavidin pellet. more importantly, no sds-resistant material was biotinylated, suggesting that the sds-resistant gmli~ constitutes an intracellular (perhaps golgi-resident) pool that is distinct from the sds-sensitive protein, and that only a portion of gml~, is transported to the plasma membrane where it can be biotinylated. this result strongly suggested that oligomer formation occurs before cell solubilization. the time course of gml oligomerization was determined. hela cells expressing gml were metabolically labeled for min, chased for the indicated times, solubilized, and immunoprecipitated with anti-vsv g antibody (fig. ) . oligo- figure . sds-resistant oligomers are not subject to cell surface biotinylation. ceils were metabolically radiolabeled for min, and then chased for rain. cell surface-specific biotinylatiun was performed at "c using sulfo-nhs-biotin, and the cells were solubilized and immunopreeipitated with anti-vsv antibody. the bound protein was eluted from s. aureus pellets and divided in half. one aliquot was solubilized in sample buffer (t), and the remainder was incubated with streptavidin-coupled agarose beads. the supematant (is], unbound material) was collected and tca precipitated, and the streptavidin beads (p) were washed and solubilized before sds-gel electrophoresis. merization was not detected immediately after synthesis of gml, but occurred gradually with a lag of ~ min. the kinetics of gml oligomer formation were consistent with the time at which newly synthesized proteins arrive at the cis-golgi. to determine if oligomerization occurred in a post-er compartment, we asked whether the sdsresistant oligomer would form when er to golgi traffic was blocked by incubation at °c. at this temperature, newly synthesized proteins accumulate in the intermediate compartment (saraste and kuismanen, ) . hela cells were transfected with constructs encoding gml or vsv g, metabolically radiolabeled for min, and then chased at or °c as indicated for up to min (fig. ) . no oligomer formed when cells expressing gml were chased at c; however, if cells chased at °c were subsequently transferred to c, oligomer formation occurred. in separate experiments, we found that the kinetics of oligomer formation at °c were unaffected by the inclusion of a c pretreatment step (data not shown). in addition, treatment with . mm cccp during the chase period also inhibited sds-resistant oligomer formation (not shown). this treatment blocks transport of newly synthesized proteins from the er. these experiments suggested that gml oligomer formation normally occurred in a post-er compartment. however, brefeldin a pretreatment (or treatment during chase) did not block oligomer formation (not shown), although it did cause redistribution of gml to an er-like pattern (machamer et al., ) . to ask if gml oligomerization was an artifact generated during sds-page, we analyzed oligomer formation using velocity gradient sedimentation in sucrose (doms et al., % gml does not occur at °c. transfected hela cells were metabolically radiolabeled for min, and then chased at or °c for up to rain. one set of dishes was incubated for min at °c, then transferred to °c for min. vsv g and related proteins were immunoprecipitated from solubilized cells as described in materials and methods. ). hela cells were transiently transfected with vsv g or gml, and then metabolically radiolabeled for min and solubilized either immediately or after min of chase. lysates were loaded onto - % sucrose gradients, centrifuged, and analyzed as described in materials and methods. immediately after synthesis, vsv g migrated as a s .~ peak (fig. ; doms et al., ) . after a -min chase, all of the vsv g had trimerized, migrating as a discrete s ,~ peak. the behavior of gml in these gradients was markedly different. immediately after synthesis, gml migrated similarly to the vsv g monomer. after a -rain chase, however, much of the gml was found in the pellet in an sds-resistant form ( fig. ; note top of lane). the timedependent shift in the mobility of the oligomer in sucrose gradients suggests that oligomers already exist in the cells at the time of lysis and immunoprecipitation. the sdssensitive material in the gml pellet fractions may represent an intermediate form of oligomer that is not yet sdsresistant. alternatively, this material could contain protein released from the sds-resistant oligomer during sds-page. in contrast to our findings with gml, misfolded mutants of vsv g that aggregate in the er are found in the pellet (and are sds-soluble) immediately after synthesis (doms et al., ) . using different centrifugation conditions, we estimated the average size of the gml oligomer to be approximately s .~, or roughly kd (data not shown). therefore, if gml (mw kd) is the sole component of the oligomer, we estimate there would be ,o molecules per oligomer. in contrast to the chimeric gml protein, oligomers of the ibv m glycoprotein are not detected when assayed by sucrose gradient sedimentation (data not shown). this might suggest that oligomerization is not a retention mechanism for native m protein; alternatively, m oligomers might readily dissociate in detergent. the resistance of the gml oligomer to solubilization suggests a structural feature of vsv g may stabilize the complex. we asked whether the lumenal domain of vsv g, which is sufficient for trimerization (doms et al., ) , was required for oligomer formation. to do this, we replaced the lumenal domain ofvsv g or gml with a small soluble glycoprotein, the o~ subunit of hcg. when expressed alone, the ol subunit of hcg is secreted from cells as a monomer (guan et al., b) . when hcgo~ is fused to the membrane-spanning domain and cytoplasmic tail of vsv g, the chimeric protein (c~m) is membrane-bound and transported to the cell surface (guan et al., a) . when the vsv g membrane-spanning domain of c~m is replaced with the ml domain of ibv m, the resulting chimera (oemlg) is retained in the golgi complex (swift and machamer, ) . interestingly, in metabolically labeled hela cells expressing cxmlg, we observed the time-dependent accumulation of an sds-resistant species at the interface between the separating and stacking gels (fig. , arrowhead) . this species was not observed in cells expressing am. the smaller l~gure . sucrose gradient sedimentation of gml and vsv g. hela cells transfected with vsv g or gml were metabolically labeled for rain, and then solubilized immediately or after rain of chase. lysates were loaded onto - % sucrose gradients and centrifuged as described in materials and methods. fractions were collected, immunoprecipitated using anti-vsv antibody, and analyzed by sds-page. after the -min chase, all of the gml formed a large oligomer that pelleted under these gradient conditions, and much of it was sds-resistant. size of the otmlg sds-resistant oligomer is consistent with the size difference between ctmlg and gml ( vs kd). on sucrose gradients, c~mlg solubilized min after synthesis migrated as a larger species (some of which was sdsresistant) than newly synthesized oanlg. in contrast, the mobility of o~m on sucrose gradients was unaltered during the chase (data not shown). thus the vsv g lumenal domain was not required for sds-resistant oligomer formation. we used a coprecipitation assay to ask if otmlg formed hetero-oligomers with gml or other related golgi-resident proteins. hela cells were cotransfected to express both otmlg and either gml, gmlins, or vsv g. after a short metabolic labeling period ( min), cells were solubilized either immediately or after a -min chase. lysates were immunoprecipitated using anti-hcg antibody. when otmlg was expressed alone, the time-dependent accumulation of sds-resistant oligomer at the gel interface (arrowhead) was observed as described above (fig. , lane ) . interestingly, when otmlg was coexpressed with gml or gmlins, the size of the oligomer shifted dramatically, and now migated at the top of the stacking gel (fig. , lanes and ) . furthermore, some proteins appeared to be solubilized during electrophoresis, since a small amount of material migrating with authentic gml and gmlins w a s also detected in these lanes (fig. , arrow) . in contrast, no change in the size of the oligomer was observed when c~mlg was coexpressed with vsv g (fig. , lane ) or with gmlqls (data not shown), which are transported to the plasma membrane. furthermore, when cells expressing gml and c~m (containing the vsv g membrane-spanning domain) were immunoprecipitated using anti-hcg antibody, no gml was recovered (data not shown). to test the role of the cytoplasmic tail of gml in oligomer formation, we inserted a stop codon after the first residue (arg) beyond the transmembrane domain (tgml). the corresponding tailless vsv g (tmr; doms et al., ) trimerizes with normal kinetics but is transported slowly from the er to the plasma membrane. when tgml was localized by figure . amlg forms an sds-resistant oligomer. hela cells transfected with amlg or am were metabolically labeled for rain, and then solubilized either immediately or after a -min chase and immunoprecipitated with anti-hcg antibody. samples were analyzed by sds-page on % gels. note the time-dependent accumulation of an sds-resistant species at the interface between the separating and stacking gels (arrowhead). the increased mobility of amlg and am after min of chase is due to carbohydrate trimming. indirect immunofluorescence in transiently transfected cos- cells, some golgi staining was observed, although a significant amount of the protein was found in the er. unlike grid, tgml did not form an sds-resistant oligomer when expressed in cos-'/or hela cells, and did not sediment as a large oligomer in sucrose gradients (although it did apparently form trimers, not shown). however, because tgml appears to exit the er very slowly (like tmr), the kinetics of oligomer formation may have been too slow to detect. because tgml did not form detectable oligomers, we asked whether preformed gml oligomers could be disrupted by proteolysis of the cytoplasmic tall. the -amino acid figure . gml and c~mlg form hetero-oligomers. hela cells were transfected with #g each of mlllg and either calf thymus dna (ct), gml, vsv g, or gml~n~. after a -min pulse, cells were solubilized either immediately or after a -min chase. aliquots of the solubilized cells were immunoprecipitated with anti-hcg antibody and elcctrophoresed on % sds-polyacrylamide gels. the interface between the stacking and separating gels is marked with an arrowhead, and the mobility of gml in the gel is denoted with an arrow, amig forms hetero-oligomers with gml and gmlm (but not with vsv g) that could be precipitated by anti-hcg antibody. fig. a) has numerous arginines and lysines that are susceptible to proteolysis by trypsin. hela cells expressing gml or vsv g were metabolically radiolabeled for min, and then chased for or rain, and microsomes prepared as described in materials and methods. microsomes were treated with tick-trypsin for rain at °c, and then solubilized, immunoprecipitated with anti-vsv antibody, and analyzed on sds-polyacrylamide gels (fig. b) . as size standards we used radiolabeled tgml and tmr immunoprecipitated from transfected hela cells. trypsin cleaved newly synthesized vsv g protein to a species migrating slightly larger than tmr on sds-page (fig. b, compare lanes and ) . after rain of chase, two proteolytic products were detected. the upper band corresponded to cleavage of the cytoplasmic tail of mature (sialylated) vsv g, as it was resistant to endoglycosidase h, whereas the lower band was sensitive to endoglycosidase h treatment (not shown). in addition, we frequently observed some residual gml or vsv g were metabolically labeled for rain, and then chased for or rain. microst,mes were prepared as described in materials and methods and treated with #g tpck-trypsin (lanes , , , and ) or mock treated (lanes , , , and ) for min at "c. proteolysis was stopped by the addition of trypsin inhibitor ( /zg), the microsomes were solubilized, and gml (lanes - ) and vsv g anes - ) were immunoprecipitated and analyzed by sds-page. more inside-out microsomes are generated from compartments further along the secretory pathway, accounting for the loss of material recovered after trypsinization of vsv g isolated after a -min chase (lane ). tgml (lane ) and tmr (lane ) immunoprecipitated from hela cells labeled under the same conditions are included as size standards. uncleaved mature vsv g in our experiments. since the trypsin-cleaved form of vsv g migrated more slowly than t m r (whose tail contains a single amino acid), we deduced that trypsin cleaves the vsv g tall within region a marked on fig. a. the proteolytic profile generated by trypsin cleavage of gml differed from that of vsv g. trypsin treatment of figure . trypsin-treated gml is still oligomerized. cells expressing vsv g (a) or gml (b) were metabolically radiolabeled for rain, and then chased for rain. microsomes were prepared and treated with /zg tick-trypsin for rain at °c, then solubilized in mnt and centrifuged on - % sucrose gradients. fractions were collected, immunoprecipitated using anti-vsv antibody, and analyzed by sds-page. newly synthesized gml yielded a single species which migrated more slowly on sds-page than the vsv g proteolyric fragment. thus, even before oligomers are formed, the tail of gml is less accessible to trypsin than that of vsv g. interestingly, when gml was digested after a -rain chase, there was a large reduction in the amount of sds-resistant oligomer migrating at the top of the stacking gel on sds-page (fig. b, compare lanes and ) . furthermore, we saw a second, larger band in addition to the one detected immediately after the pulse label. both bands were endoglycosidase h-sensitive, indicating that the proteins they represent had not passed through the medial golgi (not shown). in more detailed kinetic experiments, the amount of this upper band increased with longer chase times, with a concomitant decrease in the amount of the lower band (not shown). based on the potential trypsin cleavage sites in the figure . trypsin treatment of gml and vsv g recovered after treatment with cytochalasin d. hela cells expressing gml or vsv g were treated with #m cytochalasin d starting h before radiolabeling. because fewer microsomes were recovered from cytochalasin d-treated cells than from untreated ceils, treated cells were radiolabeled with twice the usual concentration of radioactivity. microsomes were prepared after a -min pulse and a -min chase, and aliquots were mock treated or treated with tpck-trypsin as described in materials and methods. gml and vsv g were immunoprecipitated using anti-vsv antibody and analyzed by sds-page. cytochalasin d blocked formation of sds-resistant oligomers and accumulation of the larger trypsin-cleaved band. cytoplasmic tail and the relative mobility of these bands compared to the vsv g proteolytic fragment and tgml, we conclude that the upper and lower bands result from trypsin cleavage within regions c and b, respectively (fig. a) . the upper band may represent material generated exclusively from proteolytic digestion of sds-resistant gml. this would suggest that the last three amino acids of the cytoplasmic tail of gml are required for sds resistance of the oligomer. alternatively, trypsin may destroy another (unlabeled) component of the oligomer that is required to maintain its resistance to solubilization. trypsinization of microsomes eliminated the sds-resistant form of gml. to ask if proteolysis dissociated the oligomers as well, we subjected trypsin-treated gml-containing microsomes to sucrose gradient sedimentation. microsomes prepared from hela cells after a -min pulse and a -min chase were mock-treated or treated with tpck-trypsin ( #g) for min at °c. samples were diluted tenfold in mnt, and aliquots were loaded on sucrose gradients and centrifuged as described in materials and methods. gradient fractions were collected, immunoprecipitated, and analyzed by sds-page. the trypsin-treated samples are shown in fig. . trypsin digestion did not cause dissociation of vsv g trimers (compare fig. a with fig. ) . significantly, the larger proteolytic product of gml migrated at the bottom of the gradient, whereas some of the smaller product pelleted and some was found throughout the gradient (fig. b) . this suggested that removing part of the cytoplasmic tail of gml does not disrupt the oligomer, and further supports the possibility that digestion of sds-resistant material gives rise to the larger band. we tested the possibility of cytoskeletal involvement in sds resistance of gml by pretreating cells with drugs that disrupt microtubules or actin filaments. depolymerization of microtubules with colchicine ( /~m) had no effect on the amount of sds-resistant gml formed (not shown). interestingly, treatment with cytochalasin d ( /zm) starting h before metabolic labeling blocked formation of sds-resistant gml oligomers (fig. , lane ) . the amount of immunoprecipitated radiolabel migrating at the top of the stacking gel decreased from % of total in untreated cells to % of total in cytochalasin d-treated cells (compare fig. , lanes i and ). cytochalasin d did not block the transport of vsv g since the rate of sialylation was enhanced relative to untreated cells. on sucrose gradients, gml solubilized from cytochalasin d-treated cells migrated as an sds-sensitive oligomer (in the pellet), and vsv g trimerization was unaffected (data not shown). this observation suggests that oligomer formation and sds-resistance are separable characteristics, and furthermore implies that sds-resistance may result from interaction between gml and an actin-based matrix or cytoskeleton. because cytochalasin d blocked the formation of sdsresistant oligomers of gml, we tested whether this treatment prevented the appearance of the larger proteolytic band. hela cells expressing gml or vsv g were treated with cytochalasin d ( /zm) for h before a -min metabolic labeling. microsomes were prepared after a -min chase and mock-treated or treated with /zg of tpck-trypsin for min at °c, and then solubilized and immunoprecipitated using anti-vsv antibody (fig. ) . as mentioned above, treatment with cytochalasin d enhanced the fraction ofvsv g that became sialylated during this period (compare lanes and ). we routinely observed that trypsin degraded most of the vsv g recovered from cytochalasin d-treated cells (lane ), suggesting that the orientation or durability of microsomes was affected by treatment with this perturbant. interestingly, trypsin digestion of gml recovered from cytochalasin d-treated cells yielded predominantly the smaller proteolytic species, generated by cleavage within region b in fig. a. we therefore conclude that the larger gml species does indeed represent material digested from sdsresistant oligomers, and furthermore, that the oligomers may interact with an actin-based cytoskeleton. the experiments presented here support protein oligomerization as a mechanism for retaining the chimeric protein gml in the golgi complex. an unusual feature of the gml oligomer (its resistance to solubflization by sds) allowed us to easily assay oligomer formation. the specificity of oligomerization was mediated by the ml transmembrane domain, since mutant gml proteins that were retained in the golgi formed oligomers, while mutant proteins that were released to the plasma membrane did not. this specificity was preserved in another construct, otmlg, in which the lumenal domain of vsv g was replaced with the o~ subunit of hcg. furthermore, e~mlg was able to form hetero-oligomers with gml and other golgi-resident gml mutants, but not with mutants that move to the plasma membrane. this suggested that the lumenal domain of vsv g was not required for oligomerization. while the specificity of oligomerization was dictated by the sequence of the membrane-spanning region, the cytoplasmic tail of gml was necessary for sds-resistance and may play a role in oligomer formation. when the cytoplasmic tails of preexisting oligomers were digested with trypsin, the proteolytic products were soluble in sds but continued to migrate as oligomers on sucrose gradients. based on potential trypsin cleavage sites in the cytoplasmic tail of gml and the migration of the proteolytic fragments on sds-page, it is possible that removal of the last three carboxyterminal residues of gml destroys its resistance to solubilization by sds. alternatively, trypsin digestion could remove another component of the oligomer that is required for sds resistance. interestingly, cell treatment with cytochalasin d blocked the formation of sds-resistant oligomers, suggesting that the acquisition of sds resistance may reflect association of the gml cytoplasmic tail with an actin-based cytoskeleton. we are currently constructing a series of gml proteins with truncated cytoplasmic tails to determine if these proteins are correctly targeted and form oligomers. several observations suggest that gml oligomerization normally occurs after the protein leaves the er. oligomerization of gml was not detected until ~ min after protein synthesis, which correlates with arrival of newly synthesized proteins at the cis-golgi. oligomers were not observed when er to golgi traffic was blocked by incubation at °c or treatment with cccp. interestingly, sds-resistant oligomers did form in brefeldin a-treated cells, suggesting that oligomers can form in pre-golgi compartments under certain conditions. brefeldin a treatment may generate the conditions required to stimulate oligomer formation in the er. conditions which could trigger gml oligomerization might include its concentration in the membrane, the retrieval of glycolipids or glycoproteins typical of the golgi complex, or changes in ph or divalent ion concentrations. we were unable to solubilize the sds-resistant oligomer using a wide variety of harsh conditions. it is possible that some of the gml oligomer is covalently crosslinked to itself or to an actin-based structure via the vsv g tail. however, removal of as few as three amino acids from the cytoplasmic tail resulted in oligomers that were sds-sensitive. this indicates that the sds resistant phenotype (which requires the last three amino acids of gml) can be separated from oligomerization per se, which is mediated by the membranespanning domain. the transmembrane domains of many proteins have been shown to mediate dimerization (sternberg and gullick, ; manolios et al., ; for review see bormann and engelman, ) . the best example is glycophorin a, whose single transmembrane domain interacts to form homodimers (bormann et al., ) . this dimer is resistant to solubilization by sds at ambient temperature, although resistance is overcome by dilution or incubation at higher temperatures (furthmayr and marchesi, ) . dimerization ofglycophorin a is highly sequence specific and appears to involve interactions between hydrophobic residues in the transmembrane domain (lemmon et al., ) . the transmembrane domains of the t-cell receptor (cosson et al., ) and of class h major histocompatibility complex molecules also appear to mediate dimer formation (cosson and bonifacino, ) . in the case of major histocompatibility complex molecules, o~ and/~ chains interact to form heterodimers; dimerization appears to require a pair of oppositely charged amino acids at the lumenal edge of the apposing membrane spans. a series of glycine residues on each membrane span forms a nonpolar face which allows close packing of the transmembrane domains (cosson and bonifacino, ) . similarly, the o~ chain of the t-cell receptor assembles with the cd chain via interaction between charged transmembrane residues (manolios et al., ; cosson et al., ) . it is not clear how the transmembrane domain of gml molecules might interact to form a large oligomer. the large lumenal domain of gml may sterically prevent direct interaction between many transmembrane domains to form an oligomer. furthermore, the polar face of the ml domain itself could be expected to interact with at most one or two similar domains. the transmembrane domains of these proteins might interact to form small clusters (dimers or trimers) of gml, mutant gml proteins, and ~mlg. these clusters could be organized into large arrays by interactions between the heads and/or tails of proteins in different clusters. these structures might contain lipids and other endogenous golgi-resident proteins. association into such an array could prevent golgi-resident proteins from entering transport vesicles and thereby function as a mechanism for their retention. the targeting signals of other golgi resident proteins from different golgi subcompartments also reside in their transmembrane domains (aoki et al., ; burke et al., ; colley et al., ; munro, ; nilsson et al., ; russo et al., ; tang et al., ; teasdale et al., ; wont et al., ) , although flanking domains also contribute to retention efficiency. no obvious homologies exist between the primary sequences of these membrane-spanning domains, even in enzymes thought to be enriched in the same subcompartment. thus, the membrane composition of golgi subcompartments may be responsible for specific retention of resident proteins throughout this organelle. consistent with this, no soluble resident golgi proteins have been detected to date. nilsson et al. ( ) have proposed that the cytoplasmic tails of resident proteins in sequential golgi stacks may in-teract (perhaps via a cytoplasmic linking protein) to maintain the stacks in close apposition. the formation of gml oligomers may reflect association with such endogenous structures. the combination of overexpression and our metabolic radiolabeling conditions would not be expected to reveal any long-lived endogenous golgi-resident proteins that also reside in the oligomer. however, longer radiolabeling conditions in stable cell lines expressing gml may reveal other putative components of the oligomer. we are currently performing such experiments. golgi retention of a trans-golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring region a film detection method for tritinmlabeled proteins and nucleic acids in polyacrylamide gels synthetic peptides mimic the assembly of transmembrane glycoproteins intramembrane helix-helix association in oligomarization and transmembrane signaling the transmambrane and flanking sequences of , -n-acetylglucosaminyltransferase i specify med/a/-golgi localization the signal anchor and stem regions of the ~-galactoside a , -sialyltransferase may each act to localize the enzyme to the golgi apparatus role of transmembrane domain interactions in the assembly of class h mhc molecules membrane protein association by potential intramembrane charge pairs role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus g protein trimers differential effects of mutations in three domains on folding, quaternary structure, and intraoellular transport of vesicular stomatitis virus g protein folding and assembly of viral membrane proteins eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage t rna polymerase subunit structure of human erythrocyte glycophorin a cell biology of viruses that assemble along the biosynthetic pathway cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin a subunit effects of altered cy~-plasmic domains on transport of the vesicular stomatitis virus giycoprotein are transferable to other proteins rapid and efficient sitespecific mutagenesis without phenotypic selection cleavage of structural proteins during the assembly of the head of bacteriophage t the interaction of antibody with the major surface glycoprotein of vesicular stomatitis virus glycophorin a dimerization is driven by specific interactions between transmembrane a-helices polarized apical distribution of glycosyl-phosphatidylinositolanchored proteins in a renal epithelial cell line golgi retention signals: do membranes hold the key? a specific transmembrane domain of a coronavirus e giycoprotein is required for its retention in the golgi region influence of new glycosylation sites on expression of the vesicular stomatitis virus g protein at the plasma membrane a single n-linked ollgosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface the e glycoprotein of an avian corouavirus is targeted to the cis golgi complex retention of a cis golgi protein requires polar residues on one face of a predicted a-helix in the transmembrane domain transmembrane helical interactions and the assembly of the t cell receptor complex sequences within and adjacent to the transmembrane segment of a- , -sialyltransferase specify golgi retention. embo (eur the membrane spanning domain of - , -galactosyltransferase specifies trans golgi localization overlapping distribution of two glycosyltransferases in the golgi apparatus of hela cells a new cationic liposome reagent mediating nearly quantitative transfeodon of animal cells -galactosyltransferase: a short nh -terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for golgi retention ire-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface a sequence in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization a golgi retention signal in a membrane-spanning domain of coronavirus el protein. the transmembrane domain of n-gincosaminyltransferase i contains a golgi retention signal the signal for golgi retention of bovine , -galactosyltrunsferase is in the transmembrane domain the -residue transmembrane domain of -galactoside a , -sialyltransferase is sufficient for golgi retention key: cord- -fi pskod authors: nan title: the tgn glycoprotein contains two non-overlapping signals that mediate localization to the trans-golgi network date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: fi pskod the membrane-spanning and cytoplasmic domains of cd and cd were replaced by those of tgn . after transient expression in hela cells, the location of the hybrid proteins was determined using immunofluorescence and quantitative immuno-electron microscopy, facs analysis and metabolic labeling. the membrane-spanning domain was found to contain a signal that localized hybrid proteins to the tgn. this was in addition to the signal previously identified in the cytoplasmic domain (bos, k., c. wraight, and k. stanley. . embo (eur. mol. biol. organ) j. : - . humphrey, j. s., p. j. peters, l. c. yuan, and j. s. bonifacino. . j. cell biol. : - . wong, s. h., and w. hong. . j. biol. chem. : - ). the different properties of these two signals suggest that each operates by a different mechanism. motif results in the retrieval of soluble er proteins lost from this compartment (munro and pelham, ) . this sequence is recognized by a receptor which returns the protein to the er (semenza et al., ; lewis and pelham, ) . some membrane proteins contain a double-lysine motif in the cytoplasmic tail which ensures retrieval to the er (jackson et al., ) . golgi retention signals were first identified in a viral protein (swift and machamer, ) and in golgi glycosylation enzymes (nilsson et al., ; munro, ; aoki et al., ; burke et al., ; colley et al., ; tang et al., ; teasdale et al., ; wong et al., ) . in all cases, the retention signal lies in the membrane-spanning domain. more recently, membrane-spanning domains have been implicated in the localization of two proteins to the nuclear envelope (wozniak and blobel, ; smith and blobel, ) . so far, none of the proteins studied have been shown to contain both a retention and a retrieval signal though there are indications that more than one signal is present in some of them. when the carboxy-terminal -kdel signal is removed from the soluble bip protein, it is secreted very slowly (munro and pelham, ) . when the double-lysine motif is removed from glucuronosyltransferase, an integral membrane protein, little if any escapes from the er (jackson et al., ) . both these cases suggests that a retention signal is present in addition to the retrieval signal. to determine whether a protein can contain both signals, we focused on tgn , a resident of the trans-golgi network (luzio et al., ) . tgn has a tyrosine-based motif in the cytoplasmic domain that acts as a retrieval signal, return-ing the protein from the cell surface to the tgn (bos et al., ; humphrey et al., ; wong and hong, ). here we have looked for the second signal and have found it in the membrane-spanning domain. chemicals and reagents were obtained from the sources described by nilsson et al. ( a nilsson et al. ( , . restriction enzymes, t dna ligase, and polynucleotide kinase for recombinant dna procedures were purchased from gibco brl (gaithershurg, md), new england biolahs (bethesda, md), or pharmacia corp. (uppsala, sweden) . sequencing reagents and t polymerase were purchased from usb biochemicals. cell culture media, fetal calf serum, amino acids, and penicillin/streptomycin were from gibco brl. the tgn edna (lnzio et al., ) was mutagenized by pcr such that a bamhi site was introduced just after the stop codon. the hindiii-bamhi fragment containing the ' untranslated region and the complete coding sequence was cloned between the same restriction sites in the pcmuiv plasmid (nilsson et al., ) . the tgn edna in the mammalian expression vector puex (luzio et al., ) was used as a template for pcr amplification under the conditions described by salki et al. ( ) . oligonucleotides used in pcr reactions were generally synthesized with bamhi flanking sequences to facilitate easy cloning into plasmid vectors for sequencing. oligonucleotides were used at i mm in pcr reactions. pcr products were ethanol precipitated, digested with bamhi, and cloned into the pbs plasmid (stratagene corp., la jolla, ca) for sequencing. dna sequencing was carried out using the dideoxy chain termination method and t polymerase (tabor and richardson, ) . plasmid derivafives were digested with the appropriate enzymes to generate the correct ' and ' ends for cloning and construction of cd and cd edna chimeras. cd hybrid proteins were constructed such that the first amino acids of the extracellular domain of c~cd were preserved. this is sufficient for cd glycosylation and secretion (~y et al., ) , and should also not affect the ability of cd hybrid proteins to form homodimers (leahy et al., ; boursier et al., ) . pcr-generated dna fragments were cloned into the pcmuiv expression vector (nilsson et al., ) using dna fragments in a llgation. to construct the cd -mc and cd -mac chimeric cdnas the llgation mix contained ( ) the hindhi-apali dna fragment that encoded most of the lumenal domain of the cd molecule, ( ) an apali-bamhi dna fragment that encoded the membrane-spanning and cytoplasmic domain sequences of tgn , and ( ) the pcmuiv vector as a hindili-bamhi dna fragment. to construct the cds-m membrane-spanning chimeric edna the ligation contained ( ) the hindlli-apali dna fragment that encoded most of the lumenal domain of the cd molecule, ( ) an apali-sali dna fragment that contained the membrane-spanning domain of tgn , and ( ) the pcmuiv plasmid vector as a hindiii-sali dna fragment which also contalned the cytoplasmic domain of the cd molecule. to construct the cd -c and cd -ac chimeric cdnas the ligation contained ( ) a sali-bamhi dna fragment that contained the cytoplasmic domain of tgn , and ( ) the pcmuiv vector as a bami-ii-sali dna fragment which also contained the lumenal and membrane-spanning domains of the cd molecule. cloning of the cd edna into the pcmuiv plasmid has been described by nilsson et al. ( ) and we used this cd plasmid initially. however, there were no unique restriction sites in the cd edna at equivalent positions which would have allowed us to insert replacement membranespanning and cytoplasmic tgn sequences. we thus inserted unique restriction sites within the cd edna using pcr mutngenesis. deen et al. ( ) have shown that a cd molecule terminating after residue in the extracellular domain is efficiently secreted. as the threonine at is followed by a proline residue, this would make it unlikely that there are any structural features after thr- necessary for correct folding and transport of cd . codon (maddon et al., ) in the cd edna (which encodes a serine residue) was mutated from tcc to tca. this silent mutation created a single hincii site within the cd edna. similarly, codons and (encoding arginine residues) were mutated from cga agg to cgt cga. these silent mutations created a unique sali site in the cd edna. there were now unique restriction sites on either side of the membranespanning region in the edna. pcr was carried out on the tgn edna using ollgonucleotides similar to those described above. instead of the apali site used in cd chimera constructions, ' oligonucleotides that precede the tgn membrane-spanning domain were synthesized with a hinch site. a hinch-sali or hinch-bamhi dna fragment containing tgn sequences could be fused to cd by ligation as described previously. we thus ligated either a hindlii-hincii or hindiii-sali dna fragment (encoding the lumenal domain of the cd molecule) from pcmuiv-cd , an appropriate pcr generated dna fragment that encoded tgn sequences, and an appropriate pcmuiv vector. ligations were transformed into e. coil strain jm using standard procedures (sambrook et al., ) and clones were analyzed by restriction digestion followed by dna sequencing. large-scale preparations of the pcmuiv plasmids (for transfections) were made by alkaline lysis followed by cscl-ethidium bromide purification (sambrook et al., ) . monolayer hela cells (atcc ccl ) were grown in dmem supplemented with % fcs, non-essential amino acids, penicillin/streptomycin, and mm glutamine. cells were grown to - % conttuency before transfection by the calcium phosphate method essentially as described by nilsson et al. ( ) . for immunottuorescence, cells were seeded out into -ram, -weil plates (becton dickinson, lincoln park, nj) containing sterile coverslips. after one day, cells were transfected with ~g of plasmid dna (csci-etbr purified) and /tg of puc plasmid dna (used as a carrier to increase transfection efficiency). cells were fixed and processed for immunottuorescence - h later (nilsson et al., ; jackson et al., ) . transfected cells for electron microscopy and immunoprecipitation were prepared as described by nilsson et al. ( ) in -mm tissue culture dishes and processed - h after dna was added. cells were processed for immunofluorescence microscopy as described previously (warren et al., ; nilsson et al., ) . the rabbit antiserum to tgn has been described by luzio et al. ( ) and was diluted ( : ) in . % fish skin gelatin/pbs before use. gait was detected using either a rabbit polyclonal antiserum (roth and berger, ; nilsson et al., a ) at a dilution of : or the monoclonal antibody gt- (berger et al., ) . culture supernatants from the qs (healey et al., ) and okt (atcc crl ) mouse bybridomas were used to detect cd and cds, respectively. the secondary antibodies were horse anti=mouse conjngated to texas red (vector labs, burlingame, ca) and sheep anti-rabbit conjugated to fitc (dakopatts, copenhagen) and both were used at a dilution of : . p-,hodamine-conjngated transferrin (molecular probes, eugene, or) was dissolved in pbs and used at a final concentration of ~tg/ml; ceils were incubated in fresh media containing the ligand for rain at °c before washing with pbs and fixation. slides were examined on a zeiss axiophot fluorescence microscope using a )< objective oil immersion lens and a )< eyepiece. transfected cells were fixed in % paraformaldehyde/ . % glutaraldehyde in . m phosphate buffer ph . for h and processed for cryo-immunoelectron microscopy essentially as described by rabouille et al. ( ) . cell pellets embedded in gelatin were cut into blocks and infused with . m sucrose overnight. the blocks were frozen in liquid nitrogen, ultrathin cryosections were cut and incubated for rain with primary antibodies. polyclonal rabbit antiserum to tgn (lnzio et al., ) was used at a dilution of : and an affinity-purified rabbit antibody to cd (dr. mark marsh, mrc centre, university college london) at a dilution of : . both were diluted into . % fish skin golatin/pbs and the bound antibodies were detected using protein a-coupled to -nm gold particles used at a dilution of : (from cell biology department, ultrecht school of medicine, utrecht, netherlands). the monoclonal antibody to cd (haynes, ) was used neat as a culture supernatant and bound antibodies were detected using a secondary goat anti-mouse antibody coupled to -rim gold particles at a dilution of : (nilsson et al., ) . immunolabeling was followed by staining with uranyl acetate and embedding in methyl cellulose as described by tokuyasu ( ) . quantitation of immunolabeling was carried out by first identifying a labeled area of the cell where profiles of the crolgi stack and tgn were readily visible. the tgn was defined morphologically as described in the results. labeled cell sections were placed in one of two categories. cell sections in which no eodosomes were labeled consistently contained fewer than - gold particles whereas cell sections containing labeled endosomes contained more than - gold particles. in each experiment, - , gold particles were counted depending on the type of construct used for transfection (see fig. legend). the background observed in each experiment was extremely low and therefore ignored. the relative distribution of immunolabeling was performed by counting the gold particles over the membranes (golgi stack, tgn, endosornes, and plasma membrane) of selected cells. the linear density of the gold particles at the plasma membrane was estimated by the point hit method. briefly, pictures were printed at , × or , × final magnification and overlaid with a grid containing a point-to-point spacing of crn (plasma membrane) or . cm (tgn) (d). the number of intersections (i) of the membrane with this grid were counted. the total number of gold particles over the total membrane was counted and the linear density was then calculated using the formula:-gold particles/(i x d/magnification). transfected cells were grown in -ram wells and processed for facs analysis exactly as described by nilsson et al. ( ) . native and hybrid cd proteins were detected using the culture supernatant from the okt hybridoma. approximately × cells were incubated with the primary antibodies on ice for rain followed by gentle washing. the secondary antibody was fitc-conjugated sheep anti-mouse (vector labs., burlingame, ca) used at a dilution of : . after washing, cells were fixed in % paraformaldehyde/pbs for rain on ice. immediately before facs analysis, cells were washed three times with calcium/magnesium-free pbs and resuspended in . ml of this solution. a becton-dickinson facscan machine was used to analyze the samples. quantitation of fans data was performed by calculating two parameters: the amount of positive cells expressing each protein and the arithmetic mean intensity of fluorescence of these positive cells (~:ni.ii /~nili). the mean intensity was multiplied by the number of positive cells which gave the total fluorescence intensity (arbitrary units) detected for the positive cell population. the total fluorescence intensity of the cd positive cells was represented as % and the total fluorescence intensity of positively gated cells expressing other cd hybrid proteins were expressed as a percentage of total cd . immunopreeipitation was carried out essentially as described by jackson et al. ( ) . transfected cells in -mm dishes were labeled with . rnci [ s]methionine/cysteine for rain and either lysed in detergent immediately or chased with medium containing excess cold methionine and cysteine. after rain on ice the cells were scraped off and lysates centrifuged at medium ( , g, min at °c) and high speed ( g, rain at c) to remove particulate material. the supernatants were precleared by incubation with mouse igg bound to protein a-sepharose (pharmacia corp., uppsala, sweden) and native and hybrid proteins were immunoprecipitated using qs (anti-cd ) or okt (anti-cds) antibody bound to protein a-sepharose. immunoprecipitates were then washed sequentially twice with lysis buffer, once with lysis buffer containing . % sds, once with lysis buffer containing . m naci, twice with lysis buffer, and finally once with low ionic strength buffer ( rnm tris ph . , . % tx- ). after the final wash, immunoprecipitates were divided equally and resuspended in # of neuraminidase digestion buffer ( mm nac , ram sodium acetate ph . , mm calcium chloride and i mm pmsf) containing either mu neuraminidase (type viii; sigma) or buffer (mock treatment). samples were incubated at "c for h before analysis by sds-page and fluorography. we have used both cd and cim as the reporter molecules. these glycoproteins are members of the ig superfamily and are normally expressed on the surface of t-lymphocytes maddon et al., ) . the ~cd polypeptide consists of amino acids (littman et ai., ) and cd of residues (maddon et al., ) . the amino acid sequences of the membrane-spanning and cytoplasmic domains of cd and cd are shown in fig. a. rat tgn is a type i glycoprotein with a predicted molecular weight of kd but glycosylation increases this to - kd (luzio et al., ) . signal peptide cleavage produces a mature protein of residues. the large lumenal domain (residues - ) is followed by a single membrane-spanning domain (residues - ) and a short cytoplasmic domain (residues - ; see fig. i a) . chimeric cdnas were constructed by replacing domains in cd and cd with those in tgn at identical amino acids in the three proteins (see fig. a) . this meant that hybrid proteins containing the membrane-spanning domain of tgn also included some of the flanking amino acids. flanking sequences have been shown to influence substantiaily the ability of membrane-spanning domains to localize proteins to the golgi apparatus (nilsson et al., ; munro, ; colley et al., ) . rat tgn edna (luzio et al., ) was cloned into the pcmuiv expression vector (see materials and methods) and the plasmid introduced into cells by calcium phosphate transfection. at h after transfection, immunofluorescence microscopy showed that tgn was localized to a juxtanuclear reticulum (fig. a) very similar to that stained by antibodies to gait (fig. b) , a resident of the trans-golgi cisterna (roth and berger, ) and the tgn (lucocq et al., ; taatjes et ai., ; nilsson et al., a) . higher levels of tgn led to an accumulation of the protein in punctate structures in the cytoplasm (fig. a) and at the cell surface (data not shown; see later). these data are consistent with the localization of endogenous tgn in nrk cells and expressed protein in transfected cv- cells (luzio et al., ) . the membrane-spanning (m) and cytoplasmic (c) domains of tgn were grafted on to the lumenal domain of cd (see fig. b) . cd was mostly present on the cell surface though a small amount was present in small punctate structures, especially at high levels of expression (fig. c) . the hybrid protein (cd -mc), however, was restricted to a compact juxtanuclear reticulum (fig. d) that was very similar to that stained by antibodies to gait (data not shown). the membrane-spanning and cytoplasmic domains of tgn were also grafted on to the lumenal domain of cd . transiently expressed cd was mostly localized to the plasma membrane (fig. e) , but the cd -mc hybrid protein was again localized to a juxtanuclear compartment (fig. f) , very similar to that stained by antibodies to gait (data not shown). there was also faint staining of the nuclear envelope, suggesting some accumulation of both hybrid proteins within the er (arrows in fig. , d and f). to show that the membrane-spanning and cytoplasmic domains of tgn contained distinct localization signals, each of these domains in both cim and cd was replaced by that domains of cd , cd , and tgn . the membrane-spanning domain of cim is that defined by maddon et al. ( ) although it could inelude an additional two amino acids (-cv-) on the cytoplasmic side of the membrane. amino acid residues at which domains were replaced in the different hybrid proteins are numbered. (b and c) line sketches show cds, cim, and hybrid proteins containing the membrane-spanning and cytoplasmic domains of tgn (shaded and boxed). hybrid proteins are defined by suffixes which denote the domains of cd or cd that were replaced by equivalent domains of tgn . m = membranespanning domain and flanking residues; c = cytoplasmic domain; and ac = truncated cytoplasmic domain lacking residues from the carboxyterminus. in tgn (fig. , b and c). the tgn membranespanning domain alone localized the hybrid proteins to the golgi apparatus. both cd -m (fig. a) and cd -m (fig. e) gave a discrete juxtanuclear staining pattern very similar to that for gait, especially at low levels of expression (fig. , b and f,, respectively). at higher levels of expression, staining was more diffuse suggesting accumulation in vesicles and at the cell surface (data not shown). this pattern was more frequently observed for cd -m than for cds-m. the cytoplasmic domain of tgn contained, as expected, the other golgi localization signal (bos et al., ; humphrey et al., ; wong and hong, ) . both cd -c (fig. c) and cim-c (fig. g) were detected in a juxtanuelear reticulum very similar to that containing gait (fig. , d and h, respectively) . at all levels of expression staining of small punctate structures (fig. , c and g) was also observed and these were shown to be endosomes by immunoelectron microscopy (see below). replacing the membrane-spanning domain of either cim or cd with that of tgn could have activated or corrupted a localization signal in the cytoplasmic domain of the hybrid proteins. cd has a tyrosine residue in the cytoplasmic domain but it is the penultimate amino acid that would not norreally be expected to operate as a retrieval signal (fig. a) . cd contains a signal for endocytosis (shin et al., (shin et al., , but this does not normally direct the protein to the tgn. nevertheless, to eliminate the possibility of activating or corrupting a cytoplasmic signal, we exploited the fact that the localization signal in the cytoplasmic domain of tgn has been mapped to the sequence, sdyqrl (bos et al., ; humphrey et al., ; wong and hong, ) . comparison of the hybrid protein cd -mc (fig. a) with cd -mac (fig. b) , which lacked this sequence, showed that it had no significant effect on the localization of the protein to the golgi apparatus. both were localized to a juxtanuclear reticulum very similar to that stained by antibodies to gait (data not shown). expression of the cd -mac hybrid protein gave similar results (fig. e) . to show that we had in fact eliminated the localization signal in the cytoplasmic domain of tgn , we expressed a cd or cd hybrid protein containing the truncated cytoplasmic domain from tgn (cd -ac or cd -~c; fig. b) . unlike cd -c (see fig. c) , the cd -ac hybrid protein was not in the golgi apparatus but exhibited a cell surface staining pattern and additional accumulation in intracel- * the two categories of transfected cells, low "expressers" (<) and high "expressers ~ (>), were defined as described in materials and methods. the total number of gold particles counted was crgn ), (cd -m), and (cd -c). * the linear density at the plasma membrane and in the tgn was estimated for the expressed proteins as described in materials and methods. the number of gold particles counted as the plasma membrane was (tgn ), (cim-m), and (cd -c). note that for tgn and cim-m, gold labeling at the plasma membrane correlated with labeling of endosomes. lular punctate structures (fig. c) . some of these were shown to be early endosomes (arrowheads in fig. , c and d) by preincubating the cells with rhodamine-conjugated transferrin for min before fixation and processing. cd -ac was found to be expressed largely on the cell surface of transfected cells (fig. f) . to determine the precise location of the hybrid proteins, cryosections of transfected cells were labeled with antibodies which were detected by appropriate gold conjugates. the tgn is defined morphologically as a tubulo-reticular network, part of which is closely apposed to the trans side of the golgi stack. budding and vesicle profiles coated by clathrin are often observed (orci et al., (orci et al., , griffiths and simons, ) . transiently expressed tgn was found in the tgn, the golgi stack, endosomes, and the plasma membrane (fig. a) . the distribution of tgn depended on the level of expression. at low levels (< gold particles/cell section) tgn was found exclusively in the golgi apparatus (table i) with most of the protein ( + %) being found in the tgn (fig. ) . none was detected in either endosomes or on the plasma membrane (table i) . at higher levels of expression (> gold particles/cell section) the percentage of labeling over the golgi apparatus fell (to : %) and rose over endosomes (to : %) and the plasma membrane (to + %) ( table i) . even at high levels of expression the linear density of labeling over the tgn was four times that over the plasma membrane showing that the protein was still concentrated in the tgn. hybrid proteins containing both localization signals gave patterns of labeling almost indistinguishable from tgn both morphologically and quantitatively. both cd -mc (fig. b) and cd -mc (fig. d) were present only in the golgi apparatus at all levels of expression and more than % of the label was present in the tgn (fig. ) . hybrid proteins containing only the membrane-spanning domain also gave results very similar to those for tgn . cd -m was present only in the golgi apparatus at low levels of expression (< gold particles/cell section; table i ) with : % being present in the tgn (fig. ) . at higher levels of expression (> gold particles/cell section) the percentage of total labeling over the golgi apparatus fell (to : %) and rose over endosomes (to + %) and plasma membrane (to + %) ( table i) . this distribution was not affected by pretreatment of the cells for h with cycloheximide (fig. f) . the linear density of labeling over the tgn was times that over the plasma membrane, even higher than that for tgn (table i) . the other hybrid protein, cds-m, was also present exclusively in the golgi apparatus (fig. c) and most ( : %) was present in the tgn (fig. ) . however, more of the protein was present in the golgi stack than for other hybrid proteins. we also examined cells expressing cd -m after incubation in cycloheximide for h and found no significant change in the pattern of labeling (data not shown). the cd hybrid protein containing the cytoplasmic domain (cd -c) was also localized using immunoelectron microscopy. cd -c could not be detected by immuno-electron microscopy for reasons that are still unclear. more than % of the cd -c in the golgi apparatus was present in the tgn (fig. ) the journal of cell biology, volume , e). however, cd -c differed from other hybrid proteins and tgn in three respects. first, more than % of the total protein was present in endosomal structures at low levels of expression and this increased to nearly % at high levels. second, the type of endosomal structure labeled also changed. at low levels, all of the endosomal structures were multivesicular bodies which are late endosomal compartments (geuze et al., ; hopkins et al., ) . at high levels of expression, labeling was also present in tubular endosomes. because of the difficulties of distinguishing between tubular endosomes and the tgn (geuze et al., ; klumperman et al., ) , definitive identification of these structures will have to await further analysis using stably transfected cell lines. third and last, the plasma membrane was labeled at both high and low levels of expression ( table i ). the linear density was the same ( . + . gold particles/#m) suggesting that cd -c was as efficiently removed from the cell surface and delivered to endosomes. the protein was still concentrated in the tgn since the linear density was - times higher than that in the plasma membrane ( table i) . as a final control, cd was also expressed transiently in hela cells. all of the protein was found in endosomes and on the plasma membrane after treatment with cycloheximide and in agreement with studies carried out by pelchen-matthews et al. ( ) . little if any was found in the tgn (data not shown). this shows that accumulation of the hybrid proteins in the tgn was not a consequence of the cd ectodomain. facs analysis was used to determine the amount of cd and cd hybrid proteins on the cell surface. fixed, nonpermeabilized cells transfected with the appropriate edna were stained with the okt antibody to cd and visualized using a secondary antibody conjugated to fitc (fig. ) . mock-transfected cells were used as the negative control and the gate was set such that % of these cells scored positive. the profile of cd expression was characteristic of that in a population of transiently transfected cells (nilsson et al., ; jackson et al., ) and % scored positive (fig. a, table i ). when both localization signals were present (cds-mc) only % of the cells showed surface staining (fig. b, table ] i) and this rose to % when the cytoplasmic signal was deleted (cd -mac) (fig. d, table i ) suggesting that the membrane-spanning signal alone could retain the hybrid protein. this was confirmed by expression of cd -m. only % of the cells scored positive (fig. c, table ii). the cytoplasmic signal alone could also retain the hybrid protein. only . surface expression of cd and cd hybrid proteins analyzed using facs analysis. hela cells were fixed and stained with monoclonal antibody to cd and visualized using a secondary antibody conjugated to fitc. the gate on the x-axis (fluorescence units) was set using mock-transfected cells as a negative control. cells scoring to the right of this point were regarded as positive for cell surface fluorescence (see table i ). * the gate was set using moek-transfeeted cells and % were positive. , cells were analyzed in each experiment. * the total fluorescence intensity was calculated by integrating the area under the positive cells and expressing the results as a percentage of the value for cd . ( fig. e, table ii ). removal of the tyrosine-based motif in this cytoplasmic signal destroyed its ability to retain hybrid proteins. more than % of cells expressing cd -ac scored positive (fig. f,, table ii ), slightly higher than the number expressing cd alone ( %, fig. a, table ii ). the total fluorescence of positive cells for each protein was also calculated by integration of the results presented in fig. . these calculations, presented in table ii , are in close agreement with the percentage of cells scoring positive and provide further evidence that both the membrane-spanning and cytoplasmic signals can, independently, retain hybrid proteins inside the cell. these data were also supported by immunofluorescence microscopy on non-permeabilized cells (data not shown). there was no surface staining of cells expressing cd -mc or cd -c. a few of the transfected cells expressing cd -m showed low levels of cell surface staining. cells expressing cd -ac showed high levels of cell surface staining, in agreement with the facs analysis. cd contains two n-linked glycosylation sites (maddon et al., ) one of which is completely processed to the complex bi-antennary sialylated structure. terminal sialic acid residues are added by ot , -sialyltransferase which is located in the trans-golgi cisterna and the tgn (roth et al., ) . neuraminidase cleaves off these sialic acid residues producing a small mobility shift on sds-page (nilsson et al., ; jackson et al., ) that can be used to show that proteins have reached this part of the golgi apparatus. cells expressing cim or cd hybrid proteins were labeled with [ s]methionine/cysteine for min (see materials and methods) and either lysed immediately or chased with excess cold methionine and cysteine for h. immunoprecipitates were treated with neuraminidase or mocktreated and analyzed by sds-page (fig. ) . newly synthesized cim was insensitive to neuraminidase (cf. lanes i and ) but completely sensitive after a -h chase (cf. lanes and ). hybrid cim proteins containing either the membranespanning (cim-m) or cytoplasmic signal (cim-c) were also completely siaiylated after a -h chase period (lanes - ). , , , , , , and ) or digested with neuraminidase (lanes , , , , , , , and ) and analyzed by sds-page and fluorography. the molecular size of c-radioactive markers are indicated on the left hand side of the panels. however, cd -mc, which contains both signals, was incompletely sialylated after the -h chase (lanes and ) suggesting that transport to the trans part of the golgi apparatus was slower. the reason for this is unclear but was not the consequence of using cd as the reporter molecule. cd -mc was also incompletely sialylated (data not shown) in contrast to hybrid cd proteins containing either tgn signal (fig. ) . metabolic labeling of cd hybrid proteins could not be used to study arrival in the trans cisterna and the tgn because the bound oligosaccharides are all o-linked . sialylation of o-linked oligosaccharides is thought to occur in the medial-golgi cisterna (locker et al., ) . however, pulse-chase analysis combined with neuraminidase digestion showed that cd -m and cd -c hybrid proteins had kinetic properties similar to those for native cd (fig. ) . in all cases the half-time for acquisition of terminal sialic acid residues was between - min. taken further with the results for the cd hybrid proteins, these results show that the presence of either signal had little, if any, effect on synthesis, transport, and processing of the reporter molecules. tgn was originally identified as a resident of the tgn (luzio et al., ) but more recent work has shown that it cycles between the tgn and the cell surface (jones et al., ; reaves et al., ) . movement to the cell surface occurs via exocytic vesicles (jones et al., ) and is followed by retrieval which requires a signal present in the cytoplasmic domain that has been localized to the sequence sdyqrl (bos et al., ; humphrey et ai., ; wong and hong, ) . retrieved tgn first moves to the endosomes and then to the tgn (bos et al., ; humphrey et al., ) . using plasma membrane glycoproteins as reporter molecules, we have now shown that there is an additional signal for localizing the protein to the tgn. this signal is located in the membrane-spanning domain. four independent lines of evidence support this conclusion. first, immunofluorescence microscopy showed that cd -m and cd -m hybrid proteins localized to a juxtanuclear reticulum that could also be stained by antibodies to gait, a marker for the trans cisterna and tgn (roth and berger, ; taatjes et al., ; nilsson et al., a) . this staining pattern was not affected by pretreamaent with #g/ml cycloheximide for up to h (data not shown) showing that these hybrid proteins were not simply moving slowly along the exocytic pathway. in addition, it is unlikely that a cryptic localization signal in the cytoplasmic domain of either cd or cd was activated as a result of generating hybrid proteins. both cd -mac and cd -mac hybrid proteins which lack most of the cytoplasmic domain (including the cytoplasmic signal for tgn ) were also localized to the golgi apparatus. second, quantitative immunoelectron microscopy showed that cim-m had almost the same distribution as tgn at both high and low levels of expression. at low levels of expression, all of the cd -m hybrid protein and tgn was present in the golgi apparatus, % being present within the tgn. at higher levels of expression, cim-m and tgn were also present in endosomes and on the cell surface, again to a similar extent. even at these higher levels of expression, both proteins were still concentrated in the tgn as compared to the plasma membrane. for tgn , the linear density in the tgn was four times that in the plasma membrane; for cd -m it was even higher, being times higher in the tgn than in the plasma membrane. the distribution of cd -m was also similar to that for tgn being exclusively in the golgi apparatus at low levels of expression. there was, however, more of this hybrid protein in the golgi stack for reasons that are still unclear. third, facs analysis showed that the membrane-spanning domain reduced the percentage of positive cells from % figure . metabolic labeling of cd and cd hybrid proteins. transfected hela ceils were labeled with [ s]methionine/cysteine for min and chased for , , , , , or min. immunoprecipitates were mock-digested (-) or digested (+) with neurarninidase and analyzed by sds-page and fluorography. (for cd ) to % (for cd -m), a . -fold decrease. this effect was not the consequence of activating a cryptic signal in the cytoplasmic domain of cd since removal of most of this domain (including the tgn cytoplasmic signal) still reduced the percentage of positive ceils to % (for cd -mac), a threefold decrease. fourth and finally, metabolic labeling studies showed that all molecules of cd -m received sialic acid, showing that they had all reached the trans cisterna/tgn. the present studies are at variance with previous studies that identified only the cytoplasmic domain of tgn as a localitation signal. removal of most of the domain led to the appearance of the truncated protein on the cell surface (luzio et al., ; bos et al., ) . it should be noted that the earher study was carried out before any evidence existed for retention by membrane-spanning domains, a phenomenon first described for a viral protein (swift and machamer, ) and golgi enzymes (nilsson et al., ; munro, ) . the expectation was that the signal would be found in the cytoplasmic domain and this appeared to be borne out by the appearance of protein on the cell surface. the likely reason for this, however, was that the levels of expressed protein were so high that they saturated the retention mechanism that operates on the membrane-spanning domain. even cells expressing the wild-type protein (tgn ), containing both retention and retrieval signals, showed aberrant localization. after h transfection, % of the cells had either fallen off the coverslip or showed cell surface staining (luzio et al., ) . the more recent study by bos et al. ( ) overcame this problem by using a similar expression vector (psvl ) at a low transfection efficiency (~ . %) to ensure more constant levels of expression in each cell. unfortunately, their only assay was immunofluorescence microscopy and it was impossible to determine how much of the protein was still present in the golgi apparatus. these problems did not arise in our system since the expressed levels of hybrid protein were lower and the distributions were quantitated in several independent ways. two different reporter molecules were also used. however, it is conceivable that other small differences in the systems used are responsible for the apparent discrepancies. wong and hong ( ) , for example, found that the serine residue in the sdyqrl motif was important for golgi localization, while humphrey et al. ( ) found that an identical mutation in their system had no effect. further studies using stable cell lines will be needed to resolve these differences. though the cytoplasmic and membrane-spanning signals can act independently to localize hybrid proteins to the tgn, it is likely that they operate in different ways. the cytoplasmic domain is a clear example of a retrieval signal. this acts after a protein has left the compartment in which it norreally functions and returns them from a later compartment. all of the retrieval signals so far characterized have been located in the cytoplasmic domains of proteins and many are based on tyrosine as the critical amino acid set in a particular motif (for review see collawn et al., ; pearse and robinson, ; kornfeld, ; matter et ai., ) . the critical motif in the cytoplasmic domain of tgn is sdyqrl (bos et al., ; humphrey et al., ; wong and hong, ) and it operates once the protein reaches the cell surface. tgn is then returned to the tgn via endosomes. the membrane-spanning domain is a typical golgi retention signal. these act to keep proteins in the golgi and, so far, all golgi proteins (machamer and swift, ) and enzymes (nilsson et al., ; munro, ; aoki et al., ; burke et al., ; colley et al., ; tang et ai., ; teasdale et al., ; wong et al., ) have been found to be retained by the membrane-spanning domain. the mechanism of retention is still unclear though two possibilities have been proposed. tgn molecules could interact with their neighbors through the spanning domains forming kin oligomers so large that they can no longer enter vesicles budding from the tgn (nilsson et al., ) . alternatively, the length of the membrane-spanning domain might prevent forward movement of the protein past the tgn (bretscher and munro, ) . the latter seems less likely since the length of the membrane-spanning domain ( amino acids) is longer than the estimated average for golgi proteins ( amino acids) but further work will be needed to determine whether either of these mechanisms, or a completely different one, are responsible for retention in the tgn. our results suggest that the membrane-spanning domain is an effective retention signal since alone it can confer localization properties on cd that make it virtually indistinguishable from tgn . the predicted effect of its absence would be an increased flux of protein through the tgn to the cell surface. in other words, its absence should put a greatly increased load on to the retrieval system. as fast as the protein is retrieved and returned to the tgn, it would escape to the plasma membrane and need to be retrieved again. this would explain why cd -c, which has the retrieval but not the retention signal, is present in both endosomes and at the plasma membrane even at low levels of expression. the rate-limiting step in retrieval is not endocytosis because the linear density of cd -c at the plasma membrane was the same at both low and high levels of expression. the limiting step must be between endosomes and the tgn since cd -c accumulates in endosomes as the expression level increases. the only result that is a little puzzling is the presence of cd -m in endosomes at high expression levels. this hybrid protein would be expected to leak very slowly to the cell surface, but, in the absence of a retrieval signal, it should accumulate there. one possibility is that the endocytic signal in the cd cytoplasmic domain (shin et al., ) triggers movement to endosomes; another possibility is that the protein in the endosome is destined for degradation in lysosomes. this latter possibility is more likely for cds-m which lacks an endocytic signal yet is found in small amounts in what appear to be endosomes by immunofluorescence microscopy. it is likely that other proteins on the exocytic pathway have both retention and retrieval signals. the sum of the two processes acting on these signals would determine the amount of protein found in a particular compartment. for resident proteins which should spend as much of their time as possible in a particular compartment one would expect retention rather than retrieval to be the dominant process. for recycling proteins, such as receptors, one would expect the opposite so that substantial amounts of protein would be moving between two compartments. it is not yet clear whether tgn is best considered as a resident or a recycling protein (luzio and banting, ; stanley and howell, ) . that will have to await discovery of its function. golgi retention of a trans-golgi membrane protein, galactosyltransferase, requires cysteine and histidiue residues within the membrane-anchoring domain monoclonal antibodies to soluble, human milk galactosyltransferase actose synthase a protein) tgn is maintained in the trans-goigi network by a tyrosine-containing motif in the cytoplasmic domain evidence for an extended of the t-cell co-receptor cdga as deduced from the hydrodynamic properties of soluble forms of the extracellular region cholesterol and the golgi apparatus the transmembrane and flanking sequences of fll, -n-acetylglucosaminyltransferase i specify medial-goigi localization transferrin receptor internalization sequence yxrf implicates a tight turn as the structural recognition motif for endocytosis the signal anchor and stem regions of the fl-galactoside ot , -sialyltransferase may each act to localize the enzyme to the golgi apparatus tubulovesicular processes emerge from tmas-golgi cisteroae, extend along microtubules, and interlink adjacent trans-golgi elements into a reticulum soluble form of cd ('i" ) protein inhibits aids virus infection protein folding in the cell intracellular site of aalaloglycoprotein receptor-ligand uncoupling: doublelabel immunoelectron microscopy during receptor mediated endocytosis sorting of mannose -phosphate receptors and lysosomal membrane proteins in endocytic vesicles the trans golgi network: sorting at the exit site of the golgi complex the dynamic nature of the golgi complex summary of t cell studies performed during the second international workshop and conference on buman leukocyte differentiation antigens novel anti-cd monoclonal antibodies separate human immunodeficiency virus infection and fusion of cd + cells from virus binding movement of internalized ligand-receptor complexes along a continuous endosomal reticuinm localization of tgn to the trans-golo network: involvement of a cytoplasmic tyrosine-contalning sequence identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum retrieval of transmembraue proteins to the endoplasmic-reticulum a cytosolic complex of p and rab associates with tgn / and is involved in budding of exocytic vesicles from the trans-golgi network differences in the endosomal distributions of the two mannose- -phosphate receptors structure and function of the mannose -phosphate/insulinlike growth factor ii receptors cry.stal structure of a soluble form of the human t cell coreceptor cd at . a resolution ligand-induced redistribution of a human kdel receptor from the golgi complex to the endoplasmic reticulum the isolation and sequence of the gene encoding t : a molecule defining functional classes of t lymphccytes o-glycosylation of the coronavirus m protein. differential localization of sialyltransferases in n-and o-linked glycosylation mitotic golgi fragments in hele cells and their role in the re, assembly pathway eukaryote membrane traffic-retrieval and retention to achieve organelle residence identification, sequencing and expression of an integral membrane protein of the trans-golgi network (tgn ) golgi retention signals: do membranes hold the key? the isolation and nucleotide sequence of a cdna encoding the t cell surface protein t : a new member of the immunoglobulin gene family basolateral sorting of ldl receptor in mdck cells: the cytoplasmic domain contains two tyrosinedependent targeting determinants the golgi complex: in vitro veritas? sequences within and adjacent to the transmembrane segment of ot- , -sialyltransferase specify golgi retention a c-terminal signal prevents secretion of luminal er proteins short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum the membrane spanning domain of fll, -galactosyltransferase specifies trans golgi localization kin recognition between medial golgi enzymes in hela cells overlapping distribution of two glycosyltransferases in the golgi apparatus of hela cells kin recognition: a model for the retention of golgi enzymes. febs (fed. fur perrelet. . a clathrin-coated, golgi-related compartment of the insulin secreting cell accumulates proinsulin in the presence of monensin clathi~-immunoreactive sites in the golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells clathrin, adaptors, and sorting differential endoeytosis of cd in lymphoid and non-lymphoid cells control of protein exit from the endoplasmic reticulum segregation of mhc class ii molecules from mhc class i molecules in the golgi complex for transport to lysosomal compartments the differential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immnnocytocbemistry thrze-dimensional electron microscopy: structure of the golgi apparatus tgn / recycles between the cell surface and the tgn: brefeldin a affects its rate of return of the tgn immunocytochemical localization of galactosyltransferase in hela cells: codistribution with thiamine pyrophosphatase in trans-golgi cisternae demonstration of an extensive trans-tubular network continuous with the golgi apparatus stack that may function in glycosylation molecular dissection of the secretory pathway primer-directed enzymatic amplification of cdna with a thermostable dna polymerase molecular cloning: a laboratory manual erd , a yeast gene required for the receptor-mediated retrieval of luminal er proteins from the secretory pathway structural features of the cytoplasmic region of cd required for internalization phosphorylationdependent down-modulation of cd requires a specific structure within the cytoplasmic domain of cd the first membrane spanning region of the lamin b receptor is sufficient for sorting to the inner nuclear membrane tgn / : a molecule on the move a golgi retention signal in a membrane spanning domain of coronaviros e protein immunocytochemical localization of/~ , galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies dna sequence analysis with a modified bacteriophage t dna polymerase the transmembrane domain of n-glucosaminyltransferase i contains a goigi retention signal. ' the signal for golgi retention of bovine/ , -galactosyltransferase is in the transmembrane doromn irmnnnochemistry on ultrathin frozen sections recycling of transferrin receptors in a cells is inhibited during mitosis the sxyqrl sequence in the cytoplasmic domain of tgn plays a major role in trans-golgi network localization the -residue membrane domain of/~-galactoside tx , -sialyltransferase is sufficient for golgi retention the single transmembrane segment of gp is sufficient for sorting to the pore membrane domain of the nuclear envelope we thank dr. mark marsh for helpful advice and antibodies, dr. eric berger, dr. peter beverly, and dr, nancy hogg for antibodies, and dr. per peterson for the cd cdna. we also thank rose watson, felicia hunte, and the imperial cancer research fund (icrf) facs unit for invaluable technical assistance. we thank the icrf oligonucleotide synthesis facility for high quality oligonuclootides and the photography department for their help. we thank dr. kathryn howell, dr. wanjin hong, and dr. keith stanley for preprints of papers before publication and helpful advice. we also thank members of the warren lab for advice and critical reading of the manuscript.received for publication october and in revised form december . key: cord- - zvwu bd authors: bui, quynh trang; golinelli-cohen, marie-pierre; jackson, catherine l. title: large arf guanine nucleotide exchange factors: evolution, domain structure, and roles in membrane trafficking and human disease date: - - journal: mol genet genomics doi: . /s - - - sha: doc_id: cord_uid: zvwu bd the sec domain adp-ribosylation factor (arf) guanine nucleotide exchange factors (gefs) are found in all eukaryotes, and are involved in membrane remodeling processes throughout the cell. this review is focused on members of the gbf/gea and big/sec subfamilies of arf gefs, all of which use the class i arf proteins (arf - ) as substrates, and play a fundamental role in trafficking in the endoplasmic reticulum (er)—golgi and endosomal membrane systems. members of the gbf/gea and big/sec subfamilies are large proteins on the order of kda, and they possess multiple homology domains. phylogenetic analyses indicate that both of these subfamilies of arf gefs have members in at least five out of the six eukaryotic supergroups, and hence were likely present very early in eukaryotic evolution. the homology domains of the large arf gefs play important functional roles, and are involved in interactions with numerous protein partners. the large arf gefs have been implicated in several human diseases. they are crucial host factors for the replication of several viral pathogens, including poliovirus, coxsackievirus, mouse hepatitis coronavirus, and hepatitis c virus. mutations in the big arf gef have been linked to autosomal recessive periventricular heterotopia, a disorder of neuronal migration that leads to severe malformation of the cerebral cortex. understanding the roles of the arf gefs in membrane dynamics is crucial to a full understanding of trafficking in the secretory and endosomal pathways, which in turn will provide essential insights into human diseases that arise from misregulation of these pathways. the distinguishing feature of eukaryotic cells is their internal membrane organization. membrane-bound organelles such as the endoplasmic reticulum (er), the golgi apparatus, and endosomes, ensure that specialized functions are carried out in the appropriate delimited environment. each organelle possesses a characteristic lipid and protein composition, but is highly dynamic and linked to other organelles via traycking pathways. there are two major membrane traycking systems in eukaryotes, the secretory and the endocytic systems. the secretory pathway transports secreted and membrane proteins synthesized in the er to their wnal destination (plasma membrane (pm), cell exterior or intracellular organelle). endocytosis pathways transport material from the cell exterior to intracellular organelles such as the lysosome (or vacuole in yeasts). the golgi apparatus is a structurally complex and highly dynamic organelle that is found at the crossroads of the secretory and endosomal membrane traycking pathways. the mechanisms that generate and maintain golgi structure are not well understood. however, activation of the adpribosylation factor (arf ) small g protein is absolutely required to maintain golgi structure and function in eukaryotic cells, since inhibiting arf activation by drugs or mutations causes complete disassembly of the golgi apparatus and a complete block in traycking pathways through the golgi (klausner et al. ; jackson ). membrane traycking in eukaryotic cells is mediated by transport vesicles that bud from a donor compartment, then are targeted to and fuse with an acceptor compartment (bonifacino and glick ; behnia and munro ) . arf is required for vesicle budding, where its activation results in recruitment of evectors such as coat complexes to membranes. coat complexes deform membranes and concentrate cargo into a membrane domain to form a vesicle that carries the cargo from the donor to acceptor compartment. targeting of a vesicle to its correct acceptor compartment membrane requires tethering molecules that link the vesicle and target membranes at a distance. a series of steps ensues that result in engagement of snare proteins, which mediate membrane fusion (bonifacino and glick ) . the proteins involved in vesicle budding, cargo sorting, tethering, and fusion are highly conserved in evolution (behnia and munro ) . recent advances in genome sequencing evorts have allowed dewnition of six major supergroups of eukaryotes (adl et al. ) . interestingly, phylogenetic studies have indicated that the last common eukaryotic ancestor (the lcea) possessed a complex endomembrane system and that many of the major families of proteins involved in traycking arose prior to the divergence of the eukaryotic supergroups (jekely ; dacks and field ; gurkan et al. ; kloepper et al. ; pereira-leal ) . the lcea probably already had small g proteins including primordial arf and rab proteins, coat complexes, membrane tethering complexes, and snares (gurkan et al. ; koumandou et al. ; kloepper et al. ; pereira-leal ; dacks et al. ). there are nine subfamilies of sec domain arf guanine nucleotide exchange factors (gefs) in eukaryotes (cox et al. ) . in this review, we will focus on two of these subfamilies, the gbf/gea and big/sec gefs. other wellstudied subfamilies of arf gefs include the arno/cytohesin, syt , syt , efa , and brag subfamilies, which are in general smaller proteins, and which function primarily in endosomal-pm traycking pathways casanova ; gillingham and munro ) . of the nine subfamilies of arf gefs, only those of the gbf/gea, big/sec , arno/cytohesin, and ralf subfamilies have signiwcant activity on the class i arfs, arf -arf . ralf is the founding member of a family of bacterial arf gefs, wrst found in legionella pneumophila and rickettsia prowazekii (nagai et al. ; amor et al. ). these proteins do not arise from the bacteria themselves, but were incorporated into these bacterial genomes through horizontal transfer from their eukaryotic host (nagai et al. ). we will not discuss this special subfamily of arf gefs in this review. among the eukaryotic arf gefs, there has been some controversy as to whether arno/ cytohesin gefs actually use class i arfs as substrates in vivo, as they localize primarily to the endosomal-pm system where arf functions. however, a key paper from the donaldson laboratory has provided new insights into this issue through identiwcation of a gtpase cascade involving both arf and arf in cells (cohen et al. ) . cohen et al. demonstrated that interaction of arf -gtp with the ph domain of arno is essential for its recruitment to the pm, where it then activates arf . structural and biochemical analyses showing release of an autoinhibitory interaction within arno by arf -gtp provide further molecular insight into this regulatory cascade (dinitto et al. ) . hence, although the arno/cytohesin proteins are bona wde arf gefs, we will focus this review on the gbf/gea and big/sec arf gefs, which function in golgi and endosomal membrane traycking pathways. the gbf/gea and big/sec subfamilies are related, with members sharing sequence similarity principally in wve homology domains (mouratou et al. ) . the majority of these arf gefs are high molecular weight proteins, on the order of kda, and for this reason they have been referred to as the large arf gefs. the gbf/gea and big/ sec arf gefs function in internal membrane systems such as the golgi apparatus, the trans-golgi network (tgn) and endosomal pathways (zhao et al. ; park et al. ; casanova ; gillingham and munro ) . although the gbf/gea and big/sec arf gefs share a common domain structure, they clearly form two distinct subfamilies (cox et al. ; mouratou et al. ) . members of each subfamily have diverent localizations and functions, with gbf/gea gefs in general functioning in the er-early golgi membrane system and big/sec gefs functioning in the late-golgi and endosomal membrane systems. mammalian gbf localizes primarily to the intermediate compartment between the er and the golgi (ergic) and to cis-golgi membranes (kawamoto et al. ; zhao et al. ; , and yeast gea p and gea p localize to the cis-golgi (chantalat et al. ) . in plants, all the arf gefs fall into the gbf/gea and big/sec subfamilies, so the situation is more complicated. like its yeast and mammalian counterparts, the a. thaliana arf gef gnl localizes to and functions at the cis-golgi, but another member of the gbf/gea subfamily, gnom, localizes to and functions in the endosomal membrane system, although it still retains a capacity to function in the early golgi (richter et al. ). to compensate for the lack of endosomal-pm arf gefs, plants have thus diversi-wed the functions of the gbf/gea subfamily members, and possibly those of the big/sec subfamily as well. in mammals and yeast, the big/sec proteins localize to and function at the trans-golgi, the tgn and endosomes (franzusov et al. ; shinotsuka et al. a; shinotsuka et al. b; zhao et al. ; charych et al. ; . in this review, we will discuss the evolution of these arf gefs, and present an analysis of conserved domains that are common to both subfamilies as well as those that are speciwc to either the gbf/gea or big/sec proteins. we will describe the interacting partners of these arf gefs, indicating which of the homology regions of these proteins are involved in each interaction. finally, we will describe studies that have implicated the arf gefs in human disease, including recent studies describing speciwc inhibitors of members of the gbf/gea subfamily of arf gefs. the number of complete genome sequences has increased dramatically in the past few years, and now are available in public databanks for a wide variety of both prokaryotes and eukaryotes. analyses of these genome sequences have indicated that there are six major supergroups of eukaryotes that diverged from the lcea (adl et al. ) . these supergroups are the excavata, the chromalveolata, the archaeplastidia, the opisthokonta, the amoebozoa, and the rhizaria. fungi, including the well-studied model system saccharomyces cerevisiae and mammals, are part of the same supergroup, the opisthokonta. complete genome sequences from members of wve of the six eukaryotic supergroups are available, with the rhizaria being the one exception. we searched the complete genome sequences of representative members of these wve supergroups for members of the two subfamilies of large arf gefs, and found members of both the gbf/gea and the big/sec proteins (tables , ). humans have two big/sec proteins, called big and big , and only one gbf/gea subfamily member, gbf . arabidopsis, which like other plant species of the archaeplastida, have only gbf/gea and big/sec arf gef subfamilies and no others, have wve bigs and three gbfs. paramecium tetraurelia, a protozoan that belongs to the chromalveolata supergroup, has nine big/sec members, but no gbf/gea sequences. the model organism saccharomyces cerevisiae has one member of the big/sec subfamily, sec p, and two members of the gbf/gea subfamily, gea p and gea p (likely due to a recent genome duplication event). the human pathogen cryptosporidium parvum, a member of the apicomplexa, has no big/sec proteins, and only one gbf/gea sequence. the c. parvum gbf protein shares a high level of homology with plant gbf proteins such as the arabidopsis thaliana gnom protein. the apicomplexa are characterized by the apicoplast, an organelle which is thought to be the remnant of an endosymbiotic algal cell (abrahamsen et al. ; huang et al. ) . it is likely that the c. parvum gbf gene, like other plant-like genes in this organism, was transferred from the primordial endosymbiosed algal cell to the nucleus of the apicomplexan ancestor that engulfed it (huang et al. ). the closely related cryptosporidium hominis species has a very divergent arf gef that appears neverthe-less to be a member of the gbf/gea subfamily (xu et al. ) . phylogenetic analysis of the big/sec and gbf/gea subfamilies of arf gefs using members from wve eukaryotic supergroups indicates a clear separation of these two subfamilies (fig. ) . the entire arf gef sequences were used for the phylogenetic analysis, not just the sec domains as in previous studies (cox et al. ; mouratou et al. ) . using three diverent programs, the same tree topology was obtained, with high conwdence levels for a separation of the big/sec and gbf/gea sequences into two diverent clades (fig. ) . hence the presence of two subfamilies of large arf gefs is ancient, and it is likely that both gbf/gea and big/sec proteins existed within the lcea before the separation of the diverent eukaryotic lineages. the nine protozoan paramecium tetraurelia arf gefs, including the wve denoted ggg - , clearly fall into the big/sec subfamily (fig. ). as described previously, the large arf gefs have wve major sequence homology domains that are common to members of both the gbf/gea and the big/sec subfamilies (mouratou et al. ) . these domains are the dcb and the hus domains upstream of the catalytic sec domain, and hds , hds , and hds that lie downstream of the sec domain (fig. ) . this analysis was based on sequence data from organisms found primarily within two eukaryotic supergroups. when representative members of wve of the six eukaryotic supergroups are analyzed, the wve common homology domains are present in the arf gefs of most of the organisms examined, in both gbf/gea and big/sec subfamily members (figs. , , , , , ) . however, hds and hds are the least conserved domains, particularly for the gbf/gea proteins, and we were unable to wnd evidence for these domains in the gbf protein of the chromalveolata supergroup member c. parvum. because the gbf/gea and the big/sec proteins have distinct localizations and functions, it is pertinent to ask the question of whether there exist homology regions speciwc to each of the two subfamilies of large arf gefs. examination of homology regions both upstream and downstream of the sec domain indicated that among the representatives of all wve eukaryotic supergroups, only one domain, speciwc to the big/ sec subfamily, lies outside of the common domains ( fig. ). all other regions of high sequence conservation speciwc to either the gbf/gea or the big/sec subfamilies that were present among members of all the eukaryotic supergroups we examined were within the common homology regions dcb, hus, hds , hds , and hds . hence it appears that there is a highly conserved function and/or structure of the large arf gefs that is preserved in both the subfamilies, and speciwc functions are superposed on this common structural organization. overall, there is a higher level of sequence homology between the big/sec subfamily members than among gbf/gea sequences. outside of the catalytic sec domain, which is the most highly conserved domain of the arf gefs, the n-terminal domains dcb and hus have the next highest levels of homology, particularly among gbf/gea family members. as described previously (ramaen et al. ), the dcb domain was redewned to include residues upstream of the originally dewned domain (mouratou et al. ) (fig. ) . the most highly conserved region outside of the catalytic sec domain is the "hus box," a region of seven amino the sequences used for the analysis are shown in tables and . branches of the tree are color coded to indicate the eukaryotic supergroup that each sequence belongs to: opisthokonta (red), archaeplastidia (green), amoebozoa (blue), chromalveolata (violet), and excavata (beige) . acids near the end of the hus domain ( fig. ) (mouratou et al. ; park et al. ) . including a wide range of the eukaryotic sequences conwrms the high level of homology of this region, which has the consensus sequence y/f n y/f d c d/e/n (where stands for hydrophobic). the highly conserved aspartic acid residue within this motif is invariant in all but one of the sequences examined (fig. ) . in vivo, this residue plays an important role in traycking and in gbf/gea function in both mammalian cells and in yeast (park et al. ; ramaen et al. ; deng et al. ). on either side of this motif, there are residues conserved within each subfamily of the arf gefs, speciwc either for gbf/gea or big/sec proteins (fig. ) . in the catalytic sec domain, the loop between helices and in yeast, plant, and mammalian large arf gefs has the invariant sequence frlpge, where the wnal residue is the glutamic acid wnger that is essential for exchange activity (goldberg ; beraud-dufour et al. ) . when members of other eukaryotic families are included, only residues f and ge of this motif are invariant (fig. ) . signiwcantly, the catalytic glutamic acid residue is invariant even in this wide range of eukaryotic organisms. within the sec domain itself, there are several residues that are highly conserved within each subfamily (fig. ) . again, there are more conserved residues among big/sec subfamily members than among gbf/gea sequences. in the c-terminal homology regions hds , hds , and hds , the big/sec sequences had a signiwcantly higher level of homology than the gbf/gea sequences (figs. , , ) . both hds and hds are more highly conserved among the bigs than among the gbfs (figs. , ) , with the second half of hds showing a particularly high number of conserved residues among big/sec proteins of diverse eukaryotic origin (fig. ) . indeed, the ncbi conserved domain database has identiwed a portion of the big/sec hds domain as highly conserved and named it duf . in contrast, there are few if any residues in the second half of hds that are conserved among all the gbf/gea proteins (fig. ) . the hds domain was described previously, but our analysis has indicated that this domain is - amino acids longer than that proposed by mouratou et al. ( ) , with the additional homology region located at the c-terminal end of the originally dewned hds domain (fig. ) . including representatives of the wve eukaryotic super families indicates that there is only weak homology within this domain (fig. ) , but when representative members of only fungi, plants, and mammals are included, a signiwcant level of homology is found throughout the hds domain among members of both big/sec (not shown) and gbf/gea subfamilies (fig. ). there is one homology region speciwc to a subfamily of the large arf gefs, which we call hds , and which is present only among big/sec proteins (fig. ) . no equivalent homology region can be found in the gbf/gea proteins. we found hds sequences in eukaryotic supergroups other than the opisthokonta (animals, fungi) and archaeplastida (plants, algae), but with less homology. both tetrahymena thermophila and paramecium tetraurelia big sequences have a region homologous to amino acids , - , of the human big hds domain (fig. , data not shown). however, we did not wnd evidence for an hds domain in the amoebozoa or excavata supergroup members dictyostelium discoideum and trichomonas vaginalis, respectively. the highly conserved sequence homology domains of the gbf/gea and the big/sec arf gefs suggest that they have important, conserved functions in evolution, and are likely to be involved in protein-protein interactions. indeed, studies from a number of laboratories have fig. is conserved in the chromalveolata members that we analyzed (amino acid residues , - , of the tetrahymena thermophila big protein, corresponding to amino acids , - , of human big ). the orange box within the hus domain represents the highly conserved hus box, sequence y/f n y/f d c d/e/n ( : hydrophobic) fig. conserved residues within the n-terminal region of the big/ sec and gbf/gea subfamilies of arf gefs containing the dcb domain. multiple sequence alignment showing conserved residues speciwc to the big/sec subfamily (pink), speciwc to the gbf/gea subfamily (blue), or both subfamilies (yellow). invariant residues in all the sequences are shown in red. the most highly conserved portion of this region contains the dcb domain. secondary structure prediction of alpha-helical regions is shown above alignment in pink for big/sec sequences, and below alignment in blue for gbf/gea sequences. deletions in sequences are indicated by red xs, and correspond to: s -g , s -p in sec _sacc, r -m , s -p in sec _klul, p -g in big_dicd, v -h in big_ostt, v -n in big_chlr, and k -d in big_tett. protein sequence alignments were created using clustal w . (thompson et al. ) and t-covee (notredame et al. ) with default parameters. the multiple alignments were manually adjusted and edited using bioedit version . . (http://www.mbio.ncsu.edu/bioedit/bioedit.html). the gbf/gea and big/sec alignments (done separately) were imported into bioedit, then these were manually corrected to correspond to the combined alignment. aligned sequences were displayed with espript (gouet et al. ) using the blosum matrix with a similarity global score of . and a diverence score between conserved groups of . . secondary structure predictions on multiple alignments were performed at the pôle bioinformatique lyonnais (http://pbil.univ-lyon .fr/) and the consensus of three diverent programs (the phd, predator, and gor iv) is indicated. green box indicates akap domain of big (table ) . it is important to point out that many of these interacting partners have been identiwed by yeast two-hybrid screens or by co-immunoprecipitation of proteins from cells, which demonstrate a physical associa-tion, but not a direct protein-protein interaction. further studies are required to determine whether these interactions are direct or via a bridging protein. many of the interactions described in table have been previously reviewed (cox et al. ; ; mouratou et al. table . for big , these include the exocyst subunit exo (xu et al. ) , the myosin ixb molecular motor (saeki et al. ) , and the big protein itself (ramaen et al. ). the interactions with exo and myosin ixb suggest a role for big in targeting secretory vesicles to the pm. the big dcb domain has been demonstrated directly to form a dimer through puriwcation of this domain from e. coli and gel wltration analysis (ramaen et al. ) . this data conwrmed the initial observations for the arabidopsis thaliana gnom arf gef that the dcb domain is involved in dimerization (grebe et al. ) . for mammalian gbf , two proteins have been shown to interact directly, the small g protein rab (monetta et al. ) and the copi coat subunit gamma-cop (deng et al. ). rab plays an essential role in traycking between the er and cis-golgi (behnia and munro ) . like the arf small g proteins, rabs cycle between gdp-and gtpbound forms, and target evector proteins to membranes when in their active gtp-bound conformation. rab proteins are primarily involved in membrane fusion, with invariant residues (red). secondary structure prediction of alpha-helical regions is shown above alignment in pink islam et al. ( islam et al. ( , roles in regulation of tethering complexes and the pairing of snares which directly mediate fusion (behnia and munro ) . gbf was shown to be an evector for rab in vitro, and active rab in cells was shown to increase the association of both gbf and copi to membranes (monetta et al. ). interestingly, another rab evector, the tethering protein p , has also been shown to interact with gbf (garcia-mata and sztul ). p not only acts to tether membranes in er-golgi traycking, but also interacts directly with and promotes pairing of the er-golgi snare proteins, which are required for membrane fusion (short et al. ) . a precise molecular explanation for the rab -gbf -p series of interactions has not been demonstrated, but suggests a role for gbf in coordination of vesicle budding and fusion processes. the interaction between gbf and the gamma-cop subunit of the copi coat complex is conserved from yeast to humans. this interaction is a direct protein-protein interaction in both systems (deng et al. ; mpg-c and clj, unpublished data). studies from several laboratories have indicated that gbf is involved speciwcally in recruitment of copi to early golgi membranes. knockdown of gbf by rnai inhibits copi association with membranes with-out avecting other coats such as ap- (ishizaki et al. ; manolea et al. ; deng et al. ). in contrast, knockdown of the big and big proteins prevents ap- association with tgn membranes, without avecting copi (ishizaki et al. ; manolea et al. ) . a model explaining this speciwcity has been proposed, whereby copi interacts with the arf gef gbf (gea p and gea p in yeast) prior to nucleotide exchange on arf , so that the evector copi is in place at the time arf -gtp is formed (deng et al. ). this model overs an explanation for the observation that a single arf protein, arf , can recruit at least wve diverent coat complexes, including copi, ap /clathrin, gga/clathrin, ap , and ap , to diverent sites within cells (bonifacino and lippincott-schwartz ) . interestingly, all these coat complexes share sequence homology and are structurally similar (bonifacino and lippincott-schwartz ; mcmahon and mills ) , and function in a speciwc traycking pathway (bonifacino and lippincott-schwartz ; mcmahon and mills ) . this is also true of the large arf gefs, whose function is required to recruit these coat complexes to membranes. one of the regions of gamma-cop that interacts with the arf gefs is the appendage domain (deng et al. ), a region that is structurally conserved in all wve coats recruited to membranes by arf -gtp (mcmahon and mills ) . these results suggest the possibility that the diverent arf gefs within these two subfamilies regulate coat assembly via a common mechanism involving gef-coat interactions. an interaction between gbf and gga coat complexes has been proposed, although it has not been determined if this interaction is direct (lefrancois and mccormick ) (table ) . gbf functions and localizes to the cis-golgi, whereas the gga proteins function and localize primarily to the trans-golgi and tgn (hirst et al. ; puertollano et al. ; mattera et al. ; nakayama and wakatsuki ; puertollano et al. ) . severe perturbations of the early golgi caused by interfering with gbf function also avect late-golgi functions, where the gga proteins act. hence, the results of this study are likely due to a more indirect mechanism than by direct interaction of gbf and the ggas. an important role for the arf gefs in cell signaling has emerged with the identiwcation of big and big as a-kinase anchoring proteins or akaps. akaps are pka regulatory subunit binding proteins that act as scavolds to compartmentalize signaling cascades. this function is accomplished through formation of multi-protein complexes that spatially restrict the pka-camp signaling cascade at speciwc membrane sites within cells. big and big both contain akap domains that mediate binding of the regulatory subunit rii of protein kinase a (pka) (figs. , ) , and big accumulates in nuclei upon increase in camp levels in cells (li et al. ; padilla et al. ; citterio et al. ) . both big and big immunoprecipitated from hepg cells can be phosphorylated by recombinant pka, and this results in a decrease in gef activity (kuroda et al. ) . one pka phosphorylation site in big has been identiwed, at the c-terminus of the sec domain (li et al. ) (fig. ) . furthermore, endogenous protein phosphatase -(pp -) was found in a complex with big or big in microsomal fractions, indicating that a phosphatase of this signaling pathway is physically associated with big and big gefs (kuroda et al. ) . a recent study from this group has shown that another regulatory enzyme in this cascade, phosphodiesterase pde a, is functionally associated with big and big (puxeddu et al. ). pde enzymes break down camp to terminate camp signaling, reversing its evects on big and big . puxeddu et al. showed that pde a (but not pde ) co-immunoprecipitates with both big and big , and that knockdown of pde a led to a partial redistribution of big and big from golgi membranes to cytosol. in addition to the camp-pka regulatory proteins themselves, an akap-binding protein amy- has also been found to associate with the rii -binding (akap) domains of both big and big (ishizaki et al. ) . in cells, amy- tgn localization is abrogated in cells depleted of big , but not of big , indicating that big is the physiological partner of amy- at the tgn (ishizaki et al. ) . what are the functional consequences of big and big involvement as akaps in camp-pka signaling? for big , phosphorylation by pka results in its redistribution from the golgi and cytoplasm to the nucleus (citterio et al. ). the precise function of big in the nucleus is not known, but it involves interactions with nuclear proteins including nucleolin, wbrillarin and rna-binding protein la, as well as the small nuclear rna snou (padilla et al. ) . the type i tumor necrosis factor receptor (tnfr ) is one of two receptors for tnf that mediates its evects on inxammation, apoptosis, and the innate immune response. tnfr can be released from cells by incorporation into exosome-like vesicles which are shed from the surface of cells (hawari et al. ) . big has been shown to play a role in regulating this process by two mechanisms, one involving its gef activity, the other through its akap domains. activation of arf and arf by big is required for extracellular release of tnfr in exosome-like vesicles, and involves a physical association between big and tnrf (islam et al. ). in addition, camp-dependent protein kinase a signaling induces tnfr exosomelike vesicle release through binding of pka regulatory subunit rii to akap domains of big (islam et al. ). mutations in the big arf gef have been linked to autosomal recessive periventricular heterotopia (arph), a disorder of neuronal migration that leads to severe malformation of the cerebral cortex (microcephaly) and severe developmental delay (sheen et al. ) . two big disease alleles have been identiwed, including a frameshift mutation that results in truncation of the majority of the protein (sheen et al. ). the disease symptoms are a result of the failure of a speciwc class of neurons to migrate from their point of origin in the lateral ventricular proliferative zone to the cerebral cortex (sheen et al. ; ferland et al. ). this defect arises from a defect in vesicular traycking that alters the adhesion properties of these neurons (ferland et al. ). interestingly, mutations in other traycking proteins can give rise to periventricular heterotopia in humans or in model systems, indicating that traycking defects are likely the root cause of this disease (ferland et al. ). hence, the arf gefs not only serve essential roles in fundamental cell biological processes, but also play an important role in human development and neuronal function (sheen et al. ) . the arf gefs have been shown to act as host factors for pathogens mediating human disease. replication of several viruses has been shown to require gbf , including poliovirus, coxsackievirus, and coronavirus. poliovirus and coxsackievirus are enteroviruses belonging to the picornaviridae family of positive strand non-enveloped rna viruses. despite the fact that enteroviruses are not enclosed by a membrane, their replication never-the-less depends completely on host cell membranes. upon infection, enteroviruses cause a massive reorganization of the intracellular membranes of their host, including er and golgi membranes (bienz et al. ; salonen et al. ) . replication of poliovirus and coxsackievirus is completely inhibited by bfa, and it has been shown that gbf is the major target of this drug in poliovirus replication (belov et al. a; belov et al. ) . the enteroviral a protein has also been shown to block secretion in host cells, which inhibits the innate immune response of the host (doedens et al. ; wessels et al. ) . this block in secretion also occurs via gbf , through physical association of a with gbf which inhibits its function on golgi membranes in the host cell (wessels et al. ; wessels et al. ). hence, polio and coxsackieviruses subvert the host membrane by blocking gbf activity on host cell golgi membranes, reorganizing these membranes, then activating gbf function on arf for viral replication on these virally induced membrane structures. although a major role for gbf in enteroviral replication has been demonstrated, there is evidence that the big and the big arf gefs are also involved. interestingly, diverent viral proteins have been shown to recruit diverent coats to virally reorganized membranes. in an in vitro system, it has been shown that the a protein recruits gbf to membranes (belov et al. ) , whereas the viral cd protein speciwcally recruits big and big (belov et al. b) . furthermore, a recruitment of gbf results in speciwc binding of copi to membranes (belov et al. ) , whereas, cd recruits the gga coat to membranes (belov et al. b) . these observations support an intimate coupling between recruitment of speciwc gefs with speciwc coats to membranes, and provide another line of evidence supporting a connection between gbf and copi, and also a connection between big /big and the late-golgi coat gga . in addition to polio and coxsackieviruses, the rna replication of a number of positive-strand viruses is bfa-sensitive (gazina et al. ; molina et al. ; tai et al. ). among these is hepatitis c virus (hcv), which has been shown recently to depend also on the arf evectors copi and phosphatidylinositol- -kinase for its replication (tai et al. ). these results strongly suggest that gbf is a required host factor for hcv replication. bfa also inhibits replication of other families of viruses, including the enveloped positive-strand rna coronaviruses (verheije et al. ) . for one member of the coronaviruses, mouse hepatitis coronavirus, gbf and arf have been demonstrated to be essential host factors for its rna replication (verheije et al. ) . given the important roles of the arf gefs of the gbf/ gea and big/sec subfamilies in human disease, development of drugs that speciwcally target these proteins is of medical interest. as mentioned above, bfa is a natural substance that is a potent and speciwc inhibitor of members of both of these subfamilies. recently, a chemical (golgicide a) which speciwcally inhibits gbf , but not big or big , has been described (saenz et al. ). golgicide a was identiwed in a screen for inhibitors of the cytotoxicity of shiga toxin, a protein produced by the pathogenic bacterium shigella dysenteriae (saenz et al. ). this discovery, in addition to providing an important inhibitor of gbf , demonstrates a crucial role for this arf gef as a host factor in s. dysenteriae infection. complete genome sequence of the apicomplexan, cryptosporidium parvum the new higher level classiwcation of eukaryotes with emphasis on the taxonomy of protists the structure of ralf, an adp-ribosylation factor guanine nucleotide exchange factor from legionella pneumophila, reveals the presence of a cap over the active site large arf guanine nucleotide exchange factors in membrane traycking membrane association of the arabidopsis arf exchange factor gnom involves interaction of conserved domains organelle identity and the signposts for membrane trayc hijacking components of the cellular secretory pathway for replication of poliovirus rna activation of cellular arf gtpases by poliovirus protein cd correlates with virus replication a critical role of a cellular membrane trayc protein in poliovirus rna replication dual interaction of adp ribosylation factor with sec domain and with lipid membranes during catalysis of guanine nucleotide exchange intracellular distribution of poliovirus proteins and the induction of virus-speciwc cytoplasmic structures the mechanisms of vesicle budding and fusion coat proteins: shaping membrane transport regulation of arf activation: the sec family of guanine nucleotide exchange factors a novel golgi membrane protein is a partner of the arf exchange factors gea p and gea p the arf activator gea p and the p-type atpase drs p interact at the golgi in saccharomyces cerevisiae the brefeldin a-inhibited gdp/gtp exchange factor , a protein involved in vesicular traycking, interacts with the beta subunits of the gaba receptors evect of protein kinase a on accumulation of brefeldin a-inhibited guanine nucleotide-exchange protein (big ) in hepg cell nuclei active arf recruits arno/cytohesin gefs to the pm by binding their ph domains phylogenetic analysis of sec -domain-containing arf nucleotide exchangers evolution of the eukaryotic membrane-traycking system: origin, tempo and mode evolution of speciwcity in the eukaryotic endomembrane system sec p directs the transitions required for yeast golgi biogenesis a copi coat subunit interacts directly with an early-golgi localized arf exchange factor structural basis and mechanism of autoregulation in -phosphoinositide-dependent grp family arf gtpase exchange factors inhibition of endoplasmic reticulum-to-golgi trayc by poliovirus protein a: genetic and ultrastructural analysis disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia localization of components involved in protein transport and processing through the yeast golgi apparatus the membrane-tethering protein p interacts with gbf , an arf guanine-nucleotide-exchange factor adp-ribosylation factor/copi-dependent events at the endoplasmic reticulum-golgi interface are regulated by the guanine nucleotide exchange factor gbf diverential requirements for copi coats in formation of replication complexes among three genera of picornaviridae the small g proteins of the arf family and their regulators structural basis for activation of arf gtpase: mechanisms of guanine nucleotide exchange and gtp-myristoyl switching espript: analysis of multiple sequence alignments in postscript a conserved domain of the arabidopsis gnom protein mediates subunit interaction and cyclophilin binding an evolutionary perspective on eukaryotic membrane traycking release of full-length -kda tnf receptor in exosome-like vesicles: a mechanism for generation of soluble cytokine receptors ggas: roles of the diverent domains and comparison with ap- and clathrin a wrst glimpse into the pattern and scale of gene transfer in apicomplexa amy- (associate of myc- ) localization to the trans-golgi network through interacting with big , a guanine-nucleotide exchange factor for adp-ribosylation factors redundant roles of big and big , guanine-nucleotide exchange factors for adp-ribosylation factors in membrane trayc between the trans-golgi network and endosomes the brefeldin a-inhibited guanine nucleotide-exchange protein, big , regulates the constitutive release of tnfr exosome-like vesicles camp-dependent protein kinase a (pka) signaling induces tnfr exosome-like vesicle release via anchoring of pka regulatory subunit riibeta to big mechanisms of transport through the golgi complex small gtpases and the evolution of the eukaryotic cell gbf , a guanine nucleotide exchange factor for adp-ribosylation factors, is localized to the cis-golgi and involved in membrane association of the copi coat brefeldin a: insights into the control of membrane trayc and organelle structure an elaborate classiwcation of snare proteins sheds light on the conservation of the eukaryotic endomembrane system snareing the basis of multicellularity: consequences of protein family expansion during evolution control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and sm proteins regulation of brefeldin a-inhibited guanine nucleotide-exchange protein (big ) and big activity via pka and protein phosphatase gamma the arf gef gbf is required for gga recruitment to golgi membranes protein kinase a-anchoring (akap) domains in brefeldin ainhibited guanine nucleotide-exchange protein (big ) distinct functions for arf guanine nucleotide exchange factors at the golgi complex: gbf and bigs are required for assembly and maintenance of the golgi stack and trans-golgi network, respectively divalent interaction of the ggas with the rabaptin- -rabex- complex cop and clathrin-coated vesicle budding: diverent pathways, common approaches amp-activated protein kinase phosphorylates golgi-speciwc brefeldin a resistance factor at thr to induce disassembly of golgi apparatus diverential inhibition of cellular and sindbis virus translation by brefeldin a rab b interacts with gbf and modulates both arf dynamics and copi association the domain architecture of large guanine nucleotide exchange factors for the small gtp-binding protein arf a bacterial guanine nucleotide exchange factor activates arf on legionella phagosomes the structure and function of ggas, the trayc controllers at the tgn sorting crossroads t-covee: a novel method for fast and accurate multiple sequence alignment interaction of fk -binding protein with brefeldin a-inhibited guanine nucleotide-exchange protein (big ): evects of fk nuclear localization and molecular partners of big , a brefeldin a-inhibited guanine nucleotide-exchange protein for adp-ribosylation factors association of guanine nucleotide-exchange protein big in hepg cell nuclei with nucleolin, u snorna, and wbrillarin mutations in a highly conserved region of the arf p activator gea block anterograde golgi transport but not copi recruitment to membranes the ypt/rab family and the evolution of traycking in fungi functional analysis of adp-ribosylation factor (arf) guanine nucleotide exchange factors gea p and gea p in yeast the ggas promote arf-dependent recruitment of clathrin to the tgn morphology and dynamics of clathrin/ gga -coated carriers budding from the trans-golgi network interaction of phosphodiesterase a with brefeldin a-inhibited guanine nucleotide-exchange proteins big and big and evect on arf activity interactions between conserved domains within homodimers in the big , big , and gbf arf guanine nucleotide exchange factors functional diversiwcation of closely related arf-gefs in protein secretion and recycling big is a binding partner of myosin ixb and regulates its rho-gtpase activating protein activity golgicide a reveals essential roles for gbf in golgi assembly and function viral rna replication in association with cellular membranes mutations in arfgef implicate vesicle traycking in neural progenitor proliferation and migration in the human cerebral cortex big , a brefeldin a-inhibited guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin beta interaction of brefeldin a-inhibited guanine nucleotideexchange protein (big) and kinesin motor protein kif a guanine nucleotide-exchange factors for arf gtpases: their diverse functions in membrane trayc big , a guanine nucleotide exchange factor for adp-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity dominant-negative mutant of big , an arf-guanine nucleotide exchange factor, speciwcally avects membrane traycking from the trans-golgi network through inhibiting membrane association of ap- and gga coat proteins overexpression of an adp-ribosylation factor-guanine nucleotide exchange factor, big , uncouples brefeldin a-induced adaptor protein- coat dissociation and membrane tubulation golgins and gtpases, giving identity and structure to the golgi apparatus a functional genomic screen identiwes cellular cofactors of hepatitis c virus replication clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speciwc gap penalties and weight matrix choice mouse hepatitis coronavirus rna replication depends on gbf -mediated arf activation a viral protein that blocks arf -mediated cop-i assembly by inhibiting the guanine nucleotide exchange factor gbf molecular determinants of the interaction between coxsackievirus protein a and guanine nucleotide exchange factor gbf the genome of cryptosporidium hominis interaction of big , a brefeldin a-inhibited guanine nucleotide-exchange protein, with exocyst protein exo localization of large adpribosylation factor-guanine nucleotide exchange factors to diverent golgi compartments: evidence for distinct functions in protein trayc acknowledgments this study was supported by a chaire d'excellence from the agence nationale de la recherche, france. we thank jacqueline cherwls and valerie biou for assistance with bioinformatics programs and for critical reading of the manuscript. key: cord- -ucguzgdm authors: nan title: yeast kex p is a golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: ucguzgdm we have investigated the localization of kex p, a type i transmembrane carboxypeptidase involved in precursor processing within the yeast secretory pathway. indirect immunofluorescence demonstrated the presence of kex p in a punctate organelle resembling the yeast golgi apparatus as identified by kex p and sec p (franzusoff, a., k. redding, j. crosby, r. s. fuller, and r. schekman. . j. cell biol. : - ). glycosylation studies of kex p were consistent with a golgi location, as kex p was progressively n-glycosylated in an mnn - dependent manner. to address the basis of kex p targeting to the golgi apparatus, we examined the cellular location of a series of carboxy- terminal truncations of the protein. the results indicate that a cytoplasmically exposed carboxy-terminal domain is required for retention of this membrane protein within the golgi apparatus. deletions of the retention region or overproduction of wild-type kex p led to mislocalization of kex p to the vacuolar membrane. this unexpected finding is discussed in terms of models involving either the vacuole as a default destination for membrane proteins, or by endocytosis to the vacuole following their default localization to the plasma membrane. s ~.crea'eo biologically active proteins and peptides are classically produced as precursors which undergo both endo-and exoproteolytic processing to release the mature species while traversing the secretory pathway. the kex/gene product (kexlp) is a carboxypeptidase, specific for basic amino acid residues, and is responsible for processing proteins secreted by saccharomyces cerevisiae (dmochowska et al., ; cooper and bussey, ) . in conjunction with the proteases kex p and dipeptidyl aminopeptidase a (dpap a) ~ (stel p), kexlp proteolytically matures proteins such as c~-factor and k killer toxin from their precursors (for reviews see bussey, ; fuller et al., ) . proteins that enter the secretory pathway are thought to be transported to the cell surface by default via a "bulk flow" mechanism unless they contain additional targeting information (pfeffer and rothman, ; rothman, ; wieland et al., ; karrenbauer et al., ) . such targeting information is found in soluble proteins resident in the er which maintain their localization by containing a retention signal at their carboxy termini (munro and pelham, ; pelham et al., ) . deletion of the retention signal results in secretion of the soluble er resident proteins to the cell surface. soluble proteins destined for the mammalian lysosome re-a. cooper's present address is the institute of molecular biology, university of oregon, eugene, or . . abbreviation used in this paper: dpap, dipeptidyl aminopeptidase. ceive a mannose- -phosphate signal which targets them to the lysosome in a receptor-mediated manner. the absence of the mannose- -phosphate signal from these soluble proteins results in their secretion (for review see kornfield and mellman, ) . secretion also occurs when the targeting signals for yeast-soluble proteins destined for the lysosomelike vacuole are altered (vails et al., ; johnson et al., ; klionsky et al., ) . in addition to soluble proteins, the cell surface appears to be the default destination for mammalian er, golgi, and lysosomal membrane proteins as removal of their respective retention/targeting signals results in their delivery to the plasma membrane (machamer and rose, ; jackson et al., ; williams and fukuda, ) . as yet, no targeting or retention signals have been definitively assigned to yeast golgi or vacuolar membrane proteins, nor is it known where these proteins are delivered upon perturbation of their targeting signals. kexlp is predicted to be a type i transmembrane protein with a large amino-terminal protease domain in the lumen of the secretory pathway, a single membrane-spanning domain, and a smaller carboxy-terminai domain positioned cytoplasmicauy. the observation that kex/cells intracellularly retain kexlp activity prompted an analysis to determine in which secretory compartment kexlp resided, and how it achieved such retention. kexlp was found to be localized to the yeast golgi apparatus, with retention mediated via the cytoplasmic domain of the protein. removal of this domain or overproduction of wild-type kexlp resulted in delivery of the protein to the vacuole rather than to the cell surface. this surprising result raises the possibility that the vacuole is the default destination for yeast golgi membrane proteins. escherichia coli strains and associated dna manipulations were as described previously (cooper and bussey, ) . saccharomyces cerevisiae strains c abys ($ ), sc k, and sc k- (sc k + kex/-a/) have been described previously (cooper and bussey, ) . other strains used were $ (a strain sensitive to k killer toxin), tci cr (mata leu his trpl ura ), seys a (secl- leu ura gal ), xcy - d (mata ade - l ade x [a putative second ade mutation that results in white colonies] urn trpl lys leu - ,h amnnl::leu ), and lb - b (mata mnng), mnn mutants were crossed with either sc or sc -jh (isogenic to sc k except mata). the diploids were sporulated, asci dissected, and spores containing the relevant mutations selected. the resulting strains were hab (mnn/ura ), hab (ura ), and hab (mnn ura ). growth media, yeast transformation, and gane disruption have all been described previously (cooper and bussey, ) . restriction endonucleases, t dna polymerase, t dna ligase, and klenow fragment were purchased from either bethesda research laboratories, (gaithersburg, md) or new england biolabs (beverly, ma), and were used as recommended by the suppliers. unless otherwise stated, reagents used in these experiments were obtained from sigma chem. co., st. louis, mo. disruption of the kex/locus with the ura -based construct pl resulted in the allele kexl-a . an alternative disruption using the leu -containing plasmid p produced the allele kex/-a , p was constructed by digesting pl (dmochowska et ai., ) with ecorv to remove the ura gene and a portion of the kex/gene, and then replacing it with an hpai fragment carrying the leu gene. p was cleaved with hindiii and used to transform the strains $ , sc , . transformants were initially screened for a kex-phenotype on the basis of the abolition of a k toxin killing zone for the transformants of sc k and tc a, or on the basis of a smaller cr zone for $ transformants. disruption of the kex/locus was confirmed by southern blot analysis (not shown). mutagenesis of the carboxy-terminal region of kexi was performed on the same single-stranded dna template and in the same manner as described (cooper and bussey, ) . all of the mutations were confirmed by dna sequencing (sequenase, united states biochem. corp., cleveland, oh). fig. shows the extent of each of the mutations listed below. plasmid pkx - ams contains kex./with a precise deletion of the membrane-spanning domain (ams) inserted into the multicopy plasmid pvt -l (cooper and bussey, ) . the aberrant stop mutation (as) was identified while sequencing potential ams clones. the as clone contained a mutation at both bp and bp , introducing a premature stop codon at codon . the hpa mutation was to introduce an arginine residue immediately after the membranespanning domain followed by a termination codon using the mutagenic oligonucleotide ' ggagtttatgcgtatcgttaacgatagaagagtga '. to aid in identifying mutant clones an hpai site was introduced by the mntagenesis. all derivatives of the hpa mutagenesis were subsequently sequenced and found to have undergone a -bp deletion (a - bp) upstream from the inserted termination codon resulting in the mutation shown in fig. . the bcl mutation introduced a bcli cleavage site ( ' gtttat-gcgtatgattgatcagtgaggagaaaag immediately after the aspartate residue (codon no. ) which borders the carboxy-terminal portion of the membrane-spanning domain. the hinc mutation introduced a termination codon amino acid residues carboxy-terminal to the membrane-spanning domain using the mutagenic oligonucleotide ' ccaaat-aatagttaacatgacagt '. to aid in identifying mutant clones an hincli site was introduced by the mutagenesis. introduction of the various kex/mutations into pvti -l produced the following plasmids: pkxi- as, pkxl- hpa, pkxi- bel, and pkxl- hinc. a . -kb hindlh fragment containing the kex/gene was inserted into ycps , which had been digested with hindlii, to give the plasmid pkxi- wt. the . -kb xhoi-hindlh fragment of pkxi- containing the various kex/carboxy-terminal truncation mutations was isolated and ligated with the . -kb hindlii-xhoi fragment containing the amino-terminal portion of kex/to reassemble the gene, and then inserted into hindlh-digested ycp to create pkx - as, pkx - ams, pkxi- hpa, and pkx - hint. pad (dmochowska et al., ) , which contained the kex/ gene, was digested with xhoi, the site was filled in with klenow enzyme, and into this blunt-ended fragment was ligated the nhei nonsense codon linker ' ctagctagctag ' (new england biolabs). the resulting plasmid contained stop codons in all three frames in kex/at the xhoi site (+ bp). a . -kb hindiil fragment containing the mutation at the xhoi site was then inserted into hindlh-digested ycp to create pkx - xho. ycp -ct contains the adh/promoter and multiple cloning site from the plasmid pvt- -l (veruet et al., ) cloned into the plnsmid ycps . the termini of the . -kb bglli-hindiii fragment of kex/were filled with klenow enzyme and inserted into ycp -ct which had been digested with pvull. the resulting plasmid, pkxi- , results in an ~ -fold overproduction of kexlp. the various truncation mutations of kex/were placed downstream from the gal/promotor in plasmid pmb to create the following plasmids: pkx - wt, pkx - hpa, pkx - bcl, and pkx - hinc. antisera production and procedures for sodium carbonate extractions, radiolabeling ceils, and associated immunoprecipitations have been described previously (cooper and bussey, ) . $ - transformants were grown to mid-log, and • l s cells harvested, washed in i"i , and resuspended in ml of zsm ( . m sorbitol, mm cac , mm tris-hc , ph . ). /~g of zymolyase , (icn biomedicals, inc., costa mesa, ca) and/~-mercaptcethanol (final concentration . %) were added to the cells before incubation at oc for min. the resulting spheroplasts were gently pelleted, washed twice in . m sorbitol, mm tris-hc (ph z ), and resuspended in ml of ynb-sorb (yeast nitrogen base, % glucose, . m sorbitol), and incubated at oc for h. spheroplasts were then concentrated to ml, and bsa ( mg ml -l) and pmsf (boehringer mannheim corp., indianapolis, in; final concentration i ram) were added before the addition of mci of tram label (icn biomedicals, inc.). after rain, half of the cells were harvested and the remainder chased for rain as described above. after labeling, the spheroplasts were placed on ice, and sodium azide (fisher scientific co., pittsburgh, pa) was added to ram. spheroplasts were then gently pelleted and both the supernatant and pellet fractions retained. the supernatant was microfuged for rain and the resulting supernatant was concentrated with centricon (amicon, beverly, ma) to a final volume of ~tl before heating to ~ in the presence of sds ( %). the spheroplasts werv washed once in ice cold . m sorbitol, i mm tris-hc (ph . ), pelleted, resuspended in bsb + % sds (cooper and bussey, ) , and boiled. the detergent-solubilized samples were then treated as above and immum~recipitated with kexlp antiserum. immunofluorescence studies fl-galactosidase-kexlp fusion protein (cooper and bussey, ) was isolated by preparative sds-page; electmeluted into .i m ni-~co , . % sds; lyophilised; resuspended in coupling buffer ( . m nacl, . m nahco , ph . ); and then dialyzed against coupling buffer + . % sds + mg ml -~ pmsf. the extract was then coupled to g of activated cnbr-sepharose (pharmacia lkb biotechnology inc., piscataway, nj) overnight at room temperature. the coupling efficiency was judged to be > % as determined by comparing unbound protein with the stming material. the sepharose was incubated with blocking buffer ( m ethanolamine, . m nahco , ph . , . m naci) for h at room temperature. the resulting column (bed volume, ml) was then treated with successive washes of coupling buffer and acetate buffer ( . m sodium acetate, ph . , . m nac ), and finally with pbs. a similar column (bed volume, ml) was made with an extract made from the granules of e. coli cells producing/~-galactosidase. both the/~-galactosidase and ~-galactosidase-kexlp columns were washed successively with buffer a ( mm hepes, ph . , mm naci, mm edta), buffer b (buffer a + m guanidine-hc ), and buffer c ( mm hepes, ph z , mm nac , . m mgci ). the two columns were then arranged so that the eluate from the #-galactosidase column flowed into the /~-galactosidase-kexlp column. all buffers used from this point on contained in addition: mg/ml -i bsa, . % nan , mm pmse columns were equilibrated with buffer a; kexlp antiserum was loaded onto the linked columns and was circulated for h at ~ the columns were disconnected and the o-galactosidase-kexlp column was washed first with buffer b and then with buffer a. antibodies were then eluted from this column with buffer c; fractions were collected and immediately dialyzed against buffer a at c. eluted fractions were tested for the presence of antibodies with a dotblot western approach and the positive fractions tested for their ability to immnnoprecipitate [ s]methionine-labeled kexlp from yeast. these fractions were concentrated by dialysis against buffer a containing % glycerol, and stored at - ~ immunofluorescence studies were performed following the procedure of redding et al. ( ) and used either affinity-purified anti-kexlp antibodies or a mouse monoclonal ( d ) which is directed against the -kd subunit of the yeast vacuolar membrane h+-atpase (kane et al., ) . the cells were observed using an axiophot microscope (carl zeiss, inc., thornwood, ny) (equipped for epifluorescence at excitation wavelengths appropriate for the described fluors) with a x objective and photographed with t-max film (eastman kodak co., rochester, ny). mitochondria and nuclei were identified by ', -diamidino- -phenyl-indole staining while vacuoles were identified by differential interference contrast (nomarski) optics. upon translocation into the yeast er, proteins that are destined for asn-linked glycosylation receive a core oligosaccharide which can be elaborated in the golgi apparatus by the addition of further mannose residues to produce the "outer chain: the outer chain consists of a backbone of u( --~ ) linked mannose residues to which are attached mannose sidechains in ot(l~ ) linkages which then terminate in c~( --- ) linkages ( fig. ; ballou, ) . the extension of the core is heterogenous in nature with different proteins receiving varying degrees of elaboration. kexlp is a glycoprotein which receives asn-linked glycosylation in a multistep process (cooper and bussey, ) . the initial event occurs in the er where core oligosaccharides are attached to the protein. the observed difference in molecular mass of ~ - kd between kexlp labeled for min and that of kexlp produced in tunicamycintreated cells suggests that two of the three predicted lumenal asn acceptor residues are glycosylated. the ash-linked core oligosaccharides undergo a modification in a post-er compartment that increases the mass of kexlp by - kd, demonstrating that kexlp does not receive an extensive outer chain (cooper and bussey, ) . such a modification was reduced in a secl mutant which blocks intra-golgi transport, and was unaffected in a secl mutant that prevents secretory vesicle fusion with the plasma membrane (novick et al., ; cooper and bussey, ) . the yeast golgi apparatus has been functionally subdivided into several compartments on the basis of asn-linked oligosaccharide addition (franzusoff and schekman, ; graham and emr, ) . glycoproteins receive mannose outer chain modifications while traversing these golgi compartments. identification of the enzymes responsible for the (ballou, ; kukuruzinska et al., ) . a likely candidate for the mnn -independent modification was the golgi-localized ~( ~ ) mannosyltransferase which has been shown to be responsible for t~(l~ ) mannose addition to both the core and outer chains ( fig, ; nakajima and ballou, ; franzusoff and schekman, ; graham and emr, ) . this enzyme activity has been found to be deficient in an mnnl strain, and kexlp synthesized in a strain disrupted at the mnnl locus received only a small modification of its core oligosaccharide (fig. , lanes and ) . the ot(l~ ) mannosyltransferase is therefore responsible for producing the majority of the post-er glycosylation modification to kexlp. the modification of kexlp by the golgi ot(l~ ) mannosyltransferase suggested that kexlp reached the golgi compartment containing the mannosyltransferase, yet little kexlpdependent activity was detected at the cell surface (see below) and indicated that kexlp was retained within the secretory pathway. an analysis of mutant forms of kexlp was undertaken to examine which domain(s) of kexlp was responsible for its retention. the hydrophobic domain near the carboxy terminus of kexlp was previously shown to be responsible for conferring membrane association, as deletion of this -amino acid residue region resulted in a soluble protein, kexlp-ams (cooper and bussey, ) . cells expressing this protein showed a significant increase in kexlp-dependent activity in the cell medium (see below). that kexlp-ams was secreted rather than retained suggests that one of several domains may be responsible for retention of kexlp: ( ) the membranespanning domain which was absent in kexlp-ams, ( ) the carboxy-terminal domain which was no longer exposed in the cytoplasm in kexlp-ams, and ( ) a domain amino- s - /pkxl- hpa was radiolabeled for a -min pulse (p) and harvested or chased for an additional min (c). tunicamycin treatment (tcm) and high ph sodium carbonate extraction were performed as described previously (cooper and bussey, ) . the forms of kexlp were immunoprecipitated and analyzed by sds-page and fluorography. terminal to the membrane-spanning domain which is perturbed in kexlp-ams when the cytoplasmic domain was juxtaposed adjacent to it. a number of carboxy-terminal truncations of kexlp were produced to test the above possibilities. the construction of these mutations via site-specific mutagenesis or nonsense linker insertion is described in materials and methods and the predicted proteins are shown in fig. . immunoprecipitations of the mutant kexlp proteins were performed to confirm that they were correctly synthesized by the cell (fig. ) . all of the truncated proteins were smaller than kexlp by the predicted amount and entered the secretory pathway as shown by the addition of ash-linked glycosylation (data not shown). a concern was that the mutants designed to remain membrane associated may not attain a stable type i orientation and remain in the er. the most severely truncated mutant in this class, kexlp-hpa, lacks the entire endogenous cytoplasmic domain; it was further analyzed and shown to receive asn-linked glycosylation which was further modified within the golgi apparatus (fig. ) . previous sodium carbonate extractions of whole cell extracts demonstrated that kexlp fractionated with the membrane containing pellet fraction whereas the soluble protein, kexlp-ams, fractionated with the supernatant (cooper and bussey, ) . carbonate treatment of whole cell extracts showed that kexlp-hpa fractionated with the membrane pellet (fig. , lanes and ), consistent with it being an integral membrane protein. in addition, we have shown that cpy, a soluble kexlplike protein, is fully soluble under these extraction conditions (data not shown). kexlp activity was assayed in whole cells and their growth media to detect any potential mislocalization of the truncated forms of kexlp to the cell surface. to compare mutants, it was important that the kex/gene copy number be constant. the kexlp truncation constructs were, therefore, inserted into the vector ycp , a centromeric-based plasmid which is normally maintained at a single copy per cell (rose et al., the indicated strains and transformants were inoculated in either yepd (a) or yep + galaetose (b) to a high cell density and grown to stationary phase. the intact cells were washed twice in water and a portion were lysed in mm succinic acid (ph . ) with glass beads. the growth media, whole cells, and lysed cells were then assayed for kexlp activity as described (cooper and bussey, ) . a unit is defined as pmol of product produced per minute at "c. (error -i- %.) ). construction of these plasmids resulted in the deletion of an upstream portion of the k/ds this deletion resulted in reduced production of the encoded proteins relative to wild-type levels. these constructs (the pkx - series of plasmids) were transformed into the yeast strain $ - (kex/-a ), a derivative ofs in which the kex/gene had been disrupted. the transformants were grown in liquid selective media; and the media, whole cells, and solubilized lysed cells were assayed for kexlp activity (data not shown). $ - produced no activity and the plasmid-borne wildtype kex/gene partially restored activity ( % of the level produced by the genomic allele of kex/) consistent with the promoter truncation. in comparing the partitioning of kexlp activity from various truncation mutants with that of the wild-type, two separate groups became apparent. the kexlp-hpa and kexlp-hinc truncated proteins (those that remained membrane associated; fig. ) showed the same partitioning pattern as that of wild-type, whereas the proteins kexlp-xho, -as, and -ams (soluble proteins lacking the membrane-spanning domain) formed a different pattern with a -fold increase in activity at the cell surface relative to that of wild type. the total activity of each protein was approximately constant within a group. kexlp-hpa and kexlp-hinc had a total activity similar to that of wild type while the soluble forms of kexlp had ~ % of wild-type activity. although the decrease in activity may have been a direct consequence of the mutations, it was also possible that the reduced activity was due to secretion and subsequent degradation of the truncated soluble proteins. if such degradation of the soluble forms of kexlp-xho, -as, and -ams was occurring external to the plasma membrane, then the addition of bsa to the growth medium may lessen the extent of degradation. the addition of relatively high levels of bsa did not, however, alter the levels of total activity for kexlp-xho, -as, and -ams. a different approach to reduce potential degradation of truncated forms of kexlp was taken where transformants were grown in selective conditions (minimal media) and then transferred to nonselective yepd media for several generations before assay. yepd, a medium consisting primarily of yeast extract and peptone, should provide a substrate "buffer" against proteolytic degradation. although transformants were grown temporarily under nonselective conditions, plasmid loss was never > % (data not shown). activity partitioning data suggested that kexlp-xho, -as, and -ams were secreted from the cell while the other constructs (kexlp, kexlp-hpa, and kexlp-hinc) remained intracellular (table i) . all the mutant forms of kexlp had approximately the same total activity as the plasmid-borne wild type. the percentage of kexlp extracellular activity for the membrane-associated forms of kexlp was comparable to the percentage of cells that stained positively with the vital dye methylene blue, suggesting that such extracellular activity resulted from cell lysis. kex/was placed downstream from the gal/ promoter which, upon induction by growth on galactose, resulted in a -fold increase over endogenous kexlp activity. however, such overproduction did not increase the percentage of extracellular kexlp activity relative to wild-type levels (table i) . to demonstrate the secretion of the soluble forms of kexlp, $ - (kex/-a ) was transformed with pkx - (kexlp) or pkxi- ams (kexlp-ams), and cells were then spheroplasted. the spheroplasts were radiolabeled ( min), chased ( min), and solubilized with sds, and kexlp was immunoprecipitated. media, containing proteins exported beyond the plasma membrane, were concentrated and kexlp was immunoprecipitated. wild-type kexlp remained associated with the spheroplasts, whereas kexlp-ams was shown to be secreted from the cell; the kexlp-ams signal associated with the cell fraction diminished with time while the signal from the medium showed the reverse trend (fig. ) . thus, the results of the pulse-chase analysis correlated with the activity data and indicated that kexlp-ams, a soluble form of kexlp, was secreted from the cell. a stringent test of the effect of the truncations of kexlp was to determine the ability of the mutant proteins to carry out the golgi-based proteolytic processing of k killer toxin in vivo (for review see bussey, ) . sc k is a strain harboring the dsrna virus that encodes k killer toxin, and produces a killing zone on plates seeded with a strain sensitive to the killer toxin (bussey et al., ) . the pkx - series of plasmids (centromeric based) were transformed into sc k- (kex/-a/), and the transformants analyzed for their ability to process k killer toxin and produce a killing zone (fig. a) . sc k- containing the vector plasmid produced no killing zone, whereas the introduction of a plasmidbased copy ofkexi (pkx - wt) enabled the strain to produce active k killer toxin as shown by the appearance of a zone (fig. b) . this zone was smaller than that of sc k (kexi) because of the kex/promoter truncation in pkx - wt (discussed above). the truncated forms of kexlp, both soluble and membrane associated, produced killer zones significantly smaller than that produced by pkx - wt (fig. a) . this functional complementation assay was also conducted in a different k killer strain (tc ot- ) with similar results. the reduced processing of k killer toxin by the soluble forms of kexlp can be explained because of their secretion and consequent reduced concentration of kexlp within the processing compartment. the membrane-associated truncated forms of kexlp remain intracellular and, therefore, secretion cannot account for their reduced processing ability. the above results indicated that kexlp-hpa was membrane associated, had received glycosyl modifications in the golgi apparatus, gave wild-type levels of total activity, and was retained intracellularly; yet processed the k killer toxin precursor to a lesser extent than kexlp. kexlp-bcl and kexlp-hinc showed similar phenotypes to that of kexlp-hpa. a likely explanation for such observations was that these membrane-associated mutant forms of kexlp were not retained within the correct golgi compartment, but instead were mislocalized within the secretory pathway. indirect immunofluorescent detection of kexlp and kexlp-hpa was undertaken to determine if the location of the two proteins differed. the expression of the proteins was such that a kexlp signal could only be detected upon overproduction of kexlp (fig. ) . kexlp was found to be localized to a number of small punctate structures (on average, ,x, - per cell) which were not associated with mitochondria, nuclei, or vacuoles, but were characteristic of proteins localized to (hpa) , or pkx - -hinc (hinc). transformants were grown to stationary phase in minimal media, harvested by centrifugation, washed in h , pelleted, and than resusponded in h to a concentration of x cells ml -~. /~ was then placed onto a minimal plate (ph . ), seeded with $ (a strain sensitive to k killer toxin). plates were then incubated at "c for h and zones of toxin killing examined. approximate relative toxin activities, determined by comparison with a toxin dilution series were wt %, xho %, as %, ams %, hpa %, and hinc %. (b) sc k- (kex/), sc k (kex ), and sc k- /pkxi- wt (wt) were grown and spotted onto a plate seeded with a strain sensitive to k killer toxin and treated as described above. the yeast golgi apparatus (redding et al., ; cleves et al., ; franzusoff et al., ; roberts et al., ) . comparison of the kexlp-hpa localization with that of the vacuole (as determined by nomarski optics) indicated that almost all of kexlp-hpa was in the vacuole (data not shown). it is likely that kexlp-hpa was transported to the vacuole, where, in a protease-deficient strain such as $ - , the protein was not degraded and remained active. to confirm this possibility, colocalization studies were undertaken between the -kd subunit of the s. cerevisiae vacuolar membrane h § and either kexlp or kexlp-hpa. the vacuole (as determined by nomarski optics) correlated to the ring structure in which the -kd atpase subunit was localized by indirect immunofluorescence. the level of kexlp detected varied among cells due to the variable plasmid copy number ( #m based). observation of cells expressing both low and high levels of kexlp-hpa (as deter- mined by the intensity of the fluorescence) indicated that > % of the protein was located in the vacuole. a small percentage of cells (< %) showed both er and vacuolar staining. the protein was associated with the vacuolar membrane demonstrating that it remained membrane associated (fig. ) . kexlp was predominantly restricted to punctate structures indicative of a yeast golgi location (fig. ) . however, - % of the stained cells expressed high levels of kexlp (as determined by the intensity of the fluorescence) and showed kexlp in both golgi-like structures, and associated with the vacuolar membrane as defined by the -kd atpase subunit (data not shown). no kexlp or kexlp-hpa signal was detected at the plasma membrane. further indirect immunofluorescence analysis also localized the other membrane-associated forms, kexlp-bcl and kexlp-hinc, to the vacuolar membrane (data not shown). an analysis of the oligosaccharide modification of kexlp in-dicated secretory compartments to which the protein had been exposed, and the likely subcellular location of kexlp. the modification of the asn-linkod oligosaccharide cores of kexlp occurred within the golgi apparatus and likely involves several sequential steps. initially, in a pre-sec compartment, the core oligosaccharides are partially modified as judged by an increase in apparent molecular mass (cooper and bussey, ) . subsequently, in a post-sec compartment, the core is further modified in a mnnl-dependent manner. invertase produced in sec cells at the restrictive temperature does not receive c~( --' ) linked mannose residues to the core or outer chain (franzusoff and schekman, ) , most likely because of the failure of secretory proteins to reach the compartment containing the cx( --, ) mannosyltransferase. it is consistent that sec and mnnl mutations result in a similar reduction in the glycosyl elaboration of kexlp, as they would respectively either prevent kexlp from reaching, or reduce the activity of, the c~( ~ ) mannosyltransferase. the golgi-based modification of kexlp can thus be explained as a result of two processes. the first occurs before the sec block and most likely involves the addition of an ot(l~ ) mannose residue followed by the attachment of an oz( --' ) linked mannose residue (indicated by * in fig. ). the second step involves the mnn dependent addition of up to three a ( l ' ) linked mannose residues (indicated by a circle in fig. ) to the oligosaccharide (ballou et al., ) . graham and emr ( ) provided evidence for at least three functional compartments in the yeast golgi apparatus that contain, from cis to trans, the following activities: ( ) ct(l~ ) mannosyltransferase, ( ) cx(l~ ) mannosyltransferase, and ( ) kex p endoprotease. kexlp must reach the golgi compartment housing the mnnl-dependent o~( --- ) mannosyltransferase as it is modified by this enzyme. in addition, to process killer toxin, kexlp must reach the proposed third golgi compartment containing kex p as the toxin precursor substrate for kexlp is created by a kex p-mediated endoproteolytic cleavage. no post-kex p compartment before secretory vesicles has been observed (graham and emr, ) and, therefore, it is likely that kexlp resides with kex p in the same late golgi compartment (redding et al., ) . it is interesting to note that both kexlp and kex p figure . model for the retention of kexlp within the secretory pathway. a model for the retention of kexlp is presented. kexlp is retained in a late golgi compartment because of its cytoplasmically exposed domain interacting with a receptor (recpt). kexlp-as, being soluble, is secreted by default to the cell surface. kexlp-hpa is membrane associated and residues in the vacuole because of several possible reasons: ( ) kexlp-hpa is partially misfolded and is targeted to the vacuole via an unknown system; ( ) the membranespanning domain of kexlp contains a cryptic targeting signal for the vacuole; ( ) kexlp-hpa is secreted to the plasma membrane where it is endocytosed and delivered to the vacuole; and ( ) membrane-associated proteins that leave the golgi apparatus are targeted to the vacuole by default. kexlp is delivered to the vacuole when overproduced to a high level, presumably due to saturation of a golgi retention receptor. the horizontal bar protruding from the kexlp-wt box represents the membrane-spanning and cytoplasmic domain, while for kexlp-hpa the bar represents the membrane-sparming domain. (wilcox and fuller, ) undergo a slow addition ofa(l~ ) mannose units to their respective glycosyl groups. further evidence that kexlp is an enzyme that resides in the yeast golgi apparatus comes from indirect immunofluorescence results where a punctate pattern of staining is detected that is characteristic for a number of proteins localized to the golgi apparatus in s. cerevisiae: kex p (redding et al., ) , dpap a (roberts et al., ) , secl p (cleves et al., ) , and sec p (franzusoff et al., ) . of the mutations that result in a soluble form of kexlp (kexlp-xho, -as, and -ams), kexlp-as is the most informative with regard to determining which domain(s) of kexlp is involved in its localization. kexlp-as is secreted, yet contains all but residues of the -residue lumenal domain (fig. ) . the residues absent in kexlp-as are present in kexlp-ams which is also secreted. the results suggest that either the membrane-spanning or cytoplasmic domain, or both, are responsible for the retention of kexlp within the golgi apparatus. kexlp-hpa contains both the lumenal and transmembrane domains of kexlp yet is mislocalized to the vacuolar membrane. similar results were found for kexlp-bcl and kexlp-hinc which lack all or part of the cytoplasmic domain and strongly implicate this domain of kemp as being necessary for correct localization within the golgi apparatus. previous work has demonstrated the role that cytoplasmic domains of transmembrane proteins play in the retention and targeting of proteins within the secretory pathway. a consensus retention motif has been identified at the termini of the cytoplasmically exposed domain of mammalian er resident type i transmembrane proteins (nilsson et al., ; jackson et al., ) . the cytoplasmic domains of the transferrin receptor, cation-independent mannose- -phosphate receptor, and the lysosomal acid phosphatase are involved in the correct targeting or endocytosis of these proteins (rothenberger et al., ; lobel et al., ; peters et al., ) . the three proteins involved in processing a-factor, kemp, kex p, and dpap a, are all transmembrane proteins with cytoplasmic domains which are thought to be involved in retention of the respective proteins in similar if not identical compartments of the yeast golgi apparatus (fuller et al., ; roberts et al., ) . attempts to identify a consensus yeast golgi retention motif shared between these proteins have, however, showed no obvious amino acid sequence homology between the cytoplasmic domains of these three proteins. different mechanisms have been proposed for the retention of proteins within golgi compartments. interactions involving the cytoplasmic domain of kexlp may result in the aggregation of the protein in the appropriate compartment, and thereby prevent it from entering transport vesicles that are exiting the golgi compartment (pfeffer and rothman, ) . this retention via aggregation model is unlikely as it does not explain why aggregation would not occur earlier in the secretory pathway or why overproduction of a kexlp results in mislocalization to the vacuole rather than greater aggregation in the golgi compartment. a more likely explanation for the retention involves a receptor interacting with the cytoplasmically exposed domain of kexlp ( fig. ). high level expression of kexlp would saturate the receptor with the result that excess kexlp is diverted to the vacuole. removal of the cytoplasmic domain would abolish such a receptor-ligand interaction, also resulting in mislocalization to the vacuole. retention of soluble er proteins has been shown to involve a receptor-mediated retrieval of proteins from a pre-cis- golgi compartment (semenz~ et al., ; lewis et al., ) . a cytoplasmicauy based retrieval system analogous to the er system can be envisaged in which kexlp is retrieved from a trans compartment, possibly the plasma membrane, and returned to the correct golgi compartment. to determine if kexlp is retrieved from the plasma membrane an immunofluorescence experiment was performed by inducing expression of kexlp in a secl strain, secl is a conditional mutation that, at the nonpermissive temperature, prevents the fusion of secretory vesicles with the plasma membrane (novick et al., ) . the delivery of kexlp to its golgi location was not blocked by secl, and if kex/expres-the jouma! of cell biology, volume , sion was constitutive before imposition of the secl block, the staining pattern of kexlp was not altered even after h at the restrictive temperature (data not shown). therefore, if a receptor retrieval system exists, it is unlikely that kexlp is retrieved from the plasma membrane as outlined in one of several proposals presented by payne and schekman ( ) . if a retrieval system is responsible for the retention of kexlp, then the salvage compartment may instead be located between the golgi compartment and the vacuole where failure to bind the receptor would result in delivery to the vacuole. alternatively, the receptor may remain anchored within the golgi apparatus and not recycle. the clathrin heavy chain has been implicated in the retention of kex p (payne and schekman, ) , raising the possibility that the receptor-based system that recognizes the cytoplasmic domain of kexlp, and potentially kex p and dpap a, might be one of the clathrin-associated adaptin proteins known to occur in yeast (kirchhausen, ) . truncated forms of kexlp which lacked the cytoplasmic domain yet still remained membrane associated were not retained in the correct golgi compartment, and even at subwildtype expression levels v~ere diverted to the vacuole. similar mislocalization results have been found with dpap a, where overexpression of the protein results in mislocalization to the vacuole, as do mutations within its cytoplasmically exposed domain (roberts et al., ) . differing results were obtained with kex p, where a mutation that deleted the cytoplasmic domain and part of the membrane-spanning domain resulted in a significant proportion of the kex p activity being mislocalized to the cell surface (fuller et al., ) . the kex p activity study did not, however, address whether the truncated protein produced was membrane associated; if not, then the resulting soluble protein would be expected to be secreted to the cell surface. in addition, the strain used for such kex p activity studies contained wild-type activity levels of vacuolar hydrolases (pep ; jones, ) and, therefore, any kex p potentially mislocalized to the vacuole might be degraded and hence go undetected. mislocalization of membrane proteins to the lysosome does not appear to occur in mammalian systems where mutations that interfere with the localization of er, golgi, and lysosomal integral membrane proteins result in their delivery to the plasma membrane (paabo et al., ; machamer and rose, ; peters et al., ) . one exception to this observation in mammalian systems is the coronavirus e protein, a type hi membrane protein normally resident in the golgi apparatus. mutations that affect the retention of this protein resulted in delivery to the lysosome rather than to the cell surface (armstrong et al., ) . a number of models could explain the observation that the membrane-associated mutant forms of kexlp are deh'vered to the vacuole while the soluble truncated forms of kexl are exported to the cell surface (fig. ) . ( ) the first model suggests that membrane-associated truncated forms of kexlp are misfolded, and as such may be targeted, via an unknown mechanism, to the vacuole for degradation. this model seems unlikely as all of the truncated forms of kexlp, both soluble and membrane associated, have similar total pro-tease activity to that of the wild-type protein, suggesting that at a minimum the catalytic domain of the mutants has folded to a conformation similar to that of wild type. in addition, these forms of kexlp all exit the er and reach the golgi apparatus where they are both glycosylated and process killer toxin. the misfolding targeting model would be unusual in that it must explain the mislocalization of the membraneassociated mutant forms of kexlp yet allow the soluble forms to be secreted. also, such a garbage pathway for delivery of misfolded mutant forms of kexlp cannot explain the vacuolar localization of highly expressed but presumably correctly folded kexlp. ( ) the second model proposes that the membrane-spanning domain of kexlp contains a latent or cryptic targeting signal for the vacuole. studies are currently under way to address this possibility. it should be noted that although kexlp is homologous to the vacuolar protein cpy, the homology does not extend to include the vacuolar targeting signal found in procpy. ( ) the third model involves kexlp-hpa (-bcl, -hinc) being secreted to the plasma membrane by default, where it is then endocytosed and delivered to the vacuole. such a "transient appearance" of kexlp-hpa (-bcl, -hint) at the cell surface would not be detected by the activity assay used or by indirect immunofluorescence. soluble kexlp-as, having reached the cell surface, would be released into the medium and, hence, would not be endocytosed to the vacuole. ( ) the final model proposes that the vacuole is the direct default destination for membraneassociated proteins that enter the secretory pathway, such that membrane proteins lacking positive targeting/retention signals would be delivered to the vacuole. work with dpap b, a type ii vacuolar membrane protein homologous to dpap a, has shown that no domain of the protein contains positive targeting information for the vacuole (roberts et al., ) . the targeting signal for a second vacuolar membrane protein, alkaline phosphatase, has not been identified although the lumenal domain is not required for the correct localization of this protein (klionsky and emr, ) . the authors concluded that the vacuolar-sorting determinant must therefore reside in the cytoplasmic and/or membrane-spanning domain of alkaline phosphatase, but in light of the data presented here and elsewhere (roberts et al., ) , these results could be reinterpreted to suggest that the membrane association of alkaline phosphatase mediates its vacuolar delivery. given the results presented here, we favor models and which are variants of each other in that both result in the delivery of kexlp to the vacuole by default (no positive targeting signal involved) but differ in the delivery route. model , involving a direct golgi-to-vacuole route, may be the most likely as delivery of the vacuolar membrane protein dpap b does not involve transport to the plasma membrane and subsequent endocytosis (roberts et al., ) . in addition, kexlp-bcl contains no cytoplasmic tail to participate in classical endocytosis. an important implication of such default delivery is that yeast integral membrane proteins destined for the plasma membrane must have positive targeting information to either remain at the plasma membrane (proposal ), or to avoid being diverted to the vacuole (proposal ). it is pertinent that a mutation in the u-factor receptor (ste p), an integral plasma membrane protein, results in delivery of this protein to the vacuole, independent of the plasma membrane (ho-recka, j., and g. sprague, personal communication), raising the possibility that a plasma membrane targeting signal has been destroyed. further work is proceeding to determine if the delivery route of kexlp to the vacuole is via the plasma membrane, and whether plasma membrane proteins have positive targeting/retention information. lysosomal sorting mutants of coronavirus e protein, a golgi membrane protein yeast cell wall and cell surface revision of the oligosaccharide structures of the yeast carboxypeptidase y proteases and the processing of precursors to secreted proreins in yeast secretion of saccharomyces cerevisiae killer toxin: processing of the glycosylated precursors mutations in the cdpcholine pathway for phospholipid biosynthesis bypass the requiremem for an essential phospholipid transfer protein characterization of the yeast kex gene product: a carboxypeptidase involved in processing secreted precursor proteins yeast kex/gene encodes a putative protease with a carboxypeptidase b-like function involved in killer toxin and alpha factor precursor processing functional compartments of the yeast golgi apparatus are defined by the sec mutation localization of components involved in protein transport and processing through the yeast golgi apparatus enzymes required for yeast prohormone processing intracellular targeting and the structural conservation of a prohormone-proeassing endoprotease compartmental organization of golgispecific protein modification and vacuolar protein sorting events defined in a yeast secl (nsf) mutant identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease the synthesis and function of proteases in saccharomyces cerevisiae: genetic approaches assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar h+-atpase rate of bulk flow from the golgi to the plasma membrane identification of a putative yeast homolog of the mammalian ~ chains of the clathrin-associated complexes a new class of lysosomal/vacuolar protein sorting signals intraceilular sorting and processing of a yeast vacuolar hydrolase: proteinase a propeptide contains vacuolar targeting information the biogenesis of lysosomes protein glycosylation in yeast the erd gene determines the specificity of the luminal er protein retention system mutations in the cytoplasmic domain of the kd marmose -phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis a specific transmembmne domain of a coronavirus e glycoprotein is required for its retention in the golgi regiou a cooh-terminal signal prevents secretion of luminal er proteins yeast manno-protein biosynthesis: solubilization and selective assay of four mannosyltransferases short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum order of events in the yeast secretory pathway a short sequence in the cooh terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum clathrin: a role in the intracellular retention of a golgi membrane protein. science (wash, dc) sorting of soluble er proteins in yeast targeting of a lysosomal membrane prorein: a tyrosine-coutaining endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi immunolocalizatiou of kex protease identifies a putative late gotgi compartment in the yeast saccharo-m ,~es cerevisiae membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment a saccharomyces cerevisiae genomic plasmid bank based on a centromerecontaining shuttle vector endccytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site protein sorting by selective retention in the endoplasmic retieulum and golgi stack erd , a yeast gene required for the receptor-mediated retrieval of luminal er proteins from the secretory pathway protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase y resides in the propeptide a family of yeast expression vectors containing the phage fl intergenic region the rate of bulk flow from the endoplasmic reticulum to the celt surface posttranslational processing of the prohormone-cleaving kex protease in the saccharomyces cerevisiae secretory pathway accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail we thank marc lussier, kathryn hill, and charlie boone for reading the manuscript; anne-marie sdicu for assistance in manuscript preparation; marc lussier for immunofluorescence quantitation data; joe horecka, steve nothwehr, chris roberts, george sprague, and tom stevens for communication of results prior to publication; chris roberts and tom stevens for providing the monoclonal antibody against the -kd subunit of the vacuolar h +-atpase; kevin redding for his immunottuorescence protocol; vivian mckay for the mnnl disrupted strain; and robert la.marcbe and guy l'i-ieureux for photographic work.this work was supported by natural sciences and engineering research council strategic and operating grams. at the time of this work a. cooper was a canadian commonwealth scholar.received for publication november and in revised form september t . key: cord- - d f k authors: mall, sanjay; malcolm east, j.; lee, anthony g. title: transmembrane α helices date: - - journal: curr top membr doi: . /s - ( ) - sha: doc_id: cord_uid: d f k this chapter discusses effects of intrinsic membrane proteins on lipid bilayers and model transmembrane α helices. incorporation of a protein into a lipid bilayer has significant effects on the properties of the bilayer. the rough surface presented by a protein to the surrounding lipid bilayer tends to produce poor packing unless the lipid fatty acyl chains distort to match the surface of the protein. in a liquid crystalline bilayer the lipid fatty acyl chains are disordered, because the chains undergo extensive wobbling fluctuations. the presence of a rigid protein surface reduces the extent of these motional fluctuations. however, the chains tilt and become conformationally disordered to maximize contact with the rough surface of the protein. the net result is that the presence of a protein leads to decreased order for the chains, with a wide range of chain orientations relative to the bilayer normal, but with reduced extent and rate of motion. because of the reduced motion, lipids adjacent to membrane proteins are often referred to as being motionally restricted. it is clear that the reasons for the disorder of the bulk lipids and the disorder of the lipids adjacent to the protein are different; for the bulk phospholipids, the disorder is dynamic, whereas, for the boundary lipids the disorder is static. bilayer of /~, about residues will be required to span the core of the bilayer. the residues in the transmembrane region will be predominantly hydrophobic. however, for membrane proteins such as transporters and ion channels the transmembrane ot helices will also have to contain polar groups; the transmembrane helices will then be amphipathic rather than just hydrophobic. an extreme case could be a transmembrane a helix totally surrounded by other t~ helices in the center of a helical bundle: such a helix would not need to be hydrophobic at all, because it is not in direct contact with the lipid bilayer. however, although small in number, the available high-resolution structures for membrane proteins show no evidence for the presence of such purely hydrophilic transmembrane ot helices. it may be that the process of insertion of membrane proteins into the membrane during biogenesis requires all the transmembrane c~ helices to be relatively hydrophobic. analysis of the compositions of a large number of membrane proteins predicted to contain single transmembrane ot helices has shown that the amino acid composition of a transmembrane ot helix is distinctly different from that of hydrophobic a helices in water-soluble proteins. as expected, hydrophobic residues make up the bulk of the residues, the most common being leu (landolt-marticorena et al., ; wallin et al., ) . amino acids essentially excluded are the basic (arg and lys) and acidic (asp and glu) amino acids and their amide counterparts (asn and gin). transmembrane c~ helices are, however, relatively rich in bulky residues, such as ile, val, and thr, which, in water-soluble proteins, are classed as membrane destabilizers (their bulky side chains interfere sterically with the carbonyl oxygen in the preceding turn of the ot helix and thus destabilize the helical conformation). thus, factors such as residue volume and packing, which are important in determining helix stability in water-soluble proteins, are not so important for transmembrane ct helices, at least for membrane proteins containing single transmembrane c~ helices; effects of large residue volume will, in the membrane, be balanced by the favorable hydrophobic interactions of a large side chain with the fatty acyl chains. in water-soluble proteins, the conformationally flexible gly residue is also classed as a helix breaker, because it is an intrinsically flexible residue with the potential to adopt most of the dihedral angles available in a ramachandran plot. the observation that gly is quite common in transmembrane ot helices suggests that its potential flexibility is constrained in the bilayer environment. because gly possesses the smallest of all the side chains, it may play a role in mediating helix-helix interactions and packing in the membrane. the polar amino acids most commonly found within transmembrane ot helices are cys, thr, and ser. these residues can be stabilized within a hydrophobic environment by hydrogen bonding between their polar side chains and the peptide backbone at positions i - and i - (eilers et al., ) . figure shows the positional preferences of the residues in the transmembrane ot helices of type i membrane proteins with a single transmembrane c~ helix oriented figure positional preferences for amino acids in the transmembrane domains of human type i membrane proteins with single transmembrane a helices. modified from landolt-marticorena et al. ( ) . with its c-terminus on the cytoplasmic side of the membrane (landolt-marticoreno et al., ) . the amino-terminal end of the transmembrane domain contains an lie-rich region followed by a val-enriched region. the carboxyl-terminal half of the transmembrane a helix is leu-rich. ala is found randomly distributed throughout the transmembrane domain. aromatic residues are found located preferentially in the boundary regions, with trp at either end of the transmembrane domain, but with tyr and phe only at the carboxyl-terminal boundary. unlike the other aromatic amino acids, phe, is also found in the hydrophobic segment as well as in the boundary region. the polar regions flanking the transmembrane domain are enriched in arg and lys on the c-terminal side; asn, ser, and pro are enriched in the n-terminal flanking region. the presence of a positively charged c-terminus (cytoplasmic) could play a role in the process of insertion into the membrane, according to the inside positive rule of von heijne ( ) . the presence of particular residues at the n-and c-terminal ends of the helices could also be important in meeting the requirement to "cap" the ends of the ot helices; the initial four --nh and final four --c=o groups of an ot helix have no hydrogen-bonding partners provided by the peptide backbone of the a helix itself, and so suitable hydrogen-bonding partners have to be provided in some other way. one way is to extend the helix by three or four residues at each end with polar residues containing suitable hydrogen-bonding partners such as pro and ash. alternatively, if the hydrophobic, nonpolar residues in the transmembrane c~ helix extend into the headgroup region, hydrogen bonds could form with suitable groups in the glycerol backbone and headgroup regions of the lipid bilayer. either way, if about residues are required to span the hydrophobic core of the bilayer, the total helix length could be up to residues. the result is that there is a degree of indeterminacy in where the ends of transmembrane helices should be drawn; the precise ends of transmembrane ~ helices are often not known. the observed preference for trp and tyr residues for the ends of transmembrane ct helices agrees with measurements of the binding of small peptides at the lipid-water interface, which show that aromatic residues have a preference for the interface (wimley and white, ) . further, a number of small tryptophan analogues have been shown to bind in the glycerol backbone and lipid headgroup region of the bilayer, stabilized partly by location of the aromatic ring in the electrostatically complex environment provided by this region of the bilayer, and partly by exclusion of the fiat, rigid ring system from the hydrocarbon core of the bilayer for entropic reasons (yau et al., ) . thus, although it is agreed that aromatic residues at the ends of transmembrane a helices probably act as "floats" at the interface serving to fix the helix within the lipid bilayer, it is unclear whether the aromatic rings are located in the hydrocarbon or the headgroup region of the bilayer. this uncertainty is also apparent in the crystal structures of a number of membrane proteins. for example, the trp residues in the bacterial potassium channel kcsa (doyle et al., ) are found clustered at the ends of the transmembrane ot helices, forming clear bands on the two sides of the membrane, as shown in fig. . however, the tyr residues clearly form a band on the periplasmic side of the membrane above the band formed by the trp residues. similarly, in the bacterial photosynthetic reaction center (rees etal., ) the majority of the trp residues are found near the periplasmic side of the protein near the ends of the transmembrane c¢ helices, as shown in fig. . however, the band of trp residues is more diffuse than in kcsa, and some trp residues are likely to be located in the hydrocarbon core and some in the headgroup region. the average number of figure the crystal structure of the potassium channel kcsa. a cross section with just two of the four identical subunits is shown. trl o residues are shown in space-fill representation and tyr residues are shown in ball-and-stick representation. two potassium ions in space-fill representation are shown moving through the channel. the separation between the two planes representing the outer edges of the trp residues is /~. (protein data bank [pdb] file ib .) p ¢ figure the structure of the l and m subunits of the photosynthetic reaction center of rhodobacter sphaeroides. trp residues are shown in ball-and-stick representation. an approximate location for the hydrophobic core of the bilayer of thickness a is shown, as defined by the surface covered by detergent. (pdb file aij.) residues in the transmembrane c~ helices of the bacterial photoreaction center is , corresponding to a length of about /~. the stretch of hydrophobic residues in these helices is, however, only about amino acids or about . /~ long (ermler et al., ; michel and deisenhofer, ) . this matches the thickness of the nonpolar region of the complex (about /~) as defined experimentally as the part covered by detergent in the crystal (roth et al., (roth et al., , . detergent is seen to cover some of the trp residues on the periplasmic side of the membrane, but not others (roth et al., ) . the distribution of trp residues on the cytoplasmic side of the complex is much less distinct than on the periplasmic side (fig. ). if the hydrocarbon core of the bilayer around the complex does have a thickness of ]~, then again the trp residues on the cytoplasmic side of the membrane will be located in both the hydrocarbon core and the headgroup regions of the bilayer (fig. ) . in the ca +-atpase of skeletal muscle sarcoplasmic reticulum the situation is more complex, as shown in fig. (toyoshima et al., ) . many of the transmembrane o~ helices extend above the membrane surface to form a central stalk linking the transmembrane region to the cytoplasmic head of the protein. as a consequence some of the helices are very long; helix m , for example, contains residues. a ring of trp residues can be seen on the cytoplasmic side of the membrane helping to define the location of the membrane surface (fig. ) . a lys residue (lys- ) in transmembrane c~ helix m can be seen pointing up from the hydrophobic core of the bilayer like a snorkel. because the cost of burying a charged residue in the hydrophobic core of a bilayer is very high (about kj mo - for a lys residue ; engelman et al., ) , it is likely that the amino group on lys- will be located at the interface; the trp residues in the ca +-atpase will then be located in the headgroup region of the bilayer. the structure of the ca +-atpase is also unusual in that the first transmembrane ot helix contains two polar residues, asp- and arg- , pointing out into the hydrocarbon core; presumably, stacking of asp- against arg- allows formation of an ion pair. the distribution of trp residues on the lumenal face of the ca +-atpase is much more diffuse than on the cytoplasmic side. the hydrophobic thickness of the ca +-atpase could be expected to be about /~, because that is the hydrophobic thickness of a bilayer of di(c : )pc, the phospholipid that supports highest activity for the atpase . however, as shown in fig. , this definition locates the lumenal loops between transmembrane ~ helices m and m i phospholipid designations are pc, ps, and pa for phosphatidylcholine, phosphatidylserine, and phosphatidic acid, respectively. fatty acyl chains are given in the format m:n, where m is the number of carbon atoms and n is the number of double bonds. thus, for example, dioleoylphosphatidylcholine is di(c : )pc. and between m and m within the hydrocarbon core. further, it locates a lys residue (lys- ) totally within the hydrocarbon core, which seems unlikely. the hydrophobic thickness of the bilayer would have to be about a to locate the two interhelical loops and lys- at the lumenal surface (fig. ) ; this is close to the thickness of a bilayer of di(c :i)pc ( a). the crystal structure shown in fig. corresponds to the ca +-bound, e conformation of the atpase. it has been shown that the e conformation of the atpase is favored by di(c : )pc, whereas di(c : )pc favors the other major conformation of the atpase, e (starling et al., ) . thus, it is possible that conformational changes within the transmembrane region of the ca +-atpase lead to changes in the interhelical loops and thus to changes in the effective hydrophobic thickness of the atpase (lee and east, ) . incorporation of a protein into a lipid bilayer can be expected to have significant effects on the properties of the bilayer. the rough surface presented by a protein to the surrounding lipid bilayer will tend to produce poor packing unless the lipid fatty acyl chains distort to match the surface of the protein. in a liquid crystalline bilayer the lipid fatty acyl chains are disordered, because the chains undergo extensive wobbling fluctuations. the presence of a rigid protein surface would be expected to reduce the extent of these motional fluctuations. however, the chains will have to tilt and become conformationally disordered to maximize contact with the rough surface of the protein. the net result is that the presence of a protein will lead to decreased order for the chains, with a wide range of chain orientations relative to the bilayer normal, but with reduced extent and rate of motion. because of the reduced motion, lipids adjacent to membrane proteins are often referred to as being motionally restricted. it is clear, therefore, that the reasons for the disorder of the bulk lipids and the disorder of the lipids adjacent to the protein (the boundary or annular lipids) are different; for the bulk phospholipids the disorder is dynamic, whereas for the boundary lipids the disorder is static. an example is provided by the bacteriorhodopsin trimer, whose crystal structure is unusual in showing a few well-defined lipid molecules (belrhali et al., ; luecke et al., ) . figure shows some of the lipids located at the surface of the trimer. the electron densities for the chains are well defined, but the headgroups are disordered, so that the headgroups could not be identified; the lipids were therefore modeled simply as , -di-o-phytanylsn-propane (belrhali et al., ) . the considerable static disorder of the chains is clear in fig. , the rotational disorder of the chains being necessary to obtain good van der waals contacts with the molecularly rough surface of the bacteriorhodopsin trimer. lipids on the extracellular side of the membrane are better resolved than ~ figure structures of four phospholipid molecules identified in x-ray crystallographic studies of bacteriorhodopsin (belrhali et al., ) . the lipids have been modeled as , -.di-o-phytanyl-snpropane. (pdb file lqhj.) those on the cytoplasmic side; the degree of order of the lipids parallels that of the protein, which is also greater on the extracellular side (grigorieff et al., ) . the average distance between the glycerol backbone oxygens for phospholipids on the two sides of the membrane was . /~ (mitsuoka et al., ) . as expected, this closely matches the hydrophobic length of the transmembrane helices of bacteriorhodopsin; the mean helix length is residues, corresponding to a length of about a. the crystal structure also makes clear the very different conformations adopted by the various lipid molecules located on the surface of the tfimer. for example, one lipid molecule forms a hydrogen bond from its ether oxygens to a tyrosine --oh group at the end of a transmembrane a helix (fig. ; belrhali et al., ; essen et al., ) . the result is that the strength of the interactions of individual boundary lipid molecules with the protein will be different. the disorder of the chains seen in fig. is consistent with the results of molecular dynamics simulations of the bacteriorhodopsin trimer in a bilayer of diphytanyl phosphatidylglycerophosphate (edholm et al., ) . the molecular dynamics simulation agrees with experiment in predicting higher order for both the lipids and the protein on the extracellular side of the membrane; fluctuations in the loops and the ends of helices on the cytoplasmic side of the membrane are greater than on the extracellular side. this is also seen in fluctuations of the lipids, with lipids on the cytoplasmic side of the membrane fluctuating more strongly than those on the extracellular side (edholm et al., ) . the calculated order parameters for the chains are low, mainly due to a static tilt of the chains necessary to allow them to nestle against the rough surface of the protein. the chains in the purple membrane behave more like parts of the protein than parts of a fluid lipid phase, consistent with the idea of boundary lipids (edholm et al., ) . the boundary lipids and the bulk lipids in a membrane can be distinguished experimentally in many systems, because of the static disorder of the boundary lipids and the dynamic disorder of the bulk lipids. static and dynamic disorder give rise to very different electron spin resonance (esr) spectra for spin-labeled lipids, and esr spectra for membrane protein systems usually show two components, one "immobilized," corresponding to boundary lipid, and one relatively mobile, corresponding to bulk lipid (devaux and seigneuret, ; marsh, ) . studies with oriented samples have confirmed a wide range of orientational distributions for the boundary lipid, in contrast to the bulk lipid phase, where motion of the lipid long axis is about the bilayer normal (jost et al., ; pates and marsh, ) . a particularly important feature of a membrane protein as far as the lipid bilayer is concerned is the thickness of the transmembrane region of the protein. the cost of exposing hydrophobic fatty acyl chains or protein residues to water is such that the hydrophobic thickness of the protein should match that of the bilayer. the question then is how the system responds when these do not match. most models of hydrophobic mismatch assume that the lipid chains in the vicinity of the protein adjust their length to that of the protein, with the protein acting as a rigid body. when the thickness of the bilayer is less than the hydrophobic length of the peptide the lipid chains must be stretched. conversely, when the thickness of the bilayer is greater than the hydrophobic length of the peptide the lipid chains must be compressed (fig. ) . stretching the fatty acyl chains will effectively decrease the surface area they occupy in the membrane surface, and, conversely, compressing the chains will increase the effective area occupied in the surface (fig. ) . thus, figure the result of a mismatch between the hydrophobic length of a peptide and the hydrophobic thickness of a lipid bilayer. left: positive hydrophobic mismatch (dp > dl). right: negative mismatch (dp< de). the top shows a "side" view of the chain packing around the peptide and the bottom shows a top view, illustrating the variation in chain cross-sectional area with distance from the peptide. figure based on fattal and ben-shaul ( ) . changes in fatty acyl chain order are linked to changes in average interfacial areas per lipid molecule. a number of terms have been suggested to contribute to the total free energy cost of deforming a lipid bilayer around a protein molecule (fattal and ben-shaul, ; nielsen et al., ): . loss of conformational entropy of the chains imposed by the presence of the rigid protein wall . bilayer compression/expansion energy due to changes in the membrane thickness . surface energy changes due to changes in the area of the bilayer-water interface . splay energy due to changes in the cross-sectional energy available to the chains along their length, resulting from curvature of the monolayer surface near the protein a number of models have been proposed to estimate these terms. fattal and ben-shaul ( ) calculated the total lipid-protein interaction free energy as the sum of chain and headgroup terms. for the chains the loss of conformational entropy imposed by the rigid protein wall was positive (unfavorable) even for perfect hydrophobic matching. the other contribution to the chain term arose from the requirement for hydrophobic matching and the consequent stretching or compression of the chain. the term due to the headgroup region was treated as an interracial free energy, including an attractive term associated with exposure of the hydrocarbon core to the aqueous medium and a repulsive term due to electrostatic and excluded-volume interactions between the headgroups (excluded-volume interactions signify that no two atoms can occupy the same position in space). the resulting profile of energy of interaction as a function of hydrophobic mismatch was fairly symmetrical about the point of zero mismatch. the lipid perturbation energy f (in units of kt per angstrom of protein circumference) calculated by ben-shaul ( ) fits to the equation where dp and dl are the hydrophobic thicknesses of the protein and lipid bilayer, respectively, and the unperturbed bilayer thickness is . /~. the hydrophobic thickness of a bilayer of phosphatidylcholine in the liquid crystalline phase is given by where n is the number of carbon atoms in the fatty acyl chain (lewis and engelman, ; sperotto and mouritsen, ) . a simple, but crude calculation gives an idea of the size of the effect that can be expected from hydrophobic mismatch. it is assumed that the protein is very large and so appears fiat to a lipid molecule. it is also assumed that all the lipid perturbation energy is concentrated in the first shell of lipids around the protein. equation ( ) then shows that if a lipid occupies a of the protein circumference, the lipid-protein interaction energy would change by . kj tool - for a hydrophobic mismatch of a, corresponding to an increase in fatty acyl chain length of carbons, and by . kj mol -z for a hydrophobic mismatch of . a, corresponding to an increase in acyl chain length of carbons. changes in interaction energies of . and . kj mo - correspond to decreases in the lipid-protein binding constant by factors of . and n, respectively. if the change in lipid-protein interaction energy were to propagate out from the protein surface to affect more than the first shell of lipids, effects of hydrophobic mismatch would be reduced. for example, if effects were averaged over three shells of lipids, changes in fatty acyl chain lengths by and carbons from that giving optimal interaction would decrease lipid-protein binding constants by factors of . and , respectively. energies of the magnitude calculated by ben-shaul ( ) are easily sufficient to result in conformational changes on a protein. if a protein conformational change results in a change in the hydrophobic thickness of the protein, the change will result in a deformation of the adjacent bilayer. because the equilibrium constant describing the equilibrium between two conformational states of a protein is determined by the total free energy difference between the two states, the energetic cost of the membrane deformation will contribute toward the equilibrium constant. the approach adopted by nielsen et al. ( ) came to rather similar conclusions. the most important energy terms were found to be the splay energy and the compression-expansion term, the splay energy term being most important close to the protein, with the compression-expansion term being more import'ant further from the protein. even though the bilayer deformation was calculated to extend some a from the protein, most of the deformation was found concentrated in the component immediately adjacent to the protein. an alternative model for mismatch is the mattress model of bloom ( , ) . this again expresses mismatch as two terms. the first is an excess hydrophobic free energy associated with exposing either lipid chains or the protein surface to the aqueous medium. the second is proportional to the contact area between the lipid chains and the hydrophobic surface of the protein. the calculations showed that, for a protein of hydrophobic thickness a, which matches a bilayer ofdi(c : )pc in the liquid crystalline state, the binding constant for di(c : )pc is about a factor of . greater than for a bilayer of di(c : )pc, which will give a bilayer too thick by a (sperotto and mouritsen, ) . thus, effects of mismatch calculated in this way are similar to those calculated using the approach of fattal and ben-shaul ( ) . the importance of hydrophobic matching has been confirmed in a number of experimental studies (dumas et al., ; killian, ) . for example, although bacteriorhodopsin has relatively little effect on the phase transition temperatures of di(c : )pc or di(c : )pc (alonso et al., ) , it increases the transition temperature of di(c : )pc and decreases that of di(c : )pc (piknova etal., ) . this is consistent with hydrophobic matching models; because di(c : )pc gives a too thin bilayer in the liquid crystalline phase, bacteriorhodopsin favors the gel phase, whereas di(c : )pc gives a too thick bilayer in the gel phase, so that bacteriorhodopsin favors the liquid crystalline phase. hydrophobic matching could also be the explanation for the unexpected observation that, in mixtures of di(c : )pc and di(c : )pc at temperatures where the mixture contains both gel and liquid crystalline phases, bacteriorhodopsin partitions equally between the two phases (piknova et al., ; schram and thompson, ) . this contrasts with the observed exclusion of bacteriorhodopsin from gel-phase lipid in mixtures containing single species of phospholipid (alonso et al., ; cherry et al., ) . it has been suggested that this shows that the requirements of hydrophobic matching are of prime importance; the hydrophobic thickness of bacteriorhodopsin is intermediate between the hydrophobic thickness of di(c : )pc in the gel phase and di(c : )pc in the liquid crystalline phase, so that bacteriorhodopsin shows little preference between the two. in mixtures of di(c : )pc and di(c : )pc, where, at low temperatures, two separate gel phases are formed, one enriched in di(c : )pc and one enriched in di(c : )pc, bacteriorhodopsin partitions very strongly into the di(c : )pc-enriched domains; this could be because the hydrophobic thickness of bacteriorhodopsin is better matched to gelstate di(c : )pc than to gel-state di(c : )pc (dumas et al., ) . however, in studies of the ca +-atpase of sarcoplasmic reticulum using either spin-labeled (london and feigenson, ) or brominated phospholipids , strengths of binding of liquid crystalline-phase phospholipids to the atpase were found to be independent of fatty acyl chain length. these results are not consistent with the expectations of hydrophobic matching theory and suggest that, in liquid crystalline bilayers, u-helical membrane proteins are not rigid, but, in fact, can distort to match the thickness of the bilayer. such a distortion could explain why bilayer thickness affects the activity of membrane proteins such as ca +-atpase (lee, ) . the structural distortion could take the form of a change in the tilt of the transmembrane ~ helices with respect to the bilayer normal or could be a change in the packing of the transmembrane c~ helices. an alternative approach to these questions, avoiding the complexity of real membrane proteins, is to use simple model transmembrane ~ helices, which can be synthesized chemically in large quantity. a number of studies have used peptides of the type ac-k -g-ln-k -a-amide (pn) consisting of a long sequence of hydrophobic leu residues capped at both the n-and c-terminal ends with a pair of charged lysine residues. the poly(leu) region forms a maximally stable u helix, particularly in the hydrophobic environment of the lipid bilayer. the charged lys caps were chosen both to anchor the ends of the peptides in the lipid headgroup region and to inhibit the aggregation of the peptides in the membrane. the peptide has been shown to adopt the expected u-helical structure in both liquid crystalline-and gel-phase bilayers (davis et al., ; huschilt et al., ; zhang et al., a) . rates of hydrogen/deuterium exchange for the peptide p in lipid bilayers suggest that at least % of the peptide is in an u-helical conformation in the bilayer, meaning that the whole of the poly(leu) core must be u-helical (zhang et al., b) . rates of hydrogen/deuterium exchange were greater at the n-and c-termini of the peptides than in the middle, suggesting some unraveling of the peptide at its ends (zhang et al, b) . experiments with the peptides of this type suggest that about lipid molecules are required for complete incorporation of the peptide into a bilayer of the appropriate thickness (webb et al., ) . this agrees with estimates from molecular modeling that about - lipid molecules will be required to form a complete bilayer shell around the peptide. at molar ratios of lipid less than this, non-bilayer phases can be induced, particularly when the hydrophobic length of the peptide is less than the hydrophobic thickness of the bilayer and when the peptide contains interfacial aromatic groups (de planque et al., ; killian et al., ; morein et al., ) . effects of single transmembrane u helices on lipid bilayers are likely to be less than those of a protein containing a bundle of transmembrane u helices. the cross-sectional area of a single transmembrane u helix is not much greater than that of a phospholipid molecule in the liquid crystalline phase, so that the hydrophobic surface presented to the lipid molecules is rather small. the structure of the lipid bound to bacteriorhodopsin shown in fig. shows the two chains interacting predominantly with two different transmembrane u helices. this kind of interaction will obviously not be possible with a single transmembrane u helix. less extensive interactions between lipids and single transmembrane u helices than between lipids and membrane proteins is suggested by esr experiments. whereas esr spectra of spin-labeled lipids in the presence of membrane proteins typically show two-component spectra, as described above, esr spectra for lipid bilayers containing the peptide l and for a tryptophan-containing peptide of the type aw (la)nw a are single-component (de planque et al, ; subczynski et al., ) . this means either that the lipid fatty acyl chains are not "immobilized" on the peptide surface or that the rate of exchange between bulk and boundary lipid is fast on the esr time scale (i.e., exchange is faster than s-l). effects of peptides on chain order in di(c : )pc or di(c : )pc measured using deuterium nuclear magnetic resonance (nmr) methods are small in both the liquid crystalline and the gel phases (davis et al., ; de planque et al., de planque et al., , roux et al., ) . thus, addition of a peptide aw (la)tw a to bilayers of di(c : )pc in the liquid crystalline phase resulted in only a . -/~ increase in thickness (de planque et al., ) , whereas about an -/~ increase would be necessary for the bilayer thickness to match the hydrophobic length of the peptide. similarly, nezil and bloom ( ) estimated that the peptide p increased the thickness of a bilayer of (c : ,c : )pc by just . /~, despite the hydrophobic mismatch between the peptide and the lipid bilayer being ca. a. increases in chain order caused by p in (c : ,c : )pc in the liquid crystalline phase were detected using esr, but again the effects were small (subczynski et al., ) . effects of peptides on chain order will depend on the relative hydrophobic length of the peptide compared to the hydrophobic thickness of the bilayer, with long peptides decreasing order and short peptides increasing order, and such effects have been detected using infrared (ir) spectroscopy, but again effects were small (zhang et al., a) . thus it appears that lipids will distort slightly to improve the match between the hydrophobic length of the peptide and the hydrophobic thickness of the bilayer, but the extent of these modifications is very limited and much less than required to produce full matching. a number of studies have been published on the effects of these peptides on the phase transition properties of lipid bilayers. addition of the peptide pi to bilayers of di(ci : )pc both broadens the main gel-to-liquid crystalline phase transition and decreases the enthalpy of the transition (morrow et al., ) . similar effects have been seen on incorporation of membrane proteins such as bacteriorhodopsin and ca +-atpase (alonso et al., ; gomez-fernandez et al., ) . the decrease in enthalpy of the transition has often been taken to mean that the lipids adjacent to the protein (the boundary lipids) are very strongly perturbed by the peptide and so are unable to take part in the normal phase transition: they are effectively withdrawn from the transition. however, deuterium nmr spectra of mixtures of p and di(c : )pc above and below the phase transition are typical of liquid crystalline-and gel-phase lipids, respectively, with no evidence for any "special" lipid unable to take part in the phase transition . similarly, as already described, esr spectra of spin-labeled lipids show the presence of a single type of lipid in the system, not separate bulk and boundary lipids in slow exchange (subczynski et al., ) . thus the peptides (or proteins) do not remove lipid from the main transition, but, rather, perturb the whole lipid bilayer. the peptide decreases the enthalpy difference between the liquid crystalline and gel phases, whereas the lipids in the bilayer remain recognizably liquid crystalline or gel (morrow et al., ) . morrow et al. ( ) showed that mixtures of lipids and peptides can be modeled in terms of regular solution theory (lee, ) . unfortunately, the number of free parameters in fitting to regular solution theory is high, so that little useful information is obtained from the analysis, apart from showing that the data are consistent with regular solution theory. effects of peptides or proteins on phase transition properties have therefore been interpreted qualitatively in terms of a twocomponent model in which one component is more or less unperturbed bulk lipid and the other is highly perturbed boundary lipid, which undergoes a broad phase transition of low enthalpy; this approach works for peptides of the poly(leu) type, but, for some reason, does not work with peptides of the k (la)nk type (zhang et al., a (zhang et al., , . differential scanning calorimetry (dsc) thermograms for mixtures with the poly(leu) peptides have been fitted to two components, attributed to the phase transitions of peptide-free and boundary lipid, respectively. the phase transition temperature for the peptide-free component is slightly less than that for pure lipid; this is probably due to the normal colligative effects that will follow from mixing the "pure" lipid phase with the boundary lipid, the latter acting as an "impurity." the phase transition temperature for the boundary lipid is higher than the bulk transition temperature for short-chain lipids, but is lower for long-chain lipids (zhang et al, a) . the same observation has been made for membrane proteins (dumas et al., ; piknova et al., ) . this is consistent with the idea that short fatty acyl chains have to stretch to match the hydrophobic thickness of the membrane protein, whereas long fatty acyl chains have to compress. however, the experimental changes are much smaller than expected from models ofhydrophobic matching. further insights into how transmembrane ot helices might interact with lipid molecules in a bilayer have come from molecular dynamics simulations. one study was of a transmembrane ot helix of alanine residues in a bilayer of di(c : )pc in the liquid crystalline state (shen et al., ) . the peptide is not an ideal model for a transmembrane ot helix, because it lacks charged groups at each end to interact with the polar headgroups of the phospholipids. nevertheless many features of the simulation are informative. the simulation was started with the peptide as a pure ot helix. the central residues (ala- - ), which interacted just with the lipid fatty acyl chains, remained as a stable t helix. the n-and c-terminal regions of the ot helix, located in the lipid headgroup region, were less stable and fluctuated more, because of transient hydrogen bonding between the peptide bond amide hydrogen and the phosphate or fatty acyl ester oxygen atoms and the water; as a result, the ends of the helices become frayed. the length of the central helical region oscillated slightly about a -a average expected for an ot helix, varying between and a. the helix was tilted up to ° with respect to the bilayer normal. because the helix contains no resides that would make strong contacts in the headgroup region or with water, there is no reason for it not to tilt (shen et al., ) . tilting in fact allows more hydrophobic contact by allowing more of the ala residues to be located in the core of the bilayer. the presence of the peptide had little effect on the calculated properties of the bilayer. the average bilayer thickness was not significantly changed, although the average order parameter for the ch groups in the chains decreased in the presence of the peptide. many different lipid molecules contributed to the immediate surroundings of the peptide (shen et al., ) . even if the fatty acyl chain of a particular lipid was immediately adjacent to the peptide, the headgroup of the lipid could be a substantial distance away. given the diameter of the helix and the size of the phosphate group, a phosphate immediately adjacent to the helix would be between and a away from the center of the helix (shen et al., ) . on average, five lipid molecules having their chains adjacent to the helix also had their headgroups adjacent, using this definition. however, the headgroup, as given by the position of the phosphorus atom, could be up to - a away. since the average distance between lipid headgroups is a, this puts these lipids in the "second" shell around the peptide. other lipids existed between these extremes, suggesting a very diffuse environment around the protein rather than a discrete set of well-ordered shells of lipid. only rarely did an entire lipid molecule pack tightly around the helix. the shell around the helix contains contributions from a large number of lipid molecules, each contributing a small number of atoms (shen et al., ) . thus there is no evidence for a distinct shell of lipids around the peptide and any perturbation of the lipids extends out just a few angstroms. the lack of a clear shell of lipids around the poly(ala) peptide contrasts with the boundary lipids observed for membrane proteins and illustrated for bacteriorhodopsin in figs. and . in part this could be an intrinsic feature of single transmembrane a helices, which will not be able to present a large surface area on which fatty acyl chains could be immobilized. the regular structure of a poly(ala) peptide compared to the rough surface of a typical or-helical peptide might also contribute to the lack of an immobilization shell of lipids. however, a significant factor is also likely to be the lack of polar groups at the ends of the peptide able to interact with the phospholipid headgroups. the importance of polar groups at the ends of the peptides has been shown in a simulation of isolated helices from bacteriorhodopsin (woolf, ) . the simulations show that a small number of the lipids surrounding a helix interact with it much more strongly than other lipids, due to a combination of van der waals (mediated by chains) and charge interactions (mediated by beadgroups) (woolf, ) . a molecular dynamics simulation of the peptide el in a bilayer of di(c : )pc in the liquid crystalline phase showed that the peptide tilted with an average angle of . ° with respect to the bilayer normal, even though the thickness of the hydrophobic region of the bilayer ( a) was a good match to the length of the ~ helix, a (belohorcova et al., ) . a molecular dynamics simulation has been reported for pf coat protein in di(c : )pc (roux and woolf, ) . the coat protein contains an amphipathic a helix on the bilayer surface and a hydrophobic transmembrane a helix. fatty acyl chains next to the transmembrane helix were slightly more ordered than bulk lipids (roux and woolf, ) . similarly, in a simulation of the seventh transmembrane c~ helix of the serotonin ht receptor in di(c : )pc, lipids in contact with the peptide had slightly higher order parameters than bulk lipids (duong et al., ) . the theories described above show that there will be an energetic cost associated with any change in the thickness of a bilayer. this would be reflected in values for the equilibrium constant describing the binding of lipids to the protein. a lipid that can bind to a protein without a change in bilayer thickness would bind more strongly to the protein than one for which binding required a change in bilayer thickness. the strength of interaction between a peptide and a phospholipid in a bilayer can be measured using a fluorescence quenching method (webb et al, ) . peptides used are of the type kkgl wl kka (l ) and kkgl wl kka (l ) containing a central trp residue as a fluorescence reporter group. the peptide is incorporated into bilayers containing the brominated phospholipid dibromostearoylphosphatidylcholine (di(br c : )pc); di(br c : )pc behaves much like a conventional phospholipid with unsaturated fatty acyl chains, because the bulky bromine atoms have effects on lipid packing similar to those of a cis double bond . contact between the bromine atoms in the lipid and the trp residue in the peptide leads to fluorescence quenching. in mixtures of brominated and nonbrominated phospholipids, the degree of quenching of the fluorescence of the tryptophan residue is related to the fraction of the surrounding (boundary) phospholipid molecules that are brominated, and thus to the strength of binding of the nonbrominated lipid to the peptide. an example of the method is shown in fig. . the fluorescence intensity for the peptide l incorporated into bilayers of di(br c : )pc at a molar ratio of lipid to peptide of : is % of that in di(c : )pc, demonstrating highly efficient quenching of the tryptophan by the bromine-containing fatty acyl chains (fig. ) . the fluorescence intensity in mixtures of di(br c : )pc and di(c : )pc decreases with increasing content of di(br c : )pc, reflecting the increasing number of boundary lipids that will be di(br c : )pc. as shown in fig. , fluorescence quenching curves for l in mixtures of di(br c : )pc and (c : )pc show more fluorescence quenching at intermediate mole fractions of di(br c : )pc than in mixtures with di(c : )pc. this shows that di(c : i)pc binds more strongly to the peptide than does di(c : )pc. the results can be analyzed to give relative lipid-binding constants, as described in webb et al. ( ) . these lipid-binding constants for l and l are given in table i. for l strongest binding is seen with di(c : )pc, for which the relative binding constant is about double that for di(c : )pc (table i ). the hydrophobic length of the peptide l is about a, calculated for a stretch of hydrophobic residues in total, with a helix translation of . a per residue. thus, strongest binding is seen when the hydrophobic length of the peptide matches the hydrophobic thickness of the bilayer, as expected from theories of hydrophobic mismatch. however, relative binding constants do not continue to decrease with decreasing chain length from di(c : )pc to di(c : )pc as would have been predicted (table i ). an even larger deviation from theoretical predictions is observed with the short peptide l (table i ). in this case strongest binding is observed with di(c : )pc, with binding decreasing with decreasing chain length to di(c :i)pc, as expected. however, the peptide was found not to incorporate at all into bilayers of di(c :l)pc, instead forming aggregates of peptide separate from the bilayer. ren et al. ( ) obtained very similar results, except that under their conditions, unincorporated peptide bound to the surface of the lipid bilayer, with the long axis of the peptide parallel to the surface. similarly, l was found to be only partly incorporated into ~bilayer hydrophobic thickness d calculated from d = . (n - ), where n is the number of carbon atoms in the fatty acyl chain (sperotto and mouritsen, ) . bestimated hydrophobic length is a for li and a for l . bilayers of di(c : )pc (webb et al., ) . thus, a short peptide cannot incorporate into a too-thick bilayer. it is suggested that a too-thin bilayer can match to a too-long peptide both by a slight stretching of the lipid and by tilting of the long axis of the helix with respect to the bilayer normal so that its effective length across the bilayer is reduced. however, a too-thick bilayer can only match a too-thin peptide by compression of the lipid, which becomes energetically unfavorable when the difference between the bilayer thickness and the peptide length exceeds about /~ (webb et al., ) . possible effects of aromatic residues at the ends of transmembrane ~ helices have been studied using peptides k gfl wl fk a (f l ) and k gyl wl yk a (y li ), in which one leu residue at each end of the poly(leu) stretch is replaced by either a phe or a tyr (mall et al., ) . in contrast to the results with l , peptide f l incorporated fully into bilayers of di(c : )pc, and y li partitioned partially into di(c : )pc. the fluorescence quenching method was again used to obtain binding constants for phosphatidylcholines to the peptides, measured relative to the binding constant for di(c : )pc (table ii) . the effective hydrophobic length of the peptide y li might be expected to be somewhat greater than that of l ; if the peptide is modeled as an c~ helix with the two tyr residues oriented to be roughly parallel to the long axis of the c~ helix, the distance between the two tyr--oh groups is ca. a, about /~ greater than the hydrophobic length of l . the hydrophobic length of y l calculated in this way matches the hydrophobic thickness of a bilayer of di(c : )pc, whereas the relative lipid-binding constants increase from di(c : )pc to di(c : )pc (table ii) . similarly, relative binding constants for f li increase with increasing chain length from di(c :i)pc to di(c : i)pc. the results with y l and f li show that introduction of aromatic "hydrophobic thickness of the bilayer calculated from d = . (n - ), where n is the number of carbon atoms in the fatty acyl chain (sperotto and mouritsen, ). residues at the two ends of the hydrophobic sequence increases the ability of the short peptides to partition into thick lipid bilayers. the observation that the highest relative binding constant is obtained with bilayers considerably thicker than the calculated hydrophobic length of the peptides suggests that the presence of aromatic residues at the ends of the helices could lead to marked thinning of the bilayer around the peptides (mall et al., ) . the chain length dependence of lipid binding to y l is much less marked than for the shorter peptides; the relative binding constant increases from di(c : )pc to di(c :i)pc, but then hardly changes with increasing chain length between di(c :i)pc and di(c :)pc (table ii) . this contrasts with l , which shows markedly stronger interaction with di(c :l)pc than with phospholipids with shorter or longer chains. this again suggests that the introduction of the two tyr residues leads to an increase in the thickness of the bilayer with which optimal interaction of the peptide is observed. interactions between transmembrane c~ helices and the phospholipid headgroups also have to be considered. using the fluorescence quenching method, it was shown that a small number of anionic phospholipid molecules (possibly just one) bound strongly to the peptide l , the remaining molecules binding with an affinity equal to that of phosphatidylcholine. the binding constant for the strongly bound phosphatidic acid molecule relative to phosphatidylcholine in a medium of low ionic strength was . (in mole fraction units), corresponding to a difference in unitary binding energies of - . kj mo - . at ph . , phosphatidic acid bears a single negative charge (cevc, ) . the binding constant for phosphatidic acid changed little with ionic strength, suggesting that the interaction with the positively charged peptide did not follow simply from a high positive potential in the vicinity of the positively charged lys residues on the peptide, increasing the local concentration of anionic phospholipid. the energy of interaction between two ions u is given by where zl and z are the charges on the two ions eo is the permittivity of a vacuum, er is the relative permittivity (dielectric constant) of the medium, and r is the distance between the two ions. assuming a dielectric constant of . (water), we find that an energy of interaction of . kj tool - corresponds to a distance of separation between two monovalent ions of . /~. this therefore suggests that strong interaction requires the anionic headgroup of phosphatidic acid to be in close contact with one of the lys residues on the peptide. once this strong interaction with a single phosphatidic acid molecule has been made, other phosphatidic acid molecules will then interact with el relatively nonspecifically, with a binding constant relative to phosphatidylcholine close to . the relative binding constants for phosphatidylserine were less than for phosphatidic acid and are more sensitive to ionic strength . for phosphatidylserine, the presence of the positively charged ammonium group as well as the negatively charged carboxyl group in the headgroup region may reduce interaction with the positively charged peptide. in contrast to l , the binding constants for anionic phospholipids to l are very similar to those for zwitterionic phospholipids, with a relative binding constant close to . it could be that tilting of l in the bilayer, necessary to match the hydrophobic length of l to the hydrophobic thickness of a bilayer of di(c : )pc, locates the lys residues on the peptide too far from the lipid headgroup region to allow a strong interaction between the anionic phospholipid and the peptide. both phosphatidylserine and phosphatidic acid bind more strongly to the peptides yell and yel than does phosphatidylcholine, the effect of anionic phospholipid decreasing slightly with increasing ionic strength. however, in this case the experiments are consistent with a model in which the binding constants for all the anionic phospholipid molecules binding to the peptide are increased slightly (mall et al., ) . this suggests that the presence of the tyr residues prevents close association of the anionic phospholipid group with the cationic lys residues. these results suggest that the effects of charge on the interactions between phospholipids and transmembrane ot helices will often be rather small and will be strongly dependent on the detailed structure of the peptide and its orientation in the membrane. this picture is consistent with the results of the molecular dynamics simulations of individual ~ helices of bacteriorhodopsin in bilayers of di(ci : )pc, which showed that a small proportion of the lipid molecules interacted with the o~ helices much more strongly than the others, and that these strong interactions were dominated by electrostatic terms rather than van der waals terms (woolf, ) . in general, binding constants for phospholipids to membrane proteins also show relatively little selectivity for anionic phospholipids. for example, binding constants for phosphatidic acid and phosphatidylserine relative to phosphatidylcholine are close to for the caz+-atpase (dalton et al., ) , and binding constants for phosphatidic acid and phosphatidylserine for the (na+-k+)-atpase are about twice those for phosphatidylcholine (esmann and marsh, ) . however, there is evidence for the presence of a small number of "special" anionic phospholipids binding to some membrane proteins, acting as "cofactors." an example is provided by cytochrome c oxidase, whose crystal structure shows the presence of a lipid molecule bound between the transmembrane a helices (iwata et al., ) . interaction between an anionic phospholipid and a binding site on a membrane protein would be specific if strong binding requires close interaction between the anionic headgroup and a positively charged residue on the protein, as suggested by the results presented here. the phase of the phospholipid is important in determining interactions with transmembrane ot helices. as shown in fig. , fluorescence quenching is much more marked in mixtures of di(br c : )pc and di(c : )pc at temperatures where both liquid crystalline and gel phases are present than in mixtures of di(brac : )pc and di(c : )pc (mall et al., ) . thus, l is excluded from regions of lipid in the gel phase and accumulates in regions in the liquid crystalline phase. the binding constants of l| and l z for di(c : )pc in the gel phase relative to di(c :i)pc in the liquid crystalline phase are ca. . (mall et al., ) . this is consistent with the expectation that van der waals contacts between an all-trans fatty acyl chain and the molecularly rough surface of a peptide will be poor; one way that this poor packing can be overcome is by exclusion of the peptide from the gel phase. quenching plots for y l are very similar to those of l (fig. ) , showing that the presence of bulky aromatic residues does not have any significant effect on the selectivity for liquid crystalline-over gel-phase lipid (mall et al., ) . further, since y li shows a preference for longer chain phospholipids than l , y li might have been expected to show a greater preference for gel-phase lipid than l , because phospholipid in the gel phase gives a thicker bilayer than the corresponding lipid in the liquid crystalline phase. because y l and l show equal preferences for liquid crystalline-over gel-phase lipid, any effects of hydrophobic matching between the peptide and the bilayer must be small compared to effects of lipid phase on the interaction energies between the peptides and the lipid (mall et al., ) . preferential partitioning of proteins from domains in the gel phase into domains of liquid crystalline lipid has been demonstrated for a variety of membrane proteins, including bacteriorhodopsin (cherry et al., ) and ca +-atpase kleeman and mcconnell, ) . effects of sphingomyelin at °c are very similar to the effects of gel-phase di(c : )pc ( fig. ; mall et al, ) . mixtures of bovine brain sphingomyelin and di(c : )pc are in a two-phase region at °c, with gel-phase domains enriched in sphingomyelin (untracht and shipley, ) . thus, partitioning of the peptides between gel-and liquid crystalline-phase lipid shows little dependence on the structure of the phospholipid. it has been suggested that plasma membranes of mammalian cells contain domains or "rafts" enriched in sphingomyelin and that particular enzymes, particularly those associated with cell signaling, are concentrated within the rafts (simons and ikonen, ) . the results presented here suggest that membrane proteins containing transmembrane ot helices will tend to be excluded from these rafts, and it may therefore be significant that many of the signaling proteins suggested to be contained within the rafts are anchored to the membrane by glycosylphosphatidylinositol anchors (harder and simons, ) . the presence of cholesterol has a marked effect on incorporation of the peptides into phospholipid bilayers (webb et al., ) . incorporation of cholesterol at a : molar ratio to phospholipid leads to a general reduction in incorporation of the peptides l and l , but superimposed on this effect is a chain length effect. in the presence of cholesterol, the binding constant of p for di(c : )pc relative to di(c : )pc increased from . to about , as expected if the presence of cholesterol increases the effective chain length of the c chain so that it more nearly matches the hydrophobic length of the peptide (fig. ) . consistent with this interpretation, the presence of high molar ratios of cholesterol prevented the incorporation of p into bilayers of di(c :i)pc. nezil and bloom ( ) showed that incorporation of cholesterol at mol% increases bilayer thickness by about ,~. studies with brominated analogues of cholesterol showed that cholesterol binds to the peptides with a binding constant only a factor of about two less strongly than di(c : )pc . this is rather surprising, given the relatively rigid structure of the steroid ring of cholesterol and the molecularly rough surface of the peptide. in other studies, it has been shown that cholesterol binds relatively weakly at the lipid-protein interface of the atpase (simmonds et al., (simmonds et al., , ; comparison with the peptide studies reported here suggests that weak binding of cholesterol to the atpase involves interactions in the lipid headgroup region rather than interactions between the sterol ring and the hydrophobic transmembrane helices. the requirement to match the hydrophobic thickness of a membrane protein to that of the surrounding lipid bilayer could be important in a number of ways. targeting of proteins to their correct final destinations in a cell is essential in maintaining cell integrity. in the bulk flow model, the vast majority of proteins synthesized in the endoplasmic reticulum (er) are believed to leave the er by default and flow along the exocytic pathway until they reach the plasma membrane (nilsson and warren, ) . some proteins, however, have to be retained at particular points along the exocytic pathway. compartmental localization could be achieved in one of two ways. the first involves a retention signal in the protein, which, at the appropriate point in the exocytic pathway, prevents forward movement of the protein by denying it access to budding transport vesicles of the onward pathway. the second involves a retrieval signal, leading to recapture of the protein after it has left the compartment in which it resides. the classical retrieval signal is the kdel sequence found in many er-resident proteins; the situation appears to be different for golgi-resident proteins, where membrane-spanning domains act as retention signals (nilsson and warren, ) . despite the extensive flux of proteins through the golgi, the golgi maintains its own distinctive population of resident proteins. furthermore, the distribution of enzymes within the golgi is organized according to function, so that, for example, the distributions of glycosyltransferases and glycosidases, although overlapping, are distinct (colley, ; roth, ) . many of the proteins in the golgi membrane are predicted to contain a single transmembrane ot helix, oriented with the n-and c-termini on the inner and outer faces of the membrane, respectively. the golgi retention signal in such proteins has been shown to involve the membrane spanning domain (munro, (munro, , nilsson et al., ; swift and machamer, ) . however, the membrane-spanning domains show no sequence homology, and it has not been possible to identify any particular motif leading to retention (bretscher and munro, ; colley et al., ; munro, ) . thus, sialyltransferase remains localized in the golgi even when its -amino-acid transmembrane domain is replaced by leu residues (munro, ) . however, a longer stretch of leu residues did not provide an efficient retention signal (munro, ) . similarly, a -residue insertion into the transmembrane domain of galactosyltransferase reduced its retention in the golgi (masibay et al., ) . the reverse effect has been shown with the influenza virus neuraminidase, which shifted from the plasma membrane to the golgi and er when the number of residues in the transmembrane domain was reduced (sivasubramanian and nayak, ) . the lack of a clear retention motif, together with the inability to saturate the mechanism for golgi retention by overexpression, suggests that retention is not a receptor-mediated event (nilsson and warren, ) . one possible model is then retention by preferential interaction with membranes of optimal thickness (nilsson and warren, ) . both bretscher and munro ( ) and masibay et al. ( ) showed that transmembrane domains of golgi proteins are shorter (average residues) than transmembrane domains of plasma membrane proteins (average residues). it has therefore been suggested that if the golgi membrane is thinner than the plasma membrane, membrane proteins with short transmembrane domains will interact "more strongly" with the lipid bilayer of the golgi than with that of the plasma membrane, leading to retention in the golgi (bretscher and munro, ; masibay et al., ) . the studies with model peptides described above show that a protein containing a transmembrane ot helix with a hydrophobic length greater than the hydrophobic thickness of the golgi membrane will be able to move out of the golgi into the plasma membrane. however, a protein whose transmembrane ot helix has a hydrophobic length less than the hydrophobic thickness of a particular membrane will not be able to enter that membrane, and such a protein would then be retained in the golgi (webb et al., ) . studies of targeting of proteins in yeast are also consistent with a lipid-based model (rayner and pelham, ) . the length of the transmembrane domain is important in targeting with long helices ( residues), ensuring transport to the plasma membrane. however, for proteins with shorter transmembrane domains, the relative hydrophobicity of the transmembrane domain has been suggested to be important as well as its length, this determining targeting to the golgi and the vacuole (rayner and pelham, ) . retention of some membrane proteins in the er could also depend on the length of the transmembrane domain of the protein. an important class of er membrane proteins are those with an n-terminal catalytic domain exposed to the cytoplasm and a c-terminal membrane anchor. such proteins are inserted into the er membrane post-translationally by a signal-recognition-particle-independent pathway. no er retrieval signals have been identified in these proteins. instead, it has been observed that the hydrophobic domain is rather short. for example, cytochrome bs, a protein of this type, has a transmembrane domain containing just hydrophobic amino acid residues (pedrazzini et al., ) . if the length of the hydrophobic stretch is increased to residues, the protein is transported out of the er along the secretory pathway (pedrazzini et al., ) . it could therefore be that matching of the thickness of the lipid bilayer and the transmembrane length of the protein is important in retention in er, as was suggested for the golgi complex. although the length of the transmembrane domain appears to be the most important factor, the structure of the c-terminal, lumenal, region has also been shown to contribute to retention (honsho et al., ) . experiments with another c-terminalanchored protein, the ubiquitin-conjugating enzyme ubc from yeast, suggest that the thickness requirements of the er and golgi membranes may be different, explaining targeting between these two organelles (yang et al., ) . whereas ubc containing the wild-type -residue transmembrane domain targets to the er, increasing the length of the transmembrane domain to residues results in movement to the golgi, and increasing the length further to residues allows movement to the plasma membrane. these experiments show that the length of the transmembrane ot helix is often an important factor in targeting, although it is likely to be only one of a number of important factors. the lengths of the transmembrane ot helices are also likely to be important in the proper function of membrane proteins containing multiple transmembrane ot helices. an example already described is that of the ca +-atpase, which shows highest atpase activity in di(c : )pc and lower activities in bilayers of phospholipids with longer or shorter fatty acyl chains (lee, ) . changes in the atpase underlying these changes in atpase activity are complex (lee, ) , but all must be mediated by the transmembrane ot helices, because these are the parts of the atpase that can "sense" the change in bilayer thickness. in the case of the ca +-atpase it seems that, as described above, the two major conformational states of the atpase (el and e ) have different preferences for bilayer thickness, the e conformation favoring thin bilayers and the e conformation favoring thick bilayers (lee, ) . changing the bilayer thickness could change the tilt of the transmembrane u helices in the ca +-atpase, it could change the packing of the helices, and, possibly, it could lead to changes in the structures of the loops connecting the helices, changing the effective lengths of the helices. all these changes could be linked to changes in the phosphorylation domain of the ca +-atpase, located well above the surface of the membrane. if, as seems likely, the various membranes in a cell have different thicknesses because of their different lipid compositions, the structure of each membrane protein will have evolved to match the thickness of the membrane in 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equilibria in an amphiphilic peptidephospholipid model membrane by deuterium nuclear magnetic resonance difference spectroscopy orientation of u-helical peptides in a lipid bilayer structure at . /~ resolution of cytochrome c oxidase from paracoccus denitrificans identification and extent of fluid bilayer regions in membranous cytochrome oxidase hydrophobic mismatch between proteins and lipids in membranes induction of nonbilayer structures in diacylphosphatidylcholine model membranes by transmembrane alpha-helical peptides: importance of hydrophobic mismatch and proposed role of tryptophans interactions of proteins and cholesterol with lipids in bilayer membranes non-random distribution of amino acids in the transmembrane segments of type single span membrane proteins calculation of phase diagrams for non-ideal mixtures of lipids, and a possible nonrandom distribution of lipids in lipid mixtures in the liquid crystalline phase how lipids interact with an intrinsic membrane protein: the case of the calcium pump what the structure of a calcium pump tells us about its mechanism lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles fluorescence quenching in model membranes. . determination of local lipid environment of the calcium adenosinetripfiosphatase from sarcoplasmic reticulum structure of bacteriorhodopsin at , ~ resolution lipid-protein interactions in the membrane: studies with model peptides effects of aromatic residues at the ends of transmembrane alpha-helices on helix interactions with lipid bilayers specificity of lipid-protein interactions mutational analysis of the golgi retention signal of bovine t- , -galactosyl transferase the photosynthetic reaction center from the purple bacterium rhodopseudomonas viridis: aspects of membrane protein structure the structure of bacteriorhodopsin at . a resolution based on electron crystallography: implication of the charge distribution influence of membrane-spanning 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lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane hydrophobic mismatch and long-range protein/lipid interactions in bacteriorhodopsin/phosphatidylcholine vesicles fluorescence quenching and electron spin resonance study of percolation in a two-phase lipid bilayer containing bacteriorhodopsin transmembrane domain-dependent sorting of proteins to the er and plasma membrane in yeast membrane protein structure and stability: implications of the first crystallographic analyses control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length hydrophobicity of the peptide c=o. • -h--n hydrogen bonded group subcellular organization of glycosylation in mammalian cells detergent structure in crystals of a bacterial photosynthetic reaction centre structure of the detergent phase and proteindetergent interactions in crystals of the wild-type (strain y) rhodobacter sphaeroides photochemical reaction 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reconstituted with short and long chain phosphatidylcholines molecular organization and dynamics of -palmitoyl- -oleoylphosphatidylcholine bilayers containing a transmembrane alpha-helical peptide a golgi retention signal in a membrane-spanning domain of coronavirus e protein crystal structure of the calcium pump of sarcoplasmic reticulum at . /~ resolution molecular interactions between lecithin and sphingomyelin principles of membrane protein assembly and structure architecture of helix bundle membrane proteins: an analysis of cytochrome c oxidase from bovine mitochondria hydrophobic mismatch and the incorporation of peptides into lipid bilayers: a possible mechanism for retention in the golgi experimentally determined bydrophobicity scale for proteins at membrane interfaces molecular dynamics simulations of individual alpha-helices of bacteriorhodopsin in dimyristoylphosphatidylcholine. ii. interaction energy analysis the transmembrane domain of a carboxyl-terminal anchored protein determines localization to the endoplasmic reticulum the preference of tryptophan for membrane interfaces interaction of a peptide model of a hydrophobic transmembrane a-helical segment of a membrane protein with phosphatidylcholine bilayers: differential scanning calorimetric and ftir spectroscopic studies ftir spectroscopic studies of the conformation and amide hydrogen exchange of a peptide model of the hydrophobic transmembrane a-helices of membrane proteins peptide models of helical hydrophobic transmembrane segments of membrane proteins. . differential scanning calorimetric and ftir spectrooscopic studies of the interaction of ac-k -(la) -k -amide with phosphatidylcholine bilayers we thank the biotechnology and biological sciences research council (bbsrc) for financial support of the original studies reported here. key: cord- -zh cjk authors: ferraro, francesco; costa, joana r.; ketteler, robin; kriston-vizi, janos; cutler, daniel f. title: modulation of endothelial organelle size as an antithrombotic strategy date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: zh cjk it is long-established that von willebrand factor (vwf) is central to haemostasis and thrombosis. endothelial vwf is stored in cell-specific secretory granules, weibel-palade bodies (wpbs), uniquely rod-like exocytic organelles generated in a wide range of lengths ( . to . µm). it has been shown that wpb size responds to physiological cues and pharmacological treatment and that, under flow, vwf secretion from shortened wpbs produces a dramatic reduction of platelet and plasma vwf adhesion to an endothelial surface. wpb-shortening therefore represents a novel target for antithrombotic therapy acting via modulation of vwf adhesive activity. to this aim, we screened a library of licenced drugs and identified several that prompt wpb size reduction. these compounds therefore constitute a novel set of potentially antithrombotic compounds. summary the size of the endothelial secretory granules that store von willebrand factor correlates with its activity, central to haemostasis and thrombosis. here, human-licenced drugs that reduce the size of these secretory granules are identified, providing a set of novel potential anti-thrombotic compounds. endothelial von willebrand factor (vwf) plays a fundamental role in haemostasis, with deficiencies in its activity causing von willebrand disease (vwd), the most common inherited human bleeding disorder (sadler, ) . vwf is a large multi-domain glycoprotein, whose function in haemostasis depends on its multimeric status. vwf multimers act as mechano-transducers, which respond to shear forces in the circulation by stretching open and exposing binding sites for integrins, collagen, platelets and homotypic interaction (i.e., between vwf multimers) (ruggeri and mendolicchio, ) . endothelial cells secrete vwf in a highly multimerized form, known as ultra-large (ul)-vwf, highly sensitive to haemodynamic forces and thus very active in platelet binding (zhang et al., ) . ul-vwf's potential to cause spontaneous thrombus formation is controlled by a circulating protease, adamts , which generates the less-multimerised, less active forms of vwf seen in plasma (zhang et al., ) . persistence of ul-vwf in the circulation leads to microvascular thrombosis and the highly morbid and potentially life threatening clinical manifestations observed in a host of infectious and non-infectious diseases, such as sepsis and thrombotic thrombocytopenic purpura (ttp) (chang, ; tsai, ) . although these may be the most extreme examples of excess vwf function, many common disorders including hypertension and diabetes are characterised by increased vwf plasma levels (apostolova et al., ; westein et al., ) . while vwf is stored in the secretory granules of both platelets and endothelial cells, most of the vwf circulating in plasma derives from endothelial wpbs and is fundamental to haemostasis (kanaji et al., ) . vessel injury, in either the macro-or microcirculation, triggers localized stimulated exocytosis of wpbs, mediated by a variety of agonists (mccormack et al., ) . ul-vwf secreted from activated endothelium forms cable-like structures built of multiple multimers, both in vitro and in vivo (arya et al., ; rybaltowski et al., ) . these vwf "strings" provide scaffolds for the recruitment of circulating platelets and soluble plasma vwf, contributing to the formation of the primary haemostatic plug, but also potentially promoting microangiopathy (nicolay et al., ; ruggeri and mendolicchio, ) . the length of vwf strings generated upon exocytosis reflects the size of the wpbs in which vwf was stored. wpbs are cigar-shaped organelles, whose length ranges ten-fold, between . and . µm. their size depends on the structural status of the endothelial golgi apparatus where they form, and experimental manipulations causing golgi fragmentation consistently result in the formation of only short wpbs (ferraro et al., ) . short wpbs also form when vwf biosynthesis by endothelial cells is reduced, or following statin treatment, via golgi fragmentation-independent and -dependent mechanisms, respectively (ferraro et al., ; ferraro et al., ) . the metabolic status of endothelial cells also regulates wpb size through an ampk-mediated signalling pathway (lopes-da-silva et al., importantly, in vitro experiments have revealed that reducing wpb size results in the shortening of the vwf strings they generate and in much-reduced recruitment of platelets and soluble circulating vwf to the endothelial surface (ferraro et al., ; ferraro et al., ) . conversely, endothelial cells respond to raised glucose levels (mimicking hyperglycemia) by producing longer wpbs, suggesting a link between long vwf strings and thrombotic manifestations in diabetes (lopes-da-silva et al., ) , which is often associated with high levels of plasma vwf and microangiopathy (westein et al., ) . wpb size is, therefore, plastic and responds to physiological cues and pharmacological treatments. such findings suggest that drug-mediated reduction of wpb size might provide alternative or coadjuvant therapeutic approaches to current clinical interventions in thrombotic pathologies where dysregulated formation and/or prolonged persistence of vwf strings on vascular walls play a triggering role. we designed a screen to identify drugs that reduce wpb size and thus can potentially reduce endothelial pro-thrombotic capacity. out of human licensed drugs we found compounds fitting our criteria, with a variety of mechanisms of action consistent suggesting a number of pathways that influence biogenesis of wpbs. a quantitative high-throughput microscopy-based workflow, dubbed highthroughput morphometry (htm), allows rapid quantification of the size of tens to hundreds of thousands of wpbs within thousands of endothelial cells (ferraro et al., ) . htm has been applied for analytical purposes and in phenotypic screens (ferraro et al., ; ferraro et al., ; ketteler et al., ; lopes-da-silva et al., ; stevenson et al., ) . in the present report, htm was deployed to identify compounds that can induce a reduction of wpb size in human umbilical vein endothelial cells (huvecs). wpbs longer than µm represent ~ % of these vwf-storing organelles, but contain roughly % of all endothelial vwf. thus, while a minority, these long wpbs are disproportionally important with respect to secreted endothelial vwf (ferraro et al., ) . for the purpose of the screen, we quantified wpb size as the ratio between the area covered by wpbs longer than µm and the area covered by all these organelles; we define this parameter as "fractional area (fa) of long wpbs" (figure a ; (ferraro et al., ) . the effect of each compound in the library on wpb size was compared to two controls: treatment with dmso and nocodazole, the negative and positive controls, respectively, for wpb shortening (ferraro et al., ; ferraro et al., ) (figure b and c) . huvecs were incubated with the compounds of the prestwick library at µm for h in single wells of -well plates, in duplicate (two separate plates; figure d ). after fixation, immuno-staining and image acquisition, the fa of long wpbs in treated cells was quantified. per plate and inter-plate normalization to dmso-controls was implemented in order to rank the effects of the library compounds on wpb size by z-score, (figure e ). hit selection. statins are cholesterol-lowering drugs that inhibit the enzyme -hydroxy, methylglutaryl coa reductase (hmgcr) in the mevalonate pathway, upstream of the biosynthesis of cholesterol. we have previously shown that treatment of endothelial cells with two statins, simvastatin and fluvastatin, induces wpb size shortening, resulting in reduced adhesive properties of the vwf released by activated endothelial cells (huvec), measured by the reduced size of platelet-decorated vwf strings and by the recruitment of vwf from a flowing plasma pool (ferraro et al., ) . fluvastatin and simvastatin are present in the prestwick library. we therefore used their z-score to establish a stringent cutoff for selection of positive hits. this approach identified compounds, . % of the library ( figure e , orange box, simvastatin and fluvastatin). of note, aside from simvastatin and fluvastatin, the other three statins present in the prestwick library were among the selected hits ( table ) , indicating that both our screening approach and the criterion for hit selection are robust. hit pharmacology. forty-one ( %) of the drugs identified could be allocated in ten pharmacological classes, each including at least three compounds ( table ; and supplemental table ), consistent with a variety of mechanisms of wpb size control. some of the compounds are shared by more than one class (table and figure a) ; an indication that, aside from their known molecular targets, these drugs may exert their effect on wpb size through a yet unidentified common cellular pathway. among the pharmacological classes capable of inducing wpb shortening, we found microtubule (mt) depolymerizing agents, histone deacetylase (hdac) inhibitors, topoisomerase inhibitors and, as mentioned earlier, statins (hmgcr inhibitors). compounds with these mechanisms of action have been shown induce unlinking of the ribbon architecture of the golgi apparatus, i.e. golgi "fragmentation" (farber-katz et al., ; ferraro et al., ; gendarme et al., ; thyberg and moskalewski, ) . since an intact golgi ribbon is required for the biogenesis of long wpbs (ferraro et al., ) (see nocodazole in figure b ), identification of these classes of molecules in our screen was expected. work from our lab also showed that neutralization of the acidic lumen of wpbs disrupts the tubular structure of vwf, shifting the organelle shape from cylindrical to spherical, therefore detected as shortening (michaux et al., ) ; and, indeed, we identified transmembrane ph gradient depleting agents. reduction in vwf biosynthesis results in shorter wpbs without affecting the golgi architecture (ferraro et al., ) and one of the screen hits, cycloheximide, is a classic protein synthesis inhibitor. aside from those expected, entirely novel wpb-shortening drug classes were also identified, including neurotransmitter receptor antagonists and cardiac glycosides. interestingly, several compounds, beside their known mechanism of action, also inhibit multidrug resistance protein , mrp /abcb (table , figure a and supplemental table ), which may hint at the common cellular pathway discussed above. the cardiac glycosides and their cardenolide precursors were prominent among these novel pharmacological classes. these molecules, which inhibit na + /k + -atpase (table and (figures b and a) instead of its fragmentation, suggestive of a novel wpb size-reducing mechanism. in most cases, sub-micromolar concentrations of these compounds were sufficient to reduce wpb size ( figure b ). upon exocytosis, endothelial ul-vwf self-assembles into strings, which serve as recruiting platform for platelets and circulating vwf, thus promoting the formation of the primary haemostatic plug (ferraro et al., ; ruggeri, ; varga-szabo, ) . vwf-strings also mediate pathological processes such as tumour metastasis, endocarditis and microangiopathies (bauer et al., ; nicolay et al., ; pappelbaum et al., ) . interventions reducing the persistence and/or activity of vwf-strings are therefore of interests as potential anti-thrombotic therapies. modulation of organelle size has been suggested as a potential strategy to regulate biological functions and correct pathological states (marshall, ) . in this context, wpbs represent a paradigmatic example. reduction of wpb size has no effect on ul-vwf formation, but blunts generation of long platelet-decorated vwf-strings following exocytosis and recruitment of plasma vwf to the endothelial surface (ferraro et al., ; ferraro et al., ) . interventions that shorten wpbs could therefore provide alternative or coadjuvant therapies to clinical interventions in thrombotic pathologies where dysregulated formation and/or prolonged persistence of vwf-strings play a triggering role. apart from pharmacological treatments and other experimental manipulations disrupting the integrity of the golgi apparatus (ferraro et al., ; ferraro et al., ) , formation of short wpbs is mediated by endogenous signalling pathways. we have uncovered a pathway involving ampk-dependent regulation of the arf-gef gbf , which is (dekker et al., ; fledderus et al., ; kumar et al., ; lin et al., ) . treatment with statins also up-regulates klf expression; and their antiinflammatory, anti-coagulant and antithrombotic effects are believed to be mediated by this transcription factor sen-banerjee et al., ) . however, wpb size reduction induced by statin treatment does not require klf (ferraro et al., ) . altogether, a significant body of experimental evidence indicates that the size of wpbs is subject to regulation and represents a target for pharmacological intervention in haemostatic function and thrombotic risk. we therefore screened human-licenced drugs with the aim of identifying wpb sizereducing molecules that could be rapidly repurposed as antithrombotics. we found fifty-eight drugs with this activity, the majority of which can be grouped into pharmacological classes. some of these classes, such as microtubule depolymerizing agents and statins, have been identified by previous work (ferraro et al., ; ferraro et al., ) . others might be expected, due to their effects on the golgi ribbon, as in the case of hdac and topoisomerase inhibitors (farber-katz et al., ; gendarme et al., ) . our screen also identified compounds with pharmacology previously unknown to affect wpb biogenesis and size. together, these findings suggest that several cellular pathways can modulate the size of wpbs produced by endothelial cells. multidrug resistance protein (mrp ), is an organic anion transporter. its upregulation is responsible for the development of tumor resistance to chemotherapy, hence its name. while this activity towards xenobiotics was the first to be identified, it has become clear that mrp is also involved in the cellular efflux of endogenous molecules, mediating pro-inflammatory signalling pathways and may act as an oxidative stress sensor (cole, ) . interestingly, twenty-two compounds, with varied mechanisms of action (table and supplemental table ), have also been described as mrp inhibitors. this suggests the possibility that, in addition to their main molecular target, these drugs could affect the efflux of endogenous mrp signalling substrates, which regulate wpb size; a mechanism worth future investigation. since the screen endpoint was h, the drugs listed are relatively fast-acting. except for statins (ferraro et al., ) and cardiac glycosides (see above), drug activity was documented at the single concentration used in our screen ( µm). while such concentrations are unlikely to be used for patient administration, it is worth noting that submicromolar concentration treatment with simvastatin for h does reduces wpb size with dramatic effects on both platelet recruitment and plasma vwf adhesion to the stimulated endothelium (ferraro et al., ) . it therefore cannot be ruled out that several of the drugs identified by the screen would maintain wpb size-reducing activity at lower concentrations, compatible with their use in the clinic. statins rapidly produce anti-inflammatory and anticoagulant effects on the endothelium (greenwood and mason, ) and their acute administration in the context of percutaneous angioplasty greatly reduces post-operative myocardial infarctions (leoncini et al., ) . the compounding of these fast-acting effects, wpb size-reduction included, suggests that statins may represent a promising tool for acute, emergency treatment in endotheliopathies involving inflammation, coagulation and thrombosis. as a class, the most potent wpb-shortening drugs identified in the screen, active also at sub-micromolar concentrations (figure b and b) , were the cardiac glycosides and cardenolides. with the caution due to their known dose-dependent cytotoxicity (kanji and maclean, ) , cardiac glycosides may therefore be worth exploring in acute and chronic antithrombotic therapies. further to the potential toxicity associated with administration of drugs at high concentrations, we note that in vitro combination of wpb-size reducing treatments, acting through different mechanisms, can display synergy in the abatement of plasma vwf recruitment to the endothelial surface (supplemental figure ) . in a clinical setting, wpb shortening and its consequent antithrombotic effects might therefore be achieved by administering combinations of drugs at lower, non-toxic concentrations. in conclusion, here, we report a set of licenced drugs with potential antithrombotic activity, via a novel mechanism: the reduction of wpb size and its consequence in terms of reduced adhesion of platelets and circulating plasma vwf to the ul-vwf they release. µg/ml endothelial cell growth supplement from bovine neural tissue and u/ml heparin (both from sigma-aldrich). cells were cultured at °c, % co , in humidified incubators. reagents. the library compounds ( fda-approved drugs), from prestwick chemical, were stored at - °c as mm stock solutions in dmso. for the screen, compounds were transferred to echo qualified -well low dead volume microplates using the echo® acoustic dispenser (both from labcyte). antibodies used in this study were: rabbit polyclonal anti-vwf pro-peptide region (hewlett et al., ) huvecs were seeded in -well plates and pre-processed as described for the screen. cardiac glycosides were added to cells using the echo® acoustic dispenser (labcyte) and cells were treated as described above. concentration of each compound ranged from nm to µm in -fold increments across the wells of the plate column. duplicate plates were prepared. each plate contained dmso and nocodozale controls as detailed for the screen. treatment was for h, at the end of which cells were fixed as described for the screen. immunostaining and image acquisition. fixed cells were processed for immunostaining as previously described (ferraro et al., ) . wpbs were labelled using a rabbit polyclonal antibody to the vwf pro-peptide region. the golgi apparatus was labeled with an anti-gm mab. primary antibodies were detected with alexa fluor dye-conjugated antibodies (life technologies). nuclei were counterstained with (life technologies). images ( fields of view per well) were acquired with an opera high content screening system (perkin elmer) using a x air objective (na . ). high-throughput morphometry (htm) workflow. image processing and wpb extraction of morphological parameters (high-throughput morphometry, htm) have been described in detail elsewhere (ferraro et al., ) . wpb size was expressed per well (i.e., summing the values measured in the fields of view) as the fraction of the total wpb area covered by wpbs > µm. dose-response data analysis was done in r language, using the drc package by christian ritz and jens c. strebig (https://cran.r-project.org/package=drc). the drm () function was used to fit a dose-response model, a four-parameter log-logistic function (ll. ), applied to each dataset; four parameter values were calculated: slope, lower limit, upper limit and ec value. plasma vwf recruitment assays. sirna nucleofections, drug treatment and human plasma perfusion experiments on huvec monolayers were carried out as previously described (ferraro et al., ) . table . licenced drugs with wpb-shortening activity. individual drugs were assigned to pharmacological classes, indicated by numbers, based on their mechanism of action (see appendix table) . , cardiac glycoside or cardenolide (na + /k + -atpase inhibitor); , statin ( -hydroxy, -methylglutaryl coa reductase, hmgcr inhibitor; , topoisomerase inhibitor/ dna-intercalating agent; , serotonin ( -ht) receptor antagonist; , histone deacetylase (hdac) inhibitor; , dopamine receptor antagonist; , histamine receptor antagonist; , microtubule depolymerizing compound; , ph gradient-depleting compound; , multidrug resistance protein (mrp /abcb ) inhibitor. two plates with identical drug layout (biological duplicates) were analyzed. e. screen results. z-scores of the library drugs are plotted. hit drugs were selected based on the effects of fluvastatin and simvastatin, which we previously showed to reduce wpb size. fluvastatin, with the lowest z-score, was used as cut-off for the selection of hit compounds. graphical summary of the known mechanism of action (moa) of the hits reported in the supplemental table . each pharmacological class is depicted by a square, whose area is proportional to the number of compounds it includes. lines connecting the squares represent common drugs between pharmacological classes. thickness of the lines indicates the number of drugs shared. b. drug classes ranked by potency, using their median z-score in the screen. each data-point represents one drug and classes are color-labeled based on their know, inferred from the literature and previously unknown (novel) effects on wpb size. table pharmacology of hit compounds. information regarding the mechanism of action of hit drugs was searched in pubmed and drugbank (https://www.drugbank.ca/). forty-one compounds can be assigned to ten pharmacological classes, each containing at least drugs (see table ). no drugbank record. it is an ionophore antibiotic (na+/h+ antiporter) produced by the fungus streptomyces cinnamonensis. depletes the transmembrane ph gradient of the golgi apparatus and acidic organelles. drugbank; agonist of serotonin receptor ht . antagonist of ht a, ht b and ht c serotonin receptors. inhibitor of sodium-dependent serotonin transporter slc a . drugbank: it inhibits tubulin beta- chain and tubulin beta chain(tubb , tubb) and microtubule polymerization. inhibits several cyp enzymes and transporters. inhibits multidrug resistance protein (abcb ) drugbank: cardiac glycoside. it consists of three sugars and the aglycone digoxigenin. inhibits na/k atpase alpha- (atp a ). substrate, inhibitor and inducer of multidrug resistance protein (abcb ). drugbank: antiemetic, it is a substance p/neurokinin (nk ) receptor antagonist. drugbank: a non-benzodiazepine that is used in the management of anxiety. binds to benzodiazepine receptors, which interact allosterically with gaba receptors, potentiating the effects of the inhibitory neurotransmitter gaba. binds and is an agonist of transporter protein (tspo) which promotes the transport of cholesterol across mitochondrial membranes and may play a role in lipid metabolism (pubmed: ), but its precise physiological role is controversial. tspo is apparently not required for steroid hormone biosynthesis, was initially identified as peripheral-type benzodiazepine receptor and can also bind isoquinoline carboxamides drugbank; mucolytic agent. excessive nitric oxide (no) is associated with inflammation of airways. no enhances the activation of soluble guanylate cyclase and cgmp accumulation. ambroxol has been shown to inhibit the nodependent activation of soluble guanylate cyclase. wikipedia; ambroxol is a potent inhibitor of the neuronal na+ channels. it also activates lysosomal enzyme glucocerebrosidase. ambroxol can also diffuse into lysosomes and induce ph neutralization. ambroxol and its parent drug bromhexine have been shown to induce autophagy in several cell types. adenosine 'monophosphate drugbank; several functions, as it is a central molecule in metabolism and signalling. activates ampk methyldopa (l,-) drugbank; agonist of alpha- adrenergic receptor with both central and peripheral nervous system effects. its primary clinical use is as an antihypertensive agent. targets alpha- a adrenergic receptor (adra a) and inhibits aromatic-l-amino-acid decarboxylase (ddc) and the transporter solute carrier family member (scl a ) specific for dipeptides. drugbank; semisynthetic derivative of podophyllotoxin. exhibits antitumor activity. it inhibits dna synthesis by forming a complex with topoisomerase ii and dna, inducing breaks in double stranded dna and preventing repair by topoisomerase ii binding. accumulated breaks in dna prevent entry into the mitotic phase of cell division and lead to cell death. inhibits dna topoisomerase -alpha (top a) and dna topoisomerase -beta (top b). cyclosporin a drugbank: lipophilic cyclic polypeptide formed by amino acids with immunosuppressive and immunomodulatory properties. essentially, it is a calcineurin inhibitor and this activity allows for inhibition of t cell activation. it binds to the intracellular receptor cyclophilin- forming a ciclosporincyclophilin complex that inhibits calcineurin preventing the dephosphorylation and activation of the nuclear factor of activated t cells (nf-at). the nf-at is a transcription factor that regulates the production of pro-inflammatory cytokines such as il- , il- , interferon-gamma and tnf-alpha. cyclosporin inhibits calcineurin regulatory subunit b type (ppp r ) and binds peptidylprolyl cis-trans isomerase a and f (ppia, ppif), calcium signal-modulating cyclophilin ligand camlg. it is a substrate, inhibitor and inducer of multidrug resistance protein (abcb ). drugbank: hydroxymethylglutaryl coenzyme a (hmg-coa) reductase inhibitor. also inhibits dipeptidyl peptidase (dpp ). agonist of aryl hydrocarbon receptor (ahr). inhibits multidrug resistance protein (abcb ). it has been shown that other statins inhibit this transporter (pubmed: ). inhibits solute carrier organic anion transporter family member a , b (slco a , slco b ). it is a substrate of several transporters. drugbank: inhibitor of hydroxymethylglutaryl coenzyme a (hmg-coa) reductase (hgmcr). the first statin discovered. isolated from penicillium citinium. it also inhibits liver carboxylesterase , ces (a triglyceride lipase). drugbank: triazole antifungal agent. inhibits cytochrome p- -dependent enzymes resulting in impaired ergosterol synthesis. it has been used against histoplasmosis, blastomycosis, cryptococcal meningitis & aspergillosis. inhibits lanosterol -alpha demethylase (cyp a ) and several other cyp enzymes. inhibits multidrug resistance proteins (abcb ) and slco b . wikipedia. among the triazole antifungal agents is the only one shown to inhibit the hedgehog pathway and angiogenesis. the antiangiogenic activity was shown to be linked to inhibition of glycosylation, vegfr phosphorylation, trafficking and cholesterol biosynthesis pathways. like cyclosporine, quinidine and clarithromycin (and statins), can inhibit p-glycoproteins (multidrug resistance proteins, abcbs) causing drug-drug interactions by reducing elimination and increasing absorption of organic cation drugs. drugbank: vorinostat, known also as suberoylanilide hydroxamic acid (saha), is currently under investigation for the treatment of cutaneous t cell lymphoma (ctcl), a type of skin cancer. it is the first in a new class of agents known as histone deacetylase inhibitors. inhibits histone deacetylase , , , and (hdac , hdac , hdac , hdac and hdac ). hdac inhibitors and dna-damaging (dna-intercalating) agents were identified as novel golgi disruptors (see pmid: ). note; clemizole has been identified as a potential anti-epileptic through its action on serotonin receptors (see annotation of clemizole for reference); among the papers citing this study, one identifies vorinostat as an anti-epileptic (pmid: ) clomiphene drugbank: it is an estrogen agonist or antagonist depending on the target tissue. estrogen receptor (esr ) agonist or antagonist. inhibits some cytochrome p (cyps) enzymes. drugbank: long-acting, non-sedating second generation antihistamine used in the treatment of allergy symptoms. antagonist of histamine h receptor (hrh ). inhibitor of potassium voltage-gated channel subfamily h member (kcnh ). also acts (with unclarified pharmacological action) on potassium voltage-gated channel subfamily h member (kcnh ) and microtubuleassociated protein tau (mapt). inhibits multidrug resistance protein (abcb ) perhexiline drugbank: coronary vasodilator used especially for angina. it may cause neuropathy and hepatitis. inhibits carnitine o-palmitoyltransferase , liver isoform (cpt a), carnitine o-palmitoyltransferase , mitochondrial (cpt ). acts (no established pharmacology) on potassium voltage-gated channel subfamily h member (kcnh ). drugbank: monocationic surface-active agent with surfactant and detergent properties. it is widely used to enhance dispersion and penetration of cellular debris and exudate, thereby promoting tissue contact of the administered medication. inhibits v-atpase, v-type proton atpase subunit c (atp v c ). no drugbank record alclometasone drugbank: synthetic glucocorticoid steroid for topical use in dermatology as anti-inflammatory, antipruritic, antiallergic, antiproliferative and vasoconstrictive agent. agonist of glucocorticoid receptor (nr c ). drugbank: anthelmintic effective for pinworms; no targets reported. wikipedia: it is an anthelmintic and has been shown that the pamoate salt has preferential toxicity for various cancer cell lines during glucose starvation. drugbank: a medication used as a photosensitizer for photodynamic therapy to eliminate the abnormal blood vessels in the eye associated with conditions such as the wet form of macular degeneration. no molecular targets reported. no drugbank record. wikipedia: antagonist of hrh . recently, it was identified through a phenotypic screen as described as a ht receptor antagonist (pmid: ) fluvastatin drugbank: hydroxymethylglutaryl coenzyme a (hmg-coa) reductase inhibitor. inhibits -hydroxy- -methylglutaryl-coenzyme a reductase (hmgcr). figure . synergistic effects of wpb-shortening treatments on plasma vwf adhesion. wpb size was reduced by two treatments, simvastatin incubation and reduced vwf synthesis (ferraro et al., ; ferraro et al., ) , alone or in combination. huvecs were nucleofected with sirnas targeting luciferase (negative control) or vwf and seeded in µ-slides vi (ibidi). at h postnucleofection, cells were treated with dmso or . µm simvastatin. after h drug treatment, and h post-nucleofection, cells were exposed to histamine to stimulate vwf secretion, while perfused with pooled human plasma and then fixed under flow (as described in ferraro et al., ) . vwf on the endothelial surface was detected by immunofluorescence; the area covered by its signal measures the extent of its adhesion. each data-point represents the quantification of a field of view; median and interquartile ranges are shown. ****, p < . , mann-whitney test. blue asterisks: platelet aggregation in malignant melanoma of mice and humans sepsis and septic shock: endothelial molecular pathogenesis associated with vascular microthrombotic disease multidrug resistance protein (mrp , abcc ), a "multitasking" atpbinding cassette (abc) transporter klf provokes a gene expression pattern that establishes functional quiescent differentiation of the endothelium dna damage triggers golgi dispersal via dna-pk and golph a two-tier golgi-based control of organelle size underpins the functional plasticity of endothelial cells weibel-palade body size modulates the adhesive activity of its von willebrand factor cargo in cultured endothelial cells prolonged shear stress and klf suppress constitutive proinflammatory transcription through inhibition of atf image-based drug screen identifies hdac inhibitors as novel golgi disruptors synergizing with jq statins and the vascular endothelial inflammatory response temperature-dependence of weibel-palade body exocytosis and cell surface dispersal of von willebrand factor and its propolypeptide contribution of platelet vs. endothelial vwf to platelet adhesion and hemostasis cardiac glycoside toxicity: more than years and counting image-based sirna screen to identify kinases regulating weibel-palade body size control using electroporation tumor necrosis factor alphamediated reduction of klf is due to inhibition of mef by nf-kappab and histone deacetylases statin treatment before percutaneous cononary intervention kruppel-like factor (klf ) regulates endothelial thrombotic function a gbf -dependent mechanism for environmentally responsive regulation of er-golgi transport organelle size control systems: from cell geometry to organelledirected medicine weibel-palade bodies at a glance the physiological function of von willebrand's factor depends on its tubular storage in endothelial weibel-palade bodies cardiac glycosides as novel cancer therapeutic agents cellular stress induces erythrocyte assembly on intravascular von willebrand factor strings and promotes microangiopathy ultralarge von willebrand factor fibers mediate luminal staphylococcus aureus adhesion to an intact endothelial cell layer under shear stress statins exert endothelial atheroprotective effects via the klf transcription factor the role of von willebrand factor in thrombus formation interaction of von willebrand factor with platelets and the vessel wall in vivo imaging analysis of the interaction between unusually large von willebrand factor multimers and platelets on the surface of vascular wall biochemistry and genetics of von willebrand factor kruppel-like factor as a novel mediator of statin effects in endothelial cells g protein-coupled receptor kinase moderates recruitment of thp- cells to the endothelium by limiting histamine-invoked weibel-palade body exocytosis microtubules and the organization of the golgi complex identification and characterization of cardiac glycosides as senolytic compounds pathophysiology of thrombotic thrombocytopenic purpura the shear stress-induced transcription factor klf affects dynamics and angiopoietin- content of weibel-palade bodies cell adhesion mechanisms in platelets thrombosis in diabetes: a shear flow effect? mechanoenzymatic cleavage of the ultralarge vascular protein von willebrand factor affects (no defined mechanism) integrin beta- (itgb ) and inhibits integrin alpha-l (itgal) high affinity nerve growth factor receptor (ntrk ), macrophage colony-stimulating factor receptor (csf r), platelet-derived growth factor receptor alpha (pdgfra), platelet-derived growth factor receptor beta (pdgfrb) it is a substrate and inhibitor of multidrug resistance protein (abcb ) and other transporters of the acb family and slc family has no effect on the adrenergic system or central nervous system, but may antagonize histamine and interfere with acetylcholine release locally wikipedia: a vasodilator that acts as an adenosine reuptake inhibitor. used for the treatment of cardiopathy and renal disorders used in treatment of leukaemia and other neoplasms. targets dna (dna intercalating agent). inhibits dna topoisomerase -alpha (top a) and dna topoisomerase -beta (top b) inhibitor of multidrug resistance-associated protein (abcc ) and other acb transporters also used as an anthelmintic and in the treatment of giardiasis and malignant effusion and in cell biological experiments as an inhibitor of phospholipase a . targets dna (dna intercalating agent). inhibits / kda calcium-independent phospholipase a (pla g ), cytosolic phospholipase a (pla g a) antagonist of d( ) dopamine receptor (drd ), d( a) dopamine receptor (drd ), -hydroxytryptamine receptor a (htr a), -hydroxytryptamine receptor a (htr a), alpha- a adrenergic receptor (adra a), alpha- b adrenergic receptor (adra b) fluspirilene drugbank: long-acting injectable antipsychotic agent used for chronic schizophrenia fluphenazine drugbank: phenothiazine used in the treatment of psychoses with properties and uses generally similar to those of chlorpromazine ciclesonide drugbank: glucocorticoid used to treat obstructive airway diseases. agonist of glucocorticoid receptor (nr c ) antagonist of -hydroxytryptamine receptor a (htr a), -hydroxytryptamine receptor b (htr b), -hydroxytryptamine receptor c (htr c) doxorubicin drugbank: cytotoxic anthracycline antibiotic isolated from cultures of streptomyces peucetius var. caesius. targets dna (dna intercalating agent). inhibits dna topoisomerase -alpha (top a). inhibitor and inducer of multidrug resistance protein (abcb ) and other abc transporters antagonist of d( ) dopamine receptor (drd ), neuron-specific vesicular protein calcyon (caly; interacts with clathrin light chain and stimulates clathrin self assembly and endocytosis), alpha- a adrenergic receptor (adra a) azithromycin drugbank: broad-spectrum macrolide antibiotic with a long half-life and a high degree of tissue penetration. like other macrolides, it blocks bacterial protein synthesis. in human cells inhibits protein-arginine deiminase type- (padi ; involved in arginine modification of histones epirubicin drugbank: '-epi-isomer of doxorubicin. the compound exerts its antitumor effects by interference with the synthesis and function of dna. targets dna (dna intercalating agent). inhibits dna topoisomerase -alpha (top a) inhibits cytosolic phospholipase a (pla g a) inhibits nadh dehydrogenase [ubiquinone] subunit c (ndufc ), potassium voltage-gated channel subfamily h member (kcnh ), vascular cell adhesion protein (vcam ), e-selectin (sele) and multidrug resistance protein (abcb ). modulates hypoxia prochlorperazine drugbank: a phenothiazine antipsychotic used principally in the treatment of nausea, vomiting and vertigo antagonist of d( ) dopamine receptor (drd ), -hydroxytryptamine receptor a (htr a), -hydroxytryptamine receptor c (htr c), -hydroxytryptamine receptor (htr ) key: cord- -pqxkyg z authors: reggiori, fulvio title: membrane origin for autophagy date: - - journal: curr top dev biol doi: . /s - ( ) - sha: doc_id: cord_uid: pqxkyg z autophagy is a degradative transport route conserved among all eukaryotic organisms. during starvation, cytoplasmic components are randomly sequestered into large double‐membrane vesicles called autophagosomes and delivered into the lysosome/vacuole where they are destroyed. cells are able to modulate autophagy in response to their needs, and under certain circumstances, cargoes, such as aberrant protein aggregates, organelles, and bacteria can be selectively and exclusively incorporated into autophagosomes. as a result, this pathway plays an active role in many physiological processes, and it is induced in numerous pathological situations because of its ability to rapidly eliminate unwanted structures. despite the advances in understanding the functions of autophagy and the identification of several factors, named atg proteins that mediate it, the mechanism that leads to autophagosome formation is still a mystery. a major challenge in unveiling this process arises from the fact that the origin and the transport mode of the lipids employed to compose these structures is unknown. this compendium will review and analyze the current data about the possible membrane source(s) with a particular emphasis on the yeast saccharomyces cerevisiae, the leading model organism for the study of autophagosome biogenesis, and on mammalian cells. the information acquired investigating the pathogens that subvert autophagy in order to replicate in the host cells will also be discussed because it could provide important hints for solving this mystery. in eukaryotic cells, the principal locations where protein catabolism occurs are the proteasome and the lysosome. the proteasome mostly recognizes and degrades cytosolic factors that have been specifically marked with polyubiquitin chains (roos-mattjus and sistonen, ) . the lysosome in contrast, requires active transport in order for the diverent substrates destined for elimination to reach its interior where the proteases are located. four diverent pathways can deliver intracellular proteins into the lysosome lumen: endosomal transport routes, chaperone-mediated autophagy (cma), microautophagy, and macroautophagy, the latter generally referred to as autophagy katzmann et al., ; klionsky, ; majeski and dice, ) . the endosomal transport routes and cma are mostly devoted to the transport of polypeptides, whereas microautophagy and autophagy deliver other cellular constituents because these pathways are the only ones able to internalize entire organelles and bacteria. eukaryotes, in particular fungi, can use microautophagy to eliminate peroxisomes and is the only cellular function that has indisputably been assigned to this pathway . autophagy, on the other hand, can deliver various cargoes to the lysosome interior and has multiple physiological roles. the hallmark of this catabolic pathway is the sequestration of cargoes by large cytosolic double-membrane vesicles called autophagosomes (reggiori and klionsky, ) . the autophagosomes successively dock and fuse with mammalian lysosomes or the yeast and plant vacuoles releasing the inner vesicles into the lumen of these organelles (reggiori and klionsky, ) . the biogenesis and consumption of these structures can be divided into six discrete steps: induction, expansion, vesicle completion, docking, fusion, and breakdown ( fig. ). autophagosomes are generated by the elongation of a small template membrane, termed the isolation membrane or phagophore (fengsrud et al., ; mizushima et al., ; noda et al., ; reggiori and klionsky, ) . there are several of these structures per cell but it still remains unknown where they are derived from. the surface of this small compartment is decorated with atg and atg , and its formation requires phosphatidylinositol (ptdins)- -kinase activity (mizushima et al., , . there are two ways of triggering the expansion of the isolation membrane, and they diver depending if the process of autophagy is selective or nonselective (section i.b) (reggiori and klionsky, ) . when this figure conceptual model for autophagy. the basic mechanism of autophagy is the sequestration of the cargo material (bulk cytoplasm, protein aggregates, organelles, or pathogens) by a cytosolic double-membrane vesicle named an autophagosome. extracellular stimuli or the recognition of a specific intracellular cargo induce the expansion of the isolation membrane. upon vesicle completion, the autophagosome docks with the lysosome/vacuole and successively fuses with it. in this way the inner vesicle is liberated inside the vacuole where it is finally consumed together with the cargo by resident hydrolases. this schematic represents nonspecific autophagy and does not show specific types of autophagy including the cvt pathway. pathway is selective, the binding to the isolation membrane of the cargo that has to be specifically eliminated (or in the case of resident hydrolases, activated) leads to the expansion of this structure (ogawa et al., ; shintani and klionsky, b) . in contrast to selective autophagy, which is induced by intracellular components, the nonselective process is governed by extracellular stimuli such as nutrients or cytokines (gutierrez et al., ; lum et al., ; shintani and klionsky, a) . in both cases, covalent conjugation of the ubiquitin-like atg -atg seems to be the step that initiates the expansion of the isolation membrane . the expansion of the isolation membrane is basically the simultaneous elongation and nucleation of this little cisterna (fig. ) . it is not known how the atg -atg complex recruits additional membranes, but the crescent autophagosome acquires more atg -atg and atg along with a second ubiquitin-like molecule, atg /lc , that is unconventionally linked to phosphatidylethanolamine (pe), and probably the rest of the atg proteins (mizushima et al., , . two expansion mechanisms are possible, one that relies on delivery of lipid bilayer by vesicular trayc (vesicular expansion) and one based on the fusion of small compartments (cisternal expansion) (reggiori and klionsky, ) . in addition, it has been suggested that retrograde trayc balances double-membrane vesicle biogenesis by recycling some atg proteins, such as atg , and also recovering from the forming autophagosome membrane components specific to the compartment(s) of origin (meiling-wesse et al., ; nazarko et al., ; nice et al., ; reggiori et al., reggiori et al., , a . when the two extremities of the forming autophagosomes reach each other, they fuse together sealing the vesicle (fig. ). this fusion event, at least in yeast, appears to be snare-independent and triggers an uncoating reaction where the externally localized components dissociate from the vesicle surface (ishihara et al., ; reggiori and klionsky, ; reggiori et al., b) . in particular, the ubiquitin-like protein atg -pe is proteolytically released from its lipid moiety by the atg protease, whereas the transmembrane protein atg is completely retrieved (kirisako et al., ; reggiori et al., a) . it is still unknown which factor senses completion of the double-membrane vesicle and initiates this disassembly. once uncoated, the double-membrane vesicle docks with the lysosomes/ vacuoles (fig. ) . in mammalian cells, this association is facilitated by microtubules and seems to require dynein whereas in yeast it is independent of these structures (aplin et al., ; fengsrud et al., ; kirisako et al., ; punnonen and reunanen, ; ravikumar et al., ; webb et al., ) . the fusion between the autophagosome and the lysosome/ vacuole occurs as soon as these organelles dock and it is mediated by a set of proteins also used for other fusion reactions with the lysosome/vacuole (section i.c). during this event, the external membrane of the autophagosome becomes part of the lysosome/vacuole surface whereas the inner autophagosomal vesicle is liberated in the interior of this organelle and now called an autophagic body (fig. ) . the limiting membrane of the autophagic body is immediately attacked and consumed by resident lysosomal/vacuolar hydrolases allowing these enzymes to gain access to the content of this vesicle. as a result, the cargoes are also degraded into their basic constituents (or in the case of certain resident hydrolases, processed to their active form; fig. ). autophagy has been known for a long time as an adaptation response to starvation and as the major factor in the turnover of long-lived proteins. but in recent years, it has become evident that autophagy plays an active role in several other physiological tasks highlighting its versatility and adaptability. we now know that this catabolic pathway participates in cellular processes such as development, cellular diverentiation and rearrangement, elimination of aberrant structures, lifespan extension, mhc class ii presentation of cytoplasmic antigens, and type ii programmed cell death, as well as protecting against pathogens (both viruses and bacteria) and tumors (cuervo et al., ; debnath et al., ; deretic, ; thompson, , ; kirkegaard et al., ; komatsu et al., ; kondo et al., ; levine and klionsky, ; paludan et al., ; rubinsztein et al., ; shintani and klionsky, a) . as a result, this degradative transport route plays a relevant role in the pathophysiology of neurodegenerative, cardiovascular, muscular, and autoimmune diseases, and some malignancies thompson, , ; kondo et al., ; rubinsztein et al., ; shintani and klionsky, a; towns et al., ). autophagy provides one evective way to adjust and cope with these various situations by rapidly delivering large fractions of the cytoplasm, aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens into the lysosome/vacuole interior where they are destroyed by resident hydrolases (reggiori and klionsky, ) . the adaptability of this pathway is due to its ability to select specific cargoes when forced by circumstances. it has been believed for a long time that autophagy was a nonspecific process because when induced by starvation, cytoplasmic components and organelles were randomly sequestered into autophagosomes; however, this pathway can also be selective (table i) (reggiori and klionsky, ) . in the yeast saccharomyces cerevisiae, for example, aminopeptidase i (ape ) and -mannosidase (ams ) form a large oligomer that is unconventionally delivered from the cytoplasm directly to the vacuole interior through a process known as the cytoplasm to vacuole targeting (cvt) pathway (kim et al., ; shintani et al., ) . this transport route is specific and biosynthetic. precursor ape (prape ) is packed into double-membrane vesicles called cvt vesicles, which are four to eight times smaller in surface area than autophagosomes scott et al., ) . in the same organism, dysfunctional mitochondria are preferentially eliminated by autophagy (mitophagy) as well as superfluous peroxisomes (pexophagy) ( table i) (hutchins et al., ; priault et al., ) . the specific sequestration of peroxisomes into double-membrane vesicles and their subsequent degradation has also been very well described in other fungi such as pichia pastoris, hansenula polymorpha, and yarrowia lipolytica . mammalian cells on the other hand, seem not to possess a transport route similar to the cvt pathway, but there are indications that mitophagy could occur (bota and davies, ; elmore et al., ; rodriguez-enriquez et al., ) . pexophagy has also been report ed (lui ken et al. , ; yokota , ; yoko ta et al. , ) . it has lately been shown that autophagy can be selective in mammalian cells as the diverent types of selective autophagy, their specific cargoes, and the organisms that have been described are indicated. fulvio reggiori well, as evidenced by the specific recognition and disposal of invading bacteria and potentially also of intracellular viruses (table i) (deretic, ; kirkegaard et al., ; levine, ) . in addition, a study analyzing conditional knock-out mice defective for autophagy has revealed that the mutant animal accumulates numerous ubiquitinated aggregates in the cytosol, suggesting that this covalent protein modification could serve to specifically target to autophagosomes large structures that have to be eliminated (komatsu et al., ) . the process of autophagy has been known for at least years, but because none of the specific components involved in this pathway were known, the studies about this degradative transport route were limited to morphological and phenomenological observations. in the last years, genetic screens, mostly in the yeast s. cerevisiae and fungi such as p. pastoris and h. polymorpha, have lead to the isolation of genes termed autophagyrelated (atg) genes whose products are specifically involved in this catabolic pathway (table ii) (klionsky, ; klionsky et al., ) . the extent of the conservation of this pathway between eukaryotes was first revealed by comparing the genomes once various sequencing projects were completed (reggiori and klionsky, ) . it became immediately evident that most of the atg genes had one or more homologs in higher eukaryotic organisms. the cellular role of some of them has now been explored and in all the analyzed cases, it has been demonstrated that the homologs function as orthologs (table ii) (levine and klionsky, ; reggiori and klionsky, ) . the same genetic approaches have also led to the discovery of nine atg genes dispensable for bulk autophagy but essential for the cvt pathway and/ or pexophagy (table iii) . their products are mostly involved in cargo selection and the final sealing of the double-membrane vesicle, indicating that additional components are required for the autophagosomes to be able to enwrap specific cargoes. it is important to note that these genes involved in specific types of autophagy do not have clear homologs in higher eukaryotes sustaining the idea that the cvt pathway and pexophagy are probably only present in fungi (reggiori and klionsky, ) . in addition to the atg proteins, the genetic screens in yeast have also permitted the identification of additional components required for the normal progression of autophagy that are shared with other intracellular transport routes (table iv) . the function of several of these factors in the other pathways was already known and that has helped in clarifying the mechanism of autophagy. for example, yeast vacuoles can fuse with late . membrane origin for autophagy . in all these cases, cells use an identical fusion machinery, which consists of snare proteins, sec (nsf), sec (-snap), a rab-gtpase, and the class c vps protein complex also known as the hops complex. the same components have also been found to be exploited for the fusion of double-membrane vesicles (table iv) (reggiori and klionsky, ; wang et al., ) . similarly, it is also now evident that the dissolution of autophagic bodies is mediated by the same hydrolases that degrades the a in s. cerevisiae, these proteins also catalyze the retrieval transport from early endosomes. b atg is not required for pexophagy in s. cerevisiae but is essential for the same process in h. polymorpha. c these factors have no counterparts in s. cerevisiae or the homologs do not have a role in pexophagy. ? one report has indicated that atg is essential for pexophagy, another affirms that atg is not required for this process. mvb internal vesicles once these are released into the vacuole lumen (table iv) (epple et al., ; reggiori and klionsky, ) . most of the atg components are peripheral membrane proteins that transiently associate with the nascent autophagosomes. in contrast to mammalian cells where several isolation membranes can be simultaneously activated, a single perivacuolar site of organization for double-membrane vesicle formation (named the pre-autophagosomal structure, pas) is observed in the yeast s. cerevisiae (kim et al., ; suzuki et al., ) . the pas is believed to be the yeast counterpart of a mammalian isolation membrane and in this unicellular eukaryote, most of the atg proteins appear to be primarily restricted to this location. this unique site seems also to be present in h. polymorpha (monastyrska et al., a,b) . in p. pastoris, however, several atg components are distributed to more than one punctate structure (ano et al., ; chang et al., ; kim et al., b; mukaiyama et al., ; stromhaug et al., ) . it is unclear if this represents a diverence between organisms or is due to diverent growth conditions. p. pastoris is mostly used for the study of pexophagy and therefore grown in special media containing carbon sources that induce peroxisome proliferation. it is unclear where the pas is derived from and at which point it becomes membranous. the study of the cvt pathway has provided insights into how this structure is generated. after synthesis, prape forms a large oligomer that first associates with the atg cargo receptor and then with the atg adaptor to form the cvt complex (shintani et al., ) . this large cytosolic protein aggregate then moves in close proximity to the vacuole surface, where it induces the recruitment of the rest of the atg factors, triggering the formation of the cvt vesicle yorimitsu and klionsky, ) . neither the pas nor the vesicles are eyciently formed in the absence of any of the cvt complex components, indicating that the cargo stimulates the biogenesis of these structures . this requirement is overcome when cells are nitrogen-starved (kim et al., b; shintani and klionsky, b) . because of its dynamic properties, the pas should not be seen as a static or defined organelle but more as a structure in constant remodeling. it remains unclear at which stage and how membranes are transported at the pas, but because of their association with lipid bilayers, two proteins, atg and atg , could be important for dissecting this event. atg is a soluble ubiquitin-like protein and its carboxy-terminal arginine is removed by the atg cysteine protease leaving a glycine residue at the new carboxy terminus (kim et al., a; kirisako et al., ) . atg is activated by the e enzyme atg through a thioester bond between its carboxyterminal glycine and cysteine of atg (kim et al., ; kirisako et al., ; komatsu et al., ) . atg is subsequently transferred to the e enzyme atg via a new thioester bond between these two proteins kim et al., a) . atg is finally covalently conjugated to a pe molecule, becoming tightly membrane associated . this linkage is reversible because atg can be proteolitically released from its lipid moiety by atg , an event that takes place once the double-membrane vesicles are completed (section i.a. ) (kirisako et al., ; reggiori et al., a) . it is unclear where the atg conjugation to pe occurs. this protein is normally lipidated in mutants unable to form the pas indicating that this modification takes place at a diverent subcellular location (suzuki et al., . membrane origin for autophagy ) . this is supported by the fact that atg -pe localizes to the pas but also to tiny cytosolic vesicles (kirisako et al., ) . these data, however, do not exclude the possibility that atg -pe conjugates are formed at the pas as well. the association of atg -pe with membranes prior to getting concentrated at the pas suggests that the atg -pe structures could be at least in part the source of autophagosome lipid bilayers. this idea is supported by the observation that in the absence of atg , membranes fail to be delivered to the pas and therefore the size of autophagosomes is strongly reduced (abeliovich et al., ; kirisako et al., ; lang et al., ) . it remains a mystery where the tiny atg -pe containing vesicles are derived from, but one possibility is that that they originate from early compartments of the secretory pathway, for example, the endoplasmic reticulum (er) and/or the golgi apparatus. this hypothesis is based on two experimental findings. first, atg binds two vsnares required for both anterograde and retrograde transport between the er and golgi apparatus . second, this ubiquitin homolog has been detected on autophagosome-like structures derived from the golgi complex and/or er (section ii.d. ) (reggiori et al., b) . atg is the only integral membrane protein essential for double-membrane vesicle formation . this protein is probably transported to the pas with at least part of the lipids or lipid bilayers required to create this structure. this notion is corroborated by the fact that the totality of atg is associated with membranes reggiori et al., b) . atg cycles between the pas and several unknown punctate structures dispersed in the cytosol supporting the idea that it could partially supply the forming autophagosomes with membranes (reggiori et al., a) . a fraction of these punctate structures are atg aggregates residing on the mitochondria surface (reggiori et al., b) . this suggests that this organelle could provide the nascent autophagosomes with at least part of its lipid bilayers. however, it cannot be excluded that atg traycking carries out other functions. under certain conditions, autophagy becomes one of the principal sources of energy for the cell (kuma et al., ; lum et al., ) . because the mitochondria provide the other primary supply of energy, one could imagine that atg is used to coordinate the two sources. the sorting mechanism for atg transit from mitochondria to the pas is unknown, but under growing conditions this event is induced by cvt complex assembly and requires actin (reggiori et al., a; shintani and klionsky, b) . in contrast, the retrieval transport of this transmembrane protein from the pas has been characterized in more detail and shown to be regula ted by the atg -at g signal ing complex and requ ires atg , atg , and the ptdins - -phosp hate generat ed by the atg con taining ptd ins -kinase complex ( reggiori et al. , a) . this recycl ing eve nt, howeve r, seems to be to some extent di verentl y organiz ed in p. pasto ris dur ing micro pexoph agy, possibly because other membr ano us struc tures a re used an d assem bled in a di verent way during this invaginati on pro cess ( chang et al. , ) . an initial analys is concerning the role of yeast early secretion (sec ) mutant s in autoph agy has reveal ed that several of them are essent ial for autop hagosome form ation (ishiha ra et al. , ) . this class of genes is involved in trans port out of the er (kais er an d schekman, ). successi ve studi es, howeve r, have shown that these mutant s ha ve an indir ect negati ve e vect on both the cvt pa thway and au tophagy ( hama saki et al. , ; reggiori et al. , b ) . one possibl e explanation of their phe notype is that they alter the er morpho logy and con sequently impair severa l function s of this organel le, includi ng the put ative one to supp ly membra nes for doublemembr ane vesic le formati on. for example, the er is structural ly connected wi th the mitoc hondria and the disruption of the er organ ization in the early sec mutant s causes the fragmenta tion of the mitocho ndrial reti culum ( prinz et al. , ) . as mention ed, atg partiall y local izes to mito chondria, and in this class of mutants its tray cking out of this compartmen t is severe ly impai red (re ggiori and klio nsky, sub mitted). atg , atg , tlg , tl g , trs , vps , and the subunit s of the vpsfiftythree (vft) complex are part of retrieval transp ort routes from en dosomal compart ments back to the golgi app aratus, and consequ ently they are important in maintaining certain functions of this organelle (hettema et al., ; holthuis et al., ; sacher et al., sacher et al., , siniossoglou and pelham, ) . these proteins have also been shown to be required for the cvt pathway and some of them also play an important role in doublemembr ane vesicle biog enesis during pe xophagy and autophagy (tables iii and iv reggiori et al., ) . it is unclear, however, why these pathways are impaired in the absence of these factors. one possibility is that retrograde trayc from the forming double-membrane vesicles is . membrane origin for autophagy essential for the expansion and/or completion of these structures (meiling-wesse et al., ; reggiori et al., a,b) . a second hypothesis is that similarly to what was predicted for early sec mutants, an alteration of the golgi apparatus functions could interfere with the lipid bilayer delivery essential for the creation of these large vesicles. the major diyculty in investigating the contribution to autophagy of both the er and the golgi apparatus is that these two organelles depend on each other for their proper function. mutations that avect one of these two compartments indirectly perturb the other one. along these lines, the interpretation of the block of both the cvt pathway and autophagy in the sec mutant is not simple (reggiori et al., b) . nevertheless, the analysis of this strain has led to important information. sec is a gdp/gtp exchange factor required for traycking through the golgi complex (franzusov and schekman, ; jackson and casanova, ) . the inactivation of this protein provokes the accumulation of unsealed, autophagosome-like structures that are decorated with atg (reggiori et al., b) . these membranous arrangements enwrap ribosomes and cytosol and have been previously named berkeley bodies (esmon et al., ; novick et al., ) . this surprising result indicates that potentially, double-membrane vesicles can be created in large part by altering the activity of a single enzyme; however, it cannot be excluded that this is an indirect phenomenon. vps is a protein essential for the invagination of the late endosome limiting membranes and therefore mvb biogenesis (babst et al., ; katzmann et al., ) . a unique vps allele was isolated in a screen for mutations that result in autophagy induction even in the presence of nutrients (shirahama et al., ) . this led to an initial interpretation that endosomes play a relevant role in autophagosomes biogenesis. however, reports where the functions of these compartments have been severely impaired by specific gene deletions have revealed that the integrity of the endosomal system is not essential for either the cvt pathway or autophagy (epple et al., ; reggiori et al., b) . in contrast to the late stages of the autophagosome biogenesis where lipid bilayers are derived from endosomal compartments, the origin of the mammalian isolation membrane or phagophore remains uncertain. it is still unknown if this small sequestering cisterna is formed de novo or derived from a preexisting organelle (fengsrud et al., ) . a major problem in trying to investigate its origin is that these structures and autophagosomes are mostly composed of lipids and depleted in transmembrane proteins making particularly diycult the detection of specific organelle markers (fengsrud et al., ; hirsimaki et al., ; punnonen et al., ; reunanen et al., ; stromhaug et al., ) . this unique characteristic is one line of evidence that the isolation membranes and autophagosomes diver structurally from the other subcellular organelles. this observation also implies that whatever the origin of the lipid bilayers used to form autophagosomes, integral membrane components are segregated away from them. two models could explain how protein-depleted membranes are obtained. in the first, isolation membranes are derived from a specialized organelle subdomain where autochthonous proteins are gradually excluded. a similar process has been shown to occur during peroxisome biogenesis from the er tabak et al., ; tam et al., ) . in the second model, the same cisterna is progressively emptied of integral membrane factors by retrieval transport-a phenomenon hypothesized to occur during double-membrane vesicle formation in yeast (reggiori et al., a,b) . it is also possible that both mechanisms coexist. numerous studies have been published investigating the source for autophagosome lipid bilayers in mammalian cells but their conclusions often contrast. thus various organelles, such as the er, the golgi complex, and the plasma membrane, have been suggested to be the origin of doublemembrane vesicles. because of the heterogeneity in the results, no unanimous agreement in the field has been reached. for example, several studies have reported the presence of er-marker proteins in the isolation membranes and autophagosomes but others have shown that these structures lack er-resident factors (arstila and trump, ; dunn, a; furuno et al., ; reunanen et al., ; stromhaug et al., ; yamamoto et al., ; yokota et al., ) . as with the er, the role of the golgi complex as a lipid donor for the early autophagosome intermediates remains ambiguous. some studies have shown the presence of golgi protein markers in these structures whereas others have failed to detect them (arstila and trump, ; dunn, a; frank and christensen, ; locke and sykes, ; yang and chiang, ; yokota et al., ) . the membranes of the cis-golgi network have been shown to possess the same compositional characteristics of the isolation membrane (fengsrud et al., ; locke and sykes, ; reunanen et al., ; yamamoto et al., ) . only a few reports have indicated that the autophagic cisternae are derived from the plasma membrane and their conclusions have been challenged when other investigators have failed to detect plasma membrane protein markers in these structures (araki et al., ; arstila and trump, ; bosabalidis, ; ericsson, ; fengsrud et al., ; oledzka-slotwinska and desmet, ; reunanen et al., ) . importantly, autophagosomes have a low cholesterol content validating the idea that their membranes are not derived from the plasma membrane (reunanen et al., ) . the discrepancy between all these analyses could be due, in part, to diverent experimental approaches and techniques used in the various laboratories. but one possibility that should not be discarded a priori is that autophagosomes could be a mosaic of membranes derived from more than one organelle. for example, the isolation membrane could originate from one compartment and the additional lipid bilayers required for its expansion be acquired from other sources. in addition, the diverent contributions could vary depending on the tissues with cells able to derive the membranes from the most suitable reservoirs. the atg conjugation system is highly conserved in higher eukaryotic cells (table ii) tanida et al., ) . in mammals, there are at least three atg homologs: the microtubule-associated protein (map ) light chain (lc ), the golgi-associated atpase enhancer of kda (gate- ), and the -aminobutyric acid (gaba) a -receptorassociated protein (gabarap) (mann and hammarback, ; sagiv et al., ; wang et al., ) . it should be noted that these three proteins were first isolated because of their involvement in other traycking pathways. the mammalian counterparts of atg process these three atg homologs by exposing their conserved c-terminal glycine which then interacts with mammalian atg and atg homologs before being covalently linked to a lipid (hemelaar et al., ; scherz-shouval et al., ; tanida et al., tanida et al., , . the target phospholipid has not yet been unequivocally identified, but strong evidence suggests that it is pe (kabeya et al., ; tanida et al., ) . of the three homologs, lc has been best characterized as an autophagosomal marker in mammalian autophagy. the newly synthesized lc precursor is processed cotranslationally to generate a soluble lc form (lc -i) that, upon starvation, becomes membrane-bound and has greater mobility than lc -i when resolved by sds-page . the lipidated protein, called lc -ii, localizes on both autophagosomes and autolysosomes . these in vitro results have been confirmed using transgenic mice expressing gfp-lc . unfortunately, the small amount of lc -ii generated prior to induction of autophagy is already associated with the double-membrane vesicles formed by the basal activity of this pathway and lc -i is not clearly associated with a distinct membranous structure (kabeya et al., ) . therefore, the subcellular localization of these molecules has not furnished insights about the lipid bilayer source. both gate- and gabarap possess a form ii and localize to lc positive autophagosomes that are induced by starvation (kabeya et al., ) . thus, it remains a possibility that they participate in autophagy in addition to, or instead of, their originally described functions. because the three mammalian atg homologs are diverently expressed in various tissues (tanida et al., ) , another intriguing option is that these proteins are involved in supplying the autophagosome with membranes derived from diverent compartments depending on the cell type; for example, gate- from the golgi complex and gabarap from the same organelle as well as the synaptic cisternae (kittler et al., ; kneussel et al., ; sagiv et al., ) . a report has demonstrated that the two human proteins with high homology to atg , hsatg l and hsatg l , are its orthologs (yamada et al., ) . in human adult tissues, hsatg l is ubiquitously expressed, whereas hsatg l is highly expressed in placenta and pituitary gland. importantly, the authors have also shown that these two factors are not distributed on mitochondria. instead they localize to a perinuclear region, suggesting that in higher eukaryotes atg could supply autophagy with membranes by deriving lipid bilayers from a diverent reservoir. this observation could also explain why hsatg l and hsatg l cannot substitute for the yeast atg (reggiori et al., b; yamada et al., ) . however, hsatg l possesses a nonfunctional mitochondrial targeting sequence that is also present in its closest higher eukaryote homologs (yamada et al., ) . this characteristic raises the possibility that this is an ancient localization signal. because the subcellular distribution of hsatg l and hsatg l have not been carefully examined and the preliminary localization analysis was performed with overexpressed proteins, the identification of the precise localization of these two proteins could provide insights into membrane dynamics during autophagosome biogenesis in mammals. in mammalian cells, autophagosomes, also called initial autophagic vacuoles (avi), undergo a stepwise maturation process that can be followed ultrastructurally by monitoring the disintegration status of their internal lipid . membrane origin for autophagy bilayer and cargoes (fig. ) (dunn, b; eskelinen, ; fengsrud et al., ; rabouille et al., ) . these morphological changes correlate with the increasing acquisition of lysosomal makers dunn, b; liou et al., ; tanaka et al., ) . autophagosomes, which contain intact cytosol and organelles, fuse first with endosomal vesicles and mvb turning into early degradative autophagic vacuoles (avd) or amphisomes. these structures successively fuse together or with lysosomes becoming late avd or autolysosomes. the degradation of the internal material starts in the early avd and continues in the late avd until completion. in contrast to yeast, the endosomal system plays an essential role in mammalian autophagy (see section ii.d. ). this divergence between species has been highlighted by the discovery that skd is necessary for autophagosome maturation in mouse cells (nara et al., ) . skd is the mouse homolog of yeast vps and, as its counterpart, it is also essential to maintain endosome morphology and endosomal transport . as mentioned, vps is not required for autophagy in yeast (section ii.d. ) (reggiori et al., b; shirahama et al., ) . it is unclear why mammalian autophagosomes need the additional maturation step characterized by their fusion with endosome-and/or trans-golgi network (tgn)-derived transport vesicles and mvb. in yeast, doublemembrane vesicles fuse with a much larger vacuole one after the other. therefore, their cargoes do not influence the hydrolytic capacity of this compartment by altering, for example, the ph because the volume of their contents is just a fraction of that of the entire vacuole. lysosomes, in contrast, are much smaller than vacuoles and their size is comparable to that of autophagosomes. figure autophagosome maturation in mammalian cells. once sealed, the autophagosome (or avi) fuses with endosome-and/or tgn-derived transport vesicles and the mvb becoming an amphisome (or early avd). this event leads to the acquisition of hydrolytic enzymes that initiate the consumption of the autophagosome cargo. the amphisome then fuses with a lysosome generating a new organelle termed autolysosome (or late avd) where the degradation of the content of the initial autophagosome is completed. consequently, if these two structures would immediately fuse together, an important dilution of the lysosome content could occur impairing its internal enzymatic activity. addition of extra hydrolytic enzymes prior to autolysosome formation could help to compensate for this dilution phenomenon. autophagy provides a cellular defense against invading pathogens but unfortunately, some of them have developed systems to avoid the sequestration and elimination by double-membrane vesicles (deretic, ; kirkegaard et al., ; levine, ) . in addition, there are virus and bacteria that exploit this transport route to enter and replicate inside the host cell (kirkegaard et al., ) . the study of this latter class of pathogens has furnished some indications about the possible origin of autophagosome membranes even if it should be kept in mind that these invading microorganisms are also altering other cellular pathways, and therefore autophagy could progress in part diverently in the infected cells. upon infection, positive-strand viruses disassemble and release their genomic rna into the cytoplasm of the host cell. the genomic rna is subsequently translated to produce the replicase proteins that induce the formation of the rna replication complexes. these complexes are assembled and anchored on membrane surfaces and this is an essential requisite for their virulence. some of the positive-strand viruses, such as the poliovirus, the mouse hepatitis virus (mhv), the equine arterivirus (eav), and the severe acute respiratory syndrome (sars) coronavirus, seen to use autophagosomes as a membrane platform (kirkegaard et al., ) . factors of the poliovirus rna-replication complex localize to doublemembrane vesicles that are derived from the er by the action of viral proteins bc and a by a mechanism that excludes resident host proteins (schlegel et al., ; suhy et al., ) . importantly, these structures contain lc /atg and are highlighted with the fluorophore monodansylcadaverine, a dye that specifically stains autophagosomes (jackson et al., ) . the idea that poliovirus subverts components of the cellular autophagy machinery to promote its replication is also supported by the fact that inhibition of this pathway by -methyladenine or by rna interference against mrnas that encode two diverent atg proteins (lc /atg and atg ) decrease the poliovirus yield (jackson et al., ) . coronaviruses (mhv and sars) and arteriviruses (eav) are the two families within the order nidovirales. cells infected by these viruses accumulate double-membrane vesicles and the viral rna-replication complexes are associated with them (goldsmith et al., ; gosert et al., ; pedersen et al., ; shi et al., ; van der meer et al., ) . in the case of the mhv and sars coronaviruses, it has also been shown that these structures are decorated with lc /atg , revealing that they are autophagosomes (prentice et al., a,b) . for the mhv in addition, it has been demonstrated that the autophagy machinery is required to generate these compartments and in its absence the virus replication is severely blocked (prentice et al., a) . importantly, studies about the origin of these doublemembrane vesicles generated in cells infected by nidoviruses indicate that they are derived from the er (pedersen et al., ; prentice et al., a; shi et al., ; van der meer et al., ) . after endosomal uptake of porphyromonas gingivalis and brucella abortus, by the host cell, the endosomes that contain these bacteria immediately fuse with structures resembling autophagosomes (dorn et al., ; pizarro-cerda et al., a,b; progulske-fox et al., ) . this event prevents their delivery to the lysosome where they would be eliminated. in addition containing endosomal factors, the double membranes surrounding these two pathogens are decorated with er protein markers and their formation is blocked by autophagy inhibitors such as -methyladenine and wortmannin (rich et al., ) . legionella pneumophila is a gram-negative bacterium that can replicate within human macrophages. after being taken up by phagosomes, this pathogen becomes enwrapped within a double-membrane compartment that contains the er resident chaperone bip through an unknown mechanism, and starts to replicate (coers et al., ; joshi et al., ; sturgill-koszycki and swanson, ; swanson and isberg, ) . it has been shown that this compartment also progressively becomes decorated with typical autophagosome markers such as atg and atg (amer and swanson, ) . however, it remains unclear if these structures are autophagosomes or similar conformations derived from the er that at successive stage acquire autophagosomal membranes or subvert the autophagy machinery to complete their biogenesis (kagan and roy, ; tilney et al., ) . in dictyostelium discoideum, a natural host for l. pneumophila, deletion of atg genes leads to a defect in autophagy without avecting the formation of the doublemembrane compartment and therefore the replication of this invading microorganism is unavected (otto et al., ) . but this could just reflect host-specific diverences. listeria monocytogenes is another gram-negative bacterium that after entering into host cells destroys the phagosome membrane using hemolysin to gain access to the cytoplasm where it starts to multiply. however, when infected cells are treated with chloramphenicol, an inhibitor of bacterial protein synthesis, or lack the acta gene, the bacteria become trapped into double-membrane compartments shortly after phagosome lysis (rich et al., ) . these structures are autophagosomes because l. monocytogenes sequestration is enhanced by autophagic induction through serum withdrawal and blocked by autophagy inhibitors such as -methyladenine and wortmannin (rich et al., ) . the formation of these autophagosomes seems to be mediated by the assembly of small vesicles and cisternae with variable morphology, which contain the er protein marker protein disulfide isomerase (pdi). importantly, pdi-positive vesicular structures are accumulated around the cytoplasmic bacteria during the early stages of autophagosome biogenesis but not at later stages when these structures begin to acquire endosomal makers (rich et al., ) . our knowledge about the physiological roles of autophagy has enormously increased and we have realized how important this pathway is for cell survival in several extreme situations. despite the identification and partial characterization of the atg proteins, however, the molecular mechanism of this catabolic transport route remains largely unknown. a major challenge in studying this process arises from the fact that the origin and the transport mode of the lipids employed to compose these structures is unknown. investigations on this topic seem to indicate that the er and possibly the golgi complex are involved in supplying the nascent autophagosomes with membranes. endosomal compartments, in contrast, play a relevant role only in mammalian cells and at a later stage during autophagosome maturation. the large majority of the morphological characterization of autophagosome formation was done - years ago, when specific autophagy markers were unavailable. atg proteins provide now the researchers with the longawaited markers that could be used to at least dissect this transport route at an ultrastructural level, thus solving some of the mysteries that surround the double-membrane vesicle origin and biogenesis. analysis of pathogens and their gene products has helped in the past to unveil and analyze numerous cellular pathways. the discovery of the existence of viruses and bacteria subverting autophagy will probably have a similar impact. the study of these microorganisms will help us to understand how lipid bilayers are derived from the membrane source(s) but will also potentially lead to the isolation of agents that will allow investigators to manipulate this process. cytoplasm to vacuole traycking of aminopeptidase i requires a t-snare-sec p complex composed of tlg p and vps p dissection of autophagosome biogenesis into distinct nucleation and expansion steps autophagy is an immediate macrophage response to legionella pneumophila a sorting nexin ppatg regulates vacuolar membrane dynamics during pexophagy via binding to phosphatidylinositol- -phosphate cytoskeletal elements are required for the formation and maturation of autophagic vacuoles redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane studies on cellular autophagocytosis. the formation of autophagic vacuoles in the liver after glucagon administration two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome the vps p aaa atpase regulates membrane association of a vps protein complex required for normal endosome function isolation and characterization of rat liver amphisomes. evidence for fusion of autophagosomes with both early and late endosomes developmental features of autophagy in aging secretory cells of tamarix aphylla salt glands protein degradation in mitochondria: implications for oxidative stress, aging and disease: a novel etiological classification of mitochondrial proteolytic disorders ppatg encodes a novel membrane protein that traycs to vacuolar membranes which sequester peroxisomes during pexophagy in pichia pastoris identification of icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for legionella pneumophila intracellular growth fulvio reggiori autophagy and aging: the importance of maintaining does autophagy contribute to cell death? autophagy in innate and adaptive immunity porphyromonas gingivalis traycs to autophagosomes in human coronary artery endothelial cells studies on the mechanisms of autophagy: formation of the autophagic vacuole studies on the mechanisms of autophagy: maturation of the autophagic vacuole pexophagy: the selective autophagy of peroxisomes defective autophagy leads to cancer death by design: apoptosis, necrosis and autophagy the mitochondrial permeability transition initiates autophagy in rat hepatocytes intravacuolar membrane lysis in saccharomyces cerevisiae. does vacuolar targeting of cvt /aut p avect its function? studies on induced cellular autophagy. i. electron microscopy of cells with in vivo labelled lysosomes maturation of autophagic vacuoles in mammalian cells compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast ultrastructural and immunocytochemical characterization of autophagic vacuoles in isolated hepatocytes: evects of vinblastine and asparagine on vacuole distributions ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy structural aspects of mammalian autophagy localization of acid phosphatase in lipofuscin granules and possible autophagic vacuoles in interstitial cells of the guinea pig testis functional compartments of the yeast golgi apparatus are defined by the sec mutation immunocytochemical study of the surrounding envelope of autophagic vacuoles in cultured rat hepatocytes involvement of the endoplasmic reticulum in peroxisome formation ultrastructural characterization of sars coronavirus rna replication of mouse hepatitis virus takes place at double-membrane vesicles autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages the early secretory pathway contributes to autophagy in yeast a single protease, apg b, is specific for the autophagy-related ubiquitin-like proteins gate- , map -lc , gabarap, and apg l retromer and the sorting nexins snx / / mediate distinct retrieval pathways from yeast endosomes vinblastine-induced autophagic vacuoles in mouse liver and ehrlich ascites tumor cells as assessed by freeze-fracture electron microscopy two syntaxin homologues in the tgn/endosomal system of yeast peroxisome degradation in saccharomyces cerevisiae is dependent on machinery of macroautophagy and the cvt pathway a ubiquitin-like system mediates protein lipidation autophagosome requires specific early sec proteins for its formation and nsf/snare for vacuolar fusion turning on arf: the sec family of guaninenucleotide-exchange factors subversion of cellular autophagosomal machinery by rna viruses evidence that dot-dependent and -independent factors isolate the legionella pneumophila phagosome from the endocytic network in mouse macrophages lc , a mammalian homologue of yeast apg p, is localized in autophagosome membranes after processing lc , gabarap and gate localize to autophagosomal membrane depending on form-ii formation legionella phagosomes intercept vesicular trayc from endoplasmic reticulum exit sites distinct sets of sec genes govern transport vesicle formation and fusion early in the secretory pathway receptor downregulation and multivesicular-body sorting fulvio reggiori transport of a large oligomeric protein by the cytoplasm to vacuole protein targeting pathway apg p/cvt p is required for the cytoplasm-to-vacuole targeting, macroautophagy, and peroxisome degradation pathways membrane recruitment of aut p in the autophagy and cytoplasm to vacuole targeting pathways requires aut p, aut p, and the autophagy conjugation complex cvt /gsa functions in sequestering selective cytosolic cargo destined for the vacuole convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation formation process of autophagosome is traced with apg /aut p in yeast the reversible modification regulates the membrane-binding state of apg /aut essential for autophagy and the cytoplasm to vacuole targeting pathway cellular autophagy: surrender, avoidance and subversion by microorganisms the subcellular distribution of gabarap and its ability to interact with nsf suggest a role for this protein in the intracellular transport of gaba a receptors a unified nomenclature for yeast autophagy-related genes the g-aminobutyric acid type a receptor (gaba a r)-associated protein gabarap interacts with gephyrin but is not involved in receptor anchoring at the synapse the c-terminal region of an apg p/cvt p is required for homodimerization and is essential for its e activity and e -e complex formation impairment of starvationinduced and constitutive autophagy in atg -deficient mice the role of autophagy in cancer development and response to therapy the role of autophagy during the early neonatal starvation period aut p and aut p, two novel microtubule-associated proteins are essential for delivery of autophagic vesicles to the vacuole aut p, a soluble autophagic factor, participates in multiple membrane traycking processes membrane origin for autophagy eating oneself and uninvited guests: autophagy-related pathways in cellular defense development by self-digestion: molecular mechanisms and biological functions of autophagy the autophagic and endocytic pathways converge at the nascent autophagic vacuoles the role of the golgi complex in the isolation and digestion of organelles autophagic degradation of peroxisomes in isolated rat hepatocytes growth factor regulation of autophagy and cell survival in the absence of apoptosis mechanisms of chaperone-mediated autophagy molecular characterization of light chain . a microtubule binding subunit of map a and map b trs (gsg ), a component of the trapp complexes is required for the organization of the preautophagosomal structure during selective autophagy via the cvt pathway dissection of autophagosome formation using apg -deficient mouse embryonic stem cells mouse apg l, a novel wd-repeat protein, targets to the autophagic isolation membrane with the apg -apg conjugate in vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker the hansenula polymorpha atg gene encodes a novel coiled-coil protein that is required for macropexophagy atg is essential for macropexophagy in hansenula polymorpha modification of a ubiquitin-like protein paz conducted micropexophagy through formation of a novel membrane structure skd aaa atpase-dependent endosomal transport is involved in autolysosome formation early secretory pathway gene trs is required for selective macroautophagy of peroxisomes in yarrowia lipolytica cooperative binding of the cytoplasm to vacuole targeting pathway proteins, cvt and cvt , to ptdins ( )p at the pre-autophagosomal structure is required for selective autophagy fulvio reggiori apg p/cvt p is an integral membrane protein required for transport vesicle formation in the cvt and autophagy pathways yeast autophagosomes: de novo formation of a membrane structure identification of complementation groups required for post-translational events in the yeast secretory pathway escape of intracellular shigella from autophagy two ubiquitin-like conjugation systems essential for autophagy participation of the cell membrane in the formation of ''autophagic vacuoles macroautophagy is dispensable for intracellular replication of legionella pneumophila in dictyostelium discoideum endogenous mhc class ii processing of a viral nuclear antigen after autophagy open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes virulent brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments coronavirus replication complex formation utilizes components of cellular autophagy identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast mutants avecting the structure of the cortical endoplasmic reticulum in saccharomyces cerevisiae porphyromonas gingivalis virulence factors and invasion of cells of the cardiovascular system evects of vinblastine, leucine, and histidine, and -methyladenine on autophagy in ehrlich ascites cells intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver membrane origin for autophagy the diverential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immunocytochemistry dynein mutations impair autophagic clearance of aggregate-prone proteins autophagy in the eukaryotic cell vps is part of the yeast vps fifty-three tethering complex essential for retrograde trayc from the early endosome and cvt vesicle completion the atg -atg complex regulates atg and atg retrieval transport from the pre-autophagosomal structure early stages of the secretory pathway, but not endosomes, are required for cvt vesicle and autophagosome assembly in saccharomyces cerevisiae autophagosomes: biogenesis from scratch? the actin cytoskeleton is required for selective types of autophagy, but not nonspecific autophagy, in the yeast saccharomyces cerevisiae atg cycles between mitochondria and the pre-autophagosomal structure in yeasts studies on vinblastine-induced autophagocytosis in mouse liver. v. a cytochemical study on the origin of membranes cytochemical studies on induced autophagocytosis in mouse exocrine pancreas cytoplasmic bacteria can be targets for autophagy tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes the ubiquitin-proteasome pathway autophagy and its possible roles in nervous system diseases, damage and repair identification and characterization of five new subunits of trapp trapp i implicated in the specificity of tethering in er-to-golgi transport gate- , a membrane transport modulator, interacts with nsf and the golgi v-snare gos- the cooh terminus of gate- , an intra-golgi transport modulator, is cleaved by the human cysteine protease hsapg a cellular origin and ultrastructure of membranes induced during poliovirus infection fulvio reggiori aminopeptidase i is targeted to the vacuole by a nonclassical vesicular mechanism colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells autophagy in health and disease: a double-edged sword cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway mechanism of cargo selection in the cytoplasm to vacuole targeting pathway mutational analysis of csc /vps p: involvement of endosome in regulation of autophagy in yeast an evector of ypt p binds the snare tlg p and mediates selective fusion of vesicles with late golgi membranes purification and characterization of autophagosomes from rat hepatocytes gsa encodes a unique -kda protein required for pexophagy and autophagy in pichia pastoris legionella pneumophila replication vacuoles mature into acidic, endocytic organelles remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles the pre-autophagosomal structure organized by concerted functions of apg genes is essential for autophagosome formation association of legionella pneumophila with the macrophage endoplasmic reticulum peroxisomes start their life in the endoplasmic reticulum. trayc pex p initiates the formation of a preperoxisomal compartment from a subdomain of the endoplasmic reticulum in saccharomyces cerevisiae accumulation of autophagic vacuoles and cardiomyopathy in lamp- -deficient mice the human homolog of saccharomyces cerevisiae apg p is a protein-activating enzyme for multiple substrates including human apg p, gate- , gabarap, and map-lc lc conjugation system in mammalian autophagy human apg p/aut p homologue is an authentic e enzyme for multiple substrates, gate- , gabarap, and map-lc , and facilitates the conjugation of hapg p to hapg p how the parasitic bacterium legionella pneumophila modifies its phagosome and transforms it into membrane origin for autophagy rough er: implications for conversion of plasma membrane to the er membrane sera from patients with type diabetes and neuropathy induce autophagy and colocalization with mitochondria in sy y cells orf a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex yeast homotypic vacuole fusion requires the ccz -mon complex during the tethering/ docking stage gaba areceptor-associated protein links gaba a receptors and the cytoskeleton microtubule disruption inhibits autophagosome-lysosome fusion: implications for studying the roles of aggresomes in polyglutamine diseases endothelial nitric-oxide synthase antisense (nos as) gene encodes an autophagy-related protein (apg -like ) highly expressed in trophoblast characterization of the isolation membranes and the limiting membranes of autophagosomes in rat hepatocytes by lectin cytochemistry formation of a whorl-like autophagosome by golgi apparatus engulfing a ribosome-containing vacuole in corpora allata of the cockroach diploptera punctata formation of autophagosomes during degradation of excess peroxisomes induced by administration of dioctyl phthalate degradation of excess peroxisomes by cellular autophagy: immuno-cytochemical and biochemical analysis atg links cargo to the vesicle-forming machinery in the cytoplasm to vacuole targeting pathway the mouse skd , a homologue of yeast vps p, is required for normal endosomal traycking and morphology in mammalian cells the author thanks daniel klionsky, judith klumperman, catherine rabouille, and ger strous for critically reading the chapter. the author also wishes to thank marc van peski and rené scriwanek for figs. and . key: cord- - gpije d authors: netherton, christopher; moffat, katy; brooks, elizabeth; wileman, thomas title: a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication date: - - journal: adv virus res doi: . /s - ( ) - sha: doc_id: cord_uid: gpije d virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the “cytopathic effect” that has been the hallmark of virus infection in tissue culture for many years. recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. in this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. in many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. it is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. the recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release. viruses are obligate intracellular parasites. unlike their hosts, they cannot replicate by growth or division but use their genomes to redirect host cell processes to produce all the components needed to make new viruses. virus replication and assembly are often confined within specific intracellular compartments called virus factories, viroplasm, or viral inclusions. these are thought to provide a physical platform to concentrate new genomes and proteins involved in replication and assembly, and this is likely to increase the efficiency of virus production. the formation of specialized sites of replication can involve extensive reorganization of cellular cytoskeleton and membrane compartments. this can lead to cell rounding and swelling and a ''cytopathic effect'' that has been documented for many years (reissig et al., ; robbins et al., ) . recent advances in microscopy, such as live cell imaging and tomography, combined with the power of reverse genetics, are now allowing the cytopathic effect to be redefined in terms of specific effects of viral proteins on specific cellular processes rather than an overwhelming assault on the cell in preparation for cell lysis. there is considerable interest in understanding how virus infection leads to the large changes in cellular organization required to produce complex replication sites. in the simplest model, virus replication sites would form passively through self-association of viral components and exclusion of host organelles. viruses, however, require a considerable number of host proteins to facilitate replication, and there is increasing evidence that these are specifically transported to sites of replication. host proteins may move to replication sites because they are actively recruited by binding to specific viral proteins. alternatively, viruses may transport viral and host material to replication sites by subverting host defences against infection [reviewed by kirkegaard et al. ( ) and wileman ( ) ]. the large scale changes in cellular membrane and cytoskeletal organization, which occur during the formation of replication sites, can offer further benefit to viruses. rearrangement of the cytoskeleton can, for example, facilitate virus release, and the block in the secretory pathway seen during infection with positive-stranded rna viruses can reduce release of inflammatory mediators and protect against innate and acquired immune responses. this is a broad subject of considerable interest to virologists and cell biologists, and we have benefited from excellent reviews that have been published (mackenzie, ; novoa et al., ) . in writing this chapter, we have concentrated on describing sites of virus replication in the context of the cell in which its replication takes place. we have illustrated these concepts with reference to replication sites produced by many different viruses and, where possible, described how virus replication impacts on the functioning of the host cell. virus replication sites have been studied for many years and have evolved their own terminology. early studies of poxvirus replication (dales and siminovitch, ; morgan et al., ) describe electron-dense aggregates and amorphous material induced early during infection called viroplasm. viroplasm has also been used to describe similar structures induced during infection with poliovirus (dales et al., a) . viroplasm is often concentrated within perinuclear areas that exclude host organelles. viroplasm is thought to indicate sites of virus replication, and concentrations of viroplasm have been called virosomes, or virus factories, to reflect an organelle involved in virus production. virus infection also produces inclusion bodies. as a working definition, these can be considered to form later during infection. they can form virus factories once virus production has peaked, and/or at other sites in the cell they probably arise from an accumulation of viral proteins that do not become incorporated into viruses. the positive-stranded rna viruses encode nonstructural proteins (nsp) that cause proliferation and modification of membranes of the host secretory pathway. the membranes are thought to provide a physical framework or ''replication complex'' that concentrates the cellular and viral components required for virus replication (bienz et al., ; egger et al., ; froshauer et al., ; gazina et al., ; magliano et al., ; schlegel et al., ; van der meer et al., ) . assembly of the replicase on membranes, rather than the cytosol, may also help viruses evade host defence pathways that monitor cells for double-stranded rna (dsrna) intermediates indicative of virus replication. the replicase complexes of all the positive-stranded rna viruses contain an rna-dependent rna polymerase (rdrp), a protein with ntpase and helicase activity, and in many cases a methyl transferase to cap viral rna. these proteins are generated from the viral polyproteins by viral proteases, and are then targeted to membranes in ways that differ depending on virus family (fig. ). the replicase proteins of positive-stranded rna viruses are directed to membranes by nsp with membrane-targeting information. (a) picornavirus. the replication complex contains d, the rdrp (red), and c which has ntpase and helicase motifs (purple). the d polymerases do not have membrane-targeting information but are synthesized as a abcd precursor. abcd is processed to ab by the c protease (red triangle) and a hydrophobic domain in a targets ab to the cytoplasmic face of er membranes. ab binds directly to d and this targets the polymerase to the replication complex. the replication complex also requires bc and c proteins that are targeted to membranes via their own hydrophobic domains (black lines). (b) flavivirues. the replication complex is encoded at the c-terminus of a polyprotein that is processed by the ns protease (red triangle). ns b is the rna-dependent polymerase (red), and ns acts as helicase (purple). ns b is a polytopic membrane protein inserted into the er cotranslationally. ns a, a, and b have hydrophobic domains (gray lines) that allow posttranslational insertion into the cytoplasmic face of the er membrane. ns is recruited into the complex by associating with ns a. (c) alphavirus. the nsp polyprotein is processed by a protease activity in the c-terminus of p (red triangle). the polyprotein is anchored to the cytoplasmic face of endosome and lysosome membranes membranes that lie between the er and the golgi apparatus called the er-golgi intermediate compartment (ergic), or tubulovesicular structures, and specific fusion with ergic membranes is determined by a complex of proteins called transport protein particle (trapp ). trapp proteins tether the vesicles on ergic and golgi membranes, allowing interactions between vesicle and target snare (soluble n-ethylmaleimidesensitive factor attachment protein receptor) proteins to facilitate membrane fusion. the snare interactions are controlled by vesicle-specific small gtpases called rab proteins (fig. ) . further sorting events in the ergic and early golgi involve a second complex of coat proteins called copi. the copi complex contains seven proteins (a, b, b , g, d, e, and z cop proteins), which generate vesicles that take proteins from the ergic and golgi apparatus back to the er through a retrieval pathway (fig. ) . the copi proteins are recruited from the cytosol by the arf -gtpase. activation of arf requires binding to gtp and is facilitated by gtp exchange protein, arf-gef. arf -gtp inititates coat assembly while hydrolysis of gtp by arf leads to coat disassembly. this disassembly is stimulated by an arf -gtp-activating protein (arf-gap) that promotes gtp hydrolysis by arf . a possible role for arf in the generation of vesicles during picornavirus replication has been the focus of much work following the observation that poliovirus replication is blocked by brefeldin-a (bfa), a drug that inhibits the recruitment of arf onto membranes (maynell et al., ) . membrane vesicles are also produced in cells in response to starvation. this pathway, known as autophagy, is used as a part of a quality control system that removes long-lived proteins and damaged organelles from the cytoplasm and has been shown to provide a defence against intracellular pathogens (deretic, ; kirkegaard et al., ; levine and klionsky, ; shintani and klionsky, ) . the origins of the membranes formed during autophagy are unclear but may be derived from the er (reggiori and klionsky, ) . autophagy is suppressed by the target of rapamycin (tor) kinase and is activated by conditions that lead to inactivation of tor. this leads to the production of membrane crescents in the cytoplasm, called isolation membranes, which mature into doublemembraned vesicles of -to -nm diameter called autophagosomes. this maturation engulfs small quantities of cytoplasm, and any organelles or pathogens present at sites of autophagy become trapped within autophagosomes. the autophagosomes ultimately fuse with lysosomes resulting in degradation of their content. autophagosomes are of interest because infection of cells with picornaviruses and coronaviruses (covs) can generate double-membraned vesicles that may be related to autophagosomes. in addition to supplying membrane and proteins to the secretory pathway, the er acts as a major site of lipid synthesis. as a consequence, the er contains a large quantity of membrane, and this is organized into a complex reticulum made from tubular and lamella structures (borgese et al., ) . the smooth er increases in response to a buildup of er membrane proteins and can be organized into lamellae or concentric whorls called organized smooth er (oser). structures similar to oser are also seen during virus replication. b. picornavirus replication induces numerous membrane vesicles picornaviruses are nonenveloped positive-stranded rna viruses. the genome encodes a large polyprotein that is processed to generate capsid proteins from the p region and nonstructural replicase proteins from the p and p regions. picornavirus d contains the rdrp, while c has ntpase and helicase motifs. the d polymerase does not have membrane-targeting information but is synthesized as a abcd precursor. abcd is processed to ab by the c protease, and a hydrophobic domain in a targets ab to the cytoplasmic face of the er. d binds directly to ab, and this targets the polymerase to the replication complex. the d polymerase of poliovirus is believed to self-assemble into a large ordered array on membranes, which is critical for binding rna and rna elongation (lyle et al., ) . the replication complex also requires bc and c proteins that are targeted to membranes via their own hydrop hobic dom ains ( fig. a ). the accumulation of large numbers of densely packed membrane vesicles in the cytoplasm is characteristic of a picornavirus infection (bienz et al., (bienz et al., , cho et al., ; dales et al., a; schlegel et al., ; stuart and fogh, ; suhy et al., ) . studies have suggested that vesicles induced by poliovirus are derived from the er, either from copii-coated vesicles or from er-derived autophagic double-membraned vacuoles (bienz et al., ; jackson et al., ; rust et al., ; schlegel et al., ; suhy et al., ) . however, the detection of er, golgi, and lysosomal markers in membranes induced at later stages of infection by poliovirus suggests that more than one organelle may contribute membranes to the replication complex (schlegel et al., ) . in interpreting these studies, it is important to consider if the vesicles observed are involved in replication, or if they represent a bystander response to virus infection. evidence for a role of specific membranes in replication is provided by the presence of replicase proteins, or better still dsrna or negative-stranded intermediate viral rna (egger and bienz, ) . examination of cells infected with poliovirus for the first appearance of negative-stranded rna suggests that this initial stage of replication starts on the er. this is consistent with high-resolution immunofluorescence microscopy (rust et al., ) showing the poliovirus b protein associated with eres containing the sec -sec p proteins of the copii complex. these sites exclude resident er proteins, suggesting colocalization of b with copii-coated transport vesicles. replication complexes containing negative-stranded rna then move on microtubules to a perinuclear area to initiate synthesis of positive-stranded rna (egger and bienz, ) . membrane rearrangements have been studied by expressing individual, or combinations, of picornavirus proteins in cells. most of this work has involved studies of poliovirus proteins, and membrane rearrangements are reported for the b, c, bc, a, and ab proteins. poliovirus b causes fragmentation of the golgi complex (sandoval and carrasco, ) . the bc and c proteins lead to vesiculation and tubulation and sometimes myelin-like swirls of er-derived membranes (aldabe et al., ; cho et al., ) . similar structures are induced by c and bc of hepatitis a virus (teterina et al., ) . expression of the poliovirus a protein causes swelling of er cisternae and the disappearance of vesicles budding from the er, while the ab protein also induces myelin-like swirls of er (egger et al., ) . the membrane rearrangements induced by expression of single proteins do not, however, mirror those observed in infected cells, and since myelin-like modifications to the er are also seen following overexpression of er proteins [reviewed by borgese et al. ( ) ], their relevance to viral replication is unclear. importantly for poliovirus, it is a combination of bc and a protein expression that induces membrane structures morphologically similar to those seen in infected cells (suhy et al., ) . gazina et al. ( ) have studied replication complexes formed by several different picornaviruses. encephalomyocarditis virus (emcv), parechovirus , and echovirus induce clustered vesicles containing dsrna in the perinuclear region of the cell. the precise nature of the vesicles varied with virus. parechovirus produced homogeneous vesicles of - nm, while membranes produced by emcv and echovirus were heterogeneous but more compact and associated with electron-dense material. differences for parechovirus have also been reported by krogerus et al. ( ) who suggest that replication may occur on membranes derived from the late golgi rather than early er and ergic compartments. all three viruses, however, cause loss of ribosomes from the er and lack of visible golgi apparatus. the copi coat protein b-cop was found to colocalize with echovirus replication complexes, but not with replication complexes produced by emcv, again suggesting that vesicles produced by different picornaviruses may differ. infection with foot-andmouth disease virus (fmdv) also results in loss of ribosomes from the er and an accumulation of heterogeneous vesicles to one side of the nucleus . high-pressure freezing can be used to increase the preservation of cellular ultrastructure during processing for electron microscopy. such analysis of cells infected with poliovirus shows that the vesicles have two membranes suggestive of autophagosomes ( jackson et al., ; suhy et al., ) . double-membraned structures containing electron-dense material, and possibly viruses, were also revealed by the early work on poliovirus (dales et al., a) . high-pressure freezing has been used to compare fmdv and bovine enterovirus (bev). bev produced heterogeneous membrane clusters similar to the rosettes described for poliovirus (egger et al., ) . many of the vesicle membranes have high electron density suggestive of double membranes and lie adjacent to accumulations of virus-like particles. clusters of fmd viruses were also associated with vesicles and electron-dense material, but there were fewer doublemembraned vesicles . immunofluorescence analysis of poliovirus vesicles shows colocalization of replicase protein a and autophagy marker lc , suggesting assembly of the replicase on autophagosomes. similar work suggesting the use of autophagosomes during replication of covs will be described below. for poliovirus, expression of a and bc, which produces vesicles similar to those seen in infected cells (suhy et al., ) , can induce autophagy (jackson et al., ) , and inhibition of autophagy reduces yields of extracellular virus. the results suggest that the autophagy pathway may facilitate the release of poliovirus from cells, and it will be interesting to see if this is true for other enteroviruses that are resistant to the low ph and proteases present in lysosomes and autophagosomes. evidence that different members of the picornavirus family vary in the way that they interact with host membranes is provided by studies of virus sensitivity to bfa. bfa completely inhibits poliovirus and echovirus replication (cuconati et al., ; gazina et al., ; irurzun et al., ; maynell et al., ) and partially inhibits parechovirus replication (gazina et al., ) but not other picornaviruses such as emcv (gazina et al., ) or fmdv o'donnell et al., ) . bfa prevents assembly of copi coats and this has generated considerable interest in understanding how copi and copii coats contribute to formation of the replication complex, and how bfa inhibits picornavirus replication. in cells infected with the highly bfa-sensitive virus echovirus , b-cop was recruited into the replication complex; in contrast, the replication complex formed by the bfa-resistant emcv did not contain b-cop. this correlation suggests that bfa-sensitive viruses may require copi coats for replication (gazina et al., ; mackenzie, ) . since copii coats are resistant to bfa (lippincott- schwartz et al., ; orci et al., ; , it is suggested that copii coats may provide the membranes for replication complexes formed by bfa-insensitive viruses. the observation that poliovirus replicase b protein is seen in eres containing copii proteins, but poliovirus is sensitive to bfa, can be reconciled if this association of b with eres is considered to be an early step in generation of membrane for the replication complex that precedes recruitment of copi coat proteins. this is supported by work showing the movement of poliovirus replication complexes containing negative-stranded rna from the er to perinuclear sites (egger and bienz, ) . direct evidence that copi coat proteins are required for picornavirus replication comes from studies of drosophila c virus (dcv). dcv is a positive-stranded rna dicistronic virus that is similar to poliovirus and replicates in a cytoplasmic compartment containing virus-induced membrane vesicles. a genome-wide rna silencing screen identified six (a, b, b , g, d, and z) of the seven copi coat proteins as essential for virus replication. furthermore, the formation of virus-induced vesicles required b-cop, but not copii protein, sec p. notably, small interfering rnas against a-cop, but not sec p, also slowed poliovirus replication (cherry et al., ) . the formation of copi-coated vesicles is regulated by the arf -gtpase. the observation that bfa inhibits the replication of enteroviruses such as poliovirus, and also inhibits the function of the arf -gtpase, provides a second link between virus replication and copi coats. arf proteins are regulated by arf-gefs that facilitate binding of gtp by removing gdp, and by arf-gaps that increase hydrolysis of gtp by arfs. arf -gefs are inhibited by bfa, and bfa therefore reduces levels of arf -gtp in cells. the gefs affected by picornavirus infection are golgi-associated bfaresistant protein (gbf ) and bfa-inhibited protein (big / ). work by belov et al. ( belov et al. ( , indicates that infection of cells with poliovirus increases intracellular arf-gtp levels fourfold, suggesting increased activity of arf -gefs or inhibition of arf -gap proteins. in the absence of virus, arf is concentrated in the golgi apparatus, but during infection with poliovirus arf staining fragments and colocalizes with replicase protein c. this suggests that infection leads to a redistribution of arf proteins from the golgi apparatus to the replication complex. the binding of arf proteins to membranes is dynamic, with arf-gdp being released from membranes following hydrolysis of gtp. cytosolic arf -gdp would redistribute naturally to membranes enriched for the arf -gefs that facilitate loading of new gtp. significantly, poliovirus infection causes enrichment of gefs in membranes containing replicase proteins, and this would provide a mechanism for increasing levels of arf -gtp at sites of virus replication. translation of poliovirus rna on membranes in vitro provides an alternative means of studying the role of arf proteins in virus replication. replication is inhibited by bfa and peptides that function as competitive inhibitors of arf (cuconati et al., ) , and for the most part, the assay mimics what is observed in infected cells. translation in vitro leads to recruitment of arf and arf but not arf (belov et al., ) onto membranes. suitable antibodies recognizing the er-associated arf were not available for these experiments, so it is not known if arf is also recruited to membranes during translation. membrane recruitment of arf proteins can be reconstituted by translation and expression of poliovirus a or cd. poliovirus proteins do not show intrinsic gef activity, but a and cd will induce association of gbf and big / , respectively, with membranes in vitro. this raises the possibility that recruitment of a and cd to the replication complex during infection targets arf-gef to virus-induced membranes, which in turn increases local levels of arf -gtp. this is thought to be necessary for replication because inhibition of arf -gef by bfa blocks replication, and replication can be rescued by overexpression of gbf (belov et al., ) . high levels of arf -gtp would also increase recruitment of copi proteins and be consistent with the work on dcv showing that copi proteins are required for replication and vesicle production (cherry et al., ) . a poliovirus a mutant with a serine insertion at position is unable to cause translocation of arf to membranes (belov et al., ) . poliovirus carrying the a mutation does not, however, show defects in replication, suggesting that arf -gef recruitment to membranes by a is not essential for replication. it is possible that during infection the defect in a is compensated for by cd. interestingly, a bfa-insensitive poliovirus with mutations in the c and a proteins (crotty et al., ) induces vesicles and dispersal of the golgi apparatus, which begs the questions, does this mutant use a different process for forming the replication complex, or do the mutations in a allow the proteins to compete with bfa for gbf recruitment? the role of arf proteins during coxsackievirus infection has also been studied. in common with poliovirus, coxsackieviruses are enteroviruses and their replication is inhibited by bfa. expression of coxsackievirus a causes loss of copii coats from eres, and an accumulation of a, copii and a model secreted protein in both the er, and tubular-vesicular post-er structures containing ergic marker proteins. these effects closely resemble the effects of adding bfa to cells, suggesting coxsackievirus a may affect the function of arf proteins. coxsackievirus a affects the regulation of arf proteins (wessels et al., b) . interestingly, the process differs to that described by belov et al. ( belov et al. ( , for poliovirus a translated in vitro. expression of coxsackievirus a in cells caused loss of copi and arf from membranes, and there was redistribution of big / and gbf from the golgi apparatus into the cytoplasm. this suggests that coxsackievirus a reduces, rather than enhances, levels of arf -gtp. coxsackievirus a also caused redistribution of arf -gap to punctate structures suggestive of the ergic. a block in arf -gef activity, combined with recruitment of arf -gap, would reduce the levels of arf-gtp and inhibit membrane recruitment of copi. wessels et al. ( a) examined the effects of the a proteins of other picornaviruses and found that only the a proteins of enteroviruses bound gefs. intriguingly, wessels' work contrasts with belov in that they found the interaction of a with gefs lead to a loss of arf proteins from membranes. why these differences are seen is, as yet, unknown but may be due to differences in cell type/methods used or differences in levels of a protein expression. poliovirus and coxsackievirus slow protein movement through the secretory pathway (doedens and kirkegaard, ; wessels et al., ) . expression of b, bc, and a individually were all able to slow secretion (cornell et al., ; doedens and kirkegaard, ; doedens et al., ; van kuppeveld et al., ; wessels et al., wessels et al., , a , but for both viruses the a protein was found to have the greatest impact on er-to-golgi transport. poliovirus infection, and the a protein expressed alone in cells, reduces surface expression of mhc class i, the tnf receptor, and secretion of b-ifn, il- , and il- (choe et al., ; deitz et al., ; dodd et al., ; neznanov et al., ) , and this may offer an immune evasion strategy to the picornaviruses. this is consistent with the observation that the ability of the coxsackievirus a protein to slow secretion may be important for virulence (wessels et al., b) and has led to studies of the mechanism of action of a in blocking er-to-golgi transport. deletion analysis has identified residues in the unstructured n-terminal region of poliovirus and coxsackievirus a as important for the block in host protein secretion (choe et al., ) . an n-terminal proline-rich region (particularly pro ) is important for coxsackievirus block in trafficking (wessels et al., ) . in poliovirus, lys appears important, and in the triple-proline motif (positions - ), only the pro is indispensable for inhibition of protein secretion (choe et al., ) . a serine insertion in a protein between thr and ser , creating the a- mutant virus (berstein and baltimore, ) , was found to abolish the er-to-golgi inhibition of protein trafficking but has little effect on virus replication or membrane rearrangements (dodd et al., ; doedens et al., ) . this important observation shows that the ability of a to inhibit protein secretion is separate from its role in membrane rearrangements and viral replication. there is continuing interest in understanding how picornavirus proteins block secretion. poliovirus a and cd, and coxsackievirus a, can interact with arf-gef, but the downstream events are unclear. the recruitment of arf-gef by poliovirus a and cd would increase recruitment of arf-gtp to membranes of the replication complex. this would increase recruitment of copi coat proteins into sites of virus replication and reduce the pool of copi proteins available to the ergic and golgi apparatus. alternatively, inhibition of arf-gef and recruitment of arf-gap onto ergic membranes by enterovirus a would decrease membrane association of arf-gtp and again reduce recruitment of copi onto ergic and golgi membranes. both mechanisms would reduce the formation of copi vesicles, and as seen for bfa, block secretion. poliovirus a also binds and inactivates l s , a component of the dynein-dynactin motor complex (kondratova et al., ) , which is required to move copiiderived vesicles from eres to the ergic. as seen for expression of a, mutant l s leads to disruption of the er-to-golgi traffic and reduction in plasma membrane receptors such as tnf receptor. it is possible that a may also slow er-to-golgi transport by binding l s . a. picornaviruses differ in the use of nonstructural proteins to block secretion the ability of a to inhibit er-to-golgi trafficking has not been conserved in all picornaviruses (choe et al., ; cornell et al., ; deitz et al., ; moffat et al., ) . for example fmdv infection leads to reduced surface expression of mhc class i (sanz-parra et al., ) , but the fmdv a protein does not inhibit er-to-golgi transport . a lack of inhibition of secretion has also been reported for a proteins of human rhinovirus, hepatitis a, theiler's virus, human enterovirus, and emcv (choe et al., ; wessels et al., a) . the a protein of human rhinovirus is unable to bind gbf , or inhibit copi recruitment to membranes, and this may explain its inability to slow secretion. importantly, studies on fmdv have shown that the bc protein, or a combination of the processed products, b and c, inhibits protein movement from the er to the golgi apparatus (moffat et al., , , and this may be similar for other picornaviruses with a proteins that do not block er-to-golgi transport. a lack of effect of fmdv a on secretion does not result from an inability to bind membranes. fmdv a is recovered from postnuclear membrane fractions, and when expressed alone in cells it colocalizes with resident er proteins. in common with a, picornavirus b, c, and bc proteins also contain membrane-binding sequences. sequence alignment of the b, c ( bc), and a proteins of different picornaviruses showed a high level of conservation between the c proteins, which contain an ntp-binding site and predicted helicase motifs (gorbalenya et al., ) but large variations in the sequences of the b and a proteins (choe et al., ; moffat et al., ) , and these may explain their different abilities to block secretion. the fmdv a protein is, for example, much longer than a of enteroviruses, such as poliovirus, and it does not contain the n-terminal sequences thought important for poliovirus a to block the secretory pathway. the b protein of fmdv also locates to er membranes but shows a more reticular pattern than the fmdv a protein and can be seen in punctate structures aligned along the er suggestive of eres (fig. ) . this is similar to the b of poliovirus that colocates with both figure subcellular location of foot-and-mouth disease nsp encoded in the p region of the fmdv genome. vero cells expressing fmdv b (top), bc (middle), or c (bottom) were fixed and permeabilized and processed for immunofluorescence. c and bc were located using antibodies specific for c ( f ) and b was located using an antibody raised against an epitope tag in b. cells were counterstained using antibodies against er luminal protein erp (top and middle panels), or copi protein b-cop (bottom). merged images are shown at higher magnification on the far left. see moffat et al. ( ) for more details. reprinted from moffat et al. ( ) with permission from american society for microbiology. sec p and sec p of the copii coat. as expected, fmdv c is also membrane associated. when expressed in cells, c produces faint er staining, but mainly locates to bright punctate structures in a perinuclear region close to b-cop, reminiscent of golgi staining. the b-cop staining is, however, fragmented suggesting dispersal of the golgi apparatus, and there is not complete colocalization since c structures negative for b-cop protein can also be seen (moffat et al., ) . a similar location of fmdv nsp within the area of the cell occupied by the golgi apparatus is seen in cells infected with fmdv, and again they do not colocalize with golgi markers . the bc protein of fmdv is also recovered in postnuclear membrane fractions, but when expressed in cells, bc staining differs from that seen for the processed products, b and c (fig. ) . fmdv bc locates to punctate cytoplasmic structures and larger structures surrounding the nucleus that contain er markers suggesting swelling of the er. bc shows partial overlap with luminal er markers but, unlike poliovirus bc, does not colocate with the copii marker sec p. the er markers also appeared punctate in cells expressing bc, suggesting disruption of the er . interestingly, coexpression of b and c blocks secretion within post-er compartments, similar to those containing c. the site of block therefore seems to be determined by the subcellular location of c (moffat et al., ) and is consistent with the observation that the block in the presence of b can be redirected to the er, if c is tethered to the er by an er retention sequence. sindbis virus (sbv) and semliki forest virus (sfv) are the best studied examples of alphavirus replication in mammalian cells [reviewed by salonen et al. ( ) ]. early electron microscopy studies showed that vesicular structures called cytopathic vacuoles between -and -nm diameter, accumulated in infected cells. the vacuoles contained -nm-diameter vesicles called spherules, many of which were aligned along the inside face of the vacuole and attached by a neck to the limiting membrane. the neck was often seen connected to an electron-dense matrix extending into the cytoplasm. the observation that the cytopathic vacuoles contained nsps required for rna replication, cofractionated with lysosomal enzymes, and could be labeled with endocytic markers (froshauer et al., ) , led to the conclusion that they are sites of viral replication derived from endosomes and lysosomes. in many cases, the vacuoles were also connected to the rough er by filaments and granular material containing the rna polymerase. alp havirus ns ps are syn thesized in the cytop lasm and bind to endosom es and lysos omes to generate a replication compl ex. the rep licase pro teins are syn thesized as a polyprote in (p ) . the p domai n is the rdrp wh ile p has ntpase and helicase activi ties, and p is the methytran sferase require d to cap rna ( fig. c) . the p poly protein locate s to endosome or lysosome membranes via an amphipathic peptide sequence in p (salonen et al., ) . at this stage the p polymerase is cleaved from the polyprotein and functions with the remaining p protein to generate negative-stranded rna. interestingly, once the p is processed to individual nsps, the polymerase preferentially produces positive-stranded rna. expression of individual nsps does not lead to the formation of a cytopathic vacuoles or spherules. formation of spherules requires interactions between nsp p , p , and p and the p polyprotein precursor complex (salonen et al., ) . rubella virus is a member of the togaviridae family within the alphavirus genus. cells infected with rubella virus also contain vacuoles containing spherules and these colocalize with lysosomal markers, suggesting use of lysosomes for replication. a fibrous material connects the vacuoles to the er (lee et al., ; magliano et al., ) , again suggesting strong similarities with sfv and sbv. members of the alphavirus superfamily share homologies between proteins required for rna replication, and this extends to plant viruses. alfalfa mosaic virus replicase proteins colocalize with the plant vacuole (van der heijden et al., ) , and turnip yellow mosaic virus uses the chloroplast outer envelope as a site for replication. replication of tobacco mosaic virus, a tobamovirus, is dependent on arabdopsis proteins tom and tom a that are integral membrane proteins of the tonoplast (hagiwara et al., ) . the tonoplast is a membrane compartment within plants that surrounds the vacuole/lysosome, suggesting plant alphaviruses also use the endosome/lysosome system as a site of replication. infection of plants with alphavirus-like superfamily viruses can also induce the formation of spherules (prod'homme et al., ) . there is evidence that tobacco mosaic virus also uses the er as a site of replication because the replicase enzyme and viral rna are located on the er of infected cells, and infection causes major changes in er morphology (reichel and beachy, ) , including er aggregation and formation of lamella structures. flock house virus replicates in spherules in the outer membrane of mitochondria. the rna polymerase (protein a) of flock house virus is the only protein required for rna replication and is targeted directly to the mitochondrial outer membrane by hydrophobic amino acids at the n-terminus. this sequence contains a mitochondrial localization signal and transmembrane domain that leaves the bulk of the protein exposed to the cytoplasm (miller and ahlquist, ) . brome mosaic virus replicates in yeast and has been studied extensively. the a and a replicase proteins are produced from separate viral rnas. the a protein contains a c-terminal helicase domain and an n-terminus required for rna capping. a is targeted to the cytoplasmic face of er membranes and recruits the a polymerase to the replication complex (schwartz et al., ) . importantly, replication of brome mosaic virus on the cytoplasmic face of the er in yeast induces membrane invaginations of nm that are very similar to the spherules produced in endosomes and lysosomes during alphavirus infection of mammalian cells. it has been suggested that the active formation of spherules to separate viral rna from host responses is analogous to the coordinated assembly of viral proteins, which leads to capsid assembly, genome packaging, and budding (ahlquist, ; schwartz et al., ) . the brome mosaic virus replication complex contains viral a and a pol proteins within spherules. expression of a alone produces a shell containing hundreds of copies of a on the inside of -nm spherules. in a capsid assembly model (schwartz et al., ) , vesicles of uniform size would arise if the a protein first made a planar lattice with hexameric symmetry on membranes and achieved curvature by localized rearrangement of a into pentamers. interestingly, the formation of spherules is dependent on the relative levels of a and a pol . when levels of a pol are high, the spherules are lost, and a and a pol assemble into flat lamella structures associated with the er (schwartz et al., ) . one explanation for a failure to achieve curvature is that high levels of a pol may interfere with this hexamer to pentamer transition. this is supported by the observation that when domains that allow association of a and a pol are deleted, the a pol is unable to alter the structure of spherules formed by a. the correct ratio of a and a pol is clearly important for replication complex assembly and may be maintained during infection through inhibition of translation initiation of the a rna. d. the flaviviridae replicate in vesicular packets and membraneous webs in the flaviviridae family, which includes the flavivirus, pestivirus, and hepacivirus genera, the rna genome encodes a polyprotein precursor that is cleaved by viral proteases to produce structural proteins from the n-terminal region. the replicase of the flaviviridae is made from nsps, ns a, ns b, ns b, and ns - a, found at the c-terminus. wi th the exce ption of the polytop ic ns b membr ane prote in, whic h is ins erted co transl ationall y into the er, the membr ane-an chored co mponent s of the compl ex are inserted into the cytopla smic face of the er after tran slation ( fig. b) . the ns b is the rdrp, and a c-termi nal stretch of hydrophobic amino acids directs ns b to the cytoplasmic face of the er (dubuisson et al., ; moradpour et al., ) . the ns protein has ntpase/helicase activity. ns is not a membrane protein but is recruited to the complex through association with membrane-anchored ns a. ns a is also membrane associated, and association is mediated via amino acids at the n-terminus that form an amphipathic a-helix (brass et al., ; elazar et al., ) . replication of flaviviruses (e.g., dengue, west nile, and yellow fever viruses) takes place in membrane invaginations. for historical reasons, these are called vesicular packets [reviewed in mackenzie ( ) ]. they are larger ( -to -nm diameter) than the -nm alphavirus spherules, and form from the limiting membrane of the trans-golgi network (tgn) (uchil and satchidanandam, ; westaway et al., b) . infection by kunjin virus leads to unique membrane structures thought to be derived from both the early and late secretory pathways. these include convoluted membranes and paracrystalline arrays derived from the rough er and ergic, and vesicle packets derived from the tgn (mackenzie et al., ; ng, ; roosendaal et al., ; westaway et al., b) . the detection of dsrna and viral nsps (ns , ns a, ns , and ns a) within the vesicle packets points strongly to this being the site of rna replication (mackenzie et al., ; westaway et al., b) . the vesicle packets associate closely with the convoluted membranes and paracrystalline arrays, which are thought to be the sites of proteolytic processing of ns and ns b (westaway et al., b) . these modified membranes are linked with the er, and ultrastructural studies have shown virions present in the er, cytoplasmic vesicles, golgi cisternae, and vacuoles. the results suggest that membranes containing the spherules responsible for replication may become associated with the er to facilitate delivery of genomes to viruses, budding into early compartments of the secretory pathway (mackenzie and westaway, ) . hepatitis c virus (hcv) is closely related to the flaviviruses, and its importance as a human pathogen has generated great interest in its mechanism of replication. until, recently infection models have not been available to study the replication complex of hcv, and the studies discussed here have focussed on the expression of the entire polyprotein from replicons gosert et al., ) . however, the recent production of a hcv that rep licates ef ficiently both in vivo and in cell culture (li ndenbac h et al. , ; wakita et al., ; zhong et al., ) will exp and the possi bilities for studying and understanding the viral replication cycle. hcv replication is thought to occur on membranes derived from the er as all studies of nsps have found them localized to this organelle (dubuisson et al., ; hugle et al., ; kim et al., ; wolk et al., ) . studies have also identified a ''membraneous web'' of membrane vesicles of $ -nm diameter associated with the er and a population of irregular doublemembraned vesicles. the web resembled the ''sponge-like inclusions'' seen in the liver of chimpanzees infected with hcv, suggesting it is physiologically relevant. interestingly, the great majority of the nsp synthesized by full-length genomes or subgenomic replicons may not be involved in rna replication (quinkert et al., ) . the bulk of the nsps associated with membranes isolated from cells expressing replicons is sensitive to protease, while in vitro replicase activity is resistant to protease and nuclease activity (el-hage and luo, ; quinkert et al., ) . the results suggest that replication of hcv takes place within membrane vesicles, rather than on the surface of the membraneous web. these vesicles may be associated with the membraneous web, but the similarity between hcv and the flaviviruses leaves open the possibility that the membrane invaginations responsible for replication may also form in the tgn but be closely associated with the er. studies have investigated which viral proteins are responsible for membrane rearrangements seen in cells infected with flaviviruses. the ns a of kunjin virus induces the characteristic convoluted membranes and paracrystalline arrays seen in flavivirus infections. the ns a-b protein also causes membrane rearrangement, but the highly condensed structures seen in infected cells are not produced until the ns b- protease cleaves ns a free from ns b (roosendaal et al., ) . the ns b then translocates to the nucleus (westaway et al., a) . interestingly, this contrasts with hcv where ns b (and ns a-b) konan et al., ) rather than ns a is able to induce the membranous structures. flaviviruses have been found to upregulate cell surface expression of mhc class i and ii in response to interferon (king and kesson, ; liu et al., ; lobigs et al., ) . this is not caused by effects of the ns a or ns b proteins on membrane traffic; instead flavivirus infection increases expression of the er peptide transporter, tap . this increases the supply of peptides that are necessary for the folding and export of newly synth esized mhc proteins from the er. inc reased tap expression is media ted by increas ed transcrip tional activ ity of p and can be induc ed in liver hepg cells by express ion of the hcv core/cap sid pro tein alon e ( herzer et al., ; mombu rg et al ., ) . whil e the cap sid/cor e prote in is able to in crease cell surface expression of mhc clas s i through increase expres sion of tap , exp ression of the hc v polyp rotein has been sho wn to slow the moveme nt of prote ins thr ough the secretory pathway of hos t cells ( konan et al ., ) . the rate of delive ry of mhc cl ass i to the plasm a me mbrane in cells infected with hc v was reduced three-to five fold relative to cu red contr ol cells. exp ression of the pre cursor ns a-b was fou nd to red uce er-to-g olgi traf fic two-to threefo ld (kon an et al., ) , while the ot her ns proteins of hc v inclu ding ns a and ns b, indiv idually or comb ined, were unabl e to interf ere with the traf ficking pathway . ns b a lone indu ces a memb raneo us web in ce lls , and both ns a-b and ns b indu ce, and locat e to, clust ered and aggregate d membr anes looking v ery similar to the me mbraneo us web seen in cells expre ssing rep licons. in addition to agg regated me mbrane s, ns a/b also ind uces, but does not coloca lize with , swol len vesicul ar structure s. thes es swo llen vesicl es have a similar morphology to the vesicles induced by the a protein of p o li ov ir us , wh ich swe lls er memb ranes and blocks sec retio n betwee n the er and the golgi appar atus (doe dens et al., ). konan et al . ( ) hypo thesize that the ns a/b could be func tioning in a sim ilar man ner to poliovi rus a. e. the nidovira les replicate in association with doub le-membraned vesicles . the nidovirus replicase is generated from two polyproteins the nidovi rales order comp rises the arterivi ridae, coron aviridae , and ron iviridae famil ies. the rep licase gene is co mposed of two ope n read ing fram es termed orf a and orf b. orf b is gen erated from a fram eshift in a, and both reading frame s encode co mplex poly proteins pro cessed by viral prote ases (go rbalenya et al., ; ziebu hr, ) . the arter ivirus orf b encode s nsps - , incl uding the rdrp (nsp ) and helicase (nsp ). the orf b, however , lacks hydroph obic dom ains able to target the rep licase to membr anes. intere stingl y, the hydroph obic domains necessar y for membr ane targetin g are enco ded by orf a in nsp , , and , sugge sting that orf a pr oteins produc e a scaffold to locate the viral rep lication -transcr iption compl ex to membr anes ( fig. d ) ( pedersen et al., ; van der meer et al., ) . a similar strategy is used by cov, for example mouse hepatitis virus (mhv) and severe acute respiratory syndrome-cov (sars-cov) (prentice et al., a,b) , where transmembrane domains are located in nsp , , and , and helicase and polymerase proteins are nsp and , respectively, and nsp encodes the methytransferase. the nidovirales have the largest coding capacity of the single-stranded rna viruses, and not all the nsps have been studied in detail. it is possible that other proteins encoded by orfs a and b, such as rna processing enzymes, are incorporated into the replication complex. several studies have investigated the intracellular sites of replication of equine arterivirus (eav), mhv, and sars-cov. such studies are difficult because during nidovirus infection, the processes of replication and envelopment occur on different membranes, and these may merge during encapsidation. furthermore, late during infection cells infected with mhv can form syncitia. newly synthesized mhv viral rna has been found in perinuclear sites colocalized with the rdrp (shi et al., ) , and depending on whether human or murine cells were infected, these sites colocalized with golgi or er membranes, respectively. similar studies in mouse l cells report that the polymerase and newly synthesized rna locate to late endosomes and endocytic carrier vesicles . this discrepancy is in part reconciled by later work showing that the subcellular distribution of the replicase proteins can change during the course of infection, since replicase proteins move to sites of envelopment in the ergic (bost et al., ) . this is supported by the finding that individual replicase proteins distribute differently following cell membrane fractionation (sims et al., ) . membrane fractionation has also been carried out by gosert et al. ( ) , who showed that several proteins encoded by orf a and b were associated with membranes, and when observed by immunogold electron microscopy, these were associated with rosettes of double-membraned vesicles - nm in diameter. the role of these vesicles in viral rna replication was confirmed by in situ hybridization of labeled riboprobes. double-membraned vesicles are also seen in cells infected with eav (pedersen et al., ) . eav replicase proteins accumulate in perinuclear regions containing ergic and er markers and colocalize with newly synthesized viral rna, again suggesting sites of genome replication. notably, similar structures can be produced by expression of arterivirus orf a-encoded proteins nsp - , which contain the membrane proteins thought to tether the replicase to membranes. double-membraned vesicles are usually rare in cells but are induced during autophagy. a role for autophagy during mhv infection is suggested because autophagy is induced in cells infected with mhv. furthermore, in cells lacking atg , a protein required for the formation of autophagosomes, there is a % reduction in virus yield and mhv fails to induce double-membraned vesicles (prentice et al., a) . electron micrographs show that the double-membraned vesicles induced by sars-cov extend from the er and can be labeled with antibodies specific for replicase proteins. this suggests that, in common with mhv, the vesicles are a site of replication . even though all sars-cov replicase proteins tested colocalize to punctate structures that accumulate near the nucleus, there are conflicting reports about their relationship with autophagosomes. in monkey vero cells, the replicase proteins colocalize with autophagosomes identified using antibodies against lc (prentice et al., a) . however, when autophagosomes are identified by expression of gfp-lc , the replicase proteins do not colocalize with the gfp signal . the vesicles induced by sars-cov are smaller at -to -nm diameter than autophagosomes ( - nm) and are labeled with er markers. this has lead snijder and colleagues to suggest that they are virus-induced extensions to the er, rather than bona fide autophagosomes (pedersen et al., ; snijder et al., ) . the precise origins of the membrane crescents that form at the start of autophagy are unclear, and a number of studies have suggested they may form from the er. this makes it possible that the double-membraned structures may be autophagosomes that have been modified by an accumulation of viral protein. determining if autophagy is beneficial to sars-cov replication will have to await studies in cells where key proteins in the autophagy pathway have been removed or suppressed by gene silencing. the asfiviruses, poxviruses, iridoviruses, and the phycodnaviruses are large dna viruses encoding hundreds of proteins from genomes ranging between and kbp. a comparison of protein sequences encoded by these viruses has suggested that they should be grouped together in a family of viruses called the nucleocytoplasmic large dna viruses (ncldv) (iyer et al., ) . sequence similarities are seen in the major capsid proteins, redox enzymes that maintain disulphide bonds in the cytosol, and proteins that regulate apoptosis; and the family has been extended to include the giant mimivirus isolated from the ameba acanthamoeba polyphaga (la scola et al., ) . even though these viruses infect a diverse range of hosts from different phyla, including vertebrates [poxviruses, african swine fever virus (asfv)], arthropods (entomopox, asfv, chloriridoviruses), amphibians and fish (ranavirus, megalocytivirus, and lymphocystivirus genera of the iridoviridae family), marine algae (phycodnaviruses), and protozoa (mimivirus), they all generate cytoplasmic factories as major sites of virus assembly and replication (illustrated in fig. ). the factories share many similarities with one another, again suggesting that this diverse group of viruses may be related and that the need to produce a virus factory in the cytoplasm was generated early in virus evolution. . asfv factories form next to the microtubule organizing center asfv is the sole member of the asfivirus genus, family asfarviridae but shares striking icosahedral similarity with the iridoviruses, phycodnaviruses, and mimivirus. asfv is a large double-stranded dna (dsdna) virus with a genome size ranging from to kbp. gene expression is a regulated cascade and immediate early, early, early/late, intermediate, and true late gene types have been characterized to date. the virion has multiple concentric layers with an electron-dense core at the center that contains the viral genome. a protein matrix surrounds the core, which in turn is enclosed by a lipid bilayer. finally, the bilayer is surrounded by a protein capsid layer. asfv can gain a third envelope when it buds from the plasma membrane at the tip of actin-rich projections that resemble filopodia (jouvenet et al., ) . asfv probably enters cells by receptor-mediated endocytosis, but the steps following entry are poorly understood. it is possible that a viral core is delivered into the cytoplasm intact; alternatively, cores may dissociate in endosomes requiring some mechanism of genome delivery across the endosome membrane. genome replication occurs both in the nucleus and cytoplasmic factories. transfer to the nucleus may involve microtubule transport since late gene expression is inhibited by agents that depolymerize microtubules and the dominant-negative dynein motor protein p -dynamitin (alonso et al., ; heath et al., ) . asfv does not produce nuclear inclusions analogous to those seen in herpesvirus and adenovirus infection, but there is evidence that small fragments of viral dna are synthesized in the nucleus. the major site of asfv dna replication is, however, the virus factory (rojo et al., ) . a. cytoplasmic factories formed during asfv infection are assembled at the microtubule organizing center asfv induces one principal factory in the cytoplasm during infection. electron microscopy shows that the virus factory excludes obvious cellular organelles and contains mostly viral dna, viral proteins, virus-induced membranes, and partially and fully assembled virions (table i; fig. a ; brookes et al., ; moura nunes et al., ; rouiller et al., ) . the mechanisms that target viral proteins, virus-induced membranes, and viral dna to the asfv factories are poorly understood. immunofluorescence staining for viral structural proteins generally reveals a strong signal at the factory and a weaker signal in the cytoplasm. the b lp protein (cap ), which is a viral chaperone involved in folding and membrane recruitment of the major capsid protein, p , is, for example, absent from the virus factories (cobbold et al., ; epifano et al., ) . this suggests that p is synthesized and folded in the cytoplasm and then recruited to factories. similarly, the viral dutpase, which is necessary for efficient replication, is excluded from the viral factory (oliveros et al., ) . since the bulk of viral dna synthesis occurs in the factory (garcía-beato et al., ) , it is not easy to explain how the viral dutpase edits uracil from progeny viral genomes, without being present at the site of viral dna synthesis and encapsidation. asfv factories disperse when cells are incubated with drugs that depolymerize microtubules (heath et al., ) suggesting their formation involves microtubule motors. this may involve dynein motor proteins since p -dynamitin, a dominant-negative version of the dynein motor, prevents both late asfv gene expression (heath et al., ) and vimentin recruitment to factories (see below and stefanovic et al., ) . yeast-twohybrid screens and in vitro pull-down experiments show that one asfv structural protein, p /j lp, interacts with dynein (alonso et al., ) . while direct binding of p /j lp to the motor protein has not been observed in infected cells, it is possible that the protein is involved in transporting some viral proteins into factories. the protein locates to virus factories and deletion of the e l gene encoding p /j lp generates factories that lack viral membranes, the major capsid protein p , and the polyprotein precursors (pp , and pp ) of the viral matrix (epifano et al., ; rodríguez et al., ) . p /j lp is a membrane protein with the bulk of the protein, including the dynein-binding motif, exposed to the cytosol. the p capsid protein and pp polyprotein associate with membranes before assembly into viruses (cobbold and wileman, ; cobbold et al., ; heath et al., ) . if these membranes contain p /j lp, it would provide a means of allowing recruitment to factories by retrograde transport along microtubules. the formation and morphology of asfv factories closely resemble the formation of aggresomes (heath et al., ) , a cellular response to accumulation of misfolded protein aggregates (johnston et al., ) . aggresomes are microtubule-dependent inclusions containing protein aggregates that human herpesvirus induces nuclear tegusomes (t). herpesviruses induce cytoplasmic assembly sites where envelopment and some tegument are acquired (env) in human herpesvirus , these sites include electron-dense bodies (db). iridoviruses induce multiple cytoplasmic virus factories (vf) and crystalline arrays (ca), both of which associate with mitochondria. reoviruses also induce multiple cytoplasmic virus factories (vf) and crystalline arrays (ca) that are enclosed within lysosomal membranes. alcamí et al., ; alonso et al., ; andrés et al., andrés et al., , borca et al., ; brookes et al., a,b; carrascosa et al., ; chacó n et al., ; cobbold et al., ; galindo et al., ; garcía-beato et al., ; heath et al., ; hingamp et al., ; jouvenet and wileman, ; jouvenet et al., ; martinez-pomares et al., ; moura nunes et al., ; rodríguez et al., ; rouiller et al., ; sanz et al., ; simón-mateo et al., ; sun et al., ; vigário et al., contents of cellular origin ubiquitin, hsp chaperone, g-tubulin, pericentrin, p , mdm surrounded by: er membranes, vimentin, p golgin, mitochondria, and tubulin. granja et al., ; heath et al., ; hingamp et al., ; jouvenet and wileman, ; netherton et al., netherton et al., , rojo et al., ; rouiller et al., ; stefanovic et al., poxviridae almazán et al., ; beaud and beaud, ; betakova et al., ; chiu et al., ; cudmore et al., ; da fonseca et al., ; davis and mathews, ; de silva and moss, ; domi and beaud, ; krijnse-locker et al., ; murcia-nicolas et al., ; nerenberg et al., ; ojeda et al., ; palacios et al., ; pedersen et al., ; reckmann et al., ; resch et al., ; risco et al., ; roper, ; salmons et al., ; senkevich et al., ; sodeik et al., ; szajner et al., a,b,c; tolonen et al., ; vanslyke and hruby, ; welsch et al., ; wolffe et al., ; yeh et al., ; yuwen et al., (continued) contents of cellular origin hmg a viral genome binding protein, hsp ; transient association, ubiquitin, ying-yang transcription factor, tbp transcription factor, sp transcription factor, rna polymerase ii, sumo- , ergic- c surrounded by: vimentin and mitochondria. broyles et al., ; dales and siminovitch, ; hsiao et al., ; hung et al., ; husain and moss, ; nerenberg et al., ; oh and broyles, ; palacios et al., ; risco et al., ; wilton and dales, iridoviridae, ranavirus appearance and contents f electron lucent, virus, viral dna, k early protein, k, k major capsid protein (orf r in fv ), k, k, k rana grylio virus dutpase (orf r in fv ). surrounded by vimentin, rough er, mitochondria and polysomes. chinchar et al., ; darlington et al., ; huang et al., ; goorha, , ; zhao et al., herpesviridae barnard et al ., ; de bruyn kops et al ., ; everett and maul, ; goodrich et al ., ; jahedi et al ., ; knipe et al., ; lamberti and weller, ; leopardi et al ., ; liptak et al., ; markovitz and roizman, ; olivo et al ., ; randall and dinwoodie, ; reynolds et al ., ; taus et al ., ; ward et al ., contents of cellular origin rna polymerase ii, eap ribosome component, proliferating cell antigen, retinoblastoma protein, p , dna ligase , dna polymerase a, promyelocytic leukemia (pml), dna-pkcs, ku nonhomologous end joining, bloom syndrome gene product, breast cancer-associated gene protein, msh , rad , wrn recq helicase family member, brg or brm-associated factor , brahma-related gene- protein, brahma protein, histone deacetylase , hsnf h, msin a, tata binding protein (tbp), tbp-associated factors. leopardi et al., ; quadt et al., ; taylor and knipe, ; wilcock and lane, nuclear sites of capsid assembly or assemblons contents of viral origin d ul (hhv- ), ul (hhv- ) tegument, ul capsid, ul icp major capsid protein, ul . icp dna packaging, ul dna packaging, ul vp p capsid, ul vp c capsid assembly, ul . , ul . de bruyn kops et al., ; goshima et al., ; nalwanga et al., ; nozawa et al., ; wada et al., ; ward et al., a,b; yamada et al., contents of cellular origin actin, myosin a actin motor feierbach et al., cytoplasmic assembly and envelopment site contents of viral origin d membranes, vacuoles, capsids and enveloped virus ul (hhv- ) vp major capsid protein ul (hhv- ) gb vp ul (hhv- ) icp - , tegument ul (hhv- ) tegument, ul (hhv- ) tegument kato et al., ; murata et al., ; nozawa et al., ; watanabe et al., contents of cellular origin mitochondria, g-tubulin, hsp chaperone, hsp chaperone, gm golgi marker murata et al., ; nozawa et al., (continued) becker et al., becker et al., , broering et al., ; cashdollar, ; dales et al., b; miller et al., ; sharpe et al., ; silverstein and schur, reoviridae, rotavirus appearance and contents electron-dense viroplasm, assembling and complete double-shelled particles vp , vp , vp , nsp , nsp , nsp altenburg et al., ; gonzález et al., ; petrie et al., petrie et al., , silvestri et al., silvestri et al., , a african swine fever virus gene nomenclature is based on that for the badajoz vero adapted strain with that of the malawi lil / strain in parentheses. b vaccinia virus gene nomenclature is based on that for the copenhagen strain with that of the western reserve strain in parentheses. c one report places in ergic- within the virosome (risco et al., ) , one report places it outside (husain and moss, ) . d open reading frames from human herpesvirus (herpes simplex virus ) unless specified otherwise. e open reading frames from human herpesvirus (human cytomegalovirus) unless specified. f proteins specified by frog virus unless indicated otherwise. form next to the microtubule-organizing center (mtoc). aggresomes recruit cellular components needed to deal with the problems associated with a buildup of aggregated misfolded protein. these include cellular chaperones and proteasomes to facilitate protein folding and/or degradation and mitochondria that may provide the atp required for folding and proteolysis. the most striking structural changes seen during aggresome formation are the collapse of the intermediate filament protein, vimentin, into a cage surrounding the protein aggregates and the gross fragmentation of the golgi apparatus. asfv factory formation shows many similarities with this response to protein aggregation. factory formation is preceded by clearance of cytoplasmic proteins from perinuclear areas around the mtoc. vimentin then concentrates at the mtoc where it forms an aster aligned along microtubules (stefanovic et al., ) . following the onset of virus dna replication and synthesis of late structural proteins, the vimentin aster is rearranged into a cage around the factory ( fig. b ; heath et al., ; monaghan et al., ; stefanovic et al., ) . during this period, mitochondria and cellular chaperones are recruited to the factory (heath et al., ; rojo et al., ) . formation of vimentin cages in asfv-infected cells is linked to phosphorylation of vimentin at serine by calcium calmodulindependent protein kinase ii (camkinase ii) (stefanovic et al., ) , and drugs that inhibit camkinase ii activity block late gene expression and vimentin rearrangement. as will be discussed for poxviruses and iridoviruses, the vimentin cage may form a physical scaffold within the factory, or act as a cage to prevent movement of viral components into the cytoplasm. chaperones recruited to the factory may facilitate folding of viral structural proteins during assembly, as has been shown for other viruses. the proximity of mitochondria to viral factories may provide the atp that is required for asfv assembly (cobbold et al., ) or be indicative of an antiviral response as mitochondria are effectors of apoptosis. taken together these results suggest that a cellular response originally designed to deal with the buildup of protein aggregates in cells is used by asfv to generate a site specialized for virus assembly. as will be described later, similarities between aggresomes and virus assembly sites are also seen for the iridoviruses and poxviruses. following the onset of asfv dna replication, the microtubule network becomes disorganized. microtubules are partially excluded from virus factories and form bundles and concentric rings in the cytoplasm (jouvenet and wileman, ) . asfv infection leads to disassembly of g-tubulin and pericentrin from the centrosome, and the centrosome becomes less able to nucleate microtubules. at the same time microtubules are stabilized by acetylation (jouvenet et al., ) . since pericentrin and g-tubulin play key roles in microtubule organization and nucleation at the mtoc, their loss from the centrosome, coupled with acetlylation of tubulin, may explain the rearrangement of microtubules induced by asfv. the reasons for these profound effects on microtubules are not known but they may facilitate disruption of the virus factory allowing release of assembled viruses into the cytoplasm. c. membrane rearrangements caused by asfv infection perturb the secretory pathway current models for asfv envelopment in virus factories predict that viral membranes are obtained from the er. the major structural proteins are recruited from the cytoplasm onto the cytoplasmic face of the er, and after which protein-protein interactions between these, and possibly viral proteins targeted to the er lumen, lead to constriction of er cisternae and clearance of host proteins from the er lumen prior to envelopment (andrés et al., ; netherton et al., netherton et al., , rouiller et al., ) . this is consistent with low levels of er proteins observed at asfv assembly sites by immunoelectronmicroscopy (rouiller et al., ) and standard fluorescence microscopy where er proteins appear to be actively excluded from areas of viral replication (andrés et al., ; netherton et al., ) . in addition to effects on the er, asfv also affects the structure and function of later golgi compartments of the secretory pathway (mccrossan et al., ; netherton et al., ) . golgi structure is linked to microtubule organization and the changes seen during infection may in part be related to effects of asfv infection on centrosome and microtubule function listed above. asfv infection causes dispersal of ergic marker protein ergic- , the peripheral golgi protein gm , and late golgi protein galnac-t transferase, suggesting disruption of ergic and golgi membrane compartments. most striking is the complete loss of the tgn. tgn loss is dependent on microtubules and involves dispersal of the tgn into separate vesicle populations containing either peripheral golgi proteins or the integral membrane protein, tgn . not surprisingly, this dispersal slows the transport of proteins through the secretory pathway. asfv slows the delivery of newly synthesized lysosomal enzymes to lysosomes (mccrossan et al., ) , and in macrophages reduces transport of newly synthesized mhc class i to the plasma membrane (netherton et al., ) . thus, in common with picornaviruses, disruption of the secretory pathway by asfv has the potential to slow the transport of important immunomodulatory proteins to the surface of infected cells and may mask them from immune surveillance. poxviruses are large dsdna viruses with genomes ranging from to kbp. poxvirus gene expression follows the regulated cascade of other large dsdna viruses with early, intermediate, and late transcripts described. poxvirus progeny genomes are replicated exclusively in the cytoplasm in virus factories. the virus encodes all the enzymes necessary for transcription and replication of its genome. genetic analysis has identified a minimum of five viral genes necessary for genome replication, these are a r, b r, d r, d r, and e l encoding the dna polymerase processivity factor, serine/threonine protein kinase, uracil dna glycosylase, dna-independent nucleoside triphosphatase, and the dna polymerase, respectively (de silva and moss, ; evans et al., ; millns et al., ; punjabi et al., ; rempel et al., ; sridhar and condit, ) . only the product of the d r gene, encoding the viral dna glycosylase, has been confirmed to localize to the site of genome synthesis (de silva and moss, ) , and it would be interesting to discover the subcellular location of the other members of the minimum replicase. when viewed by electron microscopy, infectious virions have a striking brick-shaped morphology, and different forms of virus are documented which vary in degree of complexity [for review, see condit et al. ( ) ]. the interior of all poxvirus particles contains the virus core which houses the viral genome. cores are enveloped in virus factories to produce the intracellular mature virus (imv), which is fully infectious. additional envelope layers gained at the tgn give rise to intracellular enveloped viruses (iev), which after budding through the plasma membrane form cell-associated and extracellular enveloped viruses (cev and eev). poxviruses induce two principal inclusions during infection, the a-type inclusion that is nonreplicative and the b-type inclusion where virus replication and assembly occur in the virus factory ( fig. ; kato et al., ). a. poxvirus a-type inclusions contain the mature intracellular virus but not enveloped viruses a-type inclusions are cytoplasmic bodies of dense homogeneous matter that contain mature virus particles and are studded with polyribosomes (fig. a) (ichihashi et al., ) . a-type inclusions are extremely rare in vaccinia, variola, and rabbit pox infections but are prominent in cowpox, ectromelia, fowlpox, and canarypox infections where they are also referred to as downie, marchal, bollinger, and burnet bodies, respectively (kato et al., ) . the major component of a-type inclusions is the product of the a l gene or its equivalents. in vaccinia, a is truncated and produces a protein of - kda whereas the fulllength gene in cowpox encodes a protein of kda (patel et al., ) , both versions are myristylated (martin et al., ) . immunfluorescence analysis of cells infected with vaccinia virus with antibodies raised against a does reveal multiple a-type inclusions in the cytoplasm, but they are much smaller than those seen in cells infected with cowpox, and do not contain virus particles (patel et al., ) . in cells infected with wild-type cowpox, only imv particles were observed within a-type inclusions, but treatment with rifampicin, a drug that blocks poxvirus maturation at an early stage in morphogenesis, caused aberrant immature virus particles to integrate into the inclusions (ichihashi et al., ) . the factor necessary for occlusion of viral particles in a-type inclusions has been identified as the c core protein (mckelvey et al., ; shida et al., ; ulaeto et al., ) . it has been hypothesized that c retains vaccinia virions within the cell as imvs in a-type inclusions preventing their transport to the tgn for envelopment and maturation to the iev types of virion (mckelvey et al., ) . a-type inclusions are predicted to protect imvs during transport between hosts akin to that of the polyhedra that occlude entomopox and baculoviruses (rohrmann, ) . therefore, eevs may be important for cell-to-cell spread, while imvs (whether occluded or not) may be more important for host-to-host spread (mckelvey et al., ) . b. poxvirus b-type inclusions are factories and are the main sites of replication and assembly b-type inclusions originally called guarnieri bodies (guarnieri, ) are the primary replication centers of the poxviruses, now generally referred to as virosomes or virus factories (fig. c) . electron microscopic analysis of b-type inclusions revealed a granular matrix that was denser than the surrounding cellular material and in a defined area of the cytoplasm called viroplasm (dales and siminovitch, ; higashi, ) . the factories also contain viral crescents consisting of membrane and viral proteins associated with viroplasm, spherical immature virus, and imvs (dales and siminovitch, ) . factories are surrounded by mitochondria, increase in number and size during the replication cycle and can occupy the majority of the cytoplasm at late times of infection (dales and siminovitch, ) . the assembly and envelopment of vaccinia virus within virus factories has been the subject of many studies and is discussed in papers and reviews (griffiths et al., ; heuser, ; hollinshead et al., ; sodeik and krijnse-locker, ) . here, we will review some of the early steps that lead up to the start of genome replication and factory production. these have also been described in a review (schramm and krijnse-locker, ) . it is generally believed that infection results in the delivery of viral cores into the cytoplasm. cores are seen associated with microtubules (carter et al., ; mallardo et al., ; ploubidou et al., ) and may use microtubules to reach perinuclear sites that will eventually house the virus factories. viral cores can transcribe as many as early mrnas before the onset of dna replication, and these early mrnas appear in discrete foci that associate with microtubules, contain polyribosomes and other translational machinery. it is unlikely that foci involved in transcribing early rnas mature into viral replication sites because they do not initiate dna synthesis (mallardo et al., ) . it is likely that each infecting virus can induce its own replication center (cairns, ) , but it is not clear where in the cell the cores initiate dna synthesis. it has been suggested that the onset of dna synthesis may occur at peripheral sites and therefore precedes delivery to the perinuclear region of the cell. when cells are incubated with hydroxyurea to prevent the onset of viral dna replication, it is possible to localize viral dna released into the cytoplasm. under these conditions, viral genomes are seen at several discrete sites that contain b protein kinase, e membrane protein, i ssdnabinding protein, and h late transcription factor (domi and beaud, ; welsch et al., ) . after removal of hydroxyurea, these foci begin to make new viral dna, showing that they are sites of dna replication. live cell imaging studies have shown that these initial sites of dna replication form in the cell periphery and then move toward the nucleus where they coalesce into large structures (schramm and krijnse-locker, ) . electron micrographs suggest that sites of dna release from cores are intimately associated with er membranes and become completely enclosed by them during the initial stages of dna replication (mallardo et al., ) . this process is likely facilitated by the e r gene product which is a membrane protein localized to the er and early golgi membranes, has dna-binding activity, and is able to capture viral genomes tolonen et al., ) . these er-enclosed genomes are short-lived structures because they are not seen once viral crescents, iv and imvs, appear in factories (tolonen et al., ) . the sites of dna replication are also separate from the foci involved in transcribing early rnas, and it is interesting to consider how the cores are separated from newly transcribed rna. viral cores and sites of rna transcription both align on microtubules and partially colocalize with the l core dna-binding protein (mallardo et al., ) . the l protein is able to bind microtubules (ploubidou et al., ) and may be involved in separating rna from cores along microtubule tracks (mallardo et al., ) . inducible recombinants or temperature-sensitive mutants grown under nonpermissive conditions can give further insight into the early stages of inclusion formation. electron micrographic analysis of the factories formed under these conditions yield striking images of distinct inclusions of homogeneous electron-dense viroplasm next to empty spherical immature virions ( fig. b and c) (szajner et al., (szajner et al., , (szajner et al., , a . a seven-protein complex comprising the gene products of the a l, a l, d l, d l, f l g l, and j r open reading frames has been identified as being necessary for association of viral membranes with the viroplasm (szajner et al., a) . consistent with this role, all of these proteins are known to localize to the virus factory except d and d (table i) ; however, these have been identified as core proteins (dyster and niles, ) so are likely to reside at viral assembly sites. localization of d l to the virus factory is sensitive to the antibiotic rifampicin (miner and hruby, ) , and treatment with this drug induces irregular shaped viral membranes instead of the well-defined hemispherical viral crescents seen in natural infection (moss et al., ; pennington et al., ) . therefore, it was suggested that d l may act as a scaffold on which the viral membrane is shaped, allowing correct association with the viroplasm (mohandas and dales, ) . deep etch electron microscopy has confirmed this role for d l, as it forms the honeycomb lattice identified as the outer coat of the viral membrane of immature virions (heuser, ; szajner et al., ) . interestingly, d l shares a structural similarity with structural proteins from many other virus families, including those of the other large dsdna viruses (benson et al., ) . it will be interesting to see if the structural similarities to d l translate to functional similarities in the assembly strategies of other viruses. to the viral factory. ying-yang (yy ), tbp, sp transcription factors, and rna polymerase ii are recruited from the nucleus to the factory (broyles et al., ; oh and broyles, ; wilton and dales, ) . yy is a nuclear transcription factor that can activate late viral promoters and although poxviruses encode most of the genes necessary for transcription, there is evidence that cellular factors may be required for intermediate and late gene expression (lackner and condit, ; rosales et al., ; wright et al., ) . the function of the other transcription factors in viral replication is unknown. they may be necessary for viral transcription like yy , or perhaps they are sequestered into the factory to divert them from their normal roles in the nucleus, or their presence may represent an antiviral response by the cell. the presence of rna polymerase ii in the viral factory is a surprise because the virus encodes its own rna polymerase activity which accounts for at least orfs and $ % of the genome capacity [western reserve (wr) strain]. another cellular protein recruited from the nucleus to the cytoplasm is the hmg a protein. this protein can bind the viral genome and has been implicated in host range restriction of vaccinia virus in chinese hamster ovary cells (hsiao et al., ) . during unproductive infection by vaccinia virus, hmg a is recruited from the nucleus to the factory where it binds viral dna. if the cowpox host range gene cp is artificially introduced into vaccinia virus then cp also enters the virus factory and binds to hmg a; the cellular protein then dissociates from the viral genome and replication proceeds (hsiao et al., ) . as seen for iridovirus and asfv replication sites, vaccinia factories are surrounded by a vimentin cage (risco et al., ; schepis et al., ) and recruit molecular chaperones (hung et al., ) , suggesting similarity with aggresomes. many proteins targeted to aggresomes are ubiqutinated, and most poxviruses encode a ring protein that is both a functional ubiquitin ligase and a virulence factor (nerenberg et al., ) . exceptions to this are the two most common laboratory strains of vaccinia, copenhagen and wr. the ring protein from the ihd-w strain of vaccinia is capable of directing transfected tagged ubiquitin to wr virus factories (nerenberg et al., ) ; however, it is unknown if native ubiquitin is localized to wr factories. the product of the a r gene of vaccinia is tagged with the ubiquitin-like protein sumo- , and this modification is necessary for a targeting to viral factories, where it associates with er membranes and may play a role in the formation of i sites (palacios et al., ) . it is not known if movement of sumolyated a and ubiquitinated protein is directed along microtubules in a manner analogous to hdac mediated targeting of misfolded proteins to aggresomes (kawaguchi et al., ) . as reported for asfv (see above) and cells infected with herpes simplex virus (avitabile et al., ) , infection of cells with vaccinia virus also leads to disruption of microtubule organization and centrosome function and dispersal of the golgi apparatus (ploubidou et al., ) . whether these are bystander effects of the production of virus factories close to the centrosome or induced deliberately to facilitate virus egress is not known. imv exit from the factory and transport to envelopment sites at the tgn is nonetheless dependent on microtubules (sanderson et al., ) and has been reported to be dependent on the a l and a l gene products (sanderson et al., ; ward, ) . following envelopment, the a l and f l gene products then regulate microtubule-dependent movement of intracellular enveloped viruses from the tgn to the plasma membrane (herrero-martínez et al., ; ward and moss, ). crystalline arrays a. iridoviruses iridoviruses are large dsdna viruses with genomes ranging from to kbp in length encoding between and proteins (williams et al., ) . much of the work on iridovirus replication has been carried out on the ranavirus frog virus (fv ). fv genome synthesis occurs in the nucleus and cytoplasm. no nuclear inclusions have been reported during fv infection, and as such it is unclear how the nuclear replication stage is mediated. however, viral dna is initially synthesized as units that are - genomes in length and then transported to the cytoplasm where multiple length concatemers are produced (goorha, ) . b. cytoplasmic factories formed during iridovirus infection resemble aggresomes infection induces two cytoplasmic inclusions. viral factories form in the cytoplasm and become the major site of viral dna replication. fv also induces large crystalline arrays of viral particles which give rise to the iridescent coloring of purified virus, and hosts, that are characteristic of iridovirus infections. virus factories are electron lucent relative to the cytoplasm and contain viral membranes, partially assembled viruses, and are surrounded by rough er membranes and polysomes. fv factories also resemble aggresomes since they recruit intermediate filaments (fig. a) and mitochondria, some of which show darlington et al. ( ) with permission from elsevier. signs of damage (darlington et al., ; granoff et al., ; huang et al., ; tripier et al., ) . crystalline arrays of virus are associated with virus factories and can induce nuclear deformations that lead to kidneyshaped nuclei similar to those seen in asfv infection (fig. b ) and after aggresome formation (darlington et al., ; heath et al., ; johnston et al., ) . as seen for asfv and poxviruses, the intermediate filament vimentin plays an important role in replication (murti and goorha, ) . vimentin is phosphorylated during fv infection, prior to factory formation (chen et al., ; willis et al., ) , and temperature-sensitive mutants that are unable to phosphorylate vimentin do not form vimentin cages and are unable to proceed to late gene expression. drug treatment with taxol or colchicine (murti et al., ) showed that recruitment of vimentin to assembly sites requires dynamic, but not polymerizing microtubules, and microinjection of anti-vimentin antibody prevented recruitment of vimentin to factories. this allowed intrusion of cell components into assembly sites and reduced virus growth by - % (murti et al., ) . vimentin may therefore provide a scaffold for iridovirus replication, maintaining a barrier between the cytoplasm and the contents of the virus factory. consistent with this hypothesis is the observation that during infection, polyribosomes and most newly synthesized viral proteins associate with intermediate filaments (murti and goorha, ) . fv factory formation may also be dependent on the early k protein, as it is recruited to factories in the absence of late protein synthesis (chinchar et al., ) . phycodnaviruses and the recently described giant virus mimivirus (la scola et al., ) induce replication complexes in the cytoplasm of infected ameba (meints et al., ; raoult et al., ) . the factories of the phycodnavirus paramecium bursaria chlorella virus (pbcv- ) are electron translucent areas of the cytoplasm and contain viral membranes, electron-dense viroplasm, and assembling viruses. unlike many viral factories, a distinct order appears to be present in pbcv- virosomes. the assembling viruses are arranged at the periphery of the virosome/ factory, giving the appearance of a rosette (meints et al., ) . phycodnavirus replication and factory formation are not affected by a wide range of pharmacological disruptors of the cytoskeleton, including microtubule depolymerization by nocodazole and taxol, and depolymerization of actin by cytochalasin d (nietfeldt et al., ) . in this way, they differ from factories formed by large dna viruses such as asfv, vaccinia, and fv . the successful cultivation of algae in the laboratory has allowed studies of the intracellular sites of replication of large icosahedral mclav- and hincv- viruses (wolf et al., (wolf et al., , . these viruses produce a latent infection that becomes apparent once the algae produce reproductive organs that become host to millions of virus particles. replication of these viruses begins in the nucleus, but the first evidence for virus assembly is provided by the appearance of electron-dense bodies next to the nucleus at sites of breakdown in the nuclear envelope. infection leads to stacking of er cisternae that may provide membranes for virus envelopment. the dense bodies remain next to the nucleus in large inclusions, and take on the angular shape characteristic of capsid assembly seen for iridoviruses and asfv. the nucleus eventually disintegrates, and the virus factory occupies most of the cytoplasm. herpesviruses are large dsdna viruses with genomes ranging in size from to kbp. herpesvirus genes are expressed in a regulated cascade starting with the immediate early a genes, then early b genes, and finally two subsets of late g genes, g and g . complete herpesvirus particles have four main layers, the core containing dna, an icosahedral capsid, a poorly defined layer of protein called tegument, and finally the viral envelope containing several glycoproteins. genome synthesis and packaging and capsid assembly occur in inclusions in the nucleus. nucleocapsids then obtain tegument in either the nucleus or the cytoplasm, or both, and the viral envelope is acquired exclusively in the cytoplasm [see mettenleiter ( ) and for more thorough analysis]. the transfer of virus from the nucleus to the cytoplasm and acquisition of tegument appears well defined for human herpesvirus (hhv- ) (roffman et al., ) but is controversial for the alphaherpesviruses (campadelli-fiume and roizman, ; mettenleiter and minson, ) . the subcellular organization of herpesvirus replication complexes formed in the nucleus during the early stages of productive infection has been described in considerable detail. the inclusions function as sites of virus replication and contain the virally encoded proteins and host proteins needed for virus replication. interestingly, nuclear inclusions formed during herpes virus replication also contain cellular proteins involved in the control of dna damage and repair. these may be recruited into inclusions in response to virus genome replication, and whether they are beneficial or detrimental to virus replication is a subject of considerable interest [reviewed by everett ( ) ]. herpesviruses enter the cell by fusing their envelopes with the plasma membrane, whereon the naked nucleocapsids migrate to nuclear pores, possibly along microtubules (granzow et al., ; sodeik et al., ) [reviewed by smith and enquist ( ) ]. nuclear inclusions housing herpesvirus dna replication are globular and can occupy the majority of the nucleus (de bruyn kops and knipe, ; randall and dinwoodie, ; taylor et al., ) . they are identified through the presence of the viral dna-binding protein encoded by the ul gene, which is also known as infected cell protein (icp ). a minimum set of seven genes, ul , ul , ul , ul , ul , ul , and ul , has been identified as necessary for viral dna replication (challberg, ) . a plasmid transfection system has shown in vitro these can form globular nuclear compartments that are sites of -bromo- -deoxyuridine (brdu) incorporation and visually are similar to those formed during infection zhong and hayward, ) . nuclear inclusions organizing viral dna replication have been followed in real time by a recombinant virus expressing a gfp-icp fusion protein. small inclusions merge with adjacent replication complexes and increase in size to form globular replication complexes, which eventually fill most of the nucleus (randall and dinwoodie, ; taylor et al., ) . replication compartments are formed from a number of different discrete foci that are induced early in infection and whose interrelatedness is not fully understood. the initial stages of productive herpesvirus infection are, however, intimately linked with nuclear structures called nd bodies (illustrated in fig. ) , maul et al. ( ) , review by borden ( ) ]. live cell studies have shown that the immediate early regulatory protein icp , which binds viral dna, forms discrete foci as early as -min postinfection (fig. a) . these initially appear close to the nuclear envelope, possibly at sites where the genome first enters the nucleus following capsid disassembly at nuclear pores (everett and murray, ) , and are then seen throughout the nucleus (everett et al., ) . icp foci are seen juxtaposed to the nd marker promyelocytic leukemia protein (pml) some -min later. the early and late regulatory protein icp is recruited to icp foci -h postinfection and facilitates efficient early gene expression (everett et al., ) . during the same period, the immediate early regulatory protein, icp , colocalizes with nd bodies, some of which are likely juxtaposed to icp bodies (everett et al., ) . icp mediates the ubiquitin and/or sumo- -targeted proteasomal degradation of nd components (chelbi-alix and de everett, ; everett and maul, ; everett et al., ) . finally, parental genomes localize to icp foci (everett and murray, ) , and the icp foci enlarge into structures that resemble early icp replication compartments (everett and murray, ; everett et al., ) . formation of icp replication compartments (taylor and knipe, ) is also known to involve redistribution of nd bodies (burkham et al., ) . the relationship between the early icp structures associated with parental genome and the later icp compartments associated with replication and production of progeny genome is not clear; however, icp and icp both localize to late replication compartments (knipe et al., ) . a description of the relative and temporal distribution of the two proteins at early times awaits live cell studies following both proteins simultaneously. a second prominent nuclear inclusion induced by herpesvirus infection is the assemblon (ward et al., b) . this is the site where capsid proteins accumulate and assemble into nucleocapsids (fig. b) . the assembly of herpesvirus nucleocapsids has been researched in great detail at the ultrastructural level facilitated by a cell-free system for reconstituting the particles (heymann et al., ; newcomb et al., newcomb et al., , . the mature herpesvirus capsid is icosahedral with a t ¼ symmetry and is composed of hexons and pentons of the major capsid protein ul . the place of the remaining penton is taken by a -mer of the portal protein ul , which by analogy with bacteriophage may be the site of genome entry. nucleocapsids mature from fragile procapsids, through b capsids that lack dna and contain the internal scaffold protein ul . , to c capsids that contain the viral genome. the relationship between assemblons and sites of viral dna replication has been a topic of some controversy as some reports show direct colocalization (taus et al., ) , whereas others have shown a proximity (nalwanga et al., ; ward et al., b) , similar to that seen between nd bodies and icp foci during the initial stages of infection. clearly, the dna has to reach the capsid in order to complete assembly, and it is likely that the different results are indicative of the dynamic interactions between different herpesvirus nuclear inclusions. the dna cleavage and packaging proteins encoded by the ul and ul genes are required for colocalization of viral dna and capsids (lamberti and weller, ; taus et al., ) . cells infected with a virus encoding a faulty ul gene exhibit nuclear localization of the capsid protein vp that is separate from replication sites (lamberti and weller, ) . similarly, in cells infected with mutants that lack functional ul , the icp protein fails to colocalize with icp and icp (taus et al., ) . actin also plays an important role in the correct nuclear subcompartmentalization of viral proteins. infection with hhv- or suid herpesvirus- causes actin filaments to assemble in the nucleus, prior to the accumulation of capsid proteins (feierbach et al., ) . depolymerization of actin with latrunculin a inhibited correct nuclear compartmentalization of a representative capsid protein (vp ). vp colocalizes with the actin motor myosin va (feierbach et al., ) , and capsid movement within the nucleus is inhibited by the myosin motor inhibitor , -butanedione monoxime (forest et al., ) . this suggests that the organization of nuclear inclusions involved in herpesvirus assembly is dependent on cellular actin filaments, and it will be interesting to see if the organization of inclusions housing viral dna replication sites is similarly dependent. other inclusion bodies have been reported in the nucleus of cells infected with herpesvirus. the tegument proteins vp and vp / localize to inclusion bodies that align closely but do not overlap icp /nd /icp pre-replication complexes or assemblon inclusions (hutchinson et al., ) . ul also localizes to structures that overlap but are distinct from assemblons and dna replication complexes (fig. b ) . ul localizes to type iv and type v intranuclear dense bodies as well as virions and cytoplasmic ribbon structures (baines et al., ) . the alkaline dnase encoded by the ul gene localizes to discrete electron-dense bodies within the nucleus that also contain b- nucleolar protein (lopez-iglesias et al., ; puvion-dutilleul and pichard, ) . it is unknown whether these different structures are related to each other, whether they are homogenous accumulations of the individual herpesvirus protein(s), or if they are simply dead-end accumulations of protein. a large number and variety of cellular proteins accumulate at nuclear sites of herpesvirus replication and assembly. a comprehensive proteomic analysis of icp interacting proteins revealed more than viral and cellular proteins that maybe recruited to dna replication sites (taylor and knipe, ) . a number of these interacting proteins were confirmed to localize to replication sites by microscopy experiments (taylor and knipe, ) , and these as well as proteins identified in other studies (leopardi et al., ; quadt et al., ; wilcock and lane, ) reveal that at least cellular proteins are known to localize to nuclear inclusions involved in dna replication during herpesvirus infection (table i) . the functions of these proteins span the expected functions of nuclear genes, including dna replication, transcription, chromatin remodeling, dna repair, recombination, and nonhomologous end joining. of particular importance is the recruitment of rna polymerase ii, which is required to transcribe the viral genome. rna polymerase ii is phosphorylated during viral infection by icp and icp , and the latter modification is required for targeting to replication complexes (dai-ju et al., ) . the role of all of these cellular genes in the viral replication cycle is poorly understood; however, cells deficient in wrn, a recq helicase family member, produced reduced virus yields while cells lacking ku , part of a nonhomologous end-joining protein complex, produced increased yields of virus (taylor and knipe, ) . the implication therefore is that some cellular proteins may be actively recruited to replication complexes to aid viral replication, and some may be recruited by the cell as part of an antiviral response or sequestered by the virus in inclusions to subvert their antiviral nature. pml is induced by interferon, suggesting an antiviral role. many of the genes shown to be required for recruitment of pml to viral pre-replication sites are part of the minimal set of genes required to synthesize viral dna. recruitment of pml to viral replication sites is, for example, dependent on the viral dna polymerase (ul ), the origin binding protein (ul gene) and the helicase-primase complex (ul , ul , and ul ) (burkham et al., ) . recent evidence has suggested that this may be the reason why icp causes dispersal of pml early in infection. pml knockdown by short interfering rnas (sirna) facilitates productive replication of icp null mutants of herpesvirus (everett et al., ; moreover, icp null mutants are hypersensitive to interferon in a manner dependent on pml (chee et al., ) . this is of particular importance because icp plays a role in determining whether herpesvirus induces a quiescent or a productive, lytic infection (mossman and smiley, ) . the tegument layer of alphaherpesviruses is composed of at least different proteins (mettenleiter, ) . us , ul , ul , ul , and ul are components of the tegument, and all are localized to the nucleus (if not exclusively) during the productive life cycle of the virus hutchinson et al., ; kopp et al., ; roller and roizman, ; taus et al., ) . ul may play a role in egress from the nucleus, though this has not been unequivocally established (mossman et al., ) . therefore, it is likely that some tegument proteins are acquired in or during viral egress from the nuclear inclusions. recently, cytoplasmic aggresome-like struct ure s have been desc ribed in ce lls infected with hhv- . these contain the major capsid protein, tegument proteins, envelope glycoproteins, and markers for the golgi complex (nozawa et al., ) . the latter finding is particularly interesting because herpesvirus envelopment involves membranes from the tgn turcotte et al., ) . hhv- is a betaherpesvirus and late during infection produces a juxtanuclear ''assembly compartment'' that again contains tegument proteins (pp , pp , and pp ), the major capsid protein, and viral envelope proteins (gb, gh, and gp ), suggesting a cytoplasmic site specialized for tegumentation and envelopment (fig. c) ; (adair et al., ; sanchez et al., ) . the precise role of the cytoplasmic assembly compartment is unclear. on the one hand, the concentration of glycoproteins and tegument proteins in one site may facilitate final stages of assembly prior to release from the cell. interestingly, in common with aggresomes induced by asfv and misfolded proteins, the cytoplasmic assembly compartment recruits chaperones and mitochondria and is dependent on microtubules and localizes to the microtubule organizing center. at present, the assembly compartments are not considered to be bona fide aggresomes because they are not surrounded by a collapsed cage of intermediate filaments (nozawa et al., ; sanchez et al., ) . it is nevertheless possible that these structures are related to aggresomes and are produced in response to a buildup of products resulting from nonproductive assembly pathways that occur late during infection. they may also contribute to the cytopathic effect seen in cells infected with hhv- . hhv- infection results in cytomegaly characterized by increased cell size and intracellular water content. cytomegaly and virus replication are both dependent on the presence of extracellular na þ , and infection results in sequestration of the plasma membrane na-k-cl-cotransporter protein in large perinuclear structures that resemble the assembly compartment/ viral aggresome (maglova et al., ) . electron-dense bodies can be seen by electron microscopy within the cytoplasmic assembly compartments induced during hhv- infection (craighead et al., ) . dense bodies are enveloped and obtain viral glycoproteins but do not contain dna and are noninfectious. as can be seen in fig. c , dense bodies bud into membranes and appear as oversized enveloped viral particles without a dna containing core. dense bodies exit the cell to become extracellular dense bodies (craighead et al., ) . interestingly, hhv- immediate early ie proteins also become associated with extracellular dense bodies despite no reported localization to their intracellular relations (tsutsui and yamazaki, ) . purified extracellular dense bodies are mostly composed of ul but have a full complement of viral glycoproteins (irmiere and gibson, ) . the function of dense bodies remain unclear, and they may represent the end point of a nonproductive assembly pathway resulting from attempts to envelope capsids lacking genomes or may be used to deliver viral components to neighboring cells. interestingly, for human herpesvirus (hhv- ), the tegument layer appears to be acquired within a dedicated structure that has been dubbed the tegusome (roffman et al., ) . this work is based on electron microscopy of cells infected with hhv- and shows tegusomes as intranuclear membrane compartments that abut the nuclear envelope (fig. d ). tegusomes may be cytoplasmic invaginations of the nuclear envelope into the nucleus because they appear to contain ribosomes and are sometimes in continuity with the cytoplasm. nucleocapsids appear to bud into the tegusome, capsids obtain a tegument layer, and then bud into cytoplasmic vacuoles where they acquire envelopes and exit the cell. adenovirus are medium-sized, nonenveloped dsdna viruses with genomes ranging from to kbp in length and virions of the order of - nm in diameter. like other dna viruses, they have an ordered cascade of transcripts, early, delayed early, and late types having been described. adenovirus transcripts are spliced to generate multiple transcripts from a given transcriptional unit. viral replication occurs in the nucleus, and adenovirus infection was utilized extensively as a model system for exploring different nuclear subcompartments. a productive infection of lytic adenovirus induces profound rearrangement of existing subcompartments and the induction of several new ones within the host nucleus. a study on the localization of the human adenovirus iva protein described distinct nuclear and nucleolar subcompartments induced or associated with virus replication (lutz et al., ) , and these are listed in table i . earlier studies carried out before markers for specific nuclear subcompartments were available have described the structures in terms of shape and location (see table i ). during the initial stage of infection, viral rna , single-stranded dna (ssdna), and dsdna (puvion-dutilleul and pichard, ) are all synthesized in small fibrillar regions termed early replication sites. by the intermediate stage of replication, the ssdna is deposited in the center of these structures, while transcription and dsdna synthesis occur on the outside and begin to form an inclusion. the inclusion has a characteristic doughnut shape, and has been called the fibrogranular network. at late stages of infection, dsdna, viruses, and trace amounts of ssdna appear in large viral inclusions (besse and puvion-dutilleul, ; puvion-dutilleul and pichard, ) . targeting of the initial replicon is dependent on a dcmp modification of the preterminal protein (ptp), which enables ptp to form a complex with the dna polymerase and the genome (temperley and hay, ) . ptp mediates targeting of the heterotrimeric complex to the nuclear matrix (fredman and engler, ) , possibly through an interaction with cad (carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase) (angeletti and engler, ) . transcription and splicing are mediated by host proteins and viral rna, and non-snp rna splicing factor, hnrnp c proteins, and rna polymerase ii all colocalize with viral rna in nuclear inclusions. splicing small nuclear ribonucleoproteins (snrnps) colocalize with viral rna but not replication foci (pombo et al., ) , and snrnps then move to interchromatin granules late in infection, which is blocked by mutations in e (bridge et al., ) . a. rearrangement of host nuclear compartments during adenovirus replication like herpesvirus described above, adenovirus infection redistributes the components of nd bodies. prior to infection, pml is associated with interchromatin granules but is redistributed to the fibrillogranular matrix within the nucleus along with sp , another nd component (carvalho et al., ) . later in infection, pml is redistributed once again from the fibrillogranular matrix to clear amorphous inclusions and protein crystals (puvion-dutilleul et al., ) . another study reported that sp and ndp , but not pml, were relocated from nd bodies to viral inclusions (doucas et al., ) . while this is confusing, it is clear that adenovirus employs multiple mechanisms to reorganize pml. the initial movement of pml, sp , and ndp to the fibrillogranular matrix occurs prior to viral dna synthesis and is dependent on the e -orf -kda protein (carvalho et al., ; doucas et al., ) . it may also be mediated by e a proteins that colocalize with pml (carvalho et al., ) . e b- -kda protein also colocalizes with pml early on in infection, then associates with the periphery of replication centers; these interactions are mediated by the orf protein of the e transcriptional unit (lethbridge et al., ) . interestingly, e b- k and e -orf target the mre -rad -nbs (mrn) complex to aggresomes for degradation (araujo et al., ; liu et al., ) . polyoma-and papillomaviruses are small double-stranded tumorigenic dna viruses with genomes of and kbp, respectively. replication and assembly of these two viruses follow similar strategies, and both involve nd bodies. the vp capsid protein of human polyomavirus jc is targeted to nd domains by vp , vp , and agnoprotein where they are assembled into virions (shishido-hara et al., ) . a similar process occurs during papillomavirus infection where the minor capsid protein, l , is responsible for targeting capsomeres of the major capsid protein, l , to nd domains (florin et al., a) . this process involves l -induced redistribution of nd bodies by targeting sp for proteasomal degradation. at this point the cellular daxx protein is recruited (florin et al., b) . daax has multiple functions in the nucleus including transcriptional activation and modulating fas-mediated apoptosis [reviewed by salomoni and khelifi ( ) ]. its role in virus replication is at present unclear. one characteristic of papillomavirus infections is the appearance of nuclear and cytoplasmic inclusions in cells contained within warts. the size and number of inclusions is dependent on the type of papillomavirus and the site of infection. human papillomavirus (hpv- ), for example, induces many small inclusions while hpv- induces one single inclusion that takes over most of the cytoplasm (croissant et al., ) . in vivo these structures label strongly with antiserum raised against e gene products which are the -kda e ∧ e and -kda e proteins (doorbar et al., ; rogel-gaillard et al., ) . inclusions can be induced in certain cell types in vitro by expressing e gene products. hpv- e staining reveals an initial association with the intermediate filament keratin and subsequent formation of inclusion bodies in the cytoplasm and nucleus (roberts et al., ; rogel-gaillard et al., ) . the hpv- cytoplasmic inclusions retain their association with keratin and appear to induce small cages surrounding e protein that are interconnected by keratin filaments (roberts et al., ) . the e gene gives rise to two proteins, the -kda e ∧ e which can induce cytoplasmic and nuclear inclusions whereas the -kda e can induce inclusions solely in the cytoplasm (rogel-gaillard et al., ) . interestingly, expression of e ∧ e gene product from hpv- induces the complete collapse of the keratin network, but not that of the microtubule or actin networks (doorbar et al., ) . it is unclear what the role of the inclusions is in viral replication or the pathology of infection. however, hpv- e expression induces the redistribution of nd to the periphery of nuclear inclusions in cells in culture, and similar signals are seen in vivo (roberts et al., ) . the temporal and functional connection between e and l redistribution of pml is unknown. members of the reoviridae family are dsrna viruses with segmented genomes and include the clinically important rotavirus and orbiviruses that cause diseases in human and animals. reoviruses are nonenveloped viruses with genome segments contained inside a virion $ nm in diameter. the genome is encapsidated by two protein shells, an outer capsid and an inner core shell. the core contains the rdrp, capping enzymes, and the dsrna genome segments [reviewed in yue and shatkin ( ) and furuichi and shatkin ( ) ]. viruses are taken up by receptor-mediated endocytosis, the outer capsid is lost and the core is delivered into the cytoplasm. the core does not disassemble on entering cells and imports ribonucleoside triphosphates and s-adenosyl-l-methionine from the cytosol to synthesize and then export viral mrnas. in this way the core particle functions as a self-contained transcriptional unit and as such represents the replication complex. viral mrna transcribed in the cytoplasm make viral proteins that eventually form large perinuclear inclusions, called virus factories that function as sites of further virus replication and assembly. the reoviridae family contains genera, and this chapter will concentrate on the two best characterized of these, the orthoreoviruses and rotaviruses. . formation of factories during orthoreoviruses replication and assembly a. the shape of orthoreovirus factories is determined by association with the cytoskeleton orthoreoviruses contain genome segments which are classed by size and then numbered, that is l is large segment . large segments encode l genes, medium (m) segments encode m genes, and small (s) segments encode s genes. virus replication occurs in the cytoplasm in virus factories, and the majority of the virus-encoded proteins have been shown to localize completely or partially with factories (table i ). early observations revealed that different strains of orthoreoviruses induced factories with different appearances; orthoreovirus type lang factories were filamentous while the factories of the dearing isolate of orthoreovirus type were globular ( fig. a and b) . this difference maps precisely to a serine-proline switch at residue of the m core protein . control of the localization of orthoreovirus factories reflects the degree of association m has with the microtubule network. filamentous virus m stabilizes microtubules to a greater relative degree than globular virus m , and depolymerizing microtubules with nocodazole convert filamentous factories to globular ones. many of the events of orthoreovirus factory formation have been successfully reconstituted in vitro. a screen of orthoreovirus proteins revealed that mns, sns, and s were the first viral proteins to localize with viral mrna prior to the synthesis of progeny dsrna (antczak and joklik, ) . subsequently, it was discovered that expression of the mns protein of isolate dearing in the absence of other viral proteins induced a phasedense structure that was indistinguishable in appearance from that observed during wild-type infection . the shape of the artificial mns inclusion could be altered from globular to filamentous by coexpressing a m protein from a filamentous virus . similar experiments showed that coexpression of l , l , and s core surface proteins with mns caused them to localize to the mns inclusion (broering et al., ) . furthermore, the shape of the mns structure that the core proteins colocalized to could be altered to filamentous by coexpressing m from a filamentous virus (broering et al., ) . mns can also recruit sns, but not s , to artificial inclusions (becker et al., ) , so other factors or conditions are necessary for complete assembly of an orthoreovirus factory. the precise domains involved in initiating factory formation are beginning to be elucidated. the minimal region of mns necessary for inclusion like body formation in vitro is the region composed of c-terminal amino acids of the residue proteins (broering et al., ) . residues - of sns are important for the interaction between sns and rna (gillian and nibert, ) , and treatment with rnase dissociates a proportion of mns from sns in coimmunoprecipiation experiments (miller et al., ) . interaction between mns and m is dependent on residues - or of mns and residues - are necessary for interaction between mns and sns (miller et al., ) . it is likely that factory formation occurs through an interaction between mns and a sns-rna complex; this can then recruit m that will determine the globular or filamentous localization of the factory and hence the localization of the other viral proteins. orthoreovirus factories are clearly intimately associated with the microtubule network (fig. c) and have also been suggested to interact with intermediate filaments. orthoreovirus type infection induces a redistribution of vimentin and viral inclusions reported to contain filamentous structures (sharpe et al., ) . it will be interesting to see if the in vitro factories induced by mns can also alter the distribution of the intermediate filament network. orthoreovirus factories are also ubiquitinated, and interestingly the nature of the factory determined the degree of ubiquitination; globular factories are prone to contain more ubiquitinated protein than filamentous ones . ubiquitination of orthoreovirus factories has been mapped to the m protein but is independent of the filamentous/globular factory determinate of m , that is converting a filamentous factory to a globular factory does not lead to an increase in ubiquitinated m . . formation of factories during rotavirus replication and assembly a. virus nonstructural proteins organize factory formation and virus assembly rotaviruses contain genome segments of dsrna and like the orthoreoviruses replicate in cytoplasmic factories. rotavirus virions are composed of three protein layers. these are the core which contains the genome and polymerase, an inner capsid layer, and an outer capsid layer. the core and inner capsid layer comprise the double-layered particle (dlp), while the addition of the third capsid layer forms the mature triple-layered particle (tlp). the acquisition of the third capsid layer occurs after the virus buds into the er, and in doing so obtains a transient envelope. rotavirus factories are composed of electron-dense viroplasm often in proximity to membranes derived from the er (fig. d) (altenburg et al., ) . viroplasm contains high levels of nsp (fig. e ) and nsp which are thought to coordinate assembly of the factory and recruitment of structural proteins such as the inner core protein vp and viral polymerase vp . the factory also contains double-layered rotaviruses, whereas the er membranes associated with the factory contain enveloped intermediates and tlp (arrowed in fig. d ). virus factories grow in size and decrease in number during the course of infection as neighboring factories merge (eichwald et al., ) . rotavirus factories appear to have an internal structure, as their centers occasionally appear more electron lucent than the periphery, giving a doughnut-like appearance (fig. e ). electron microscopy shows dlp at the periphery of the factory and this is (altenburg et al., ) consistent with fluorescent microscopy showing that the nonstructural protein nsp localizes to the center of the virus factory, whereas nsp and inner capsid protein vp localize to the periphery (eichwald et al., ; gonzález et al., ) . these different localizations could have functional relevance because vp binds the er-targeted nsp membrane protein and is implicated in the budding of dlps into er membranes associated with factories (silvestri et al., ) . therefore, a localization to the exterior of the factory may represent an organized progression of virus maturation from the interior of the viroplasm to the exterior. however, things are probably not that straightforward because vp is also part of the viral rna complex along with nsp (aponte et al., ) which, as noted above, is localized to the center of the viroplasm. virus factory-like structures can be introduced in vitro by coexpressing nsp and nsp (fabbretti et al., ) , and this is regulated by domains in the n-and c-termini (fabbretti et al., ) as well as the central portion of nsp (eichwald et al., ) . the process is also dependent on phosphorylation of nsp , possibly by cellular casein kinase ii (eichwald et al., ) . structures similar to factories can also be induced by expressing the inner capsid protein vp in vitro (nilsson et al., ) . these structures look similar to factories in the electron microscope but lack electron-lucent areas and dlps. interestingly, expression of vp of group a simian rotavirus sa- induced globular structures, whereas expression of vp from group c porcine rotavirus cowden/amc- induced filamentous structures (nilsson et al., ) analogous to the difference between type and type orthoretroviruses. it is not clear if the difference in factory shape is solely determined by vp and if this involves differences in association of the factory components with microtubules. b. virus factories organize viral rna replication and translation the factory does provide the virus with a mechanism to organize viral rnas. positive-stranded viral rna is utilized as the template for synthesizing progeny dsrna genomes and as mrna for translating viral proteins. interestingly, sirna-targeted degradation of nsp rna blocks translation of the protein but does not block genome synthesis (silvestri et al., ) . furthermore, rna synthesis occurs in factories, but viral rna transcribed in vitro and introduced to infected cells after infection does not localize to factories. the implication of these experiments are that the factory enables rotavirus to sort viral rna into separate pools, one within the factory to act as a template for the rna polymerase and genome replication, and the other outside the factory where it translated on ribosomes to make viral proteins. it likely that this organization allows the virus factory to protect dsrna genomes from antiviral responses. arenaviruses are negative-stranded rna viruses that have two singlestranded genome segments which are packaged into -to -nmdiameter enveloped virions. lassa, junín, and manchupo viruses are responsible for emerging hemorraghic fevers in humans. arenaviruses induce moderately electron-dense inclusions in the cytoplasm that are composed of -to -nm-diameter granules identical to those seen within virus particles in the electron microscope (murphy et al., ) . the granules represent host ribosomes and between and are packaged into virions (pedersen, ) . the inclusions increase in size and density during infection until cytopathic effects are observed in cells (buckley, ; buckley and casals, ) and stain positive for viral antigens (young et al., ) ; however, it is unclear if they represent true virus factories. arenavirus replication is believed to occur in the cytoplasm but also requires a nuclear step as limited growth is observed in enucleated cells (banerjee et al., ) . the viral z protein may play a role in this as it is sufficient in vitro to shuttle pml from the nucleus to cytoplasmic inclusion bodies as occurs in vivo (borden et al., ) . n protein also localizes to discrete nuclear foci, as well as in the cytoplasm (young et al., ) , but the relationship to nd bodies and z protein is unknown. rabies virus is a neurotropic lyssavirus of the rhabdovirus family. rhabdovirus virions are bullet-shaped  nm particles containing a single negative strand of rna. rabies induces two types of inclusion body in vitro, neither of which have been proven as replication sites. negri bodies are induced by street rabies viruses in infected neurons of the brain (negri, ) and are a good indicator for the presence of an infection site in tissue ( jackson et al., ) . different neuronal cell types appear to be more prone to negri bodies ( jackson et al., ) . negri bodies contain innerbodies (negri, ) and electron microscopic studies suggest the subcompartments may be cytoplasmic material engulfed by the coalescence of several smaller negri bodies (matsumoto, ) . the role of negri bodies in infection is poorly understood. initial em observations showed virions localized to some bodies in some cells (matsumoto et al., ) , and cytological staining show they contain genetic material, indicating they may be replication complexes. however, h-thymidine or h-uridine fail to label the structures, arguing against this conclusion (matsumoto, ) . fixed (brain-adapted laboratory strains) rabies can infect nonneuronal cell lines and in these cell types induce fuchsin-stained cytoplasmic structures (fcps) as well as negri-like bodies (ni et al., ) . fcps increase in size during infection that correlates with cytopathic effects and are composed of rabies glycoprotein and matrix protein, whereas negri bodies contain nucleocapsid (ni et al., ) . this chapter has described the changes to cell architecture that are induced during virus replication. we have focused on viruses that induce new cellular structures, such as inclusion bodies, virus factories, or replication complexes, to concentrate virus and host factors necessary for replication and assembly. much progress has been made in identifying which cellular components are used to generate these structures, and in some cases specific virus proteins have been identified that are able to induce them. virus inclusions often result in rearrangement of cellular membrane compartments and/or cytoskeleton. the functions of these organelles are carefully regulated in cells, and it is a challenge for the future to determine how viruses disrupt them for use as sites of replication and assembly. changes in cellular architecture may represent bystander responses to the stress associated with virus infection, and some viruses may replicate perfectly well without them. alternatively, viruses may have evolved to target key stages in the regulatory pathways that control organelle structure and function to generate sites that are essential for replication and assembly. given the coevolution of viruses with the cells that carry them, changes in cell structure induced during infection are likely to involve a combination of the two. it is also important to appreciate that many of the structures that have been studied to date have been generated by infecting tissue culture cells with attenuated viruses, often with disregard to the host range and tropism. it is possible that in the natural setting, changes in cell structure induced by viruses will be more subtle, particulary during persistent infections that occur without inflammation or cell lysis. the products of human cytomegalovirus genes ul , ul , ul and us are tegument components parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses mapping and sequence of the gene encoding the african swine fever virion protein of m r membrane permeabilization by poliovirus proteins b and bc the vaccinia virus superoxide dismutase-like protein (a r) is a virion component that is nonessential for virus replication african swine fever virus protein p interacts with the microtubular motor complex through direct binding to light-chain dynein ultrastructural study of rotavirus replication in cultured cells assembly of african swine fever virus: role of polyprotein pp african swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum african swine fever virus protease, a new viral member of the sumo- -specific protease family adenovirus preterminal protein binds to the cad enzyme at active sites of viral dna replication on the nuclear matrix reovirus genome segment assortment into progeny genomes studied by the use of monoclonal antibodies directed against reovirus proteins recovery and characterization of a replicase complex in rotavirus-infected cells by using a monoclonal antibody against nsp adenovirus type e orf protein targets the mre complex to cytoplasmic aggresomes redistribution of microtubules and golgi-apparatus in herpes-simplex virusinfected cells and their role in viral exocytosis the herpes simplex virus ul proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells requirement of cell nucleus for the replication of an arenavirus expression and characterization of a novel structural protein of human cytomegalovirus, pul preferential virosomal location of underphosphorylated h r protein synthesized in vaccinia virus-infected cells reovirus sns protein is required for nucleation of viral assembly complexes and formation of viral inclusions reovirus sns and mns proteins form cytoplasmic inclusion structures in the absence of viral infection poliovirus proteins induce membrane association of gtpase adp-ribosylation factor hijacking components of the secretory pathway for replication of poliovirus rna does common architecture reveal a viral lineage spanning all three domains of life? deletion mutants of the herpes simplex virus type ul protein: effect on dna synthesis and ability to interact with and influence the intracellular localization of the ul and ul proteins poliovirus mutant that contains a cold-sensitive defect in viral rna synthesis high resolution localization of replicating viral genome in adenovirus-infected hela cells the vaccinia virus a . l gene encodes a hydrophobic -amino-acid virion membrane protein that enhances virulence in mice and is conserved among vertebrate poxviruses intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures association of polioviral proteins of the p genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography the mechanisms of vesicle budding and fusion a structural dna binding protein of african swine fever virus with similarity to bacterial histone-like proteins pondering the promyelocytic leukemia protein (pml) puzzle: possible functions for pml nuclear bodies an arenavirus ring (zincbinding) protein binds the oncoprotein promyelocyte leukemia protein (pml) and relocates pml nuclear bodies to the cytoplasm endoplasmic reticulum architecture: structures in flux nuclear factor i is specifically targeted to discrete subnuclear sites in adenovirus type -infected cells mouse hepatitis virus replicase protein complexes are translocated to sites of m protein accumulation in the ergic at late times of infection an amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis c virus nonstructural protein a adenovirus early region promotes the localization of splicing factors and viral rna in late-phase interchromatin granule clusters mammalian reovirus nonstructural protein mns forms large inclusions and colocalizes with reovirus microtubule-associated protein m in transfected cells reovirus nonstructural protein mns recruits viral core surface proteins and entering core particles to factory-like inclusions carboxyl-proximal regions of reovirus nonstructural protein mns necessary and sufficient for forming factory-like inclusions assembly of african swine fever virus: quantitative ultrastructural analysis in vitro and in vivo intracellular virus dna distribution and the acquisition of the nucleoprotein core during african swine fever virus particle assembly: ultrastructural in situ hybridisation and dnase-gold labelling characterization of african swine fever virion proteins j r and j l: immuno-localization in virus particles and assembly transcription factor yy is a vaccinia virus late promoter activator junín and tacaribe work in hela cells lassa fever, a new virus disease of man from west africa. . isolation and characterization of the virus nd protein pml is recruited to herpes simplex virus type prereplicative sites and replication compartments in the presence of viral dna polymerase interactions of herpes simplex virus type with nd and recruitment of pml to replication compartments the initiation of vaccinia infection the egress of herpesviruses from cells: the unanswered questions localization of structural proteins in african swine fever virus particles by immunoelectron microscopy vaccinia virus cores are transported on microtubules targeting of adenovirus e a and e -orf proteins to nuclear matrix-associated pml bodies characterization and structural localization of the reovirus l protein the african swine fever virus iap homolg is a late structural polypeptide herpes simplex virus dna replication promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus effects herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated pml and sp proteins interaction of frog virus with the cytomatrix. iv. phosphorylation of vimentin precedes the reorganization of intermediate filaments around the virus assembly sites copi activity coupled with fatty acid biosynthesis is required for viral replication early proteins are required for the formation of frog virus assembly sites effects of a temperature sensitivity mutation in the j r protein component of a complex required for vaccinia virus assembly membrane rearrangement and vesicle induction by recombinant poliovirus c and bc in human cells inhibition of cellular protein secretion by picornaviral a proteins the major structural protein of african swine fever virus, p , is packaged into large structures, indicative of viral capsid or matrix precursors, on the endoplasmic reticulum involvement of the endoplasmic reticulum in the assembly and envelopment of african swine fever virus biochemical requirements of virus wrapping by the endoplasmic reticulum calcium store during envelopment of african swine fever virus a virally encoded chaperone specialized for folding of the major capsid protein of african swine fever virus in a nutshell: structure and assembly of the vaccinia virion inhibition of protein trafficking by coxsackievirus b : multiple viral proteins target a single organelle nonviral microbodies with viral antigenicity produced in cytomegalovirus-infected cells specificity of cytopathic effect of cutaneous human papillomaviruses the poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein brefeldin a inhibits cell-free, de novo synthesis of poliovirus a vaccinia virus core protein, p , is membrane associated characterization of the vaccinia virus h l envelope protein: topology and posttranslational membrane insertion via the c-terminal hydrophobic tail icp interacts with the c-terminal domain of rna polymerase ii and facilitates its recruitment to herpes simplex virus transcription sites, where it undergoes proteasomal degradation during infection analysis of intracellular and intraviral localization of the human cytomegalovirus ul protein the development of vaccinia virus in earle's l strain cells as examined by electron microscopy electron microscopic study of the formation of poliovirus the uptake and development of reovirus in strain l cells followed with labeled viral ribonucleic acid and ferritin-antibody conjugates viruses and renal carcinoma of rana pipiens: ii. ultrastructural studies and sequential development of virus isolated from normal and tumor tissue acidic c terminus of vaccinia virus dnabinding protein interacts with ribonucleotide reductase formation of dna replication structures in herpes virus-infected cells requires a viral dna binding protein comparison of the intranuclear distributions of herpes simplex virus proteins involved in various viral functions origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors mhc i-dependent antigen presentation is inhibited by poliovirus protein a autophagy in innate and adaptive immunity poliovirus a protein limits interleukin- (il- ), il- , and beta interferon secretion during viral infection inhibition of cellular protein secretion by poliovirus proteins b and a inhibition of endoplasmic reticulum-to-golgi traffic by poliovirus protein a: genetic and ultrastructural analysis the vaccinia virus e r gene product: a viral membrane protein that is made early in infection and packaged into the virions' core the punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral dna synthesis identification of the human papilloma virus- a e gene products specific interaction between hpv- e -e and cytokeratins results in collapse of the epithelial cell intermediate filament network adenovirus replication is coupled with the dynamic properties of the pml nuclear structure interaction of hepatitis c virus proteins with host cell membranes and lipids genetic and biochemical characterization of vaccinia virus genes d l and d r which encode virion structural proteins intracellular location and translocation of silent and active poliovirus replication complexes reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral rna synthesis formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral rna synthesis expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex rotavirus nsp : mapping phosphorylation sites and kinase activation and viroplasm localization domains characterization of rotavirus nsp / nsp interactions and the dynamics of viroplasm formation replication of hepatitis c virus rna occurs in a membranebound replication complex containing nonstructural viral proteins and rna amphipathic helix-dependent localization of ns a mediates hepatitis c virus rna replication the african swine fever virus non-structural protein pb l is required for the formation of the icosahedral capsid of the virus particle the vaccinia virus d protein, which is required for dna replication, is a nucleic acid-independent nucleoside triphosphatase icp induces the accumulation of colocalizing conjugated ubiquitin interactions between dna viruses, nd and the dna damage response hsv- ie protein vmw causes redistribution of pml nd components relocate to sites associated with herpes simplex virus type nucleoprotein complexes during virus infection recruitment of herpes simplex virus type transcriptional regulatory protein icp into foci juxtaposed to nd in live, infected cells formation of nuclear foci of the herpes simplex virus type regulatory protein icp at early times of infection: localization, dynamics, recruitment of icp , and evidence for the de novo induction of nd -like complexes pml contributes to a cellular mechanism of repression of herpes simplex virus type infection that is inactivated by icp two non-structural rotavirus proteins, nsp and nsp , form viroplasm-like structures in vivo alpha-herpesvirus infection induces the formation of nuclear actin filaments assembly and translocation of papillomavirus capsid proteins reorganization of nuclear domain induced by papillomavirus capsid protein l active intranuclear movement of herpesvirus capsids adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes the ul tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions viral and cellular mrna capping: past and prospects characterization of the african swine fever virus protein p : a new late structural polypeptide role of the host cell nucleus in the replication of african swine fever virus dna differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae amino terminus of reovirus nonstructural protein sns is important for ssrna binding and nucleoprotein complex formation relative localization of viroplasmic and endoplasmic reticulum-resident rotavirus proteins in infected cells localization of the herpes simplex virus type -kilodalton dna-binding protein and dna polymerase in the presence and absence of viral dna synthesis frog virus dna replication occurs in two stages a new superfamily of putative ntpbinding domains encoded by genomes of small dna and rna viruses nidovirales: evolving the largest rna virus genome rna replication of mouse hepatitis virus takes place at double-membrane vesicles identification of the hepatitis c virus rna replication complex in huh- cells harboring subgenomic replicons subcellular localization of the us protein kinase of herpes simplex virus type modulation of p cellular function and cell death by african swine fever virus viruses and renal carcinoma of rana pipiens. i. the isolation and properties of virus from normal and tumor tissue ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment structure and assembly of intracellular mature vaccinia virus: thin-section analyses recherches sur la pathologie et étiologie de l'infection vaccinique et varioleuse subcellular localization of host and viral proteins associated with tobamovirus rna replication aggresomes resemble sites specialized for virus assembly membrane association facilitates the correct processing of pp during production of the major matrix proteins of african swine fever virus vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the a r protein upregulation of major histocompatibility complex class i on liver cells by hepatitis c virus core protein via p and tap impairs natural killer cell cytotoxicity deep-etch em reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic ''honeycomb'' surface coat dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy electron microscopy of viruses in thin sections of cells grown in culture a ubiquitin conjugating enzyme encoded by african swine fever virus vaccinia virus intracellular mature virions contain only one lipid membrane a poxvirus host range protein, cp , binds to a cellular protein, hmg a, and regulates its dissociation from the vaccinia virus genome in cho-k cells electron microscopic examination of the viromatrix of rana grylio virus in a fish cell line the hepatitis c virus nonstructural protein b is an integral endoplasmic reticulum membrane protein molecular chaperone hsp is important for vaccinia virus growth in cells evidence against an essential role of copii-mediated cargo transport to the endoplasmic reticulum-golgi intermediate compartment in the formation of the primary membrane of vaccinia virus sequential localization of two herpes simplex virus tegument proteins to punctate nuclear dots adjacent to icp domains biogenesis of poxviruses: role of a-type inclusions and host cell membranes in virus dissemination isolation and characterization of a noninfectious virionlike particle released from cells infected with human strains of cytomegalovirus involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin a the periphery of nuclear domain (nd ) as site of dna virus deposition common origin of four diverse families of large eukaryotic dna viruses quantitative study of the infection in brain neurons in human rabies subversion of cellular autophagosomal machinery by rna viruses colocalization of the herpes simplex virus ul protein with infected cell protein in small, dense nuclear structures formed prior to onset of dna synthesis aggresomes: a cellular response to misfolded proteins african swine fever virus infection disrupts centrosome assembly and function transport of african swine fever virus from assembly sites to the plasma membrane is dependent on microtubules and conventional kinesin african swine fever virus induces filopodia-like projections at the plasma membrane a study on the morphological and cyto-immunological relationship between the inclusions of variola, cowpox, rabbitpox, vaccinia (variola origin) and vaccinia ihd and a consideration of the term ''guarnieri body synthesis, subcellular localization and vp interaction of the herpes simplex virus type ul gene product the deacetylase hdac regulates aggresome formation and cell viability in response to misfolded protein stress subcellular localization of hepatitis c viral proteins in mammalian cells interferon-independent increases in class i major histocompatibility complex antigen expression follow flavivirus infection cellular autophagy: surrender, avoidance and subversion by microorganisms stages in the nuclear association of the herpes simplex virus transcriptional activator protein icp foot-and-mouth disease virus replication sites form next to the nucleus and close to the golgi apparatus, but exclude marker proteins associated with host membrane compartments nonstructural protein precursor ns a/b from hepatitis c virus alters function and ultrastructure of host secretory apparatus poliovirus protein a binds and inactivates lis , causing block of membrane protein trafficking and deregulation of cell division identification and characterization of the pseudorabies virus tegument proteins ul and ul : role for ul in virion morphogenesis in the cytoplasm the role of a -kda viral membrane protein in the assembly of vaccinia virus from the intermediate compartment replication complex of human parechovirus a giant virus in amoebae vaccinia virus gene a r dna helicase is a transcript release factor the herpes simplex virus type cleavage/packaging protein, ul , is involved in efficient localization of capsids to replication compartments human cytomegalovirus structural components: intracellular and intraviral localization of p characterization of rubella virus replication complexes using antibodies to double-stranded rna association of herpes simplex virus regulatory protein icp with transcriptional complexes containing eap, icp , rna polymerase ii, and viral dna requires posttranslational modification by the u(l) proteinkinase nuclear matrix localization and sumo- modification of adenovirus type e b k protein are controlled by e orf protein development by self-digestion: molecular mechanisms and biological functions of autophagy cell culture-grown hepatitis c virus is infectious in vivo and can be recultured in vitro secretory protein trafficking and organelle dynamics in living cells functional order of assembly of herpes simplex virus dna replication proteins into prereplicative site structures flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro adenovirus exploits the cellular aggresome response to accelerate inactivation of the mrn complex evidence that a mechanism for efficient flavivirus budding upregulates mhc class i herpes simplex virus type -induced modifications in the distribution of nucleolar b- protein formation of herpes simplex virus type replication compartments by transfection: requirements and localization to nuclear domain herpes simplex virus type prereplicative sites are a heterogeneous population: only a subset are likely to be precursors to replication compartments nucleoplasmic and nucleolar distribution of the adenovirus iva gene product visualization and functional analysis of rna-dependent rna polymerase lattices wrapping things up about virus rna replication assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively subcellular localization and some biochemical properties of the flavivirus kunjin nonstructural proteins ns a and ns a markers for trans-golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells rubella virus replication complexes are virus-modified lysosomes perinuclear localization of na-k-clcotransporter protein after human cytomegalovirus infection microtubule-dependent organization of vaccinia virus core-derived early mrnas into distinct cytoplasmic structures relationship between vaccinia virus intracellular cores, early mrnas, and dna replication sites small dense nuclear bodies are the site of localization of herpes simplex virus u(l) and u(l) proteins and of icp only when the latter protein is present novel acylation of poxvirus a-type inclusion proteins characterization of the african swine fever virus structural protein p . a dna binding protein rabies virus further studies on the replication of rabies and rabies-like viruses in organized cultures of mammalian neural tissues nuclear domain as preexisting potential replication start sites of herpes simplex virus type- inhibition of poliovirus rna synthesis by brefeldin a the trans golgi network is lost from cells infected with african swine fever virus identification of the orthopoxvirus p c gene, which encodes a structural protein that directs intracellular mature virus particles into a-type inclusions assembly site of the virus pbcv- in a chlorella-like green alga: ultrastructural studies herpesvirus assembly and egress egress of alphaherpes viruses herpesvirus assembly: a tale of two membranes flock house virus rna polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion reovirus sns protein localizes to inclusions through an association requiring the mns amino terminus increased ubiquitination and other covariant phenotypes attributed to a strain-and temperature-dependent defect of reovirus core protein m the vaccinia virus-encoded uracil dna glycosylase has an essential role in viral dna replication rifampicin prevents virosome localization of l , an essential vaccinia virus polypeptide effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: bc but not a blocks endoplasmic reticulum-to-golgi transport inhibition of the secretory pathway by the foot-and-mouth disease virus bc protein is reproduced by co-expression of b with c and the site of inhibition is determined by the subcellular location of c involvement of spicules in the formation of vaccinia virus envelopes elucidated by a conditional lethal mutant modulation of transporter associated with antigen processing (tap)-mediated peptide import into the endoplasmic reticulum by flavivirus infection high-pressure freezing in the study of animal pathogens the ultrastructure of the developing replication site in foot-and-mouth disease virus-infected bhk- cells membrane association of the rna-dependent rna polymerase is essential for hepatitis c virus rna replication structure and development of viruses observed in the electron microscope. ii. vaccinia and fowl pox viruses rifampicin: a specific inhibitor of vaccinia virus assembly herpes simplex virus icp and icp . counteract distinct interferon-induced barriers to virus replication evidence that herpes simplex virus vp is required for viral egress downstream of the initial envelopment event ultrastructural study of african swine fever virus replication in cultures of swine bone marrow cells mitochondrial distribution and function in herpes simplex virusinfected cells identification by mass spectroscopy of three major early proteins associated with virosomes in vaccinia virusinfected cells arenoviruses in vero cells: ultrastructural studies interaction of frog virus- with the cytoskeleton. i. altered organization of microtubules, intermediate filaments, and microfilaments synthesis of frog virus proteins occurs on intermediate filament-bound polyribosomes a functional role for intermediate filaments in the formation of frog virus assembly sites localization of adenovirus-encoded dna replication proteins in the nucleus by immunogold electron microscopy the ul gene product of herpes simplex virus is a virion protein that colocalizes with intranuclear capsid proteins 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cells by ultrastructural immunocytochemistry ultrastructural localization of rotavirus antigens using colloidal gold immunoelectron microscopy analysis of hcmv gpul (gn) localization vaccinia virus infection disrupts microtubule organization and centrosome function adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells coronavirus replication complex formation utilizes components of cellular autophagy identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins detection and subcellular localization of the turnip yellow mosaic virus k replication protein in infected cells clustered charge-to-alanine mutagenesis of the vaccinia virus a gene: temperaturesensitive mutants have a dna-minus phenotype and are defective in the production of processive dna polymerase activity simultaneous detection of highly phosphorylated proteins and viral major dna binding protein distribution in nuclei of adenovirus 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dna replication sites quantitative analysis of the hepatitis c virus replication complex intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that icp is associated with progeny virus dna the discovery and characterization of mimivirus, the largest known virus and putative pneumonia agent the vaccinia virus f r protein interacts with actin autophagosomes: biogenesis from scratch? tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum sequence of morphological changes in epithelial cell cultures infected with poliovirus temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication vaccinia virus nonstructural protein encoded by the a r gene is required for formation of the virion membrane characterization of the ul gene product of herpes simplex virus the vaccinia virus -kda protein forms a stable complex with the p a/ a major core protein early in morphogenesis endoplasmic reticulum-golgi intermediate compartment membranes and vimentin filaments participate in vaccinia virus assembly cytopathogenic effect of poliomyelitis viruses 'in vitro' on human embryonic tissues the nd component promyelocytic leukemia protein relocates to human papillomavirus type e intranuclear inclusion bodies in cultured keratinocytes and in warts african swine fever virus structural protein p is essential for the recruitment of envelope precursors to assembly sites african swine fever virus pb l protein is a flavin adenine dinucelotide-linked sulfhydryl oxidase putative site for the acquisition of human herpesvirus virion tegument cytopathic effect in human papillomavirus type -induced inclusion warts: in vitro analysis of the contribution of two forms of the viral e protein polyhedrin structure migration of mitochondria to viral assembly sites in african swine fever virus-infected cells replication of african swine fever virus dna in infected cells the herpes simplex virus rna binding protein us is a virion component and associates with ribosomal s subunits regulated cleavages at the west nile virus ns a- k-ns b junctions play a major role in rearranging cytoplasmic membranes and golgi trafficking of the ns a protein characterization of the vaccinia virus a r protein and its role in virulence functional analysis of adenovirus protein ix identifies domains involved in capsid stability, transcriptional activity, and nuclear reorganization adenovirus protein ix sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation a cellular factor is required for transcription of vaccinia viral intermediate-stage genes african swine fever virus is wrapped by the endoplasmic reticulum cellular copii proteins are involved in production of the vesicles that form the poliovirus replication complex vaccinia virus membrane proteins p and p are cotranslationally inserted into the rough endoplasmic reticulum and 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nucleus selection for temperature-sensitive mutations in specific vaccinia virus genes: isolation and characterization of a virus mutant which encodes a phosphonoacetic acid-resistant, temperature-sensitive dna polymerase vimentin rearrangement during african swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase ii micromorphology of fl cells infected with polio and coxsackie viruses remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles characterization of the african swine fever virion protein j l vaccinia virus a l protein is required for association of viral membranes with dense viroplasm to form immature virions vaccinia virus g l protein interacts with the a l protein and is required for association of viral membranes with dense viroplasm to form immature virions a complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions evidence for an essential catalytic role of the f protein kinase in vaccinia virus morphogenesis physical and functional interactions between vaccinia virus f protein kinase and virion assembly proteins a and g external scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice the herpes simplex virus ul gene is required for localization of capsids and major and minor capsid proteins to intranuclear sites where viral dna is cleaved and packaged proteomics of herpes simplex virus replication compartments: association of cellular dna replication, repair, recombination, and chromatin remodeling proteins with icp herpes simplex virus replication compartments can form by coalescence of smaller compartments recognition of the adenovirus type origin of dna replication by the virally encoded dna polymerase and preterminal proteins induction of intracellular membrane rearrangements by hav proteins c and bc vaccinia virus dna replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei frog virus morphogenesis: effect of temperature and metabolic inhibitors subcellular distribution of the major immediate early proteins of human cytomegalovirus changes during infection herpes simplex virus type capsids transit by the trans-golgi network, where viral glycoproteins accumulate independently of capsid egress architecture of the flaviviral replication complex. protease, nuclease, and detergents reveal encasement within double-layered membrane compartments the vaccinia virus c and a-type inclusion proteins are specific markers for the intracellular mature virus particle alfalfa mosaic virus replicase proteins p and p interact and colocalize at the vacuolar membrane orf a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication immunolocalization of vaccinia virus structural proteins during virion formation identification and localization of genetic material of african swine fever virus by autoradiography identification and characterization of the ul gene product of herpes simplex virus type production of infectious hepatitis c virus in tissue culture from a cloned viral genome visualization and characterization of the intracellular movement of vaccinia virus intracellular mature virions vaccinia virus intracellular movement is associated with microtubules and independent of actin tails a novel herpes simplex virus gene, ul . , maps antisense to the ul gene and encodes a protein which colocalizes in nuclear structures with capsid proteins assemblons: nuclear structures defined by aggregation of immature capsids and some tegument proteins of herpes simplex virus maintenance of golgi structure and function depends on the integrity of er export identification of nuclear export signal in ul protein of herpes simplex virus type the vaccinia virus i l gene product is localized to a complex endoplasmic reticulum-associated structure that contains the viral parental dna a proline-rich region in the coxsackievirus a protein is required for the protein to inhibit endoplasmic reticulum-to-golgi transport effects of picornavirus a proteins on protein transport and gbf -dependent cop-i recruitment a viral protein that blocks arf -mediated cop-i assembly by inhibiting the guanine nucleotide exchange factor gbf proteins c and ns b of the flavivirus kunjin translocate independently into the nucleus ultrastructure of kunjin virus-infected cells: colocalization of ns and ns with doublestranded rna, and of ns b with ns , in virus-induced membrane structures localization of p , retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells aggresomes and autophagy generate sites of virus replication a decade of advances in iridovirus research macromolecular synthesis in cells infected by frog virus . xi. a ts mutant of frog virus that is defective in late transcription relationship between rna polymerase ii and efficiency of vaccinia virus replication virus assembly in hincksia hincksiae (ectocarpales, phaeophyceae) an electron and fluorescence microscopic study assembly of a large icosahedral dna virus, mclav- , in the marine alga myriotrichia clavaeformis (dictyosiphonales, phaeophyceae) a myristylated membrane protein encoded by the vaccinia virus l r open reading frame is the target of potent neutralizing monoclonal antibodies subcellular localization, stability, and trans-cleavage competence of the hepatitis c virus ns -ns a complex expressed in tetracycline-regulated cell lines vaccinia virus late transcription is activated in vitro by cellular heterogeneous nuclear ribonucleoproteins characterization of the ul gene product of herpes simplex virus type the vaccinia virus a l gene encodes a membrane protein required for an early step in virion morphogenesis localization of an arenavirus protein in the nuclei of infected cells enzymatic and control functions of reovirus structural proteins nuclear localization of a double-stranded rna-binding protein encoded by the vaccinia virus e l gene characterization of an early gene encoding for dutpase in rana grylio virus assembly of complete, functionally active herpes simplex virus dna replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays robust hepatitis c virus infection in vitro the coronavirus replicase: insights into a sophisticated enzyme machinery key: cord- -wekvet f authors: maceyka, michael; machamer, carolyn e. title: ceramide accumulation uncovers a cycling pathway for the cis-golgi network marker, infectious bronchitis virus m protein date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: wekvet f the m glycoprotein from the avian coronavirus, infectious bronchitis virus (ibv), contains information for localization to the cis-golgi network in its first transmembrane domain. we hypothesize that localization to the golgi complex may depend in part on specific interactions between protein transmembrane domains and membrane lipids. because the site of sphingolipid synthesis overlaps the localization of ibv m, we asked whether perturbation of sphingolipids affected localization of ibv m. short-term treatment with two inhibitors of sphingolipid synthesis had no effect on localization of ibv m or other golgi markers. thus, ongoing synthesis of these lipids was not required for proper localization. surprisingly, a third inhibitor, d,l-threo- -phenyl- -decanoylamino- -morpholino- -propanol (pdmp), shifted the steady-state distribution of ibv m from the golgi complex to the er. this effect was rapid and reversible and was also observed for ergic- but not for golgi stack proteins. at the concentration of pdmp used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. pretreatment with upstream inhibitors partially reversed the effects of pdmp, suggesting that ceramide accumulation mediates the pdmp-induced alterations. indeed, an increase in cellular ceramide was measured in pdmp-treated cells. we propose that ibv m is at least in part localized by retrieval mechanisms. further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics. -propanol (pdmp), shifted the steady-state distribution of ibv m from the golgi complex to the er. this effect was rapid and reversible and was also observed for ergic- but not for golgi stack proteins. at the concentration of pdmp used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. pretreatment with upstream inhibitors partially reversed the effects of pdmp, suggesting that ceramide accumulation mediates the pdmp-induced alterations. indeed, an increase in cellular ceramide was measured in pdmp-treated cells. we propose that ibv m is at least in part localized by retrieval mechanisms. further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics. t he organelles of the classical secretory pathway must maintain their identity despite a large flux of lipids and proteins. two models have emerged to explain how proteins can be maintained in specific compartments (machamer, ; nilsson and warren, ). the retention model proposes that proteins are efficiently anchored in the appropriate compartment. the retrieval model proposes that proteins are continually recycled from later compartments. the two models are not mutually exclusive; indeed most proteins within the secretory pathway probably use both mechanisms for localization, albeit to differing extents. an example of this is the localization of the er resident protein bip, which contains a kdel retrieval signal but is only slowly secreted when this signal is removed (munro and pelham, ) . this suggests that other parts of the molecule may contain retention information. the m glycoprotein from the avian coronavirus, infectious bronchitis virus (ibv), is a model protein for studying localization to the early secretory pathway. immunoelectron microscopy showed that ibv m expressed in the absence of other ibv proteins was found in the tubulovesicular structures at the entry or cis face of the golgi stack, as well as the first or second cisterna of the golgi stack (machamer et al., ; sodeik et al., ) . we will refer to this region as the cis -golgi network (cgn; mellman and simons, ) . defined in this way, we would consider the cgn to at least partially overlap with the intermediate compartment (ic), defined by such markers as ergic- or p (schweizer et al., ; lahtinen et al., ) . the first transmembrane domain of ibv m is sufficient to target chimeric proteins to the cgn (swift and machamer, ; machamer et al., ) . although the transmembrane domains of other golgi proteins also contain targeting information, no consensus motif for localization has been identified (for review see colley, ) . we are intrigued by the possibility that the targeting of golgi membrane proteins may in part depend on interactions between their transmembrane domains and specific membrane lipids. independent studies demonstrate that the lipid compositions of membranes differ at each stage of the secretory pathway (keenan and morre, ; cluett et al., ; for review see van meer, ) . sphingolipids, including sphingomyelin (sm) and glucosylceramide (glccer), the precursor to all gangliosides, are one class of lipids thought to increase in relative concentration through the secretory pathway. ceramide, the precursor of sphingolipids, is synthesized in the er. in rat liver, ceramide is converted into the different classes of sphingolipids by enzymes localized to the cgn and the cis -and medial -golgi cisternae (futerman et al., ; futerman and pagano, ) . when expressed from a recombinant vaccinia virus, ibv m is localized to the cgn in several cell types, and its localization presumably overlaps with that of sm and glccer synthase activities. this led us to speculate that there may be a link between sphingolipid synthesis and the localization of ibv m. to address this question we tested the effects of three sphingolipid synthesis inhibitors on the steady-state localization of ibv m. we observed a dramatic redistribution of ibv m induced by one of these inhibitors, the glucosylceramide analogue d,l-threo- -phenyl- -decanoylamino- -morpholino- -propanol (pdmp). use of upstream inhibitors coupled with lipid analysis suggested that the pdmp effects are mediated by the accumulation of the precursor ceramide. because ibv m can be induced to move to the er, we propose that ibv m is at least in part localized by retrieval mechanisms. fcs was from atlanta biologicals (norcross, ga); fumonisin b was from calbiochem (la jolla, ca); chromatography plates were from e. merck (darmstadt, germany); endoglycosidase h (endo h) from new england biolabs (beverly, ma); n -hexanoylsphingosine (c cer), lipid standards, and pdmp were from matreya (pleasant gap, pa); [ h]palmitate ( ci/mmol) was from dupont-nen (wilmington, de); pro-mix ( Ͼ , mci/mmol [ s]methionine) was from amersham intl. (arlington heights, il); dme, tissue culture-grade trypsin, and penicillin-streptomycin were from gibco brl (gaithersburg, md); all other reagents were from sigma chemical co. (st. louis, mo). antibodies were obtained as follows: monoclonal anti-bip, stressgen (victoria, bc, canada); monoclonal anti-giantin and monoclonal anti-ergic- , hans-peter hauri (basel, switzerland); polyclonal anti-␤ -cop, jennifer lippincott-schwartz (national institutes of health, bethesda, md); polyclonal ␣ -mannosidase ii, marilyn farquhar (university of california at san diego, la jolla, ca) and kelley moreman (university of georgia, athens, ga); polyclonal antibodies to ibv m and vesicular stomatitis virus (vsv) were prepared as described (machamer and rose, ; weisz et al., , respectively) ; texas red-and fitc-conjugated secondary antibodies, jackson immunoresearch (west grove, pa). cell culture. tissue culture cells were grown in dme supplemented with % (bhk- ) or % (vero) fcs. cells were grown at Њ c in an atmosphere of % co . pulse-chase labeling. pulse-chase experiments were performed as previously described (rosenwald et al., ; weisz et al., ) . briefly, cells were plated in -mm dishes the night before the experiment to be % confluent the next day. vsv (san juan strain, indiana serotype) was adsorbed for min in . ml of serum-free dme. at h after infection, the cells were starved for methionine for min. cells were then pulsed for min with ci s-pro-mix and chased for the indicated amount of time in the presence or absence of the indicated drugs. the isopropanol carrier alone had no effect on the rate of transport of vsv g protein. after the chase, cells were washed once with cold pbs and lysed in detergent. vsv g protein was then immunoprecipitated, treated with endo h as described (rosenwald et al., ) , separated by sds-page, and visu-alized by fluorography. endo h-sensitive and -resistant forms of the vsv g protein were quantitated by densitometry. indirect immunofluorescence microscopy. these experiments were performed as previously described (swift and machamer, ) . briefly, cells were infected for min with a recombinant vaccinia virus encoding ibv m, and the indicated treatments were begun h after infection. for experiments using exogenous ceramides, the soluble short-chain analogues of ceramide were added to cells as a complex with . mg/ml defatted bsa as described (pagano and martin, ) . after treatment, cells were fixed, permeabilized, and stained with the appropriate antibody. images were acquired using a microscope (axioskop; zeiss, inc., thornwood, ny) equipped with epifluorescence and a ccd camera (photometrics sensys, tucson, az) using ip lab software (signal analytics corp., vienna, va). all images shown are the raw data collected at ϫ binning with a gain of . lipid synthesis assays. cells were seeded onto -cm plastic dishes d before the experiment so that they were % confluent the day of the experiment. cells were incubated in dme supplemented with g/ml cycloheximide, and half of the dishes were also incubated with mm ␤ ca. h later, fresh medium containing cycloheximide and [ h]palmitate ( ci/ dish) was added, along with either % isopropanol (control and ␤ ca) or m pdmp and mm ␤ ca if indicated. h later, cells were trypsinized and washed off the plate in . ml cold pbs. to normalize lipid levels, l were removed for a protein assay by the method of bradford ( ) . lipids were then extracted by the method of bligh and dyer ( ) . labeled samples were doped with g cold ceramide to allow for visualization. samples were run on ϫ cm high performance thinlayer chromatography plates with a mobile phase of chloroform/glacial acetic acid ( : ; abe et al., ) . plates were sprayed with water to visualize the ceramide bands that were scraped and counted after the addition of scintillation fluid. alternatively, plates were dipped in % , -diphenyloxazole in chloroform for visualization by fluorography (henderson and tocher, ) . to test whether ongoing sphingolipid synthesis was necessary for the correct localization of the ibv m protein, we used three inhibitors of sphingolipid synthesis: ␤ -chloroalanine ( ␤ ca; medlock and merrill, ) , fumonisin b (fb ; wang et al., ) , and pdmp (vunnam and radin, ; inokuchi and radin, ) . the biosynthetic pathway for sphingolipids with the sites of inhibition of these drugs is shown in fig. . both ␤ ca ( mm) and fb ( m) inhibited the incorporation of radiolabeled precursors into sphingolipids in bhk- cells by Ͼ % (data not shown). consistent with previous results (rosenwald et al., ) , we found that pdmp ( m) inhibited glccer synthesis by ‫ف‬ % and sm synthesis by ‫ف‬ % in bhk- cells (data not shown). to express the ibv m protein, bhk- cells were infected with a recombinant vaccinia virus encoding ibv m (machamer and rose, ) . h after infection the cells were treated with cycloheximide for h to chase newly synthesized m protein out of the er. sphingolipid synthesis inhibitors were then added and the cells incubated for another h before processing for immunofluorescence ( fig. ) . in control cells, ibv m exhibited a tight, juxtanuclear staining pattern that colocalized with golgi markers. the golgi localization pattern of ibv m was unchanged by treatment with either ␤ ca or fb , suggesting that ongoing sphingolipid synthesis is not required for proper localization of ibv m. in contrast, pdmp-treated cells showed a marked change in the staining pattern of the ibv m pro-tein after h. the localization of ibv m in the presence of pdmp changed from golgi to er based on the absence of strong juxtanuclear staining and the presence of nuclear rim staining and a tubulo-reticular staining pattern that colocalized with er markers. treatment of infected cells with lower concentrations of pdmp had no effect on ibv m localization and very little effect on sm synthesis (data not shown). we next tested the kinetics of pdmp-induced mislocalization of ibv m. cells were infected as before and treated for h with cycloheximide. then cells were treated with pdmp for varying lengths of time up to h (fig. ) . redistribution was first observed at min and became maximal at min. this redistribution was rapidly reversible, such that min after washing out pdmp, ibv m had moved back to the golgi region. we next asked if pdmp mislocalized other golgi markers. cells were infected and treated as described above and then stained with antibodies to two integral membrane proteins of the golgi stack, giantin (linstedt and hauri, ) and mannosidase ii (man ii; moremen and robbins, ; velasco et al., ) . treatment of cells with m pdmp for h had no effect on the localization of either protein (fig. ) , suggesting that the morphology of the golgi was not greatly altered. we also examined the localization of ␤ -cop, a peripheral membrane protein of the stack and cgn involved in vesicular traffic (oprins et al., ) . as seen in fig. , the distribution of ␤ -cop appeared unaffected in treated cells. we then looked at a protein which cycles through the cis -golgi. as we did not have access to antibodies that crossreact with known markers in bhk- cells, we examined ergic- in vero cells. ibv m localizes to the cgn in these cells and is also redistributed to the er by pdmp (data not shown). ergic- is an ic protein that cycles between the er, ic, and golgi stack (lippincott-schwartz et al., ; schindler et al., ) . vero cells were treated with g/ml cycloheximide for h. pdmp ( m) was added, and dishes were fixed at the indicated times and prepared for indirect immunofluorescence. after h in pdmp, the remaining dishes were washed three times with ml dme with serum and incubated with cycloheximide-containing medium for the indicated time before being prepared for indirect immunofluorescence. for each experimental series, the nomarski image is shown on the left and the fluorescence image on the right. bar, m. with cycloheximide for h and then with or without m pdmp for an additional hour before being fixed and prepared for immunofluorescence (fig. ) . similar to the effects on ibv m, pdmp treatment shifted the steady-state distribution of ergic- to the er. in addition, neither ␤ ca nor fb had any effect on the localization of er-gic- (data not shown). the above immunofluorescence results suggested that pdmp might generally alter the distribution of proteins that have a dynamic localization mechanism. interestingly, it has been shown that pdmp slows the rate of both anterograde vesicular traffic (rosenwald et al., ) and endocytosis (chen et al., ) in cho cells. we tested whether pdmp also slowed anterograde traffic in bhk- cells. we used a plasma membrane protein, the well-characterized g protein from vsv. a pulse-chase experiment was performed either in the presence or absence of pdmp, ␤ ca, or fb . the rate of accumulation of endo h-resistant vsv g was used as an assay for the rate of arrival at the medial -golgi. neither ␤ ca nor fb had any effect on the rate of anterograde traffic. however, pdmp increased the halftime for transport by ‫ف‬ . -fold from to min (fig. ). fig. shows that neither ␤ ca nor fb had any effect on the transit rate of vsv g, suggesting that the decreased rate with pdmp was not due to a block in the ongoing synthesis of sphingolipids. pdmp inhibits the conversion of ceramide into glycosphingolipids and sphingomyelin. one possible explanation for the effects observed with pdmp was an accumulation of the precursor ceramide. ␤ ca and fb act upstream of pdmp (fig. ) , and the intermediates in sphingolipid synthesis between these steps and ceramide production are thought to be short lived (merrill and wang, ; medlock and merril, ) . to ask if ceramide accumulation might mediate the pdmp-induced slowing of anterograde traffic, cells were treated with ␤ ca or fb before pdmp. we performed a pulse-chase labeling experiment with cells pretreated with either ␤ ca or fb for h before being chased in the presence or absence of ␤ ca or fb and pdmp (fig. ) . pretreatment with the earlier inhibitors reduced the pdmp-induced slowing of anterograde traffic by about half, suggesting that accumulation of newly synthesized ceramide at least partially mediates this effect. to test whether ceramide was likely to mediate the pdmpinduced mislocalization of ibv m, indirect immunofluorescence was performed as above with cells pretreated with either ␤ ca or fb for h before the addition of pdmp. to simplify the quantification, the localization of ibv m was classified into one of three staining patterns. class was the most commonly seen pattern in untreated cells, i.e., tight juxta-nuclear staining that colocalized with golgi markers. class was the most commonly seen pattern in pdmp-treated cells, i.e., diffuse staining with prominent nuclear envelope staining that colocalized with er markers. class was intermediate between the class and class . whether this pattern represents an overlap of er and golgi staining patterns or an increase in ic staining is not clear. for these experiments, coded samples were quantified by counting at least cells for each sample. the experiment was performed five times, with one representative experiment shown (fig. ) . interestingly, when cells were quantified in this way, we observed that both ␤ ca and fb caused a slight shift from class to class . the significance of this observation is not clear. pdmp dramatically shifted the distribution of ibv m from mainly class in the control cells to mainly class . pretreatment with either ␤ ca or fb significantly reduced this shift in the staining pattern of ibv m, suggesting that accumula- figure . pdmp induced the redistribution of the endogenous ic protein, ergic- , to the er. vero cells were incubated with cycloheximide for h and then incubated for h in the presence either % isopropanol (control) or m pdmp. cells were then fixed and prepared for indirect immunofluorescence with antibodies to ergic- . for each experimental group, the nomarski image is shown on the left and the fluorescence image on the right. bar, m. the pretreatment experiments suggested that increased levels of ceramide mediate the pdmp-induced effects on ibv m localization. ceramide can act as a second messenger (for review see hannun, ) and has been suggested to be the mediator of some of the effects of pdmp in cho cells (rosenwald and pagano, ) . furthermore, in several systems, cellular levels of ceramide increased upon treatment with pdmp or one of its more soluble analogues (for example see rani et al., ; posse de chaves et al., ) . we measured the levels of ceramide after various treatments by pulse-labeling with [ h]palmitate. after labeling and treatment, cellular lipids were extracted and run on chromatography plates. the appropriate spots corresponding to ceramide were scraped and counted. consistent with our hypothesis, fig. a shows that pdmp treatment increased the levels of ceramide . -fold over control levels. pretreatment with ␤ ca reduced pdmpinduced ceramide accumulation to roughly half of control levels, but this level was still threefold higher than cells treated with ␤ ca alone. fig. b is a representative fluorograph showing the ceramide region that was quantified in fig. a. interestingly, we noticed the appearance of a lower migrating ceramide band in pdmp-treated cells. the pretreatment experiments as well as lipid analysis implicated ceramide as the mediator of pdmp effects. if this were true, addition of a soluble, short-chain analogue of ceramide might mimic the effects of pdmp. indeed, one such cell-permeable analogue of ceramide, n -hexanoylsphingosine (c cer), reproduces the effects of pdmp on anterograde traffic of vsv g in cho cells (rosenwald and pagano, ) . we first asked whether c cer slowed anterograde traffic in bhk- cells. a pulse-chase labeling experiment with vsv-infected cells was performed, with cells chased in the absence or presence of m c cer. c cer slowed the rate of anterograde traffic in bhk- cells ‫- . ف‬fold (data not shown). we went on to use c cer in our indirect immunofluorescence assay. as expected, c cer had no effect on the localization of either man ii or ␤-cop (fig. ) . however, contrary to our expectation, c cer did not alter the localization of either ibv m or er-gic- . this suggests that exogenously added c cer and ceramide generated by treatment with pdmp do not have the same effects. two other soluble analogues of ceramide, c cer and c cer, were also tested and found to have no effect on the localization of ibv m (data not shown). there are two possible mechanisms to maintain the steady-state localization of proteins to specific sites along the secretory pathway: (a) retention in the particular compartment, and (b) retrieval from other compartments (machamer, ; nilsson and warren, ) . the redistribution of ibv m induced by pdmp was surprising, as we expected that if we disrupted its localization, it would move with "bulk flow" to the plasma membrane. that ibv m was redistributed to the er suggests that it has specific targeting information for retrieval to the er. coupled with earlier work in our laboratory on the role of oligomerization of ibv m chimeras in cgn targeting (weisz et al., ) , our findings suggest that ibv m maintains its steady-state distribution by both retention and retrieval ( fig. a) . we propose two models to explain the redistribution of ibv m protein induced by pdmp. the simplest model (fig. b, left arrow) is that the m protein normally cycles between the er and the golgi at a significant rate, as has been shown for ergic- (lippincott-schwartz et al., ; schindler et al., ) . the slowing of anterograde traffic would cause the protein to shift its steady-state distribution to the er, assuming that retrograde traffic is unaffected or perhaps increased by pdmp. a second possibility is that pdmp disrupts retention of the m protein, which then cycles back to the er by normal retrieval mechanisms (fig. b, right arrow) . these two models are not mutually exclusive, and both effects of pdmp may be necessary to redistribute ibv m to the extent observed. implicit in both models is the idea that ibv m contains retrieval information. pdmp-induced er redistribution was also observed for ergic- , a dilysine-containing ic marker known to cycle through the er (lippincott-schwartz et al., ; schindler et al., ) . double-label immunoelectron microscopy in hela cells showed that the distri-bution of ergic- and ibv m overlapped significantly, though not completely (sodeik et al., ) . this suggests that ibv m may in part use the same, as yet unknown, cellular machinery used by ic proteins to maintain its steadystate localization. using deletion mutants and chimeric molecules, dissection of ibv m retention and retrieval information should be possible using pdmp as a tool. the pathway followed by ibv m during retrieval will also be important to decipher. experiments in nontreated cells suggest that the protein may move through later golgi compartments even though its steady-state distribution is the cgn, because its oligosaccharides are slowly processed (machamer et al., ) . interestingly, we did not observe redistribution of two golgi stack markers (man ii and giantin) to the er with pdmp treatment. golgi membrane proteins were recently suggested to be highly mobile (cole et al., b) and to cycle through the er (cole et al., a) . our results with pdmp suggest that cycling of these proteins does not occur during the time scale of our experiments, and/or that its predominant effect on ibv m is a loss of retention. interestingly, it has been shown that myriocin, an inhibitor of sphingolipid synthesis thought to inhibit the same step as ␤ca, reduces the rate of transport of a glycosylphosphatidylinositol-linked protein but not other proteins out of the er in yeast (horvath et al., ) . it would be interesting to test whether inhibition of ceramide synthesis in mammalian cells also slows the rate of transport of glycosylphosphatidylinositol-linked proteins in mammalian cells. however, neither ␤ca nor fb had an effect on the rate of anterograde traffic of vsv g or the localization of ic, cgn, or golgi stack proteins. when we quantified the effects of these two inhibitors on the immunofluorescence figure . exogenous c cer did not redistribute ibv m or er-gic- . bhk cells (ibv m, man ii, and ␤-cop) or vero cells (ergic- ) were infected with a recombinant vaccinia virus expressing ibv m. h after infection the cells were treated with g/ml cycloheximide. at h after infection cells were incubated for h in serum-free dme with . mg/ml defatted bsa with or without m c cer. cells were then prepared for indirect immunofluorescence and stained with the indicated antibodies. for each experimental series, the nomarski image is shown on the left and the fluorescence image on the right. bar, m. figure . current models for how ibv m maintains its steady-state distribution and how pdmp might alter this distribution. (a) evidence presented here shows that ibv m can be induced to redistribute to the er, indicating that ibv m has information necessary for traffic to the er. we propose that ibv m normally maintains its steady-state distribution by cycling through the er at some basal rate. (b) if ibv m is cycling through the er normally, then its localization would depend on the balance of anterograde and retrograde traffic rates. as pdmp slows anterograde traffic, ibv m could accumulate in the er when the balance of membrane traffic is disrupted (left arrow). alternatively, pdmp may act by disrupting the retention of ibv m, causing it to move out of the golgi (right arrow). it is also possible that redistribution of ibv m requires both effects of pdmp. staining pattern of ibv m, we did detect a slight shift toward the intermediate staining pattern (fig. ) . whether this represents an altered distribution pattern or subtle effects on golgi structure caused by these inhibitors is unclear. from these results we conclude that ongoing sphingolipid synthesis is not required for either normal rates of anterograde traffic or proper localization of golgi proteins. however, the glucosylceramide analogue pdmp, at a concentration that inhibits both glccer and sm synthases, causes a redistribution of ibv m to the er and a slowing of anterograde traffic in bhk- cells. why does pdmp have these effects while ␤ca and fb do not? it is likely that pdmp exerts its effects by causing the accumulation of ceramide. other groups have found that the various effects of pdmp and its active analogues were concomitant with (uemura et al., ; shayman et al.; rani et al., ) or actually caused by increases in ceramide concentrations (abe et al., ; posse de chaves et al., ) . in bhk- cells treated with m pdmp for h we measured an increase in newly synthesized ceramide (fig. a) , showing at least a correlation between levels of newly synthesized ceramide and the effects of pdmp on protein traffic and localization. the fact that pretreatment with either ␤ca or fb , both of which block the synthesis of ceramide, can ameliorate the effects of pdmp demonstrates that ceramide is at least in part the mediator of these effects. however, the effects of pdmp are not completely abolished by pretreatment, even though ␤ca pretreatment reduced the level of newly synthesized ceramide by % compared to control. one explanation is that ceramide accumulation is only one of the ways that pdmp exerts its effects. another possibility is that ceramide is being generated by the degradation of preexisting sphingolipids, e.g., at the cell surface or in lysosomes. this ceramide cannot be made back into sphingolipids because the enzymes sm and glccer synthases are inhibited in the presence of pdmp. interestingly, the fluorograph of the ceramide bands generated after the various treatments (fig. b) shows different relative amounts of ceramide species. we suspect that these ceramides differ in acyl chain lengths (abe et al., ) and that this may be an indication of different sources of ceramide, i.e., de novo synthesis or sphingolipid degradation. we are currently investigating this possibility. based on the pretreatment results, we expected that soluble analogues of ceramide would mimic the effects of pdmp. rosenwald and pagano ( ) showed that like m pdmp, m c cer decreased the rate of transit of an itinerant protein to the medial-golgi in cho cells. consistent with these results, m c cer also slowed anterograde traffic in bhk- cells. however, m c cer had no effect on the localization of either the m protein or ergic- . other short-chain ceramide analogues (c and c -ceramide) also had no effect on ibv m localization. how does this fit with our model that ceramide mediates pdmp effects? one explanation is that the effect on protein localization is independent of the effect on antero-grade traffic rates. in this case, it may be that c cer and endogenous ceramide have similar ability to bind and affect proteins that regulate anterograde traffic, but c cer is unable to bind or affect proteins that regulate protein localization. it has been shown that some short chain analogues of ceramide do not have the same effects as endogenous ceramide (e.g., wolff et al., ) . a second possibility is that effects of ceramide are limited to the bilayers in which its concentration increases, and the difference seen between endogenous ceramide and exogenous c cer is due to the different intracellular distribution of these molecules. c cer would be unlikely to accumulate in compartments where ibv m and ergic reside, because it is rapidly converted to sphingolipids there (rosenwald et al., ) . effects on membrane traffic might be more dramatic in compartments where c cer accumulates, possibly the trans-golgi and tgn (pagano et al., ) . consistent with this idea, the rate of movement of vsv g through the late golgi (measured by sialylation) was slowed to a much greater extent by c cer than by pdmp (rosenwald and pagano, ) . it is not yet clear how ceramide induces the changes in anterograde traffic and protein localization. recent studies have shown that ceramide (hannun, ) and its metabolites (hakomori, ) play a major role as second messengers. preliminary experiments with both the ceramidase inhibitor n-oleoylethanolamine and exogenously added sphingosine suggest that the ceramide breakdown product sphingosine does not play a role in the localization of ibv m (maceyka, m., and c. machamer, unpublished observations). ceramide activates a cytosolic protein phosphatase (dobrowsky and hannun, ; wolff et al., ) that can be inhibited by okadaic acid. preliminary experiments suggest that okadaic acid does not block the pdmpinduced changes (maceyka, m., and c. machamer, unpublished observations). ceramide could be activating a protein kinase (mathias et al., ) , but this kinase has not been fully characterized. another possibility is that increased ceramide within the lipid bilayer directly affects the localization of ibv m. earlier work from our laboratory showed that the first transmembrane domain of ibv m can target chimeras to the cgn (swift and machamer, ; machamer et al., ) . it is possible that cgn bilayers have distinct lipid domains, and that these domains contain different sets of proteins, analogous to glycosphingolipid rafts. segregation of membrane proteins involved in vesicular traffic, such as snares, would lead to mobile and immobile domains. we hypothesize that under normal conditions, ibv m would be targeted to an immobile domain and escaped molecules would be cycled back through the er. elevated levels of ceramide could disrupt these immobile lipid domains, inducing ibv m to cycle. alternatively, perhaps ceramide binds to ibv m transmembrane domains, preventing an interaction with immobile lipid domains. metabolic effects of shortchain ceramide and glucosylceramide on sphingolipids and protein kinase c inhibition of glucosylceramide synthase by synthase inhibitors and ceramide a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding a rapid method of total lipid extraction and purification ceramide as a modulator of endocytosis heterogeneous distribution of an unusual phospholipid in the golgi complex golgi dispersal during microtubule disruption: regeneration of golgi stacks at peripheral endoplasmic reticulum exit sites diffusional mobility of golgi proteins in membranes of living cells golgi localization of glycosyltransferases: more questions than answers ceramide stimulates a cytosolic protein phosphatase determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the golgi apparatus bifunctional role of glycosphingolipids. modulators for transmembrane signaling and mediators for cellular interactions the sphingomyelin cycle and the second messenger function of ceramide thin-layer chromotography ceramide synthesis enhances transport of gpi-anchored proteins to the golgi apparatus in yeast preparation of the active isomer of -phenyl- -decanoylamino- -morpholino- -propanol, inhibitor of murine glucocerebroside synthetase phospholipid class and fatty acid composition of golgi apparatus isolated from rat liver and comparison with other cell fractions molecular cloning and expression of a -kda cis-golgi and intermediate compartment protein giantin, a novel conserved golgi membrane protein containing a cytoplasmic domain of at least kda microtubule-dependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway golgi retention signals: do membranes hold the key? a specific membrane-spanning domain of a coronavirus e glycoprotein is required for its retention in the golgi region the avian coronavirus e protein is targeted to the cis golgi retention of a cis golgi protein requires polar residues on one face of a predicted ␣-helix in the transmembrane domain characterization of a ceramide-activated protein kinase: stimulation by tumor necrosis factor ␣ inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact chinese hamster ovary cells by ␤-chloroalanine the golgi complex: in vitro veritas? biosynthesis of long-chain (sphingoid) bases from serine by lm cells. evidence for introduction of the -trans-double bond after de novo biosynthesis of n-acylsphinganines isolation, characterization and expression of cdnas encoding murine ␣-mannosidase ii, a golgi enzyme that controls conversion of high mannose to complex n-glycans a c-terminal signal prevents secretion of luminal er proteins retention and retrieval in the endoplasmic reticulum and golgi apparatus ␤-cop localizes mainly to the cis-golgi side in exocrine pancrease use of fluorescent analogs of ceramide to study the golgi apparatus of animal cells molecular trapping of a fluorescent ceramide analogue at the golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-golgi marker for both light and electron microscopy elevation of ceramide within distal neurites inhibits neurite growth in cultured rat sympathetic neurons cell cycle arrest induced by an inhibitor of glucosylceramide synthase. correlation with cyclin-independent kinases inhibition of glycoprotein traffic through the secretory pathway by ceramide effects of a sphingolipid synthesis inhibitor on membrane transport through the secretory pathway ergic- , a membrane protein of the er-golgi intermediate compartment, carries an er retention motif identification, by a monoclonal antibody, of a -kd protein associated with a tubulovesicular compartment at the cis-side of the golgi apparatus modulation of renal epithelial cell growth by glucosylceramide. association with protein kinase c, sphingosine, and diacylglycerol assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the golgi stacks a golgi retention signal in a membrane-spanning domain of coronavirus e protein effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts transport and sorting of membrane lipids cell type-dependent variations in the subcellular distribution of ␣-mannosidase i and ii analogs of ceramide that inhibit glucocerebroside synthetase in mouse brain inhibition of sphingolipid biosynthesis by fumonisins: implications for diseases associated with fusarium moniliforme oligomerization of a membrane protein correlates with its retention in the golgi complex role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction we thank drs. e. cluett, d. raben, and a. hubbard for useful discussions and the members of the machamer lab for critical reading of the manuscript. we also thank h.-p. hauri, m. farquhar, k. moremen, and j. lippincott-schwartz for antibodies. this work was supported by grant gm from the national institutes of health.received for publication august and in revised form october .the journal of cell biology, volume , key: cord- - tbgn b authors: nan title: the -residue transmembrane domain of beta-galactoside alpha , - sialyltransferase is sufficient for golgi retention date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: tbgn b beta-galactoside alpha , -sialyltransferase (st) is a type ii integral membrane protein of the golgi apparatus involved in the sialylation of n-linked glycans. a series of experiments has shown that the -residue transmembrane domain of st is sufficient to confer localization to the golgi apparatus when transferred to the corresponding region of a cell surface type ii integral membrane protein. lectin affinity chromatography of chimeric proteins bearing this -residue sequence suggests that these chimeric proteins are localized in the trans-golgi cisternae and/or trans-golgi network. further experiments suggest that this -residue sequence functions as a retention signal for the golgi apparatus. transferase i (nti), ß- , -galactosyltransferase (gt), and c , -sialyltransferase (st)have beenlocalizedto themedial-, trams-cisternae, and the trams-cistemae/tgn, respectively (dunphy et al ., ; roth and berger, ; roth et al., ; chege and pfeffer, ) . despite our current knowledge about the morphology, organization, function, and composition of the golgi apparatus, the mechanism for establishing and maintaining the highly ordered golgi structure is poorly understood, and very little is known about the signals that mediate the golgi localization of its resident proteins. previous studies have shown that a catalytically active fragment of st is secreted jamieson, , ; paulson and colley, ) . further experiments also showed that a -- kd fragment of st could be released from golgi membranes by endogenous cathepsin d-like protease (lammers and jamieson, ; weinstein et al ., ) . this - ) fragment is not anchored in the membrane but is still enzymatically active . protein sequencing analysis has established that this fragment is released by cleavage between residues and (weinstein et al ., ) . it seems that the secreted form of st is also the result of cleavage around this site . when fused to a cleavable signal sequence, an enzymatically active st fragment from residue to the cooh terminus is secreted in transfected cells . these observations suggest that the nh -terminal region of st is necessary for membrane anchorage and golgi localization paulson and colley, ) . in this report, we have fused different regions of the nh -terminal st sequence to the ectodomain of dipeptidyl peptidase iv (dppiv), a type surface membrane protein (hong and doyle, ; ogato et al ., ) to study their abilities in conferring golgi localization ofthe chimeric proteins. we found that the -residue transmembrane domain of st is sufficient for golgi localization and that this se-native and chimeric cdna dppiv. a . -kbp ecori cdna fragment encoding for dppiv in the pgem- z vector (its ' end facing the sp promoter) has been described (hong and doyle, ) . tb facilitate the construction of chimeric cdna, the recombinant plasmid was digested with the restriction enzymes xhoi and bamhi, blunt ended, and then self-ligated . this resulted in the deletion of most of the '-untranslated region of dppiv cdna . the resulting construct was referred to as pgem/d s) . this process resulted in the creation of a unique ecorl site at the ' end and unique xhol, xbai, sall, and hindiii sites at the ' end of the dppiv coding region . there is a unique nod site in the codons for residues - in the dppiv cdna . the ' unique ecorl site and this nod site were used to replace the dna sequence encoding for the n -terminal -residue sequence of dppiv with various dna sequences encoding for different n -terminal st sequences . st a cdna clone of unknown identity was obtained by screening a rat kidney kgtll cdna library with polyclonal antibodies against rat golgi wgabinding proteins . limited sequence analysis established that it encodes for st. to facilitate the analysis, oligonucleotides and were used to retrieve the st coding region by polymerase chain reaction (pcr) . the pcr product was digested with xhol and inserted into the xhol site of pgem- z(+) with its ' end facing the sp promoter. s d. oligonucleotides and were used to retrieve st cdna encoding for the n -terminal -residue sequence by pcr . the pcr product was digested with ecori and nod and used to replace the ' ecori-noti sequence of dppiv in pgem/d s. the resulting chimeric cdna thus encodes for the fusion protein s d . s d. oligonucleotides and were used to create chimeric cdna for fusion protein s d as described above for s d. the journal .of cell biology, volume , s d. chimeric cdna coding for s d was similarly constructed by using oligonucleotides and . s d. this was constructed as above using oligonucleotides and . s d. oligonucleotides and were used to construct chimeric cdna for s d the same way as described above for s d. s d. this was created as above using oligonucleotides and . s d. oligonucleotides and were used to construct the chimeric cdna for s d. s d. oligonucleotides and were used to modify dppiv cdna by pcr (dppiv cdna as template) . the pcr product was digested with xhoi and inserted into the xhol site of pgem- z(+) . this process resulted in the substitution of dppiv cytoplasmic domain by that of st. s - d. oligonucleotides and were used by pcr (st cdna as template) to create a cdna fragment that encodes a protein fragment in which the nh -terminal -residue cytoplasmic domain of dppiv was fused to an st sequence from residue to . the resulting pcr fragment was digested with ecorl and nod and used to replace the ' ecori-noti region of dppiv cdna . s - d. oligonucleotides and were used by pcr (st cdna as template) to create a cdna fragment . after digesting with ecorl and nod, this pcr fragment was used to replace the ecori-notl region of dppiv cdna . this created the chimeric cdna encoding for s - d. s - d. oligonucleotides and were used by pcr (dppiv cdna as template) to create a dna fragment . the pcr product was digested with ecori and nod and used to replace the ecori-notl region in pgem/d s . the resulting chimeric cdna encodes for s - d. chimeric cdnas (mainly the st and fusion regions) were all confirmed by dna sequencing in pgem vectors . the resulting chimeric cdnas all have a unique xhoi site in both ' and ' ends . for insertion into prsn, they were digested with xhol to retrieve the chimeric cdna and ligated into the xhol site of prsn. the right orientation was verified by restriction enzyme digestion and direct dna sequencing of the ' region ( - -bp sequence) with oligonucleotide no. , which hybridizes to the sequence before the xhol site of the prsn vector. insertion of dppiv cdna into prsn has been described previously (low et al ., a,b) . cell culture mdck (strain ) cells were a generous gift from dr. k . simons (european molecular biology laboratory) . mdck and the transfected cells were cultured as before (low et al ., b) . this was performed as described previously (low et al ., b) . cells grown on coverslips were washed twice with pbs containing mm cac and mm m c (pbscm), and then fixed with . % paraformaldehyde in pbscm at °c for min. the fixed cells were washed once with pbscm, twice with pbscm containing mm nh cl, and then three times with pbscm ( - min each) . the cells were either not permeabilized for surface staining or permeabilized with . % saponin in pbscm for min for total staining (surface plus intracellular) . mabs (ascitic fluid), diluted : , in fluorescence dilution buffer (pbscm with % normal goat serum, % fbs, and % bsa, ph . ) were added to the cells and incubated at room temperature for - min . after washing six times with pbscm ( - min each), the cells were then incubated with goat anti-mouse igg conjugated with fix ( pg/ml) for - min . after washing, the cells were mounted in % glycerol in pbs, ph . , containing mg/ml p-phenylenediamine, observed using the axiophot microscope (carl zeiss, inc ., thornwood, ny) equipped with epifluorescence optics and photographed with kodak tri-x film . diluted mabs and goat anti-mouse igg conjugated with fix were spun down at °c for min ( krpm in a microfuge) before use . for colocalization with wga, cells were first incubated with . mg/ml wga for min on ice to block surface wga binding sites . after a brief rinse with pbscm, the cells were fixed and permeabilized as above . cells were then incubated with rhodamine-wga ( . mg/ml) in pbscm for min . after washing, the cells were fixed again to immobilize the bound rhodamine--wga . after wga staining, the cells were sequentially incubated with monoclonal antibodies against dppiv and goat anti-mouse igg-fitc. the coverslips were then processed and observed as above . cells were washed twice with pbscm and then incubated for nun at °c inmethionine-freemedium supplemented with % dialyzed fbs (labeling buffer). the cells were then pulse-labeled with [ s]methionine ( mci/ml in labeling buffer), washed, and chased in medium containing excess cold methionine ( mg/liter) for various times as detailed in each figure. for assessing the surface expression, cells were grown on transwell filters so that the entire surface was maximally exposed for biotinylation. cells grown on transwell filters were similarly labeled with ( s]methionine. this was performed as described (le bivic et al., a,b; low et al., a,b) . briefly, cells grown on transwell filters were washed four times with pbscm ( - min each) on ice. the entire surface was biotinylated by adding to both the upperand lower chambers ail of pbscm containing s-nhs-biotin ( . mg/ml diluted from mg/ml stock in dmso) . the biotinylation was performed twice on ice ( - min each) and stopped by repeated washing with pbscm containing mm nh cl and medium with % fbs. these were performed exactly as described previously (low et al., a,b) . the immunocomplex on sepharose beads was resuspended in al of . m sodium citrate, ph . , . % triton x- , . % -mercaptoethanol, mg/ml aprotinin, and mm cac . mu of endo h was then added. after overnight incubation at °c, the beads were recovered and lysed in al sds sample buffer followed by sds-page. immunoprecipitated proteins were eluted from beads by boiling in al sds sample buffer (without ß-mercaptoethanol) followed by dilution in several milliliters of lectin buffer ( mm tris-hci, ph . , mm nacl, mm cac , mm mgc , and mm mnc ). an aliquot of this was incubated withthe various lectin-agaroses ( al slurry) for - min. after extensive washing, the bound materials were eluted by boiling in sds sample buffer and analyzed by sds-page. in vitro translation and sodium bicarbonate treatment these were performed as described previously (hong and doyle, ). these were carried out exactly as described before (low et al., a) . localization ofs d and s d to the golgiapparatus st is a - -kd integral membrane protein oftype ii orientation (single transmembrane domain with its nh terminus in the cytosol and the cooh terminus in the lumen of the golgi apparatus) weinstein et al., ; our unpublished observations) . the nh -terminal -residue sequence of rat st is shown in fig. . to determine the minimum sequence ofthe n -terminal region of st that is sufficient for membrane anchorage and golgi localization, we have constructed a series of chimeric cdnas which encode different fusion proteins ( fig. b) , with decreasing lengths of the n -terminal st sequence fused to the ectodomain of dppiv . the nh -terminal residue sequence of dppiv is also shown in fig . a (d ) . previous studies have shown that the n -terminal residue sequence of dppiv is the signal/anchor sequence (hong and doyle, ) . the first set of experiments resulted in the construction of chimeric cdnas encoding for fusion proteins s d, s d, s d, s d, s d, and s d in which the same ectodomain of dppiv (from residue to the cooh-terminus) was fused to the n -terminal -, -, -, -, -, and -residue sequence of st ( fig. b) . in vitro translation studies demonstrated that all ofthem, except for s d, were efficiently anchored in the membrane. the results for s d and s d are shown in fig. . results similar to s d were obtained for s d, s d, s d, and s d (data not shown) . these results demonstrated that the nh -terminal -but not -residue sequence of st is sufficient for membrane anchorage. since s d was not anchored in the membrane, it was not pursued further. to examine the role in golgi localization of the st sequence in the remaining five fusion proteins, the respective chimeric cdnas were inserted into the eukaryotic expression vector prsn (low et al., a,b) and transfected into mdck cells . stably transfected cells were found to express the desired fusion proteins. since similar results were obtained for all the five fusion proteins, only the results obtained for fusion proteins s d and s d are shown. pulse-chase experiments were performed to follow the intracellular transport of s d and s d (fig. a) . as shown, a -kd protein was specifically detected in the pulse-labeled cells expressing s d. as chase proceeded, this d form was converted into a -kd form. this conversion was very efficient, as all newly made -kd molecules were converted into the -kd form within min of chase . in cells expressing s d, a -kd form was initially detected in pulse-labeled cells and this form was converted into a -kd form during the chase period. a majority of the -kd form was converted into the -kd form after min of chase, although a small amount of the -kd form was still observed . endo h treatment ofimmunoprecipitated proteins (fig. b) showed that the -kd form ofs d and the -kd formofs dwere mainly converted into -and -kd polypeptides, respectively, which were of the same size as the respective polypeptides without any glycosylation (produced by in vitro translation without microsomes) . this result suggests that all the n-linked glycans (about - n-linked glycans, but no detectable -linked glycans, were present in the ectodomain of dppiv [hong and doyle, ; hong et al., ] ) attached to the -kd form of s d and the -kd form of s d were essentially sensitive to endo h and that they represent the newly made proteins in the er bearing n-linked glycans of the highmannose type. the size of the -kd form of s d was reduced by - -kd after endo h treatment, suggesting that about two of the n-linked glycans in the polypeptide were of the high-mannose type while the remaining n-linked glycans (n - ) were resistant to endo h. for the mad form residue numbers from the nh terminus are indicated above the sequence. (b) summary of fusion proteins used in this study and their properties in membrane anchorage and cellular localization . residue numbers corresponding to st are indicated above while those to d are indicated below each fusion protein. for fusion proteins, they were named sxd (in which the nh -terminal x-residue sequence of st was fused to the ectodomain of dppiv) or sx-yd (in which the st sequence from residue x to y was used to replace the corresponding region of dppiv) . for membrane anchorage, + and -indicates that the majority of the protein is membrane anchored or not, respectively, as assessed by in vitro translation experiments. for cellular localization, sand grepresent surface or golgi localization, respectively, as examined by indirect immunofluorescence microscopy, while nd indicates "not determined" because of theirnonanchorage in the membrane. figure . membrane anchorage of selected fusion proteins . the mrna for the fusion protein was translated in rabbit reticulocyte lysate supplemented with microsomes as described previously doyle, , ) . pl of total translation reaction was diluted with ,u of sodium bicarbonate, ph . , and the membrane-associated proteins (p) were separated from those extractable by this treatment (s) . samples equivalent to gl of original translation were analyzed forboth p and s fractions. the -kd polypeptide is the untranslocated form, while the -kd polypeptide is the translocated n-glycosylated form for the respective fusion proteins . of s d, a - kd size decrease was observed after endo h treatment, suggesting that one of the n-linked glycans was a high-mannose type while the rest (- - ) of the n-linked glycans were endo h resistant . these results demonstrate that the majority of the n-linked glycans in the Ád form of s d and the mad form of s d had been modified by enzymes in the medial-golgi cisternae . these results therefore show that both fusion proteins were transported efficiently from the er to the golgi apparatus and that they were localized in the medial-and/or post-medial-golgi compartment. indirect immunofluorescence microscopy was used to determine the cellular localization of these fusion proteins cell lysates were immunoprecipitated with monoclonal antibodies to dppn and the immunocomplexes were analyzed by sds-page and fluorography. the smaller-and the larger-sized bands represent the er and golgi forms, respectively. (b) endo h treatment of fusion proteins s d and s d. cells expressing the respective fusion proteins were labeled with pis]methionine for min followed by min of chase . after being immunoprecipitated, half of the sample was treated with endo h (+ ) while the remaining half was processed in an identical way except that no endo h was included (-) . ( fig . a) . an intense staining signal was detected on the surface of mdck cells expressing normal dppn (a) . a similar staining pattern was observed for normal dppiv in permeabilized cells (b) except that some perinuclear signal was also detected . the perinuclear staining most likely represents molecules in transit through the golgi apparatus . in contrast, only background staining was observed on the surface of cells expressing s d (c) and s d (e) . when the cells were permeabilized, intense perinuclear vesiculartubular staining was detected in the cells expressing s d (d) and s d (f) . this pattern of intracellular staining is typical for the golgi apparatus (lipsky and pagano, ; louvard et al., ; saraste et al., ) , suggesting that s d and s d are predominantly localized to the golgi apparatus . to further confirm the golgi localization of s d and s d, colocalization of s d or s d with intracellular wga binding sites was carried out . intracellular wga binding sites are present predominantly in the golgi apparatus (lipsky and pagano, ; tartakoff and vassalli, ) . as shown in fig . b, the staining for s d (a) and s d (c) colocalized well with that for wga binding sites (b and d). these results demonstrated that s d and s d were indeed localized to the golgi apparatus . localization ofs d and s d to the trans-golgi cisternae and/or tgn the results obtained from pulse-chase and endo h treatment experiments suggest that s d and s d were present in the medial-and/or post-medial-golgi cisternae . the indirect fluorescence microscopy demonstrated that they were localized in the golgi apparatus . to further determine the compartment of their localization, their n-linked carbohydrate structures were examined by lectin affinity chromatography (haselbeck et al ., ; lis and sharon, ) (fig. ) . cells expressing s d were pulse labeled with [ s]methionine followed by min of chase such that both the -kd er form and the -kd golgi form of s d were present in the cells . similar amounts of labeled s d were incubated with various lectin-agarose beads . the material bound to the beads was analyzed . as shown in fig. a, both the -and ) forms could be quantitatively recovered with con a-agarose (mannose-binding lectin) (lane ), consistent with the presence of n-linked glycans of high-mannose type in both -and -kd forms of s d as concluded from the endo h experiment . the -but not the -kd form was also recovered from wga-, erythrina cristagalli, (eca)-, sambucus nigra (sna)-, and maackia amurensis (maa)-agarose beads. since eca binds specifically to the gal- , -g cnac structure in n-linked glycans, this result demonstrates that the -kd form has been modified by trans-golgi gt. sna and maa bind a- , -and a- , linked sialic acids, respectively. thus the -kd form of s d has been modified byboth a- , -and a- , -sialyltransferases, both of which are localized to the trans-golgi cisternae and/or tgn . these results demonstrate that s d had been modified by glycosyltransferases of the trans-golgi cisternae and the tgn and suggest that it is localized to the trans-golgi cisternae and/or tgn . wga binds both sialic acid and n-acetylglucosamine while succinylated wga (swga) binds only n-acetylglucosamine (monsigny et al., (monsigny et al., , . the binding of -kd s d to wga but not swga not only confirmed the presence of sialic acid on the n-linked glycans but also demonstrated that no n-acetylglucosamine residues were exposed on the glycans . both the -and the -kd forms of s d failed to bind lotusagarose, a fucose-specific lectin . this result suggests that no significant fucose residues were added to the n-linked glycans of s d. similar experiments performed with cells expressing s d (fig . b) revealed that the -kd golgi form of s d binds con a-, wga-, eca-, sna , and maa-, but not swga-or lotus-agarose (identical pattern as the -kd form of s d). the -kd form of s d has thus been modified by both gt and sialyltransferases . the -kd form of s d was quantitatively recovered from con ( )) and golgi ( )) forms were present . similar amounts of the immunocomplex were analyzed directly (control) or incubated with various lectin-agaroses and the bound fraction was then analyzed . (b) lectin-binding properties of s d. cells expressing s d were analyzed as described for s ). con a, concanavalin a; swga, succinylated wheat germ agglutinin; wga, wheat germ agglutinin; eca, erythrina cristagalli; lotus, lotus tetragonolobus ; sna, sambucus nigra; maa, maackia amurensis. a-agarose beads . interestingly, a small but detectable portion of the -kd form of s d was also capable of binding wga, eca, and sna (but not swga, lotus, or maa) . this observation suggests that a small fraction of the -kd form might have undergone modifications by -gt and st, although the possibility that the observed binding of the -kd form of s d to wga, eca, and sna was due to nonspecific background binding could not be excluded yet . the small amount of -kd form present in pulse-labeled cells after min of chase (fig . a, lane ) may therefore have been transported to the golgi apparatus . these results, taken together, suggest strongly that both s d and s d are localized in the trans-golgi cisternae and/or tgn . we have thus demonstrated that both s d and s d are predominantly present in the golgi apparatus . similar results were obtained for s d, s d, and s d (data not shown) . these results demonstrate that the nhz-terminal -residue sequence of st is sufficient for golgi localization and that the stem region of st (sequence from residue to ) is apparently not essential for golgi localization . the sequence in this stem region is in fact the most divergent be- tween rat and human st. the nh -terminal -residue sequence ofhuman st (grundmann et al ., ) is also shown in fig . a (m) . incontrast to the stem region, the sequence of the cytoplasmic domain is totally conserved between the rat and human st. the transmembrane domain is also highly conserved . the sequence conservation of the cytoplasmic and transmembrane domains but not the stem region is consistent with our finding that the nh -terminal -residue sequence of st is sufficient for golgi localization . this residue sequence can be further divided into three regions : the cytoplasmic domain, the transmembrane domain, and the -residue sequence flanking the cooh-terminal side of the transmembrane domain. to further narrow down the sequence required for golgi localization, an additional five chimeric cdnas were constructed which encode fusion proteins s d, s d, s - d, s - d, and s - d (fig. b) . in vitro translation studies demonstrated that s d, like s d, was not anchored in the microsomal membrane (fig. ) . interestingly, s - d, which is identical to s d except that the st cytoplasmic domain was replaced by that of dppiv, was mainly (n %) anchored in the membrane (fig. ) . for s d, s d, and s - d, the translocated form in the supernatant was ofa smaller size than the translocated form in the membrane, implying that the nonanchorage in the membrane of s d, s d, and, to a much lesser extent, sio- d might be due to a cleavage of (or part of) the signal/anchor sequence ofst by the signal peptidase, although this explanation will have to be verified by future experiments . replacing the cytoplasmic domain of st by that of dppiv in s - d significantly enhanced the membrane anchorage . in connection with the results obtained with s d and s d (fig. ) , it can be concluded that the signal/anchor sequence of st is within its nh -terminal -residue sequence, comprising the -residue hydrophobic core and flanking hydrophilic sequences on both sides. the nature of the hydrophilic sequences on either side of the hydrophobic core significantly affect its efficiency in acting as a signal/anchor sequence. results similar to s d (fig. ) were obtained for s d, s - d, and s - d (data not shown) . based on these in vitro translation results, chimeric cdnas encoding s d, s - d, s - d, and s - d (s d was not pursued further) were inserted into the prsn expression vector and transfected into mdck cells. the cellular localization ofthe fusion proteins was examined by indirect fluorescence microscopy. as shown in fig . , intense surface staining was detected for s d (a) and s - d (c), but not for s - d (e) or sio- d (g) . when the cells were permeabilized, intense intracellular staining ofperinuclear vesicular-tubular network was observed for sio- d (f) and s - d (h), typical of the golgi apparatus . this was further confirmed by their colocalization with intracellular wga binding sites (data not shown) . s - d and s - d have lectin-binding properties similar to those of s d and s d, demonstrating the modification of their n-linked glycans by gt and sialyltransferases (data not shown) . s - d and s - d thus reside in the trans-golgi cisternae and/or tgn . to obtain quantitative data on the intracellular golgi localization of the various fusion proteins, we used a biochemical approach to determine the levels of surface expression of respective proteins (fig. ) (bretscher and lutter, ; le bivic et al ., a,b ; low et al., la,b) . for convenience, we arbitrarily defined the surface expression of dppiv as % . the extent of surface expression for other proteins was normalized with that ofdppiv and defined as a percentage of surface expression . as shown, the surface expression of s d, s d, s - d, and s - d was between and % as compared with dppiv. the loss of surface expression demonstrates that the golgi localization signal of st is highly effective in targeting the fusion proteins to the golgi apparatus. in contrast, high levels of surface expression, comparable to dppiv, were observed for s d and s - d. these biochemical results are consistent with and further extend the indirect immunofluorescence data. newly made molecules of s d, s d, s - d, and s - d were hardly detectable on the cell surface after h of chase (fig. ) . in connection with the predominant golgi staining of these proteins, these results suggest that they are specifically and efficiently retained in the golgi apparatus . however, the possibility that they may be initially transported to the cell surface and then rapidly and effectively endocytosed and sorted to the trans-golgi cisternae and/or tgn could not be ruled out because only a single chase point was examined. if endocytosis followed by sorting is the mechanism for their golgi localization, a peak in surface expression should be expected during a series of chase intervals after a single pulse-labeling . cells expressing s d, s - d, and dppiv were selected for further examination . cells were pulse-labeled with ['ss]methionine for min. after chasing for min, min, min, h, h, and h, cells were surface biotinylated with nhsbiotin and cell lysates were immunoprecipitated . one tenth of the immunoprecipitate served as a measure of the total amount of labeled fusion protein ( / total), while nine tenths was absorbed with streptavidin-agarose to recover the biotinylated surface proteins (surface) (fig. ) . all were initially made as the -kd form followed by rapid conversion into the -kd form, demonstrating that they were efficiently transported from the er to the golgi apparatus . strong surface expression was detected for dppiv, and an almost maximal level of surface expression was detected after min of chase. since s d and s - d were transported to the golgi apparatus as efficiently as dppiv, a peak expression on the surface at - min of chase should be ex- ]methionine for min followed by h of chase. proteins exposed on the cell surface were biotinylated with nhs-biotin . cell lysates were immunoprecipitated with monoclonal antibodies against dppiv. one tenth of the immunocomplex was analyzed directly and served as a measure ofthetotal amount of the respective protein ( / ). the remainingnine tenths was absorbed to streptavidin-agarose to recover the biotinylated proteins and analyzed (surf) . this served as a measure of the amount that was expressed on the cell surface. (b) quantitation of the results in a. the cell surface expression of each fusion protein was normalized to that of dppiv (arbitrarily defined as %). transfected cells a, b, c, d, e, f, and g represent cells expressing dppiv, s ), s - , s d, s d, s - ), and s - ), respectively. pected if their golgi localization was mediated by initial surface expression followed by specific retrieval and endocytosis. this was not observed for either s d or s - . the surface expression for s d and s - was undetectable after min of chase and only nl- % (normalized to dppiv surface expression) was visible after h of chase. longer chase up to h revealed a slight increase (up to - %) in surface expression for s d and s - this was most likely due to the slight leakiness in their golgi retention. interestingly, surface expression was undetectable after h of chase, which couldbe explained by surface deg-the journal of cell biology, volume , radation and/or slow internalization with the bulk membrane endocytosis. these results suggest that both s d and s - d are specifically retained intracellularly in the golgi apparatus. the i -residue transmembrane domain of st is sufcientfor golgi localization previous observations have shown that the n -terminal re-gion of st is required for membrane anchorage and golgi localization jamieson, , ; paulson and colley, ; colley et al ., ; weinstein et al ., ) . we thus constructed a series of chimeric cdnas encoding fusion proteins in which the n -terminal st sequence of different sizes were fused to the ectodomain of a cell surface protein dppiv (hong and doyle, ; ogato et al ., ; low et al., a,ó) . when expressed in transfected mdck cells, fusion proteins bearing nhz-terminal -, -, -, -, and -residue st sequences were all localized to the golgi apparatus as assessed by indirect fluorescence microscopy and other biochemical methods. the results with two fusion proteins, s d and s d, are presented in this report . pulse-chase experiments demonstrated that both s d and s d wereefficiently transported from the er to the golgi apparatus and have passed beyond the medial-golgi cisternae . affinity chromatography with several lectin-agaroses demonstrated that both s d and s d were extensively modified by gt and st. since gt has been previously localized to the trans-golgi cisternae (roth and berger, ) and st has been localized to the trans-golgi cisternae and tgn (both et al ., ) , these results suggest that s d and s d are localized in the trans-golgi cisternae and/or the tgn . because the actual localization of st in mdck cells has not been established, our conclusion for the trans-golgi cisternae and/or tgn localization of s d and d d is based on the assumption that st is localized in the same compartments in mdck cells . our results, nevertheless, demonstrated that s d and s d are localized in the golgi subcompartments in which they have extensive interaction with endogenous gt and st. since s d and s d (as well as s d, s d, and s d) were localized to the golgi apparatus with comparable efficiencies, it was concluded that the n -terminal -wong et al . signal for golgi retention figure . s d and s - d are predominantly retained intracellularly. cells expressing dppiv, s d, or s - d were pulse-labeled with [ s]methionine for min followed by a series of chase intervals as indicated . the cell surface proteins were biotinylated at each chase point . cell lysates prepared from each chase interval were immunoprecipitated . one tenth of the immunocomplex was analyzed as a measure of total newly made protein ( / total) . the remaining nine tenths was absorbed to streptavidinagarose to recover the surface biotinylated proteins as a measure of surface expression (surface) . intense surface expression was detected for dppiv while the surface expression ofthe two fusion proteins was hardly detectable . residue st sequence is sufficient for golgi localization and that the stem region from residues to is not required for golgi localization . the n -terminal -residue sequence was further divided into three regions : the -residue cytoplasmic domain, the -residue transmembrane domain, and the -residue sequence flanking the cooh-terminal side of the transmembrane domain . more chimeric cdnas were thus constructed encoding additional fusion proteins. the transmembrane domain alone (in s - d) or in combination with its hterminal flanking sequence (in s - d) could confer golgi localization, while the cytoplasmic domain alone (in s d) or the cooh-terminal flanking sequence alone (in s - d) was not capable of conferring golgi localization . these results thus demonstrate that the -residue transmembrane domain of st is sufficient for golgi localization . further characterization of the fusion protein s - d by lectin affinity chromatography demonstrated thatthe -residue sequence can confer the same golgi subcompartment localization as the nhz-terminal -and -residue sequence of st. recently, a common peptide stretch was described for golgi-localized glycosyltransferases (bendiak, ) . this stretch (ser-gln-glu-lys) is located between residues and (residue - ) in st. our results clearly demonstrate that this stretch is not critical for golgi localization. furthermore, the sequence of this stretch is not conserved in the human st (ser-leu-gly-lys) (grundmann et al ., ) . since glycosyltransferases are localized to distinct subcompartments of the golgi apparatus, it is hard to envision a common sequence for distinct subcompartment localization . further studies with other glycosyltransferases will reveal the structural features required for localization to other subcompartments of the golgi apparatus . since our study involved extensive "cut-and-paste ex-periments for the fusion of domains derived from two different proteins, one may argue that conformational changes might have contributed to the results observed in our system . this seems highly unlikely because the chimeric proteins were transported from the er to the golgi apparatus as efficiently as normal dppiv and were modified by enzymes in all subcompartments of the golgi apparatus. if there was any conformational abnormality, the fusion proteins would have been retained in the er and/or pre-golgi compartments. many misfolded chimeric proteins have been described and all failed to be transported beyond the cis-face of the golgi apparatus (rose and doms, ) . furthermore, preliminary experiments have shown that all the fusion proteins possessed similar enzymatic activity as the normal dppiv, suggesting no global conformational change in the ectodomain of dppiv in the chimeric proteins . additional experiments demonstrated that the golgi localization of the chimeric proteins by the st transmembrane domain is a specific event, because the replacementof the transmembrane domain of dppiv by that of another type ii surface protein resulted in a chimeric protein that was transported to the cell surface (data not shown) . the golgi localization of the fusion proteins could be mediated by their specific retention in the golgi subcompartment (in this case, the golgi localization signal functions as a retention signal). alternatively, they could be initially transported to the cell surface followed by their efficient retrieval and transcytosis back to the final golgi localization . to distinguish between these two possibilities, we used the membrane-impermeable reagent nhs-biotin to selectively tag the proteins present on the cell surface after a single pulse and several chase intervals. the biotinylated proteins were specifically recovered by absorption to streptavidin-agarose and served as a measure of surface expression . efficient surface expression of dppiv was detected . studies with s d and s - d demonstrated that they were hardly detectable on the cell surface. these results established that the majority of s d and s - d were specifically retained in the golgi apparatus and that the -residue transmembrane domain of st functions as a retention signal for the golgi subcompartment. our studies demonstrated that the membrane-spanning region of st, in addition to its passive role in membrane anchorage, plays a dominant role in golgi retention. our observations raised the question of whether the transmembrane domains of other glycosyltransferases play similar roles in specifying their subcompartment golgi localization . comparison of the primary sequences of gt revealed that the entire transmembrane domain is identical in human and bovine and that of the residues in the transmembrane domain are also conserved in mouse (dagostaro et al ., ; masri et al ., ; russo et al ., ; shaper et al., ) . furthermore, the sequence of the transmembrane domain of nti is totally conserved between human and rabbit (kumar et al ., ; sarkar et al ., ) . the high degree of conser- the journal of cell biology, volume , vation in the transmembrane domain sequence of gt and nti suggests that they might also be involved in other functions in addition to their role in membrane anchorage. whether the transmembrane domain of gt and/or nti is similarly involved in golgi localization needs further investigation . our recent studies with nti suggest that its transmembrane domain is indeed involved in golgi localization (tang et al ., ) . the sequence of the trans-membrane domain varies greatly between st, gt, and nti, and this sequence divergence of the transmembrane domain among different glycosyltransferases might be related to their distinct subcompartment localization in the golgi apparatus. further studies will shed more light on this issue. the golgi localization specified by the transmembrane domain of st could potentially be mediated by two different but related mechanisms . the membrane spanning domain may interact with specific lipid components (or specific lipid microenvironment) in the golgi membrane . this interaction may immobilize the proteins in a particular microdomain in the membrane. proteins in this microdomain may be specifically prevented from entry into the budding vesicles for subsequent transport. alternatively, the transmembrane domain may interact with a specific membrane-spanning region of other proteins that are already immobilized in the golgi membrane through unknown mechanisms. furthermore, interaction of the transmembrane domains among different molecules of st (self interaction) induced by the golgi environment may also be involved . at present, we are unable to propose a detailed molecular mechanism to explain the golgi localization mediated by the st transmembrane domain . future studies with point mutations in the st transmembrane domain and other glycosyltransferases will enable us to gain more understanding about the cellular targeting mediated by the transmembrane domain . cellular localization determined by a membrane anchoring region has been described for other proteins. recent studies have established that the gpi lipid anchor functions as a targeting signal for the apical plasma membrane domain in epithelial cells lisanti et al., ) . furthermore, the transmembrane domain of the t cell antigen receptor a-subunit has been sown to be sufficient for er degradation and subunit assembly of the t cell antigen receptor (bonifacino et al ., ) . studies with the el protein of avian coronavirus infectious bronchitis virus, a cis-golgi protein (machamer et al ., ) , demonstrated that the first of its three transmembrane domains is necessary and sufficient for golgi localization (machamer and rose, ; swift and machamer, ) , although different results were obtained with the el protein of another coronavirus (mouse hepatitis virus a ) (armstrong et al ., ; armstrong and patel, ) . these observations suggest that the subcellular destination of some integral membrane proteins is signaled by their membrane-spanning regions. during the review of this manuscript, two papers were published which demonstrated that the membrane-spanning domain of gt (nilsson et al ., ) and the transmembrane domain and its flanking sequences of st (munro, ) could specify golgi localization . although similar conclusions were reached by munro in his study, there were a few significant differences in the approach and results presented from our study. (a) munro used a transient expression system in cos cells while our study was performed in stably transfected mdck cells. (b) our results clearly demonstrate that the -residue transmembrane domain itself was sufficient for golgi localization of the chimeric protein and its flanking sequences did not contribute much to the efficiency of golgi localization . this difference could be due to the different expression systems used and the fact that our chimeric proteins were derived from two well-defined cellular proteins of the same membrane topology while those in munro's study involved a tripartite fusion among three proteins. (c) in addition to confirming biochemically the golgi localization of the chimeric proteins, we also used surface biotinylation to demonstrate that the golgi-localizedchimeric proteins were not initially transported to the cell surface followed by recycling back to the golgi, providing the first evidence that they were selectively retained in the golgi complex. the golgi sorting domain of coronavirus el protein lysosomal sorting mutants of coronavirus el protein, a golgi membrane protein a common peptide stretch among enzymes localized to the golgi apparatus : structural similarity of golgi-associated glycosyltransferase colocalized transmembrane determinants for er degradation and subunit assembly explain the intracellular fate of tcr chains a new method for detecting endocytosed proteins mechanism of membrane anchoring affects polarized expression of two proteins in mdck cells compartmentation of the golgi complex : brefeldin-a distinguishes trans-golgi cistemae from the trans-golgi network conversion of a golgi apparatus sialyltransferase to a secretory protein by replacement of the nh -terminal signal anchor with a signal peptide cloning of cdna encoding the membrane-bound form of bovine , -galactosyltransferase mannose- -phosphate receptors and lysosomal enzyme targeting involvement of -cop in membrane traffic through the golgi complex attachment of terminal n-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cistemae of the golgi stack the golgi apparatus (complex)-( - ) -from artifact to center stage endocytotic membrane traffic to the golgi apparatus in a regulated secretory cell line the trams-golgi network : sorting at the exit site of the golgi complex complete cdna sequence encoding human -galactoside a- , -sialyltransferase structural characterization of glycoprotein carbohydrate chains by using digoxigenin-labeled lectins on blots membrane orientation of rat gp as studied by in vitro translation molecular dissection of the nh -terminal signal/anchor sequence of rat dipeptidyl peptidase iv expression of enzymatically active rat dipeptidyl peptidase iv in chinese hamster ovary cells after transfection a recycling pathway between the endoplasmic reticulum and the golgi apparatus for retention of unassembled mhc class i molecules sorting and traffic in the central vacuolar system assembly of asparagine-linked oligosaccharides the biogenesis of lysosomes cloning and expression of n-acetylglucosaminyltransferase i, the medial golgi transferase that initiates complex n-linked carbohydrate formation the role of a cathepsin d-like activity in the release of gal - glcnac a - sialyltransferase from rat liver golgi membranes during the acute-phase response studies on the effect of lysosomotropic agents on the release of gal - g cnac a - sialyltransferase from rat liver slices during the acute-phase response biogenetic pathways ofplasma membrane proteins in caco- , a human intestinal epithelial cell line vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in mdck cells a human homologue of the yeast hdel receptor a vital stain for the golgi apparatus lectins as molecules and as tools a glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells antibodies to the golgi complex and the rough endoplasmic reticulum involvement of both vectorial and transcytotic pathways in the preferential apical cell surface localization of rat dipeptidyl peptidase iv apical cell surface expression of rat dipeptidyl peptidase iv in transfected madin-darby canine kidney cells a specific transmembrane domain of a coronavirus el glycoprotein is required for its retention in the golgi region the el glycoprotein of an avian coronavirus is targeted to the cis golgi complex identification of the fulllength coding sequence for human galactosyltransferase ( -n-acetylglucosamine : , -galactosyltransferase) properties of succinylated wheat germ agglutinin sugar-lectin interactions : how does wheat germ agglutinin bind sialoglycoconjugates sequences within and adjacent to the transmembrane segment of a- , -sialyltransferase specify golgi retention the membrane spanning domain of fl- , -galactosyltransferase specifies trans golgi localization primary structure of rat liver dipeptidyl peptidase iv deduced from its cdna and identification of the nh -terminal signal sequence as the membrane-anchoring domain the trans-most cistemae of the golgi complex: a compartment for sorting of secretory and plasma membrane proteins glycosyltransferase : structure, localization, and control of cell type-specific glycosylation evidence that luminal er proteins are sorted from secreted proteins in a post-er compartment recycling of proteins between the endoplasmic reticulum and golgi complex biosynthetic protein transport and sorting by the endoplasmic reticulum and golgi morphogenesis of the polarized epithelial cell phenotype regulation of protein export from the endoplasmic reticulum immunocytochemical localization of galactosyltransferase in hela cells : codistribution with thiamine pyrophosphatase in trans-golgi cisternae demonstration of an extensive trans-tubular network continuous with the golgi apparatus that may function in glycosylation bovine ßl- -galactosyltransferase : two sets of mrna transcripts encode two forms of the protein with different amino-terminal domains : in vitro translation experiments demonstrate that both the short and the long forms of the enzyme are type ii membrane-bound glycoproteins antibodies to rat pancreas golgi subfractions : identification of a -kd cis-golgi protein molecular cloning and expression of cdna encoding the enzyme that controls conversion of high-mannose to hybrid and complex n-glycans : udp-n-acetylglucosamine : a- -d-mannoside ß- , -nacetylglucosaminyltransferase i of -kd protein associated with a tubulovesicular compartment at the cis-side of the golgi apparatus characterization of the full length cdna for murine ß- , -galactosyltransferase : novel features at the y-end predict two translational start sites at two in-frame augs polarized sorting in epithelia intracellular movement of cell surface receptors after endocytosis : resialylation of asialo-transferrin receptor in human erythroleukemia cells a golgi retention signal in a membrane-spamting domain of coronavirus el protein the transmembrane domain of n-acetylglucosaminyl-transferase i contains a golgi retention signal lectin-binding sites as markers of golgi subcompartments : proximal-to-distal maturation of oligosaccharides sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal primar y structure of ß-galactoside a , -sialyltransferase : conversion of membrane-bound enzyme to soluble forms by cleavage of the nhrterminal signal anchor transferrin receptors recycle to cis-and middle as well as trans-golgi cisternae in ig-secreting myeloma cells this work was supported by a research fund from the institute of molecular and cell biology, national university of singapore, to w . hong .received for publication november and in revised form january . key: cord- - pv zu g authors: nan title: o-glycosylation of intact and truncated ribophorins in brefeldin a- treated cells: newly synthesized intact ribophorins are only transiently accessible to the relocated glycosyltransferases date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: pv zu g ribophorins i and ii are type i transmembrane glycoproteins of the er that are segregated to the rough domains of this organelle. both ribophorins appear to be part of the translocation apparatus for nascent polypeptides that is associated with membrane-bound ribosomes and participate in the formation of a proteinaceous network within the er membrane that also includes other components of the translocation apparatus. the ribophorins are both highly stable proteins that lack o- linked sugars but each contains one high mannose n-linked oligosaccharide that remains endo h sensitive throughout their lifetimes. we have previously shown (tsao, y. s., n. e. ivessa, m. adesnik, d. d. sabatini, and g. kreibich. . j. cell biol. : - ) that a cooh-terminally truncated variant of ribophorin i that contains only the first amino acids of the luminal domain (ri ), when synthesized in permanent transformants of hela cells, undergoes a rapid degradation with biphasic kinetics in the er itself and in a second, as yet unidentified nonlysosomal pre-golgi compartment. we now show that in cells treated with brefeldin a (bfa) ri molecules undergo rapid o-glycosylation in a multistep process that involves the sequential addition of n-acetylgalactosamine, galactose, and terminal sialic acid residues. addition of o-linked sugars affected all newly synthesized ri molecules and was completed soon after synthesis with a half time of about min. in the same cells, intact ribophorins i and ii also underwent o-linked glycosylation in the presence of bfa, but these molecules were modified only during a short time period immediately after their synthesis was completed, and the modification affected only a fraction of the newly synthesized polypeptides. more important, these molecules synthesized before the addition of bfa were not modified by o-glycosylation. the same is true for ribophorin i when overexpressed in hela cells although it is significantly less stable than the native polypeptide in control cells. we, therefore, conclude that soon after their synthesis, ribophorins lose their susceptibility to the relocated golgi enzymes that effect the o-glycosylation, most likely as a consequence of a conformational change in the ribophorins that occurs during their maturation, although it cannot be excluded that rapid integration of these molecules into a supramolecular complex in the er membrane leads to their inaccessibility to these enzymes. sequential addition of n-acetylgalactosamine, galactose, and terminal sialic acid residues. addition of o-linked sugars affected all newly synthesized ri molecules and was completed soon after synthesis with a half time of about rain. in the same cells, intact ribophorins i and ii also underwent o-linked glycosylation in the presence of bfa, but these molecules were modified only during a short time period immediately after their synthesis was completed, and the modification affected only a fraction of the newly synthesized polypeptides. more important, these molecules synthesized before the addition of bfa were not modified by o-glycosylation. the same is true for ribophorin i when overexpressed in hela cells although it is significantly less stable than the native polypeptide in control cells. we, therefore, conclude that soon after their synthesis, ribophorins lose their susceptibility to the relocated golgi enzymes that effect the o-glycosylation, most likely as a consequence of a conformational change in the ribophorins that occurs during their maturation, although it cannot be excluded that rapid integration of these molecules into a supramolecular complex in the er membrane leads to their inaccessibility to these enzymes. ibophorins i and ii are two well characterized, highly stable er resident glycoproteins that have a type i (n, luminal; c, cytoplasmic) transmembrane disposition and bear high mannose oligosaccharides in their luminal segments (rosenfeld et al., ; harnik-ort et al., ; crimaudo et al., ) . these proteins are segregated to the rough domains of the er (kreibich et al., a, b; macantonio et al., ; amar-costesec et al., ) and yung shyeng tsao's present address is schering-plough research, union, nj. akira takatsuki's present address is riken, the institute of physical and chemical research, wako, saitama, japan. appear to be part of the apparatus that effects the transit)cation of polypeptides synthesized on membrane-bound ribosomes across the er membrane (yu et al., (yu et al., , . ribophorins and other components of the translocation apparatus, such as the signal peptidase and the receptors for the signal recognition particle and for the signal peptide itself, form a suprarnolecular complex, or proteinaceous network, within the er membrane that, after treatment of rough microsomes with neutral detergents, can be recovered together with associated ribosomes in a rapidly sedimenting fraction (kreibich et al., ; marcantonio et al., ; amar-costesec et al., ; wiedmann et al., ) . the incorpo-ration of ribophorins and other components of the translocation apparatus into this network may be responsible for the characteristic cisternal morphology of the rough portions of the er, that is quite distinct from the tubular arrangement of membranes in the smooth portions of the organelle (kreibich et al., a,b) . moreover, retention of the components of the translocation apparatus in the er may simply result from the fact that, when assembled into the intramembranous network, they cannot gain access to the transport vesicles that normally flow from the er to the golgi apparatus. we have previously shown (tsao et al., ) that a truncated variant of ribophorin i that contains only the first amino acids of the luminal domain (ri ), when synthesized in permanent transformants of hela cells, is rapidly degraded by a nonlysosomal pathway with biphasic kinetics. the first phase of degradation of ri begins immediately after synthesis is completed and takes place in the er itself. the second phase, however, appears to require vesicular transport of the remaining molecules from the er to a second compartment, where degradation takes place at an accelerated rate. the two degradative compartments appear to fuse in cells treated with brefeldin a (bfa) ~, in which the truncated ribophorin is degraded with monophasic kinetics at a rate intermediate between those of the two normal degradative phases (tsao et al., ) . bfa is an antibiotic (h/irri et al., ) that profoundly affects the structure of the golgi apparatus and causes the backflow of golgi enzymes to the er (fujiwara et al., ; lippincott-schwartz et al., , doms et al., ; ulmer and palade, ) . in this report we show that in the presence of bfa both the newly synthesized normal ribophorins and the truncated ribophorin i variant undergo posttranslational modifications which do not take place in untreated cells. both ribophorins undergo o-glycosylation and the n-linked oligosaccharide chain in ribophorin ii, but not that in ribophorin i, is converted into an endoglycosidase h (endo h) resistant form. however, whereas the truncated ribophorin i molecules remain accessible to the relocated golgi enzymes throughout their lifetime, the intact ribophorins i and ii are susceptible to the modifying enzymes only during or immediately after their synthesis. it would appear that only during this brief period the intact newly synthesized molecules are in a conformational state or location, which, after bfa treatment, permits access to the relocated golgi enzymes. only during a subsequent maturation process do the intact ribophorins become assembled and sequestered in a supramolecular complex from which the truncated variant is excluded. bfa was isolated in crystalline form from fl- fungi (tamura et al., the permanent hela cell transformant (hela-ri ) that expresses a truncated version of ribophorin i, consisting of the nh -terminal amino acids of the luminal domain of this type i transmembrane glycoprotein, has been described (tsao et al., ) . procedures for cell culture, radioactive labeling, immunopreeipitation, endo h treatment, and page, in either or - % gradient gels, were also described previously (tsao et al., ) . in one experiment (see legend to fig. ) in which cells were labeled with tritium-labeled sugars or [~ s]methionine, methionine-and glucose-free mem was used. before use, the tritium-labeled sugars were dried in a svci h savant speed vac concentrator (savant instruments, inc., farmingdale, ny) and dissolved in medium to a concentration of ~cilml. unless otherwise indicated, cultures treated with bfa were preincubated in methionine-free medium containing the drug for rain before pulse labeling, and the drug was present in the culture medium throughout the labeling and chase periods at a concentration of ~g/ml. to overexpress ribophorin i the rat ribophorin i edna was excised from the pgemi vector (harnlk-ort et al., ) and ligated into the eukaryofic expression vector pmt (kaufman, ) using standard procedures (sambrook et al., ) . hela cells were transiently transfected with this construct by the calcium phosphate precipitation method ( - /zg dna per -ram dish) and the synthesis, modification, and stability of ribophorin i were analyzed at - h after transfection. glycosidase treatment of immunoprecipitated proteins was carried out according to protocols provided by the suppliers. protein a-sepharose beads carrying the immunoprecipitates were boiled for rain in a buffer containing . % sds and mm na-phosphate, ph . . the beads were then sedimented and the supernatanls containing the eluted protein diluted with the same volume of a np- containing buffer to final concentrations of . % np- , mm edta, mm na-phosphnte, mm na-acetate, ph . , u/ml trasylol. aliquots ( ~d) were then treated for h (or mock treated) with the following glycosidases before gel electrophoresis: o-glycosidase (endo-n-acetyl-a-d-galactosaminidase from diplococcus pneumoniae, . mu/sample), ~ -d-galactosidase (from bovine testes, mu/ sample), endo-o-d-galactusidase (from bacteriodes frasilis , mu/sampie), c~-l-fucosidase (from beef kidney, mu/sample in a dilation buffer containing mm na-acetate, ph . , and no na-phosphate), n-acetyl-o-d-glucosaminidase (from beef kidney, mu/sample) and neuraminidase (from clostridium perfringens, mu/sample). subconlluent cultures of hela-ri cells were incubated with fresh media with or without /~g/rnl bfa at c for h and fixed in % glutaraldehyde in . m sodium cacodylate buffer, ph . , for vain on ice. the cells were then scrap~ off the dish, sedimented, postfixed with glutaraldehyde and oso , and processed for routine em. thin sections were stained with uranyl acetate and lead citrate and examined in an electron micro= scope operated at kv (model ; philips electronic instruments co., mahwah, nj). we have previously shown (tsao et al., ) that in permanently transfected hela cells expressing ri , a truncated form of ribophorin i that lacks the membrane and cytoplasmic segments and consists of only the amino-terminal amino acids of the luminal domain the truncated molecules are degraded with biphasic kinetics. furthermore, in cells treated with bfa, the degradation of ri proceeded with monophasic kinetics and a rate constant intermediate between the two rates observed in control cells (tsao et al., ) . upon careful analysis it was apparent that in contrast to control cells, in the presence of bfa the apparent molecu- figure . the bfa-induced modification of ri and ribophorin i does not affect its n-linked oligosaccharide, which remains endo h sensitive, and also occurs in cells treated with tunicamycin. (a) hela-ri cells were incubated in methionine-free medium for min, pulse labeled with [ ss]methionine for min, and chased for up to h. bfa ( /zg/ml) was present during all these incubations. immunoprecipitates from samples taken at different times of chase were incubated with endo h or in the absence of the enzyme, as indicated, and analyzed by sds-page followed by fluorography. r/%z indicates the position of the polypeptide generated from ri after removal of the n-linked oligosaccharide by endo h. the asterisks alongside lane a and on the band in lane f mark the position of ri' .~, the polypeptide generated by endo h digestion of ri .m. the position of the latter is marked with dots in lane e and alongside lane a. (b) hela-ri cells were preincubated with tunicamycin ( tzg/ml) for . h and in methionine-free medium containing tunicamycin and bfa ( ~g/ml) for min. they were then pulse labeled with [ s]methionine for min, and chased for up to min in the presence of both drugs (c'-g'). control cultures, not treated with the drugs, were harvested immediately after the pulse (a'), or after min of chase (b'). immunoprecipitates were obtained and analyzed by sds-page, followed by fluorography. (m), a modified form of the ribophorin i and ri polypeptides observed in the presence of bfa; (*) a form lacking the n-linked oligosaccharide. lar weight of the labeled ri molecules underwent a progressive increase throughout the chase ( fig. a, lanes a, c, e, g, i, k, and m) . after min of chase • % of the ri molecules are modified (lane c), and after min, the posttranslational modification is essentially completed (lane g). after prolonged chase periods, the decaying pool contained a heterogenous population of ri molecules, represented by several closely spaced electrophoretic bands ( fig. a, lanes k and m). both ribophorin i and its truncated variant (ri ) contain a single n-linked high-mannose oligosaccharide chain attached to asn s, which can be cleaved off by endo h digestion (rosenfeld et al., ; harnik-ort et al., ; tsao et al., ) . to determine whether the apparent increase in the molecular masses of the truncated ribophorin i molecules that appear in bfa-treated cells reflects a processing of the n-linked oligosaccharide chain, the effect of endo h treatment on this protein was examined. as is shown in fig. a, endo h digestion increased the electrophoretic mobility of all the labeled truncated ribophorin i molecules produced in bfa-treated cells. the same behavior was found for intact ribophorin i (not shown). this demonstrates that the n-linked oligosaccharide chains in these molecules had not undergone the trimming and addition of sugars that have been observed for other proteins as a consequence of the bfa-induced backflow of golgi enzymes to the er (lippincott-schwartz, ; doms et al., ) . however, the deglycosylated truncated products were still of higher molecular weight than those produced by endo h digestion of the labeled molecules present immediately after the pulse, which had not yet undergone the bfa-induced posttranslational modification ( fig. a, compare r/% .m and r/% ). this demonstrates that the latter modification affects parts of the ribophorin i molecule other than the n-linked oligosaccharide chain. that the bfa-induced modification of ribophorin i does not involve, or even require, the presence of the n-linked oligosaccharide chain was definitively established by the finding (fig. b) that bfa treatment led to a decrease in the electrophoretic mobility of both the normal full length and the truncated (ri ) molecules, even when these were synthesized in the presence of tunicamycin, a drug that prevents the addition of n-linked oligosaccharide chains to newly synthesized polypeptides (takatsuki et al., ) . moreover, in this experiment it is apparent that the endogenous intact ribophorin i molecules also undergo a similar decrease in electrophoretic mobility although only a fraction (< %) of the population of the newly synthesized native ribophorin i molecules was eventually modified ( fig. b , r/*m). the possibility that in bfa-treated cells the ribophorin molecules were modified by the addition of phosphate or sulfate groups was eliminated by the finding that prolonged incubation with medium containing radioactive phosphate (capasso et al., ) or sulfate (hutmer, ) did not result in detectable labeling of either the intact or truncated ribophorin i molecules (results not shown). we therefore examined the possibility that the modified ribophorins had acquired o-linked oligosaccharides, which are normally not present in these molecules. first, the effect of various glycosidases that do not affect the core sugars of n-linked oligosaccharides, on the electrophoretic mobility of the modified ribophorins was investigated ( fig. ) . digestions with these enzymes were carded out after different times of chase, since o-linked chains grow by sequential addition of monosaccharide units (sadler, ) , which progressively alters their susceptibility to cleavage by specific enzymes. after min of chase, only endo-/ -galactosidase (fig. , lane e), but neither o-glycosidase, which removes o-linked oligosaccharides with terminal galactose residues (umemoto et al., ) , nor the exoenzyme/ -galactosidase (fig. , lane d) substantially increased the electrophoretic mobility of the modified ri a molecules. the apparent molecular mass of the endo-/ galactosidase-treated molecules was, however, still higher than that of the protein synthesized in control cells (fig. , compare lane e with lanes a and h). it appears, therefore, that the modified molecules contain o-linked oligosaccharides with internal galactose residues. neuraminldase, an enzyme that cleaves off terminal sialic acid residues, already had a detectable effect on molecules obtained after min of chase (fig. , lane f ) and, when applied to samples chased for min, it completely eliminated the slower migrating components of the modified ribophorin population, corresponding to the diffuse upper portion of the ri ,m band in fig. (compare lanes i and k). it is noteworthy that, even after removal of the terminal sialic acid residues by neuraminldase, the oligosaccharide chains remained resistant to o-glycosidase (fig. , compare lanes k and l). these data indicate that the galactose residues in the chains are still covered by other distal sugars. the ri molecules contain sugars that are not part of n-linked oligosaccharides could be directly demonstrated in bfa-treated cells that had been preincubated with tunicamycin to suppress n-glycosylation. in these experiments (fig. ) , the cells were incubated for min in media containing either [ h]glucosamine (a metabolic precursor of n-acetyl galactosamine), [ h]galactose, or [ h]mannose, the latter serving as a control to verify the effectiveness of the tunicamycin inhibition of n-glycosylation. in a parallel culture, cells were labeled under the same conditions with [ s]methionlne during a -min incubation period to display the full range of ri molecules at various stages of modification. fig. (lane a) shows that these molecules encompass the molecular mass range of - kd, and it seems likely that the fastest migrating component corresponds to the totally unglycosylated polypeptide. in contrast to the situation with [ s]methionlne, sugar precursors were only incorporated into the slower migrating forms of truncated ribophorin molecules that are characteristic of bfatreated cells (fig. , lanes b and c) . the two most slowly migrating species of ri were labeled with [ h]glucosamine, but only the slower of these was labeled with [ h]galactose. this labeling pattern would be expected if n-acetylgalactosamine, derived from [ h]glucosamine, is attached directly in an o-glycosidic bond to serine or threonine residues and galactose residues are subsequently added (sadler, ) . in cells incubated with [ h]mannose, only trace amounts of radioactivity were incorporated into ri molecules and this occurred in the modified molecules of lowest electrophoretic mobility. given the fact that nine mannose residues are present in the core of n-linked oligosaccharides ckornfeld and kornfeld, ) , this very low incorporation of [ h]mannose probably reflects a very low residual level of n-glycosylation in the presence of tunicamycin, or a very low degree of conversion of [ h]mannose into other labeled sugars. it is striking that in bfa-treated cells incubated for min with the sugar precursors, incorporation into the endogenous intact ribophorin i molecules was undetectable (fig. , lanes b to d), despite the fact that the amount of native protein in these cells, as measured by immunoblotting, is much higher than that of truncated molecules (not shown). since pulse-labeling experiments with [ s]methionlne in bfatreated cells (fig. ) showed that a substantial proportion of the newly synthesized intact ribophorin i molecules is modified during the subsequent chase, the lack of detectable incorporation of radioactive sugars into intact ribophorin i indicates that only newly synthesized, and not preexisting ribophorin molecules, can serve as a substrate for the o-glycosylation system. since ribophorin i is synthesized at an approximately -fold lower rate than ri , the o-glycosylated forms of the newly made ribophorin i molecules may not be easily detectable by labeling with tritiated sugars. -f and h-l) were pulse labeled with [ s]methionine for min and chased for or min as indicated. control cultures (lanes a and g) were incubated in methionine-free medium, labeled, and chased in the absence of bfa. immunoprecipitates obtained from cell lysates were incubated for h with (lanes c-f, j-l) or without (lanes a, b, and g-i) the following glycosidases: o-glycosidase (endo-n-acetyl-a-v-galactosaminidase, enzyme ); ~-d-galactosidase (enzyme ); endo-/ -d-galactosidase (enzyme ); and neuraminidase (enzyme ). after incubation, the samples were analyzed by sds-page, followed by fluorography. the behavior of ribophorin ii, a resident glycoprotein of the er membrane with the same transmembrane disposition as ribophorin i (crimaudo et al., ; pirozzi et al., ) , was also examined in bfa-treated cells. it must first be noted that in control hela cells only "~ % of the newly synthesized ribophorin ii molecules acquire an n-linked oligosaccharide chain (fig. , ri] vs rii* lane a) and are therefore sensitive to endo h treatment (lane b). a pulse-chase experiment demonstrates that in bfa-treated cells both types of ribophorin ii molecules (that is, those that bear [rii] , as well as those that lack [rh*] n-linked oligosaccharides) are convetted to more slowly migrating forms. moreover, in contrast to the situation with ribophorin i, after a -h chase period, the modified molecules were almost completely resistant to endo h digestion (fig. , compare lanes e and f). this indicates that the high mannose n-linked oligosaccharities in ribophorin ii were processed by the relocated golgi enzymes, and that o-linked sugars were added to the ribophorin ii polypeptides, whether or not they contain an n-linked oligosaccharide. the addition of o-linked sugars to ribophorin ii in bfa-treated cells was confirmed by the finding that the drug led to the appearance of more slowly migrating forms even in cells that were treated with tunica- addition of bfa ( tzg/ml) rain before labeling. one bfa-treated sample (e and f) was incubated in chase medium containing bfa for h. after lysis and immunoprecipitation with anti-ribophorin ii antibody the samples were divided into two equal aliquots that were either mock treated (a, c, and e) or treated with endo h (b, d, and f) and then analyzed by sds-page and fluorography. hela cell cultures were also pretreated with tunicamycin ( /~g/ml) for h and one culture was treated with bfa ( /~g/ml) for min before labeling for h with [ s]methionine. rii* is the membraneinserted but completely unglycosylated ribophorin ii polypeptide that, to some degree, is always found in control cells, but it is the only form present in cells treated with tunicamycin in the absence of bfa. r/i is the ribophorin ii molecule containing a high mannose (endo h sensitive) n-linked oligosaccharide chain, which is found normally in the er membrane. r/ira and r/fro are o-glycosylated forms found in bfa-treated cells that are derived from rii and rii'. in the presence of bfa the n-linked oligosaccharide in rii and in its o-glycosylated derivative (r/ ,) is converted into an endo h resistant form. mycin to suppress n-glycosylation (fig. , lanes g and h) . in this case, also in contrast to the situation with ribophorin i, two distinct modified forms of ribophorin ii were produced, which most likely correspond to molecules containing different numbers of o-linked oligosaccharide chains. the presence of galactose and sialic acid residues in the modified ribophorin i molecules indicates that after bfa treatment the respective glycosyltransferases, which are nor-mauy present only in the trans-cisternae of the golgi apparatus (kornfeld and kornfeld, ; cummings et al., ; elhammer and kornfeld, ) , are found in the er. this could result either from a relocation of preexisting golgi enzymes, as has been demonstrated for o~-mannosidase h and galactosyltransferase (lippincott-schwartz et al., , , or from the accumulation of newly synthesized glycosyltransferases that fail to leave the er in the presence of the drug. to distinguish between these possibilities, the effect of bfa was assessed in cells that, after pulse labeling with pss]methionine, were incubated with the protein synthesis inhibitor cycloheximide for min, a period sufficient to allow the egress from the er of newly synthesized proteins destined to the golgi apparatus. as shown in fig. , even under these conditions, bfa treatment resulted in the appearance of the more slowly migrating forms of ri with the same kinetics as in the absence of cycloheximide. it therefore can be concluded that the modifications induced by bfa are carried out by preexisting glycosyltransferases, relocated from the golgi apparatus to the er as a result of treatment with the drug. it may also be noted in fig. that treatment with cycloheximide appeared to slow down the degradation of ri molecules. the appearance of trans-golgi components in the er as a result of bfa treatment could also be demonstrated by em using lectin labeling techniques and immunogold detection procedures on ultrathin frozen sections (gdfliths et al., ) . in agreement with previous reports (fujiwara et al., figure . the posttranslational modification of ri observed in bfa-treated cells is etfeeted by pre-existing enzyme.s. cultures of hela-ri cells were pulse labeled with [~s]methionine for min and, after this time, two cultures were placed on ice (lanes a and n). the other cultures were incubated for rain in complete chase medium with (h-m) or without (b-g) cyclobeximide ( #g/ ml). after the chase, bfa ( /~g/ml) was added and the incubation continued for up to min. at the chase times indicated, the cells were lysed with an sds-containing buffer and processed for immunoprecipiation and sds-page, followed by fluorography. ; lippincott-schwartz et al., % ; ulmer and palade, ) , bfa led to profound changes in the structure of the golgi apparatus which, after treatment with the drug, was apparently reduced to clusters of vesicular and tubular elements located near partly rough and partly smooth er cistemae with the typical appearance of transitional elements (jamieson and palade, ) . as expected from previous studies (tartakoff and vassalli, ) , in control hela cells, figure . bfa treatment of hela cells leads to the appearance of wga binding sites in the er and nuclear envelope. ultrathin frozen sections from control hela-ri cells (a), or cells treated with bfa ( t~g/rnl) for min (b), or h (c and d) were incubated with wga. the sites of lectin binding were detectexl using anti-wga antibodies and protein a-gold. in control cells (a), the nuclear envelope (ne) and er are not labeled, but dense labeling is observed in trans-golgi cisternae (ga) and the plasma membrane (pm). within mitt after bfa treatment (b), some wga binding is observed in the nuclear envelope and er, as well as in profiles corresponding to remnants of the golgi apparatus (/arge arrows). after h of bfa treatment, the er and ne are much more intensely labeled. wga, which recognizes sialic acid and exposed n-acetyl glucosamine residues (bhavanandan and katlic, ) , labeled intensely the trans-cisternae of the golgi apparatus, but not the membranes or luminal content of the er (fig. a) . on the other hand, in cells treated with bfa ( /~g/ml) for times as short as min (fig. b) and up to h (fig. c) , wga binding situs were present throughout the er and in the nuclear envelope, as well as in the apparent remnants of the golgi apparatus (fig. d) . similar results were obtained with the lectin rca, which recognizes terminal galactose residues (baenziger and fiete, ) , or when the cells were treated with cycloheximide for h before the addition of bfa to deplete the eli of newly synthesized golgi proteins (results not shown). these findings are in accord with the preceding biochemical demonstration that bfa induces a redistribution of o-glycosylating enzymes of the trans-region of the golgi apparatus, such as galactosyl-and sialyltransferases, into the er. as shown above, all the truncated ribophorin i molecules (ri ) synthesized during a brief ( rnin) pulse eventually become posttranslationally modified (fig. ) during a chase period in the presence of bfa, even when the drug was added rain after labeling (fig. ) . on the other hand, < % of the intact ribophorin molecules synthesized during a brief pulse undergo the posttranslational modification ( fig. b) . moreover, in cells incubated with labeled monosaccharides (fig. ) , o-glycosylation of previously synthesized intact ribophorin i molecules does not appear to take place to any significant extent. this suggests that ribophorin i molecules are susceptible to o-glycosylation only during a limited period after their synthesis. it seems likely that this could correspond to the time required for the newly synthesized polypeptides to undergo a conformational change and/or eventually to become incorporated into the proteinaceous network in the rer where the mature protein has been shown to be contained (kreibich et al., a,b; yu et al., ) . to estimate the time interval during which the newly synthesized ribophorin i molecules are accessible to the o-glycosylation system, hela cells were labeled for h in the presence or absence of bfa, and the extent to which the posttranslational modification occurred in a subsequent chase period in the presence of the drug was assessed. after this relatively long labeling period, which facilitates the detection of the labeled ribophorin i molecules, modified full length polypeptides were detected only in cells labeled in the presence of the drug (fig. a) . the failure of bfa to cause the modification of previously labeled ribophorin molecules, however, could result in part from the time required for the drug to exert its effect and the golgi enzymes to reach the er. the latter has recently been estimated to be considerably less than min (donaldson et al., ) , which is in accordance with our observations of the appearance of lectin binding sites in the er within rain of bfa treatment (fig. b) . since ribophorin i is a stable protein that can only be weakly labeled during a short pulse, it is difficult to carry out this type of experiment with a shorter labeling period, to obtain accurate kinetics for the modification of the intact figure . in bfa-treated cells, intact ribophorin i and ribophorin ii molecules can be posttranslationally glycosylated only during a limited period after their synthesis. hela cells (a and c) and hela cells transiently overexpressing ribophorin i (b) were preincubated in methionine-free medium for min with (lanes a-c), or without (d-g) bfa ( t~g/ml) and then labeled with [~ss]methionine for h in the presence (a-c) or absence (d-g) of the drug. some cultures (a, d, and g) were placed on ice immediately after the labeling period, whereas the others were incubated in chase medium containing bfa for h (b and e) or h (c and f). immunoprecipitates obtained from cell lysates with antibodies against ribophorin i (a and b) or ribophorin ii (c) were analyzed by sds-page, followed by fluorography. the labeled bands are marked using the abbreviations introduced in the legends to figs. and . molecule. if we assume that, when available to the modifying enzymes, the intact and truncated molecules are modified with the same kinetics, it can be concluded that within rain or less after their synthesis is completed, ribophorin i molecules are no longer susceptible to modification by the relocated golgi enzymes. a similar experiment to examine the behavior of ribophotin ii (fig. c) showed that, like ribophorin i, only when the bfa treatment was initiated before the pulse did the newly synthesized (labeled) ribophorin ii molecules undergo the modifications carried out by the relocated golgi enzymes. therefore, both ribophorins are susceptible to the action of these enzymes only during a limited time period after their synthesis. if the ribophorins rapidly became resistant to the action of the golgi glycosyltransferases as a consequence of their incorporation into an oligomeric assembly, one would expect that overexpression of one of these proteins, so that its levels far exceeded those of other components of the assembly, would lead to much more efficient o-glycosylation. surprisingly, when the full-length ribophorin i was transiently overexpressed in hela cells no o-glycosylation was observed when bfa treatment was initiated after completion of ribophorin i synthesis (fig. b, lanes d-f) . furthermore, the level of o-glycosylation of ribophorin i observed when synthesis occurred in the presence of bfa did not markedly change (fig. b, lanes a-c) when compared with that seen in control cells ~ig. a, lanes a-c) . this result suggests that ribophorin i, before being integrated into a heterooligomeric complex, undergoes a conformational change that renders it resistant to o-glycosylation in bfa-treated cells. it has previously been observed that ribophorin i is a longlived protein of the rer (rosenfeld et al., ; see also fig. a) . a comparison of the degradation rate of ribophorin i in transfected cells to that in control cells revealed that the overexpressed molecules were much more unstable (fig. b) . since after h of chase approximately equal amounts of labeled ribophorin i remained in transfected and control cells, it would appear that the overexpressed molecules turnover with a half-life of h or less. it should be noted that in these transient transfection experiments only ~ % of the cells express the foreign protein, so that in the expressing cells, ribophorin i is being synthesized at up to times the control levels. this finding that the overexpressed portion of the ribophorin i molecules is degraded significantly faster than the endogenously expressed polypeptide is consistent with the notion that the high stability of endogenous ribophorin i results from its integration into an oligomeric assembly. ribophorins i and ii, as well as other components of the translocation apparatus, appear to represent a class of resident er membrane proteins, that, in contrast with other well studied er polypeptides, such as the el protein of adenovirus (p~bo et al., ; nilsson et al., ) and udpglucuronosyltransferase (jackson et al., ) , do not contain retention signals at their extreme cooh-termini (jackson et al., ) . ribophorins form large macromolecular assemblies or complexes within the er membrane (kreibich et al., a,b; yu et al., ) and it seems likely that their retention in the er simply results from the fact that, once incorporated into these complexes, they cannot gain access to the vesicles that mediate the transport to the golgi apparatus of other proteins synthesized in the er. thus, the segregation of proteins that form large macromolecular complexes, although resulting from a true retention mechanism, would not require a signal common to all of them. instead, the retention of each protein might only involve its interaction with another specific component of the macromolecular assembly. that a true retention mechanism, rather than a mechanism involving retrieval from the golgi apparatus, is responsible for the segregation of many proteins in the er, including the ribophorins, was first suggested by the finding that n-linked oligosaccharide chains in er proteins do not undergo conversion to complex forms, which are resistant to endo h and are capable of binding the lectins wga and rca, a process that requires the action of enzymes located in the golgi apparatus (rodriguez-boulan et al., a,b; rosenfeld et al., ; brands et al., ; yamamoto et al., ) . the validity of this argument for any particular protein, however, rests on the premise that the protein could serve as a substrate for the golgi enzymes, were it to become accessible to them. in the current work this is demonstrated for n-glycosylated ribophorin ii molecules, which normally remain sensitive to endo h but in bfa-treated cells become resistant to digestion with this enzyme. a similar behavior was recently observed for erp , another er resident protein figure . ribophorin i when overexpressed in hela ceils becomes unstable. hela ceils (a) and hela cells transiently overexpressing ribophorin i (b) were incubated in methionine-free medium for min, pulse labeled with [ ss]methionine for h, and chased for up to h. at the chase times indicated, the cells were lysed with an sds-containing buffer and processed for immunoprecipitation and sds-page, followed by fluorography. (lippincott-schwartz et al., ) . in this regard, our finding that in bfa-treated cells the n-linked oligosaccharide chain in ribophorin i remained endo h sensitive clearly demonstrates that access to the enzymes that could effect its conversion to the endo h resistant form is not sufficient to ensure that the modification takes place. indeed, the conformation of glycoproteins may very well determine the susceptibility of their sugar chains to the action of the relocated modifying enzymes. in addition, it can not be excluded that the single n-linked oligosaccharide chain of ribophorin i would acquire resistance to digestion with endo h, if the molecule would pass through the golgi stacks in an ordered fashion, instead of being exposed to the golgi glycosyltransferases in the bfa-induced er-golgi hybrid compartment. in fact, we have observed (unpublished observations) that the five n-linked oligosaccharide chains of the hemagghtinin of influenza, which normally are converted into endo h resistant forms during passage of the protein through the golgi apparatus, are not modified by relocated golgi enzymes when the protein remains in the er in bfa-treated cells. we also found that both ribophorins, which normally do not acquire o-linked oligosaccharides, do so after bfa treatment. this demonstrates that they, indeed, can serve as substrates for the enzymes that carry out this modification, and therefore, in the absence of the drug must not be exposed to them. o-glycosylation involves the addition of n-acetylgalactosamine to serine and threonine residues in the polypeptide backbone, followed by linkage to galactose and sialic acid (sadier, ) . the exact subcellular location of the n-acetyl galactosaminyl transferase that initiates growth of the o-linked oligosaccharide chain has not been established, although the relatively rapid kinetics with which this modification takes place (tooze et al., ) , as well as the fact that certain mutant low density lipoprotein (ldl) receptors that fail to transverse the golgi apparatus nevertheless acquire o-linked sugars (cummings et al., ) , has sug-gested that this enzyme could be located in a pre-golgi compartment. in fact, a recent study of the e glycoprotein of the mouse hepatitis coronavirus virus mhv-a (tooze et al., ) concluded that addition of n-acetylgalactosamine to this protein occurs in a pre-golgi compartment, which is also the site of budding of the virion into the lumen of the endomembrane system and morphologically resembles transitional elements and vesicles found between the er and the golgi apparatus. since ribophorins i and ii, as well as the truncated ribophorin i variant, ri , undergo o-glycosylation only in bfa-treated cells, we can conclude that under normal circumstances these molecules do not even reach this intermediate compartment, which may be the same as the "salvage ~ compartment from where kdel-containing luminal proteins that escape from the er are retrieved to this organelle (pelham, ) . this reinforces the conclusion that the segregation of ribophorins i and ii does not involve a retrieval mechanism. it should be noted that if, indeed, o-glycosylation takes place in the "salvage " compartment, one would expect that some luminal er proteins that recycle through this compartment would contain o-linked sugars, a prediction that can be tested experimentally. our observations that in bfa-treated ceils galactose residues are incorporated into the truncated ribophorin i variant and that neuraminidase treatment increases the electrophoretic mobility of this protein, provide biochemical evidence that oligosaccharide-modifying enzymes of the trans-region of the golgi apparatus become relocated to the er after bfa treatment. the presence of a galactosyltransferase in the er of bfa-treated cells was previously demonstrated immunocytochemically (lippincott-schwartz et al., ) , as well as by the finding that mannose- -phosphate receptor molecules synthesized in the presence of the drug bind to rca-i lectin columns (chege and pfeffer, ) . the sialic acid residue(s) that are present in the truncated ribophorin i and are removed by neuraminidase must be part of o-linked oligosaccharide chain(s), since the protein remained endo h sensitive and, therefore, its single n-linked oligosaccharide did not contain sialic acid. the striking relocation of golgi components caused by bfa was also manifested by the appearance of binding sites for the lectins rca-i and wga in the er. the former lectin binds to exposed galactose residues (baenziger and fiete, ) and the latter to exposed n-acetyl glucosamine and sialic acid residues (bhavanandan and katlic, ) . as expected from previous studies with myeloma cells (tartakoff and vassalli, ) , we found that in control hela cells intracellular binding sites for both lectins were almost exclusively present in the trans-region of the golgi apparatus and not to any significant extent in the er or nuclear envelope. after bfa treatment, however, even in cells in which protein synthesis was inhibited with cycloheximide, both lectins intensely labeled the er and the nuclear envelope. these electron microscopic results are in marked contrast with immunofluorescence observations by lippincott-schwartz et al. ( ) of cultured normal rat kidney (nrk) cells, in which bfa treatment did not lead to a relocation of wga binding sites. in later work these authors observed that bfa caused the complete relocation to the er of the trans-golgi enzyme galactosyltransferase and, therefore, suggested that wga binding sites are not relocated because they are predominantly localized in the trans-golgi network (lippincott-schwartz et al., ) . this may indeed be the case in nrk cells, but it is also possible that the lectin binding sites that appear in the er after bfa treatment do not all correspond to redistributed previously existing golgi glycoproteins. rather, to an extent which may vary with the cell type, such sites may be created by a sialyltransferase relocated from the medial or trans-golgi region, operating on er resident proteins, as well as on relocated cisand medial golgi proteins. in considering the location of lectin binding sites within the golgi apparatus, one must note that an integral membrane protein confined to cisand medial golgi cisternae has been identified that contains sialic acid residues on both its n-linked and o-linked oligosaccharide chains (yuan et al., ) . this led to the alternative suggestions that this protein is either modified in the trans-golgi network and then returned to the more proximal region of the golgi apparatus, or that an as yet uncharacterized sialyltransferase is located in cisand medial cisternae. a striking observation in this study was that, in bfatreated cells, newly synthesized, but not preexisting intact ribophorin i molecules, were susceptible to o-glycosylation by the relocated golgi enzymes. as a consequence, only a very small fraction of the total cell complement of endogenous ribophorins acquired the modification. the intact ribophorin molecules that received the modification (< % of those that were labeled during a -min pulse) did so during a brief period after their synthesis. in addition, overexpressed full-length ribophorin i molecules, synthesized in excess of the stoichiometric amounts present in untransfected cells, were also susceptible to o-glycosylation for a limited period after their synthesis. on the other hand, the truncated ribophorin molecules were suspectible to o-glycosylation throughout their lifetime and were quantitatively modified during the first -rain period after the addition of bfa. it seems likely that this difference between the intact and the truncated ribophorins results from a conformational change that only the intact polypeptide molecules undergo soon after their synthesis, and possibly also from their sequestration in the supramolecular assembly of the er membrane that is responsible for their retention within the organelle. the relatively rapid degradation of full-length ribophorin molecules expressed at levels above the stoichiometric amounts of other components of the supramolecular assembly in the rer membrane is likely to be a consequence of their failure to be assembled normally. this would be analogous to the rapid turnover of other monomeric components of multimeric proteins that is observed when individual subunits are synthesized in excess (bonifacino and lippincott-schwartz, ) . segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. i. functional tests on rat liver microsomal subfimctions structural determinants of ricinu* commun/s agglutinin and toxin specificity for oligosaceharides the interaction of wheat germ agglutinin with sialnglycoproteins. the role of sialic acid degradation of proteins within the endoplasmic reticulum retention of membrane proteins by the endoplasmic reticulum mechanism of pbosphorylation in the lumen of the golgi apparatus. translocation of adenosine '-triphosphete into golgi vesicles from rat liver and mammary gland compartrnentation of the golgi complex: brl~ldin a distinguishes trans-golgi cisternae from the trans-gotgi network human ribophorins i and ii: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins biosynthesis of n-and o-linked oligosaccharides of the low density lipoprotein receptor anti-liver-kidney microsome antibody is a marker for the rat hepatocyte andoplasmic reticulum brefeldin a redistributes resident and itinerant golgi proteins to the endoplasmic reticulum guanine nuclr modulate the effects of brefeldin a in semipermeable cells: regulation of the association of a ll -kd peripheral membrane protein with the golgi api aratus two enzymes involved in the synthesis of o-linked oligosaoeharides are localized on membranes of different densities in mouse lymphoma bw cells brefeldin a causes disassembly of the golgi complex and accumulation of secretory proteins in the endoplasrnic reticulam viral membrane proteins acquire galactose in tmns golgi cisternae during intracellniar transport isolation and characterization of edna clones for rat ribopborin i: complete coding sequence and in vitro synthesis and insertion of the encoded product into endoplasmic reticulum membranes ~ber die isoliernng neuer stoffwechselprodukte aus penicillium brefeldianum dodge tyrosine snifatation and the secretory pathway immunolabeling of frozen thin sections and its application to the study of the biogenesis of epithelial cell plasma membranes identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticnium intracellular transport of secretory proteins in the pancreatic exocrine cell. i. role of the peripheral elements of the golgi complex vectors used for expression in mammalian cells an improved procedure for immunoulectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections assembly of asparngine-linked oligosaccharides proteins of rough microsomal membranes related to ribosome binding. ii. cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites proteins of rough microsomal membranes related to ribosome binding. i. identification of ribophorins i and ii, membrane proteins characteristic of rough microsomes ribophorins i and i: membrane proteins characteristic of the rough endoplasmic reticulum rapid redistribution of golgi proteins into the er in ceils treated with brefeldin a: evidence for membrane cycling from golgi to er microtubnie-dependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. ii. rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum a short sequence in the cooh-terminus makes an adcoovirus membrane glycoprotein a resident of the endoplasmic reticulum control of protein exit from the endoplasmic reticnium rat ribophorin ii: molecular cloning and chromosomal localization of a highly conserved transmembrane glycoprotein of the rough endoplasmic reticulum spatial orientation of glycoproteins in membranes of rat liver rough microsomes. i, localization of lectin-binding sites in microsomal membranes spatial orientation of glycoproteins in membranes of rat liver rough microsomes biosynthesis and processing of ribophorins in the endoplasmic reticulum biosynthesis of glycoproteins: formation of o-linked oligosaccharides molecular cloning: a laboratory manual inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin antiviral activity of brefeldin a and verrucarin a lectin-binding sites as markers of golgi components. proximal-to-distal maturation of oligosaccharides immunochemistry on ultrathin frozen sections site of addition of n-acetylgalactosamine to el glyeoprotein of mouse hepatitis virus-a carboxy terminally truncated forms of ribophorin i are degraded in pre-golgi compartments by a calcium-dependent process targeting and processing ofglycophorins in murine erythroleukemia cells: use of brefeldin a as a perturbant of intracellular traffic purification and properties of an endo-tx-n~acetyl-o-galactosaminidase from diplocoecus pneumoniae a signal sequence receptor in the endoplasmic reticulum raembrane is cytochrome p- transported from the endoplasmic reticulum to the golgi apparatus in rat hepatocytes? reconstitution of translocation-competent membrane vesicles from detergent-solubilized dog pancreas rough microsomes antiribophorin antibodies inhibit the targeting to the er membrane of ribosomes containing nascent secretory polypeptides two integral membrane proteins located in the cis-middle and trans-part of the golgi system acquire sialylated n-linked carbohydrates and display different turnovers and sensitivity to camp-dependent phosphorylation we thank dr. r. j. kaufman (genetics institute, cambridge, ma) for the kind gift of the pmt plasmid vector. the help of j. culkin, f. forcino, and h. plesken with the preparation of the illustrations, and m. cort with the typing of the manuscript, is greatly appreciated. we also thank h. plesken for her help in the preparation of ultrathin frozen sections.this work was supported by national institutes of health grants gm and gm and a grant from the american cancer society (cd- ). n. e, ivessa was the recipient of a schrtdinger fellowship from the osterreichische fonds zur ftrderung der wissenschat~lichen forschung.received for publication july and in revised form march . key: cord- - oxzzxnd authors: salcedo, suzana p.; holden, david w. title: sseg, a virulence protein that targets salmonella to the golgi network date: - - journal: the embo journal doi: . /emboj/cdg sha: doc_id: cord_uid: oxzzxnd intracellular replication of the bacterial pathogen salmonella enterica occurs in membrane-bound compartments called salmonella-containing vacuoles (scvs). maturation of the scv has been shown to occur by selective interactions with the endocytic pathway. we show here that after invasion of epithelial cells and migration to a perinuclear location, the majority of scvs become surrounded by membranes of the golgi network. this process is dependent on the salmonella pathogenicity island type iii secretion system effector sseg. in infected cells, sseg was associated with the scv and peripheral punctate structures. only bacterial cells closely associated with the golgi network were able to multiply; furthermore, mutation of sseg or disruption of the golgi network inhibited intracellular bacterial growth. when expressed in epithelial cells, sseg co-localized extensively with markers of the trans-golgi network. we identify a golgi-targeting domain within sseg, and other regions of the protein that are required for localization of bacteria to the golgi network. therefore, replication of salmonella in epithelial cells is dependent on simultaneous and selective interactions with both endocytic and secretory pathways. salmonella enterica serovar typhimurium (s.typhimurium) is a bacterial pathogen that invades and replicates within a membrane-bound compartment (salmonellacontaining vacuole; scv) inside a variety of host cell types. the biogenesis and maturation of the scv has been studied in detail in both macrophages and epithelial cells. within a few minutes of bacterial entry, components of the early endocytic pathway, such as eea and the transferrin receptor, are recruited to the scv (early scv; me steele-mortimer et al., ) . these proteins are then gradually lost, and interactions between the scv and late endosomal compartments lead to the progressive acquisition of the vacuolar atpase and several lysosomal membrane proteins (lgps), such as lamp (intermediate scv; garcia-del portillo and finlay, ; rathman et al., ; me Âresse et al., ; steele-mortimer et al., ) . the late scv represents the third stage of maturation which begins ± h after invasion. it requires the intracellular synthesis of bacterial proteins and is characterized by the further recruitment of lgps and the initiation of sif formation. sifs are tubular structures enriched in lgps and which appear to be contiguous with the scv (garcia-del portillo et al., ) . however, the scv does not accumulate signi®cant amounts of the lysosomal enzymes that are normally delivered to a maturing phagolysosome by the cation-independent mannose- -phosphate receptor (garcia-del portillo and finlay, ; rathman et al., ) . interactions between the scv and the endocytic pathway are therefore selective, and this unique traf®cking pathway creates an environment permissive for bacterial cell division, which begins after a lag period of ± h after invasion (garcia-del portillo et al., ) . intracellular replication of s.typhimurium involves a large number of virulence proteins, including the type iii secretion system (ttss), encoded by the spi- pathogenicity island (ochman et al., ; cirillo et al., ; hensel et al., ) . this ttss translocates effector proteins into the vacuolar membrane and cytosol of the host cell. several effectors have been identi®ed in recent years, encoded both within and outside the pathogenicity island. spic is a spi- -encoded protein that prevents interactions between scvs and endocytic compartments in macrophages (uchiya et al., ) . sifa is encoded outside spi- , and is required for maintenance of the vacuolar membrane, most likely by facilitating the recruitment and fusion of vesicles with the scv (beuzo Ân et al., ) . sifa is also required for the formation of sifs (stein et al., ) . scv membrane dynamics are also in¯uenced by the action of another effector, the predicted acyl transferase ssej (ruiz-albert et al., ) . several other effectors have been described, but the functions of most of them are not understood (waterman and holden, ) . while interactions between the scv and markers of the endocytic pathway have been studied in depth, little is known about the spatial distribution of scvs within infected cells and the potential relevance of this to bacterial multiplication. we show here that the majority of scvs become surrounded by golgi membranes h after invasion of epithelial cells, a process that is dependent on the spi- ttss effector sseg. mutational analysis of this protein identi®ed a golgi-targeting domain and de®ned further regions that are required for recruitment of scvs to the golgi network. the physiological relevance of golgi targeting by salmonella is demonstrated by the requirement of both sseg and an intact golgi network for salmonella replication. therefore, scv±golgi inter-sseg, a virulence protein that targets salmonella to the golgi network the embo journal vol. no. pp. ± , ã european molecular biology organization actions represent a crucial stage in the intracellular life cycle of salmonella. to investigate the intracellular localization of salmonella, hela epithelial cells were infected with gfp-expressing s.typhimurium and analysed by confocal immuno¯uorescence microscopy. developing microcolonies usually comprised tight clusters of bacteria, positioned close to the nucleus (figures a and a ) and were often found closely associated with the golgi network. confocal x/z reconstructions revealed that these microcolonies were partially or completely enveloped by golgi membranes ( figure a ; supplementary ®gure s available at the embo journal online). this phenomenon was observed using antibodies against a variety of golgi proteins, including giantin and golgi matrix protein (gm ) ( figure a and data not shown). similar results were obtained using an antibody against tgn , a glycoprotein the subcellular localization of gfp-expressing wild-type s.typhimurium (wt-gfp, green) in relation to the cis-golgi protein giantin (red), and the host cell (dic in merged image), h after invasion. scale bar corresponds to mm. lower panel shows a °rotation on the y-axis of a d reconstruction obtained from a z-stack of the cell shown in the upper panel. scale bar corresponds to mm. (b) infected cells were labelled for tgn (red), salmonella (blue) and lamp , a marker of the scv membrane (green). points of co-localization between lamp and tgn are indicated by arrowheads. scale bar corresponds to mm. (c) transmission electron micrographs of representative hela cells showing wild-type salmonella (b) in close proximity to golgi cisternae (g). the nucleus is marked as (n). scale bars correspond to nm. localized primarily in the trans-golgi network (tgn) ( figure b ). in contrast, calreticulin, an endoplasmic reticulum (er) luminal protein, was not associated with microcolonies. as expected , there was no association between bacteria and early or recycling endosomal markers, including eea and the transferrin receptor (data not shown). at h after invasion, t % of microcolonies of gfp-expressing wild-type bacteria were associated with golgi membranes. the labelled golgi proteins were concentrated in the area around the developing microcolony, rarely between individual scvs. to determine if golgi proteins co-localized with lamp , a marker of the scv membrane beuzo Ân et al., ) , infected cells were labelled with antibodies against lamp and either giantin or tgn . neither golgi protein was found to co-localize extensively with lamp present on the vacuolar membrane, but frequent discrete points of co-localization were observed with both markers (arrowheads, figure b ). neither copi nor copii coat proteins, which mediate vesicle formation within the secretory pathway, were recruited to the scv membrane (data not shown). to determine whether scv membranes were fused with golgi membranes, infected hela cells were examined by transmission electron microscopy. scvs were frequently found within ± nm of golgi cisternae ( figure c ), and in some cases less than nm. however, there was no evidence of fusion between scv and golgi membranes in over infected cells analysed. in uninfected hela cells, the golgi network has a relatively compact appearance (asterisk, figure a ). in contrast, by h after infection, the morphology of the network was frequently distorted, with the bacterial microcolony in the centre, suggesting that golgi membranes are displaced by the presence of the growing microcolony ( figure a and arrowhead, figure a ). association of s.typhimurium with the golgi network was also observed in the human small intestinal epithelial cell line int , but not in raw . or elicited peritoneal murine macrophages, where replicating bacteria were frequently perinuclear, but neither associated in tight clusters nor surrounded by golgi membranes (data not shown). association with the golgi requires the spi- ttss effector sseg we next investigated whether bacterial virulence proteins that are released intracellularly are involved in salmonella±golgi interactions. hela cells were infected with different mutant strains, then ®xed and analysed by confocal microscopy for bacterial association with the golgi network. scvs were considered golgi-associated if they were at least partially surrounded by golgi membranes. the percentage of wild-type bacteria associated with the golgi steadily increased to~ % over a h period. however, a strain carrying a mutation in ssav, which is defective for secretion of all spi- ttss effector proteins, had a signi®cantly reduced level of association with the golgi network; < % of bacteria were associated with the golgi at any time during the experiment (figure a and b). although ssav mutant bacteria remained predominantly perinuclear, in contrast to the wild-type strain they displayed a scattered distribution ( figure a ). no differences were observed between the wild-type and strains carrying mutations in phop or spvb (data not shown), two virulence loci that are involved in growth of salmonella in macrophages (holden, ) . to identify the spi- effector responsible for this phenotype, strains carrying mutations in genes encoding different spi- -translocated proteins were examined. whereas ssej and ssei mutant strains were indistinguishable from the wild-type, an sseg mutant strain had a phenotype similar to that of the ssav mutant (figure a and c). the golgitargeting defect of the sseg mutant could be attributed to a lack of sseg because a plasmid containing the wild-type sseg allele restored association with the golgi to the level of the wild-type strain ( figure c ). inactivation of sseg leads to a replication defect in macrophages and moderate virulence attenuation in the mouse model of systemic salmonellosis . sseg is predicted to have at least two transmembrane domains; its primary amino acid sequence displays no signi®cant similarities to protein database entries, apart from a % similarity to ssef, which is encoded immediately downstream of sseg . an antibody against a c-terminal peptide of sseg was used to examine the intracellular distribution of the protein. intense labelling was typically observed outlining part of the bacterial microcolony, and also in peripheral punctate structures ( figure a and b). sseg co-localized with lamp in the scv membrane and on sifs ( figure b ). this observation is consistent with the localization of an m peptide-tagged version of sseg (kuhle and hensel, ) . however, the peripheral ssegpositive, punctate structures did not co-localize with lamp . they were found mostly within the immediate vicinity of the golgi network and occasionally colocalized with both cis-golgi and tgn markers (arrowheads, figure c ). to establish if sseg contributes to growth of s.typhimurium in epithelial cells, intracellular replication assays were carried out. the numbers of wild-type and complemented sseg mutant strains underwent an~ -fold increase over a h period. in contrast, the sseg mutant numbers increased by < -fold over the same time period, a replication defect similar to that of the ssav mutant ( figure a ). since sseg is required for both golgi localization of bacteria and intracellular replication, we hypothesized that the golgi network might be exploited by confocal immuno¯uorescence microscopy of hela cells infected for h by gfp-expressing wild-type strain or the sseg mutant as a control. cells were labelled with the anti-sseg antibody (red). scale bars correspond to mm. (b) confocal immuno¯uorescence microscopy of hela cells infected for h by wild-type s.typhimurium. boxed areas shown in higher magni®cation below show co-localization between sseg (green) and lamp (red) on a sif and scv membrane. salmonella typhimurium was labelled with an anti-salmonella antibody (blue). punctate labelling of sseg can also be seen in the vicinity of the microcolony. scale bar corresponds to mm. (c) distribution of sseg in relation to the golgi network in hela cells infected for h with wild-type bacteria (blue in merged image). arrowheads indicate points of co-localization between sseg (red) and either gm (green, upper panel) or tgn (green, lower panel). scale bars correspond to mm. salmonella for its replication. therefore, the number of wild-type bacteria in golgi-associated microcolonies was compared over time with those located elsewhere in the cell by microscopic examination. there was no signi®cant increase in numbers of bacteria not associated with the golgi network, whereas the numbers of golgi-associated bacteria underwent a signi®cant increase over a h period ( figure b ). this result suggests that bacterial replication is restricted to those associated with the golgi. to test the requirement for an intact golgi network in intracellular growth of salmonella, cells were treated min after bacterial invasion with brefeldin a (bfa). bfa inhibits nucleotide exchange on the small gtpase adp-ribosylating factor (arf ), which is required for formation of copi coated vesicles (chardin and mccormick, ) . as a result, the golgi network rapidly redistributes into the er (lippincott-schwartz et al., ) . bfa strongly inhibited intracellular growth of wild-type s.typhimurium, but had little effect on numbers of ssav mutant bacteria ( figure c ). however, bfa had no noticeable effect on markers of the maturation and traf®cking of the scv, such as lamp recruitment and cathepsin d exclusion (data not shown). therefore, the effects of bfa appear to be independent of the selective interactions between scvs and the late endocytic pathway. as a further test for the requirement of a functional golgi network in s.typhimurium replication, bacteria were examined in hela cells expressing arf t n-gfp, a dominant interfering variant of arf (dascher and balch, ) . a signi®cant reduction in intracellular bacterial numbers was observed at h after invasion, compared with bacterial replication in cells expressing gfp alone ( figure d ). sar is another small gtpase, involved in copii-mediated vesicle formation, and its inhibition by constitutively inactive sar t n prevents the formation of early secretory vesicles at er exit sites (ward et al., ) . bacterial replication was also severely inhibited in cells expressing this mutant protein ( figure d ). expression of arf t n-gfp or sar t n had no effect on bacterial internalization (supplementary table s ). together, these experiments establish that an intact and functional golgi network is required for intracellular replication of s.typhimurium. to gain further insight into the function of sseg, an epitope-tagged version of the protein (myc::sseg) was expressed in hela cells. the protein was concentrated in a perinuclear region, where it co-localized extensively with golgi markers including giantin (data not shown) and cells expressing a myc-tagged version of sseg (green) were co-labelled with an antibody against tgn (red), and examined by confocal microscopy. the majority of myc::sseg co-localizes with tgn (upper panel) and its redistribution follows that of the tgn marker upon bfa treatment (middle panel). bfa washout leads to recovery of a compact golgi structure, and the re-localization of myc::sseg (lower panel). scale bars correspond to mm. (b) cells expressing myc::sseg (green) were triple labelled with anti-myc (green), anti-tgn (red) and anti-giantin (blue) antibodies. (c) cells expressing myc::sseg (green) were treated with bfa for min, ®xed and labelled with either anti-giantin or anti-tgn antibodies. in the upper panel the arrowheads indicate tubules containing myc::sseg (green) which does not co-localize with giantin (red). scale bar corresponds to mm. in the lower panel, myc::sseg co-localizes with tgn in tubules. arrowheads indicate tubulating regions shown at a higher magni®cation in insets. (d) distribution of myc::sseg and golgi markers after exposure of transfected cells to nocodazole. cells were labelled with anti-myc (green) and either anti-giantin (red, upper panel) or anti-tgn (red, lower panel) antibodies. scale bars correspond to mm. figure a ). to verify that myc::sseg was localized to the golgi network, transfected cells were incubated with bfa for h, and either ®xed immediately or washed in medium lacking bfa, and incubated for a further h. in all transfected cells, bfa caused extensive redistribution of myc::sseg throughout the cell, where most of it co-localized with dispersed tgn ( figure a ). following bfa wash-out, tgn moved back to its original perinuclear position and was accompanied by myc::sseg ( figure a ). subcellular fractionation of transfected cells and immunoblotting showed that sseg localized to the membrane fraction, as expected for a predicted integral membrane protein (data not shown). in transfected cells that strongly overexpressed myc::sseg, the golgi network was disrupted. however, expression of other salmonella spi- secreted proteins, including two with predicted membrane-spanning domains (myc::ssec and myc::ssed), did not result in localization to the golgi or perturbation of its structure (data not shown). the distribution of myc::sseg within the golgi network was further analysed by confocal microscopy using golgi markers. cells expressing myc::sseg also showed some co-localization with the cis-golgi protein giantin. however, triple labelling with both cis-golgi and tgn markers showed that there was a greater degree of colocalization between myc::sseg and tgn than with giantin ( figure b ). expression of myc::sseg and p -gfp, a protein of the er-golgi intermediate compartment, did not result in their co-localization (data not shown). golgi membrane tubulation induced by bfa results in rapid redistribution of the cis-golgi network into the er (lippincott-schwartz et al., ) . although the tgn also undergoes tubulation, these tubules do not interact with the er but instead fuse with compartments of the early endosomal network (lippincott-schwartz et al., ; wood and brown, ) . to con®rm that myc::sseg localizes preferentially to the tgn, the kinetics of redistribution of myc::sseg were analysed after treatment with bfa. hela cells expressing myc::sseg were incubated with bfa and ®xed for immuno¯uorescence the translocation of the spi- ttss effector sifa is required for recruitment of lgp-containing vesicles and the formation of sifs, while sseg localizes scvs to the golgi network. as a consequence of this, the replicative phase of development can begin through acquisition of nutrients that enable bacterial replication, and membrane which encloses the growing population of intracellular bacteria. ee, early endosome; le, late endosome; lys, lysosome. microscopy at short time intervals thereafter. at and min after bfa treatment no signi®cant tubulation of the cis-golgi was observed. however, at the same time points, myc::sseg was found on tubulating membranes that also contained tgn (data not shown). from to min after bfa treatment, myc::sseg was still detected in tubular structures containing tgn but did not co-localize signi®cantly with giantin-containing tubules ( figure c ). as expected, a signi®cant proportion of sseg also colocalized with the early endosomal marker eea (supplementary ®gure s ). by min, the majority of giantin was redistributed in the er whereas myc::sseg remained on tgn -containing tubules (data not shown). although the two proteins did not co-localize completely on tubules (arrowheads, figure c ), the kinetics of tubulation of compartments containing myc::sseg and tgn were indistinguishable. depolymerization of microtubules with nocodazole causes slow dispersal of the golgi complex (cole et al., ) . short stacked cisternae assemble slowly at peripheral sites to form small structures that closely resemble intact golgi stacks (thyberg and moskalewski, ) . cells expressing myc::sseg were treated with nocodazole for h and then analysed by labelling myc::sseg with either giantin or tgn . there was little co-localization between myc::sseg and giantin, but a signi®cant degree of co-localization of myc::sseg and tgn was observed in many of the scattered golgi structures ( figure d ). triple labelling with anti-myc, anti-giantin and anti-tgn antibodies revealed that some of the peripheral structures containing myc::sseg were negative for both the cis-golgi and tgn markers (data not shown). collectively, these experiments show that a signi®cant proportion of sseg localizes speci®cally to the tgn when expressed ectopically in hela cells. the golgi-targeting domain of sseg sseg has two strongly predicted transmembrane domains (tmds), designated hr and hr , and a third hydrophobic region (hr ) that could also constitute a tmd ( figure a ). in addition, the n-terminal region is unusual in that of the ®rst amino acids are prolines. to identify the region(s) of sseg that confer its localization to the golgi, a series of myc-tagged truncated versions was constructed and expressed in hela cells after transfection. progressive deletions from the n-and c-termini (d , d , d , d , d , d , d ) identi®ed a amino acid region including hr that is necessary and suf®cient for golgi targeting of sseg ( figure b ). removal of either hr (d , d , d ) or the region between hr and hr (d ) resulted in a complete loss of golgi targeting and the mutant proteins were instead redistributed in a punctate manner throughout the cell ( figure b and c) . therefore, information in the second tmd and its carboxy-¯anking region are required for golgi targeting. sequence comparisons between this domain and known eukaryotic and viral golgi-targeting sequences have failed to reveal any signi®cant similarities. topology of sseg and regions required for bacterial association with the golgi the results described above show that sseg is required for the association between scvs and the golgi network, and that this protein has a novel golgi-targeting signal. therefore, one can predict that additional information must be present within sseg that localizes scvs to the golgi network. to identify regions of sseg that are exposed on the cytoplasmic face of golgi membranes, we determined the membrane topology of sseg. hela cells expressing myctagged sseg were ®rst incubated with digitonin (which renders the plasma membrane permeable to large molecules but which does not alter the integrity of secretory pathway compartments; plutner et al., ) , then labelled with anti-myc or anti-sseg antibodies in the absence of detergent. the myc epitope serves as a marker for the n-terminus of sseg and the anti-sseg antibody recognizes the c-terminus of the protein. an antibody against a luminal epitope of galactosyltransferase (galt) was used as a control for the integrity of golgi membranes. following exposure of cells to digitonin, golgi-associated sseg was readily detected by the anti-myc and anti-sseg antibodies. however, there was no labelling of galt unless the cells were exposed to triton x- , which is a non-speci®c membrane permeabilizing detergent. colabelling of galt and sseg revealed extensive co-localization of the two proteins ( figure a ). from this experiment we conclude that both n-and c-terminal domains of golgi-associated sseg are exposed on the cytoplasmic face of the membrane. it follows that sseg contains only two tmds, which are most likely hr and hr . therefore, either or both n-and c-terminal regions of the protein might be involved in localizing scvs to the golgi network. to test this, variants of sseg were expressed in hela cells and assayed for their ability to restore association of the sseg mutant strain with the golgi network. first, cells were transfected with a construct expressing full-length myc::sseg, then infected with gfp-expressing wild-type or sseg mutant bacteria for h, and examined for bacterial localization. expression of myc::sseg or mutant versions did not affect the ability of wild-type bacteria to localize ef®ciently to the golgi network ( figure b ), indicating that these proteins did not exert a dominant-negative effect on golgi targeting by wild-type bacteria. ectopically expressed myc::sseg restored golgi localization of the sseg mutant strain to the level seen with the wild-type strain ( figure b ). expression of mutants d and d , which were not golgi associated, did not rescue golgi localization to the sseg mutant. however, mutants d , d and d , which were ef®ciently golgi-localized ( figure b) , also failed to rescue the sseg mutant phenotype ( figure b ). these results demonstrate that in addition to a requirement for the golgi-targeting region of sseg, information in both the c-and n-terminal regions of the protein is required for the interaction between scvs and the golgi network. to determine if other spi- effectors might be involved in golgi-targeting, myc::sseg was tested for its ability to restore golgi localization of ssav (spi- null mutant) bacteria. in contrast to the sseg mutant, golgi localization of the ssav mutant could not be rescued by ectopic expression of sseg ( figure b ). this result suggests that other spi- translocated effectors contribute to the process of salmonella localization to the golgi network. after invasion of epithelial cells by salmonella, the scv begins to migrate to the perinuclear region, and undergoes three stages of maturation, which re¯ect selective interactions with the endocytic pathway of the host cell . in this work we have shown that these events are followed by a further, crucial phase of development, in which scvs interact with the golgi network through the action of sseg. since this stage involves bacterial replication, we refer to it as the replicative scv. therefore, maturation of the scv proceeds not only by progressive and selective interactions with the endocytic pathway but also in terms of the spatial distribution of scvs in the host cell, and their interaction with the secretory pathway. a model of scv traf®cking in relation to its intracellular localization is shown in figure c . physical continuities between scv and golgi membranes were not observed at the ultrastructural level, but it seems likely that transient interactions must occur, since intracellular bacterial replication can be inhibited either by perturbation of golgi function, or by mutation of sseg, which prevents scvs from localizing to the golgi network. the close apposition of scvs and golgi membranes, and an associated membrane fusion machinery, could enable transient fusion events between golgiderived vesicles and scvs, resulting in the acquisition of molecules required for salmonella replication. since expression of sseg in hela cells led to its concentration in the tgn, it seems likely that these vesicles are derived from late golgi compartments. in hela cells, vacuoles containing chlamydia spp. (inclusion bodies) become fusogenic with sphingomyelincontaining exocytic vesicles that traf®c between the tgn and the plasma membrane. incorporation of golgi-derived lipids is thought to provide a mechanism for growth and maintenance of the chlamydia inclusion membrane (hackstadt et al., ) . therefore it is possible that sseg could mediate a similar function in acquiring lipids for the scv membrane. bfa treatment of chlamydiainfected cells leads to smaller, morphologically abnormal inclusions, but does not affect bacterial multiplication (hackstadt et al., ) . in contrast, bfa had no effect on the integrity or morphology of the scv membrane, as judged by lamp labelling (data not shown), but strongly inhibited salmonella replication. furthermore, chlamydia inclusions do not interact with endocytic organelles (wyrick, ) , whereas the spi- effector sifa has a major role in the recruitment and probably fusion of lgpcontaining vesicles to the scv membrane (ruiz-albert et al., ) . indeed, mutation of sifa results in a strain that cannot maintain the physical integrity of its vacuolar membrane (beuzo Ân et al., ) . therefore salmonella appears to exploit late endocytic organelles as a source of membrane that is required to enclose replicating bacteria, and it seems more likely that interactions between scvs and the golgi satisfy a nutritional requirement, for example by providing carbon and/or nitrogen sources which are necessary for bacterial replication. role of sseg in bacterial association with the golgi two lines of evidence show that the spi- ttss effector protein sseg and its golgi-targeting region are necessary for s.typhimurium association with the golgi network. first, sseg mutant bacteria fail to localize to the golgi network of infected cells. second, this defect can be rescued ef®ciently by ectopic expression of full-length sseg in infected cells, but not by a version lacking the golgi-targeting domain. in infected cells, sseg was found associated with scvs and in peripheral punctate structures, which occasionally co-localized with golgi markers. how then does translocated sseg account for bacterial association with the golgi? apart from its golgi-targeting region, both the proline-rich n-terminal domain and information in the c-terminal amino acids contribute to the localization of scvs to the golgi network. furthermore, localization of sseg mutant but not spi- null mutant bacteria to the golgi can be rescued by the ectopic expression of sseg in hela cells. these results suggest the existence of another spi- effector that functions together with sseg to mediate golgi localization of scvs. alternatively the null mutant may be defective in earlier unrelated processes of vacuole maturation, on which sseg function is dependent. translocated sseg might interact with its target membrane via the golgitargeting domain, and to another effector through its n-or c-terminal regions. it is possible that sseg is a component of a multiprotein complex that mediates interactions between scvs and golgi membranes. a variety of proteins are targeted to golgi membranes by their tmds. these include integral membrane glycosyltransferases and several viral proteins. for example, the m protein of the coronavirus mouse hepatitis virus, which targets the tgn, has one signal in its tmd and a second signal in the cytoplasmic tail (gleeson, ) . in the m protein of the avian coronavirus infectious bronchitis virus, the ®rst of three tmds contains the signal that targets the protein to the golgi network (gleeson, ) . however, no similarity between sseg and either eukaryotic or prokaryotic golgi targeting sequences has been found. therefore the golgi-targeting signal present within amino acids and of sseg appears to be novel. interestingly, sseg has also been reported to be present on, and required for the formation of, sifs (guy et al., ; kuhle and hensel, ) . however, this process is likely to be independent of its interactions with the golgi network, as bfa treatment does not prevent sif formation (brumell et al., ; data not shown) . therefore, sseg might perform more than one function in the maturation of scvs. although it is clear that the salmonella spi- ttss plays an important role in intracellular replication of bacteria in epithelial cells (cirillo et al., ; bispham et al., ; beuzo Ân et al., ; paesold et al., ; this study) the functions of spi- have been mainly studied in macrophages, which are colonized during the systemic phase of the disease in mice (holden, ) . microscopic examination of s.typhimurium in primary and raw . macrophages failed to reveal signi®cant association of scvs with the golgi network. however, other evidence indicates that s.typhimurium±golgi interactions might be relevant in macrophages. first, the virulence of the sseg mutant strain is attenuated in the murine model of infection . second, the sseg mutant strain has a replication defect in raw . macrophages , and in primary peritoneal macrophages (data not shown). third, treatment of infected macrophages with bfa signi®cantly inhibited s.typhimurium replication (supplementary ®gure s ). therefore, it seems probable that s.typhimurium does interact with the golgi network in macrophages, but in a way that does not involve clustering of bacterial microcolonies within golgi membranes. it is well established that in epithelial cells, maturation of the scv is dependent on a series of selective interactions with the endocytic pathway. the major conclusion to emerge from the work described here is that while these interactions are necessary for bacterial survival, they are not suf®cient for intracellular replication: this requires scv interactions with the secretory pathway. the identi®cation of sseg as a golgi-targeting virulence protein provides the basis for further investigation into the mechanisms by which this pathogen replicates in host cells, could provide insights into the normal functioning of the golgi network and might ultimately offer novel approaches to inhibit bacterial growth in host cells. bacterial strains, plasmids and growth conditions the s.typhimurium strains used in this study were the wild-type s, and its isogenic mutant derivatives: ssav::apht (deiwick et al., ), sseg::apht ) ssei::pgp (ruiz-albert et al., and dssej (ruiz-albert et al., ) . bacteria were grown in luria bertani (lb) medium supplemented with ampicillin ( mg/ml), kanamycin ( mg/ml) or chloramphenicol ( mg/ml) for plasmidcontaining strains, as appropriate. plasmid pfvp . , carrying gfpmut a under the control of the rpsm constitutive promoter (valdivia and falkow, ) , was introduced into bacterial strains for¯uorescence visualization where indicated. the plasmids pclxsn-gfp and pclxsn-arf t n-gfp (kagan and roy, ) were provided by dr c. roy. the sar t n plasmid (ward et al., ) was obtained from dr j. lippincott-schwartz. construction of the complementing plasmid psseg and the expression plasmid pmyc::sseg are described in the supplementary data. the computer program tmpred was used to identify membrane-spanning domains within sseg (persson and argos, ) . construction of plasmids used for expression of truncated versions of sseg are described in the supplementary data. the mouse anti-lamp antibody h a was obtained from the developmental studies hybridoma bank developed under the auspices of the nichd, maintained by the university of iowa and was used at a dilution of : . anti-salmonella goat antibody csa- (kirkegaard and perry laboratories) was used at a dilution of : . the rabbit antibodies anti-gm and anti-giantin were obtained from dr a.de matteis and used at a dilution of : and : , respectively. the mouse anti-gm antibody (transduction laboratories) was used at a dilution of : . the sheep anti-human tgn antibody (serotec) was used at a dilution of : . a rabbit antibody against human tgn (ponnambalam et al., ) was obtained from dr g.banting and was used at the dilution of : . the mouse anti-c-myc antibody e (santa cruz biotechnology) was used at a dilution of : . an antibody to galactosyltransferase was obtained from dr e.berger and was used at a dilution of : (berger et al., ) . rhodamine red x (rrx)-, cyanine -, cyanine (cy )-and aminomethylcoumarin acetate-conjugated donkey anti-mouse, anti-rabbit and anti-goat antibodies (jackson immunoresearch laboratories) were used at a dilution of : . rrxand cy -conjugated donkey anti-sheep antibodies (jackson immunoresearch laboratories) were used at dilutions of : and : , respectively. bfa (sigma) was used at mg/ml. at this concentration bfa was not cytotoxic to hela cells and did not affect s.typhimurium growth in lb medium over h. nocodazole (calbiochem) was used at a ®nal concentration of mg/ml. a peptide with the sequence ssplyrllaqvtpeqrape corresponding to the last amino acids of sseg was conjugated to keyhole limpet haemocyanin and this was used to immunize rabbits. polyclonal rabbit serum was af®nity puri®ed using af®-gel (biorad), to which the peptide had been coupled, following the manufacturer's procedure. cell culture hela (clone htta ) cells were kindly provided by dr s.me Âresse. the int cell line u was a gift from dr a.l.servin. cells were grown in dulbecco's modi®ed eagle's medium supplemented with % fetal calf serum and mm glutamine at °c in % co . hela cells were infected with equivalent numbers of exponential phase s.typhimurium strains, as described previously (beuzo Ân et al., ) . in order to follow a synchronized population of bacteria, hela cells were washed after min of exposure to s.typhimurium and subsequently incubated in medium containing gentamicin to kill extracellular bacteria. for enumeration of intracellular bacteria, hela cells were washed three times with phosphate-buffered saline, lysed with . % triton x- for min and a dilution series was plated onto lb agar. for electron microscopy, samples were prepared as described previously for hela cells . for immuno¯uorescence, cell monolayers were ®xed and labelled as described previously . for the af®nity puri®ed anti-sseg antibody, coverslips were permeabilized for min with . % triton x- and then labelled as described above, in the absence of saponin. samples were analysed using a confocal laser scanning microscope (lsm ; zeiss). hela cells were transiently transfected by the calcium phosphate dna precipitation method as described previously (ruiz-albert et al., ) . bacterial infection of transfected hela cells was performed as described above, h after dna addition, and cells were ®xed h after dna addition ( h after infection). to determine the topology of myc::sseg, hela cells were transfected for h with mg of dna using polyfect (quiagen) and then permeabilized with mg/ml digitonin (calbiochem) for min on ice (plutner et al., ) . cells were then ®xed with pfa and labelled with antibodies in the absence of detergent. quanti®cation of salmonella association with the golgi network quanti®cation of the number of bacteria associated with the golgi network was performed by confocal microscopy. only bacteria that were either completely or at least % surrounded by the labelled golgi marker were counted as being golgi associated. bacterial clusters that were found adjacent to the golgi but did not ful®l the above criteria were counted as non-associated. at least host cells, corresponding to more than bacteria, were scored blind in each experiment and all experiments were repeated three times. supplementary data are available at the embo journal online. foundation for science and technology. this work was also supported by a grant from the medical research council, uk. monoclonal antibodies to soluble, human milk galactosyltransferase (lactose synthase a protein) salmonella maintains the integrity of its intracellular vacuole through the action of sifa growth and killing of a salmonella enterica serovar typhimurium sifa mutant strain in the cytosol of different host cell lines. microbiology, , ± characterization of salmonella-induced ®laments (sifs) reveals a delayed interaction between salmonella-containing vacuoles and late endocytic compartments brefeldin a: the advantage of being uncompetitive macrophage-dependent induction of the salmonella pathogenicity island type iii secretion system and its role in intracellular survival golgi dispersal during microtubule disruption: regeneration of golgi stacks at peripheral endoplasmic reticulum exit sites mutations in salmonella pathogenicity island (spi ) genes affecting transcription of spi genes and resistance to antimicrobial agents targeting of salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose -phosphate receptors salmonella induces the formation of ®lamentous structures containing lysosomal membrane glycoproteins in epithelial cells targeting of proteins to the golgi apparatus aggregation of host endosomes by salmonella requires spi translocation of ssefg and involves spvr and the fms-aroe intragenic region chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the golgi apparatus to the plasma membrane genes encoding putative effector proteins of the type iii secretion system of salmonella pathogenicity island are required for bacterial virulence and proliferation in macrophages traf®cking of the salmonella vacuole in macrophages legionella phagosomes intercept vesicular traf®c from endoplasmic reticulum exit sites ssef and sseg are translocated effectors of the type iii secretion system of salmonella pathogenicity island that modulate aggregation of endosomal compartments rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er microtubuledependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway brefeldin a's effects on endosomes, lysosomes, and the tgn suggest a general mechanism for regulating organelle structure and membrane traf®c the rab gtpase controls the maturation of salmonella typhimuriumcontaining vacuoles in hela cells genes in the salmonella pathogenicity island and the salmonella virulence plasmid are essential for salmonella-induced apoptosis in intestinal epithelial cells prediction of transmembrane segments in proteins utilising multiple sequence alignments morphological analysis of protein transport from the er to golgi membranes in digitonin-permeabilized cells: role of the p containing compartment the unique traf®cking pattern of salmonella typhimurium-containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry complementary activities of ssej and sifa regulate dynamics of the salmonella typhimurium vacuolar membrane biogenesis of salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway identi®cation of a salmonella virulence gene required for formation of ®lamentous structures containing lysosomal membrane glycoproteins within epithelial cells a salmonella virulence protein that inhibits cellular traf®cking maintenance of golgi structure and function depends on the integrity of er export functions and effectors of the salmonella pathogenicity island type iii secretion system the morphology but not the function of endosomes and lysosomes is altered by brefeldin a intracellular survival by chlamydia we are very grateful to gustavo egea, paul freemont and gareth grif®ths for helpful suggestions, and to jennifer lippincott-schwartz, antonietta de matteis, george banting, craig roy, eric berger, stephane me Âresse and alain servin for providing reagents. we thank dave goulding for electron microscopy, sarah gilliland for technical assistance and javier ruiz-albert for technical advice. we are grateful to christoph tang, emmanuelle caron and members of the holden laboratory for critical review. s.p.s. is supported by a fellowship from the portuguese golgi targeting by salmonella key: cord- -xxlcdbqi authors: nan title: the organization of endoplasmic reticulum export complexes date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: xxlcdbqi export of cargo from the er occurs through the formation of - nm copii-coated vesicular carriers. we have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of er export sites in vivo and in vitro. we find that er buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. the basic organization of an export complex can be divided into an active copii-containing budding zone on a single er cisterna, which is adjacent to budding zones found on distantly connected er cisternae. these budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain copi coats involved in retrograde transport. vesicles within these export complexes contain concentrated cargo molecules. the structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. we conclude that budding from the er through recruitment of copii is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by copi. abstract. export of cargo from the er occurs through the formation of - -nm copii-coated vesicular carriers. we have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of er export sites in vivo and in vitro. we find that er buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. the basic organization of an export complex can be divided into an active copiicontaining budding zone on a single er cisterna, which is adjacent to budding zones found on distantly connected er cisternae. these budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain copi coats involved in retrograde transport. vesicles within these export complexes contain concentrated cargo molecules. the structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. we conclude that budding from the er through recruitment of copi! is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by copi. xport of protein from the er is the first step in the vectorial movement of cargo through compartments of the secretory pathway of eukaryotic cells. pioneering studies by palade ( ) established that the site of exit from the rer in pancreatic acinar cells is through transitional elements, a region of partly rough, partly smooth tubular er juxtaposed to the cis face of the golgi stack. morphologically, er-derived vesicles are - nm in diameter and contain an electron-dense coat when viewed using transmission electron microscopy (ziegel and dalton, ) . this characteristic coat contains copii components that are assembled in response to the activation of the sarl gtpase, a machinery now recognized to be evolutionarily conserved in yeast and mammalian cells (for review see barlowe, ) . while the formation of er to golgi carrier vesicles in secretory tissues is largely confined to the transitional region facing the juxtanuclear golgi apparatus (palade, ) , studies in other cell lines have shown that export from the er can originate from multiple sites that appear randomly distributed throughout the cytoplasm and, in most instances, distant from the golgi complex. the relationship of these peripheral sites to the transitional region found in secretory cells is unknown, although they are now recognized to consist of clusters of small vesicles and tubular elements (saraste and kuismanen, ; schweizer et al., ; saraste and svensson, ; lotti et al., ) re-ferred to as vesicular tubular clusters (vtcs) . while vtcs are readily detectable at °c in vivo (saraste and kuismanen, ; saraste and svensson, ) and at °c in vitro (plutner et al., ; pind et al., a) , visualization of these structures can be markedly enhanced by incubation of cells at reduced temperature ( °- °c) (saraste and kuismanen, ) , presumably due to a rate-limiting step in membrane flow through these intermediates. elements of vtcs lack luminal continuity with the er (saraste and svensson, ; balch et al., ; connolly et al., ) , although tubular extensions of er into these structures have been observed (stinchcombe et al., ) , particularly in cells infected with certain viruses (tooze et al., ; krijnse-locker et al., ) , reinforcing their close relationship to er export. vtcs are dynamic structures with varied morphology in different cell types. in the past, vtcs have been suggested to be the site of o-glycosylation (tooze et al., ) , acylation (rizzolo et al., ) , and generation of the mannose- -phosphate signal for lysosomal protein targeting (pelham, ) . several endogenous proteins serve as useful markers for vtcs. these include the small gtpase rab (chavrier et al., ) , the transmembrane protein p in rat cells or its human homologue p (schweizer et al., ; saraste and svensson, ) , which actively cycle between the er and vtcs, and the copi subunit -cop (oprins et al., ; pepperkok et al., ; pind et al., a; aridor et al., ) . vtcs play a pivotal role in the segregation of anterograde and retrograde transported proteins (aridor et al., ; tang et al., ) . segregation is believed to involve the copi coat complex whose assembly is driven through activation of the arf gtpase (aridor et al., ; letourneur et al., ) . while vtc composition and function have been described qualitatively through use of immunofluorescence (lotti et al., ; aridor et al., ; lippincott-schwartz et al., ) and immunoperoxidase (connolly et al., ; stinchcombe et al., ) approaches, the topology of er budding sites and, in particular, their relationship to vtcs have not been studied quantitatively at high resolution. in the present paper, we use both transmission tem and immunoelectron microscopy in conjunction with quick-freeze, deep-etch methods to reconstruct these relationships morphometrically and to confirm that cargo is concentrated during vesicle budding. we find that almost all er buds detected in the cell were concentrated in regional loci facing vtcs, which had a characteristic diameter and vesicular-tubular composition. as such, the topological organization of buds and vtcs defines a morphological unit of function, which we refer to as export complexes. we conclude that budding from the er is not evenly distributed along the membrane, but is confined to specific export sites. these are highly enriched in copii and copi transport components and are likely to promote the coupling of anterograde and retrograde transport. a polyclonal antibody specific for yeast sec p that cross-reacts with a mammalian homologue (orci et al., ) was obtained from r. schekman (university of california, berkeley). affinity-purified antibodies specific for yeast secl p that cross-reacts with a mammalian homologue (shaywitz et al., ) were a generous gift of c. kaiser (massachusetts institute of technology, cambridge, ma). a hybridoma cell line expressing an mab specific for the carboxyl terminus of vesicular stomafitis virus glycoprotein (vsv-g) (p d ) was provided by t. kreis (university of geneva, switzerland) (kreis, ) . secondary antibodies were obtained from the following sources: texas red--conjugated goat anti-mouse igg from zymed laboratories (south san francisco, ca), nm gold--conjugated goat anti-mouse and anti-rabbit antibodies from jackson immunoresearch laboratories (west groven, pa), and nm gold--conjugated goat anti-mouse antibodies from amersham corp. (arlington heights, il). sarl wild type and the h g and t n mutant proteins were prepared as described (aridor et al., ) . all other reagents, except where indicated, were obtained from sigma chemical co. (st. louis, mo). normal rat kidney (nrk) and rat basophilic leukemia (rbl) cells (rbl- h ) were maintained in monolayer culture in a-mem supplemented with penicillin, streptomycin, and % (nrk) or % (rbl) fbs (gemini bioproducts, calabasas, ca) as described (plutner eta] ., ). ts -vsv (lafay, ) was propagated in bhk- ceils as described (beckers et al., ) . cells were infected with ts -vsv at a multiplicity of - plaque-forming units per cell as described (davidson and balch, ) . cells were fixed in . % glutaraldehyde (ga) in pbs for h at room temperature (rt), scraped in ga, and pelleted at , g in a microcentrifuge for rain. the tight pellet was washed in veronal-acetate buffer (ph . ) and stained in % buffered oso for h at rt. after washing in veronalacetate buffer, pellets were stained en bloc with % uranyl acetate in veronal-acetate buffer, dehydrated in alcohol and acetone, and embedded in epon (electron microscopy, sciences, fort washington, pa) . for serial sectioning, the cell pellet was cut with a glass knife followed by trimming of the resulting block to obtain an ~ × ~xm pyramid. a ribbon of - consecutive sections of nm in thickness was cut with a diamond knife on reichert ultramicrotome e and transferred to a single × . mm slot grid (electron microscopy sciences) precoated with a formvar/carbon film. sections were counterstained with a saturated solution of uranyl acetate in methanol for rain at rt and reynold's lead citrate for min. budding structures on the er and vtcs were followed in consecutive sections, and images were overlaid to reconstruct continuity between structures. stereological parameters. an estimation of the mean volume of the cells was performed by two independent methods (baddeley et al., ; griffiths et al., ) . in the first case, rbl cells were grown on glass coverslips. cells were fixed with . % ga in pbs, ph . , overnight. the cells were then viewed and photographed under phase contrast with an axiophot (zeiss, oberkochen, germany) with a × objective lens. the images were enlarged photographically times, and the mean surface of the cell projection was determined by the point-counting method (weibel, ) using a -ram square lattice grid. approximately , rbl cells were used for the estimation of the mean surface of the cell projection onto a planar surface. the cells on coverslips were stained and embedded in epon as described above. after polymerization, the coverslips were detached from the resin by plunging into liquid nitrogen. thin-strips of embedded cells in resin were then combined face to face and reembedded on top of an epon block to produce "vertical" sections (griffiths et al., ) . images of cells were taken with a transmission electron microscope ( ex; jeol usa, peabody, ma) and photoenlarged (× , total magnification), and the mean height of ~ cells in a monolayer was measured by the point-counting method (weibel, ) . the mean cell volume (vc.r.) was then determined according to formula: where sc.p. is the mean surface of the cell projection (determined by the phase-contrast light microscopy), and hc. is the mean height of the cell (determined by tem). for estimation of the mean cell volume using the second method, nrk and rbl cells were grown on -mm tissue-culture dishes (costar corp., cambridge, ma), fixed in the dish in . % ga in pbs for h at rt, and processed for tem as described above. the cell pellet was cut with a diamond knife on a reichert ultramicrotome e. - -nm sections were counterstained as described above. approximately , rbl and nrk cells were randomly chosen and photoenlarged to a magnification of , . the mean surface of the cells on sections were measured by the point-counting method (weibel, ) . the mean cell volume was found according to formula (weibel and gomez, ) : where sc.s. is the mean cell surface on thin-section, the surface to volume ratio was found according to formula: where i is the number of intersections on a grid, p is the number of points on the grid, and d is the distance between the points (weibel, ) . both procedures yielded nearly identical results for rbl cells, the values obtained from the second procedure are reported in table i . the stereological parameters of nuclei, er, and golgi apparatus were determined using the same sections. randomly taken cell contours were enlarged to a total magnification of , as described above. using the point-counting method, we determined the nucleus volume to cell volume ratio and a nucleus surface to nucleus volume ratio as described (weibel, ) . randomly chosen fields of cells containing er or golgi membranes were magnified to , to establish the er(golgi) volume to cell cytoplasm volume ratio by the point-counting method (weibel, ) . some of these images were photoenlarged to , to determine the er(golgi) surface to volume ratio. the er(golgi) surface was found by formula . corrections of bias due to section thickness were done as described (weibel and paumgartner, ) . for the er volume, the correction factor was . and . for the er surface. assuming that two-thirds of the golgi complex was composed of cisternae and one-third of tubules, the correction factors for golgi volume and surface were estimated to be . and . , respectively. estimation of number and deasity of total er-derived buds. to estimate the number er-derived buds, we used two distinct criteria: (a) a bud was considered an elevation on the surface of the er with a width of - nm and covered with a characteristic coat on the external leaflet of the membrane: and (b) buds had a direct connection with the er membrane and were extruded from the membrane by at least % of their circumference. the total number of er buds per cell was determined as described (lucocq et al., ) . to obtain this value, we multiplied the total number of er buds detected in thin-secfions of ~ cells by the mean volume of cells and divided that value by the total volume contributed by the ~ cells. the latter value was found by multiplying the thickness of a section by the total surface area of the ~ cell sections in which er buds were counted. by using relatively thick sections corresponding to the average diameter of an er bud and the above two criteria, we were able to avoid double counting most of the er buds present on consecutive thin sections. to establish this point independently, we performed a separate experiment in which the total number of er buds was counted on several stacks of serial thin-sections. by comparing the number of er buds determined by this reconstruction approach to the number determined by counting of individual sections (indirect method), we found that overestimation by the latter technique was < - %. hence, both methods could be used interchangeably. we also found that the indirect method showed a high reproducibility. in spite of the high degree of er bud enrichment in local zones, the comparison of estimates of the total number of buds per cells between groups of as few as randomly taken cells with a total surface of section of , tj.m gave a value of variance of less than % from one group to another. in addition, in four different experiments, the value of the total number of buds per cell was found to be the same. because of technical simplicity, we routinely used the indirect method to estimate the average number of buds per cell. buds in the golgiexclusion zone. the golgi exclusion zone is defined as the golgi-containing region found in the pericentrosomal region of rbl cells and includes directly adjacent bud-bearing cisternae of the er. the surface area of the golgi exclusion zone in each individual section within a stack of - consecutive sections (referred to as a disector [sterio, ; lucocq et al ] ) was determined by the point-counting method (weibel, ) , and the mean surface area in each disector was calculated for different cells (range from - ~tm ). to find the volume of the golgi exclusion zone included in the disector, the mean surface area of the golgi exclusion zone in each disector was multiplied by the thickness of the section and the number of individual sections ( to ixm for individual disectors). the total number of er buds within each disector of a given cell was found by reconstruction from serial sections. the volumetric density of er buds was calculated by dividing the number of buds found within each disector by the volume of the disector. the total volume and surface of er membranes in the golgi exclusion zone was found by the point-counting method (weibel, ) . by dividing the total number of er buds by the er surface within the golgi exclusion zone, we calculated the density of er buds in this region. buds outside of the golgi exclusion zone. the density of er buds outside of the golgi exclusion zone was found according to the procedure described above for the golgi exclusion zone. buds in export complexes. the local density of buds on individual cisternae associated with export complexes was determined as follows: using a stack of sequential serial sections, we followed one continuous bud-bearing zone of er membrane that contained at least four buds. the distances between the most distant er buds in the stack were directly measured in both the plane of the section and the depth of the stack. these two distances were multiplied by one another to get a surface area of the plane of the er bud-bearing region. the total number of er buds in such a zone (obtained by reconstruction as described above for the golgi exclusion zone) was then divided by the area of the bud-bearing zone to determine a local er bud surface density. es.lmation of number of vtcs. the number of vtcs per cell was determined using the disector method (sterio, ) as described (lucocq et al., ) . sections of random cells were photographed at a calibrated magnification of , throughout - consecutive sections in which they were present. each "end section" was designated the "lookup" section, and all clusters present in the other sections, but not in the look-up section, were counted (q). then the volume of disector (vdi~) was determined by multiplying the average surface of individual cells in the stack found by the point-counting method by the depth of the dissector, which is equal to the thickness of the section multiplied by the number of sections in the disector. the total number of clusters in individual ceils (nclustcrs/cell) was determined by the formula, ( ) to determine the number of elements in vtcs, the value reported assumes that all elements found in consecutive sections are discontinuous with one another. this value is likely to be an overestimate, as some of the tubular structures within vtcs may extend across several sections. the number of elements in vtcs was determined by direct counting of individual profiles on each serial section. these were summed throughout all sections through a given vtc to obtain a total value. probability measurements. to estimate the probability of a bud having proximity to a second bud in the cell, images of serial sections were photoenlarged to , to reconstruct a section of the cell in three dimensions. randomly chosen er buds were taken as the center of reference (referred to as reference buds), and the distance between each individual reference bud and other er buds in the same and consecutive serial sections encompassing up to . p.m distance above and below the reference bud was measured directly. a series of concentric shells with a volume equal to the volume of the most internal sphere having a . - xm diam (corresponding to a volume of . i~m ) was constructed around the reference bud. subsequently, each measured bud was assigned to a shell with its distance from the reference bud being that of the corresponding outer diameter of the shell. the probability of finding a bud within a particular shell was determined by dividing the number of positive shells by the number of reference buds, and the value was reported as a percentage. sem. statistical calculations were performed by determining the sem for the pooled stereological data for each condition as described in the results. nrk cells grown on -mm tissue-culture dishes were infected with vesicular stomatitis virus (strain ts ), postinfected for h at . °c, and permeabilized as described (plutner et al.. ) . after incubation at the permissive temperature ( °c) as described in the results, the cells were fixed for min with % paraformaldehyde and . % ga in pbs (ph . ), washed for l rain in pbs containing . m glycine, scraped, mixed with a preheated ( °c) % gelatin in pbs, and centrifuged at , g in a microcentrifuge for min. cells embedded in gelatin were cooled on ice, and a solid pellet was cut into i-ram-wide cubes. after overnight cryoprotection by infiltration with a mixture of . m sucrose in . m phosphate buffer (ph . ) containing % polyvinyl pyrrolidene, the cubes were mounted on aluminum nails and frozen in liquid nitrogen. ultrathin cryosections cut on a reichert ultracut e, equipped with a fc- cryoattachment, were picked up with . m sucrose- % bsa in pbs and collected on formvar/carbon-coated nickel grids. sections were then quenched in . m glycine in pbs, incubated for min in % fbs-pbs at rt, and for - h with primary antibodies diluted in % fbs-pbs antibody. excess primary antibody was removed by multiple rinses in % fbs-pbs, followed by transfer of the section to a drop of % fbs-pbs containing or nm gold--conjugated anti-rabbit antibodies. after a -h incubation at rt, the grids were washed in double-distilled water and stained in % neutral uranyl acetate ( rain), followed by embedment in . % polyvinyl alcohol/ . % methyl cellulose~ containing . % uranyl acetate. no labeling was observed in controls in which primary antibodies were omitted. nrk cells were infected with ts vsv as described above. after digitonin permeabilization (plutner et al., ) and incubation in vitro as described in the results, cells were fixed with . % ga/ % paraforrnaldehyde for rain. cells were washed three times with pbs, quenched with . m glycine in % fbs/pbs for rain, and incubated overnight with an anti-vsv-g cytoplasmic tail mab (p d ) (kreis, ) . cells were washed twice with fbs/pbs, followed by incubation with rabbit anti-mouse antibodies for l- h and with or nm gold conjugated to anti-rabbit antibodies for h. excess unbound antibodies were removed by washing, and the cells were fixed with . % ga for rain. cells were scraped, pelleted, and processed for epon embedding. for three-dimensional visualization using quick-freeze, deep etch replicas, immunolabeling was performed on glass coverslips. for quantitation, the epon-embedded cell pellet was cut as described above. images (xl , ) were scanned into a computer, and membrane outlines of the er and vtcs were measured using the program nih image, version . . the linear density of gold particles corresponding to vsv-g was determined as described . for determination of the distribution of vesicles and vtcs, nrk cells grown on glass coverslips were permeabilized and incubated as described in the results, fixed and labeled for vsv-g using the immunodiffusion protocol, and embedded in epon on the glass coverslip as described above. after detachment from glass, thin layers of cells embedded in epon were sandwiched against each other and reembedded in epon. vertical sections of cells were prepared and counterstained as described above. er to golgi intermediates were identified based on either the presence of gold particles corresponding to vsv-g or on their characteristic morphology as described in the results. after incubation in vitro as indicated in the results, semi-intact cells grown on × mm glass coverslips were fixed in . % ga in pbs for h, washed in pbs, and divided into small pieces (~ mm ). coverslips were rinsed exhaustively in double-distilled water, followed by a rinse with % methanol in water, and quick-frozen using a liquid nitrogen-cooled copper block gravity press (hitek, benicia, ca). the cells were fractured with a razor blade under liquid nitrogen, freeze dried in a vacuum evaporator ( ; balzers, inc., lichtenstein) and replicated with ~ nm of platinum that was rotary deposited from ° above the horizontal. the replica was then reinforced with ~ /~ of carbon using an electron gun at an angle of ° to the horizontal. a drop of % colloidion solution was applied on the replica membrane. the coverslips were detached in a % solution of hydrofluoric acid, and cells were dissolved in chlorox. after washing, replicas were transferred to formvar-coated copper grids, the colloidion film on replicas was dissolved with amylacetate, and images were examined using tem. the basic stereological parameters of the rbl and nrk cell line used in these studies are shown in table i . to morphometrically evaluate the distribution of export sites, er-budding structures were identified as an elevation on the surface of the er with a width of - nm, extruded from the membrane by at least % of their diameter, and covered with a distinctive electron-dense coat ( fig. - , arrowheads). budding sites emanate from three locations in the cell including (a) those associated with the nuclear envelope ( fig. ) , (b) those associated with the golgi apparatus that are analogous in structure to classical er transitional elements (palade, ; sesso et al., ) (fig. ) , and (c) those found in more peripheral regions that lack detectable golgi (fig. ) . buds found at the tip of tubular projections from the surface of the er had an average diameter of ± nm. although tubular projections were generally shorter than nm (fig. , section ; arrowheads), they could be as long as nm based on reconstruction from serial-thin sections. buds were covered with an ~ - -nm-thick electron-dense coat. on grazing sections and at high magnification, the coat of er buds possessed a lattice-like appearance due to a semi-regular array of - -nm elongated particles ( fig. , inset) . these coats are similar to those observed in pancreatic acinar cells (merisko et al., ) . based on analysis of random sections through ~ cells (see materials and methods), the average number of buds in a cell was found to be ± . % of budding profiles were found on er tubules located in the vicinity of golgi complexes, whereas % were located in regions without noticeable juxtaposition to golgi. % of total buds were observed emerging from the nuclear envelope. thus, it is apparent that a substantial level of membrane exiting the er appears to do so from sites distant from golgi elements. the overall average density of total er buds based on the cross-sectional volume of the cytoplasm was found to be . buds per pom , or . buds per ixm of total er surface. however, the average density of er-budding profiles found within the pericentrosomal area containing the golgi apparatus (referred to as the golgi exclusion zone) was five-to sevenfold higher ( . buds per txm of cytoplasm or . buds per ixm of er surface) than the average value found on the total er membrane. outside of the golgi region, the overall mean density was . - -fold lower ( . buds per ixm cytoplasm or . buds per ixm of er surface) than the average values found for the cell. the markedly increased budding density around the golgi apparatus is consistent with the highly focused export activity observed from er transitional elements present in the golgi region of pancreatic acinar cells (palade, ) . figure . export sites adjacent to the nuclear envelope of rbl cells. six consecutive serial sections through a vtc adjacent to the nuclear envelope. er buds (arrowheads) emerging from the nuclear envelope (section ) and parallel er membrane (stars in section -- ) are facing vtcs. (inset) higher magnification view of two er buds. the slice of the section presented in the inset encompasses either only the coat (right), or both the coat and the membrane and the luminal part of a bud (left). individual - -nm electron-dense particles arranged in a semiregular pattern (arrows.) notice the same appearance of the coat under lower magnification of serial sections that contain a honeycombed appearance consisting of a semiregular array of electron-dense particles (arrows). adjacent to buds, we observe a typical pleomorphic element (large asterisk in section ) within a vtc that has numerous tubular projections (small asterisk) in adjacent thin-sections (small asterisks in sections and ), indicative of its fenestrated structure. tubules in the fenestrated elements of the vtc possess a dark dense coat (arrows in sections and ) typical of those found on golgi compartments and are readily distinguishable from the alveolate coat associated with copii buds emanating from the er. bar, . ixm. figure . export complexes adjacent to the pericentrosomal golgi region of rbl cells. consecutive serial sections through three (a, b, and c) golgi-adjacent export complexes (encircled by dotted lines) are shown with er buds (arrowheads). export complex a contains er-derived buds, export complex b contains buds, and export complex c contains seven buds. note the characteristic cup-shaped appearance of er bud-bearing zones especially evident for export complex b. to develop a detailed understanding of the topological organization of e r export throughout the cytoplasm, we carried out a morphological reconstruction of those sites that were not adjacent to golgi elements, referred to as peripheral sites. a n analysis of peripheral sites allows us to discriminate the structure of er-derived intermediates from that of the fenestrated cis-golgi network (cgn), which is always associated with er buds near the golgi. er buds on peripheral sites (fig. ) typically emanated from short stretches of er membrane. these regions were separated by long distances from other similar budding foci. frequently, er buds found on different cisternae were closely juxtaposed and faced each other. these features are more evident in fig. (a and b) , which presents an overlapping reconstruction of serial sections of the peripheral site shown in fig. . budding profiles (blue) pro-trude from the er (green) into a central region containing a collection of vesicles and tubular elements comprising vtcs (red). this typical organization of peripheral sites is also characteristic of budding sites associated with the nuclear envelope (fig. ) and budding sites adjacent to the golgi stack (fig. ) . the close topological relationship between er buds and distinct vtcs suggests that these structures function as a compact morphological unit that we now refer to in its entirety as an export complex. morphometric analysis of peripheral export complexes revealed that these sites typically contained two to six buds emanating from the er, although this could approach a value of for some exceptional, larger clusters ( fig. a, closed circles) . these sites had an average number of buds per site of . ___ . (table ii) , a value that was slightly smaller than the average number of buds per site found in export complexes adjacent to golgi ( . +_ . buds per site; table ii) . by including only er-budding profiles facing vtcs, we were able to estimate a "local" bud density in these export complexes. on average, this value was . +__ . buds per i~m of er surface, which, on a relative scale, is times higher than the average bud density found on the total er surface ( . buds per ixm ) and approximately five times higher than the bud density found on the surface of the er within the golgi exclusion zone. the apparent nonrandom distribution of er buds led us to quantitatively estimate the probability of a given bud having proximity to a second bud in the cell. for this purpose, we constructed a series of concentric shells of equal volume that radiate outward from the center of randomly chosen buds. the radius of the first internal shell was arbitrarily assigned a value of . txm to encompass the entire tip of a budding structure. each increment in the diameter of successive shells extending outward from the first shell progressively decreased in dimension to encompass the same volume in three-dimensional space. given the average number of buds in a cell (~ ), the volume of cell cytoplasm ( i~m ), and the volume of a shell ( . × . i~m ), if buds assumed a strictly random distribution, then the probability of encountering another bud in a given concentric shell would remain equal with a value of . % ( fig. , diamonds) . however, if buds were confined to regional foci, the probability of encountering a second bud would be high in the first series of concentric shells, and then fall off very rapidly with increasing distance in threedimensional space. the results of such an analysis are shown in fig. where we have plotted the probability of encountering a bud relative to its location in sequential concentric shells of equal volume (fig. , open circles) , or relative to the diameter of the outermost surface of a given shell in which a bud is found (fig. , closed circles) . it is clear that the distribution did not follow that predicted for a strictly random budding event. the probability of detecting a second bud within the first consecutive shells having up to a . - ~m outer shell diameter markedly exceeded that of a random distribution (fig. ) . after a plateau at a value similar to that calculated for a random distribution (up to an outer diameter of . ixm or shells), the probability fell dramatically within the three-dimensional space defined by the outermost shell examined (~ . txm). we conclude that budding is not a random event along the surface of the er, but rather is remarkably restricted to regional hot spots of budding activity associated with export complexes. as illustrated in fig. , er-budding profles predomi-nately faced into an area filled with vtcs in vivo. vtcs, defined as a group of four or more - -nm vesicular profiles with a characteristic shape and coat, were never detected in regions lacking adjacent er buds. the membrane profiles of vtcs were distinct from the er and found to be more variable in size compared to the rather homogeneous appearance of buds emanating from the er surface. the average diameter of vtcs based on serialthin sections was found to be ~ . cm (table ii) with a range that varied from ~ . ~m to > ~m (fig. b) . although technical limitations do not allow us to make a complete three-dimensional reconstruction of membrane continuities between elements of vtcs, individual profiles often appeared to be continuous in several consecutive sections and consisted of short tubules that could be connected together in a more fenestrated structure (fig. , asterisks) . tubules within vtcs characteristically had a dense copi-like coat at their tips (figs. and , arrows) (melancon et al., ; orci et al., a) . the presence of a copi-like coat within vtcs is consistent with the numerous morphological studies that have demonstrated that these pre-golgi intermediates are a major site of copi localization when examined using indirect immunofluorescence (lippincott-schwartz, ; aridor et al., ) or tem (oprins et al., ; pind et al., a; griffiths et al., b) . to estimate the total number of elements within a vtc, we assumed that each profile appearing in an individual section was, in fact, a separate vesicle or tubule. from this assumption, the average number of elements was determined to be +__ (table ii) . clusters generally contained between and vesicular-tubular elements, although some of the clusters contained as many as elements (fig. c) . although these values are undoubtedly an overestimate of the total number of luminally distinct elements, given that tubules within vtcs are likely to extend across several consecutive thin-sections, they serve as a useful numerical approximation for the relative size and composition of vtcs. when we compared the number of elements found in peripheral vs golgi-adjacent vtcs, we observed very similar values, suggesting they are of comparable size (fig. c) . whereas peripheral vtcs were generally spherical in shape, those adjacent to golgi possessed a semispherical shape with the cgn occupying one hemisphere, and the other hemispheres facing er membranes. using the disector method to estimate the total number of vtcs per cell (sterio, ) (see materials and methods), we found an average value of _ clusters per cell. this level is similar to that observed using more indirect tem methods (buccione et al., ) or indirect immunofluorescence in other cell lines (plutner et al., ; aridor et al., ) . to correlate our morphological observations with previous in vitro biochemical studies, we used semi-intact nrk cells to examine the potential role of copii components in the formation of er-derived buds and vtcs (plutner et al., ; peter et al., ; balch et al., ; pind et al., b) . semi-intact cells incubated for min at °c in figure . three-dimensional reconstruction of a peripheral export complex. membrane contours shown in fig. are illustrated at a magnification of , . transparencies containing the membrane contours were scanned, overlayed, and coaligned based on the position of mitochrondria and er. green denotes er cisternae, blue denotes er buds, and red denotes tubules and vesicles of vtcs. vesicular membrane contours within the vtc whose luminal continuity to the surrounding er membranes was evident in consecutive sections were denoted in green. more intense shades of the same color reflect distance from the uppermost section. the alveolate coats of er buds, where evident, are dictated by stipples. images are presented either individually with the section number indicated (bottom two rows), or as an overlay containing four images (left) for clarity or all eight images (right). the resulting reconstruction shows the clustered structure of a typical vtc, surrounded by er-bearing buds occasionally penetrating the periphery of a vtc, as evidenced by the coated portions of er tubules (blue) adjacent to vtc tubules (red). the absence of cytosol failed to generate detectable vtcs (not shown). this result is consistent with the fact that preexisting vtcs are unstable during permeabilization (aridot et al., ) and that cytosol contains essential soluble components of the copii machinery required for the export of cargo from the e r (barlowe et al., ; kuge et al., ) . vtcs generated in vitro in the presence of cytosol are nearly identical to those observed in vivo. we have previously shown that they are composed of a compact network of tubules and vesicles that lack direct luminal connections to e r membranes and frequently label positively for b-cop using immunoelectron microscopy pind et al., a) . however, er-budding profiles, like those observed adjacent to vtcs in vivo (figs. - , arrowheads), were rarely observed in vitro. since previous studies used mild fixation conditions in conjunction with an immunodiffusion procedure to label vsv-g in vtcs , we reasoned that er buds may be labile structures. we therefore applied more stringent fixation and embedding conditions to preserve ultrastrucrural details (see materials and methods). under these conditions, er buds were observed that had a characteristic coat resembling those found in vivo and were only detected adjacent to vtcs (not shown), suggesting that even in semi-intact ceils, budding is restricted to specialized regions of the er, to examine the formation of buds and their relationship to vtcs in vitro, we made use of the nonhydrolyzable analog of gtp, gtp~/s, which permanently activates sarl and other gtpases, leading to stable coat assembly and accumulation of buds. interestingly, we observed for the first time using strong fixation conditions that nascent budding profiles generated in the presence of gtp~/s had not only a cluster appearance (pind et al., a) , but also frequently had a distinctive "beaded necklace" appearance with each vesicle being a bead (fig. , b and c) . the vesicles were nearly identical in size but lacked luminal continuity. these strings of vesicles were similar to the shorter necklaces sometimes observed in vivo under normal incubation conditions (fig. a) . these necklaces were confined to only a small fraction of the total er surface. by analyzing sections through > individual necklaces, we have never detected them to have more than one connection to the er membrane, supporting the possibility that each string grows from a local area of budding activity. these observations suggest that budding continues outer diameter of shell (lid) figure . probability of a given bud having proximity to a second bud in the cell. randomly chosen er buds present in rbl cells were assigned as the center of reference, and distances between it and any other buds present in consecutive serial sections were determined by building a series of concentric shells with a constant volume of . ixm , corresponding to the volume of the first internal shell having a diameter . ixm (to encompass a single bud) as described in the materials and methods. the probability was determined by counting the number of buds detected in each shell relative to the total number of buds detected (percentage of total). this value is plotted as shell number in which a second bud was found (open circles) or relative to the outermost diameter of a particular shell (closed circles). the calculated probability of a second bud having a completely random distribution in the cell ( . %) is presented for comparison (diamonds). in the absence of gtp hydrolysis, but that the vesicles fail to complete separation from one another. upon immunolabeling, we found accumulated vesicles to be substantially enriched in vsv-g (pind et al., a) and components of both copi ( -cop) (pind et al., a; griffiths et al., b) and copii coats (sec and sec ) (fig. , a-c) . to establish that necklaces formed in response to a specific block in copii coat disassembly, we examined the ef-fect of an activated, gtp-restricted mutant of sarl, sarl[h g], which promotes vesicle accumulation in vivo and in vitro (aridor et al., ; kuge et al., ) . examination using strong fixation conditions revealed a striking similarity to gtp~/s-formed structures. vesicles were detected as both clusters (fig. d) or as necklaces (fig. e) emerging from restricted regions of the er. as expected, coats were enriched in the copii components secl p (arrowheads in a, c, and d) and sec p (arrowheads in b, and e) as shown by the distribution of gold particles using specific antibodies. note that the cisternal portion of the golgi apparatus with the trans face labeled in c and f remains unlabeled by sec p-and sec p-specific antibodies, whereas clusters (arrows) that are closely adjacent to the golgi complex contain both secl p and sec p (c and f). bar, . txm. bannykh et al. organization of er export complexes (fig. e) and sec p (fig. f) . no copi staining could be detected using an antibody specific for -cop in these structures (not shown) (aridor et al., ) . to examine the role of copii in vsv-g concentration and the appearance of vtcs in export complexes, we used semi-intact cells infected with a temperature-sensitive form of vsv-g whose transport is blocked at the restrictive temperature ( . °c) (lafay, ; plutner et al., ) . transfer of cells to the permissive temperature ( °c) results in the migration of a synchronous wave of ts vsv-g from the er to vtcs and subsequent golgi compartments (plutner et al., ; balch et al., ) . after incubation at °c in vitro, cells were fixed and stained with an antibody specific for the cytoplasmic tail of vsv-g using the immunodiffusion protocol . as shown in table iii , incubation in the presence of wildtype sarl had little effect on er export as judged by the abundance of vtcs detectable in vitro. in contrast, the guanosine diphosphate (gdp)-restricted form of sarl (sarl [t n] ) drastically reduced the formation of vesicles and vtcs (table iii) , demonstrating the essential need to activate sarl to promote membrane flow from the er through export complexes. in contrast, incubation in the presence of the gtp-restricted mutant (sarl[h g]) caused a dramatic accumulation of vesicles in clusters and an approximate twofold increase in the apparent number of clusters per section that could be detected (table iii) . when we determined the density of vsv-g in clusters formed in the presence of the sarl-gtp restricted mutant, it was approximately five-to sixfold higher than that found in the er before incubation (table iii) . this fold-concentration was identical to that observed in vtcs present in control incubations lacking inhibitors (table iii) . the five-to sixfold increase in the density of vsv-g in vesicles that accumulate in the presence of the mutant demonstrates that vsv-g is concentrated during packaging into copii-coated vesicles. to develop a three-dimensional view of export complexes, we applied for the first time a modification of the quickfreeze, deep-etch methodologies used previously to visualize vesicles budding from the plasma membrane (heuser, ) and golgi compartments (weidman et al., ) . the approach is particularly applicable to semi-intact cells where the cytosol can be readily washed away to reveal structural features of the er membrane surface. after incubation in vitro in the presence of atp and cytosol, semi-intact cells were fixed, rapidly frozen, and fractured to expose internal membranes. after etching and replication, intracellular organelles are rendered visible as three-dimensional structures. the surface of the er was readily distinguishable from other subcellular compartments by the presence of ribosomes or, in the case of the nuclear envelope, additionally, nuclear pores. adjacent to the surface of the er, we frequently observed compact structures of similar size and apparent vesicular-tubular composition to vtcs observed in thin-sections ( fig. a) . these structures were completely absent in incubations that lacked cytosol or atp. they varied in diameter, but were generally ~ . - . ~m across and ranged from a circular to a more oblong shape under normal incubation conditions. assuming that the distinctive ~ -nm surface undulations correspond to vesicle profiles ( fig. a) and that a cluster can be represented as a sphere with a similar range of diameters, we estimate that vtcs detected in replicas could contain - individual elements. this value is compatible with the number of vesicular profiles determined by reconstruction from serial thin-sections. to identify whether the above structures formed in the presence of cytosol and atp contained er-derived cargo proteins such as vsv-g, nrk cells were infected with ts vsv at the restrictive temperature. after permeabilization and incubation in vitro at °c, cells were immunolabeled for vsv-g using the immunodiffusion protocol and replicas were prepared. while a combination of prolonged incubation using mild fixation conditions to label vsv-g and the presence of prominent "gold shadows" from the etching and replication reduces the ability of the technique to reveal surface features of these immunogold-labeled clusters, the distribution of vsv-g reveals the striking role of copii in concentrative export. before incubation of semi-intact cells at °c, gold detected on the surface of the er (fig. a, arrowheads) and nuclear envelope (fig. b, arrowheads) was distributed in an apparent random manner throughout the er cisternal network (plutner et al., ; balch et al., ) . the surface density of vsv-g in these er membranes was +- gold particles per p~m . in contrast, incubation for min in the presence of cytosol and atp led to a dramatic change in the distribution of vsv-g, rearranging gold particles to a limited number of vtcs that were well isolated from each other (fig. c) . the density of gold over vtcs projected as a flat surface parallel to the er membrane (referred to as a planar projection) was ~ -+ gold particles per p~m , a value markedly higher than that observed in the plane of the er membrane before incubation. we next used replicas to follow the effects of gtp'ys and the sarl-gtp restricted mutant on budding and concentration of vsv-g. as shown in fig. (b and c) , incu- bation of noninfected semi-intact cells in the presence of gtp~/s led to the accumulation of vesicles ~ nm in diameter that, consistent with t e m (fig. , b and c), had a zig-zag necklace-like appearance when collapsed on the surface of the er. immunolabeling of replicas formed in the presence of gtp~/s revealed that vsv-g reached a density of _ gold particles per txm ~ in planar projection within clusters. when incubations were carried out in the presence of the sarl-gtp restricted mutant, necklaces (not shown) and compact vesicular clusters were also observed on replicas (fig. d) . in the presence of the sarl mutant, vsv-g reached a density of _+ gold particles per ~m in a planar projection of the cluster (fig. e) , reinforcing our previous observations that the budding activity associated with export complexes involves concentration. we have provided the first quantitative, stereological de-scription of the three-dimensional organization of cellular structures involved in transport of cargo from the e r to the golgi apparatus. export complexes have a hierarchial organization that can be conceptually divided into three tiers (fig. a) . the first tier (fig. a, dotted box) consists of closely adjacent buds on a single e r cisterna. each can give rise to an individual string or group of erderived buds containing copii coats. these budding loci were limited to specific regions of the e r in vivo, suggesting the existence of a defined number of export sites in the living cell. the second tier (fig. a, cylindrical region outlined by dashed lines) comes from the observation that buds on one cisterna were often found in close proximity to budding profiles emanating from e r cisternae derived from distantly connected regions of the er. the third tier of organization encompassing the entire export complex ( fig. a, solid box) includes er-derived buds that face into a region housing a central vtc. tubular elements within vtcs contain distinctive copi coats and are luminally discontinuous with the er. while professional secretory figure . vsv-g is concentrated in er-derived vesicles and vtcs. nrk cells infected with ts vsv at the restrictive temperature ( . °c) to retain vsv-g in the er were permeabilized with digitonin (plutner et al., ) and either fixed immediately (a and b) or incubated for rain at the permissive temperature ( °c) in the presence of cytosol and atp (c), or additionally supplemented with ixm sarl[h g] (e). after incubation, cells were fixed and vsv-g labeled with nm (a-c, and e) or nm (d) gold particles using the immunodiffusion protocol as described in the materials and methods. cells were either prepared for thin-section tem (d) or for quickfreeze, deep-etch replication (a-c, and e). at the restrictive temperature, vsv-g was uniformly distributed within the er membrane (a) or the nuclear envelope (b). after a shift to the permissive temperature (b-e), vsv-g was concentrated in newly formed -nm vesicles associated with export complexes. (arrowheads) location of nm (d) or nm (a-c, and e) gold particles corresponding to the distribution of vsv-g. due to the use of an immunodiffusion protocol before preparation of replicas and the high density of label, extended shadows from the gold particles partially obscure membrane outlines. bar, . i~m. cells such as those found in the pancreas confine export predominately to a single transitional region juxtaposed to the cis face of the golgi apparatus (palade, ) , the two different cell lines used in the present study were found to have export complexes distributed throughout the cytoplasm. our studies provide evidence that budding from the e r occurs in areas of intense morphological specialization. each level of organization is discussed in detail below. while there is an apparent random distribution of export complexes in the cytoplasm, we found a very high degree of organization in the distribution of e r buds in the cell. we observed not only a high local density on the same stretch of e r membrane (fig. , tier i, dotted boxes), but found distantly connected e r bud-bearing zones encircling the same vtc (fig. , tier ii, dashed cylindrical region), suggestive of a regional specialization of the copii export machinery. consistent with this interpretation, buds and vesicles accumulated in semi-intact cells in the pres-ence of gtp~/s or the sar [h g] mutant strongly labeled with antibodies specific for mammalian homologues of the yeast components secl p and sec p. both the gtp-ys and the gtp-restricted sarl[h g] mutant led to the formation in vitro of er-derived vesicle clusters and strings of vesicles with a necklace-like appearance. vesicles accumulated as necklaces had no obvious continuity between the lumen of individual vesicles, suggesting that membrane fission had gone to completion. moreover, in both thin-section and replicas, clusters that formed in the presence of gtp'ys or the sarl gtprestricted mutant clearly had a more vesicular surface appearance than those formed in the absence of inhibitors. the fact that separation of vesicles appears to be blocked in the absence of gtp hydrolysis raises the distinct possibly that the function of sarl is normally required for this event. given the fact that uncoating occurs rapidly after budding (aridor et al., ) , coat disassembly could be associated with release of vesicles from necklaces. the morphological effect of gtp~s on the budding from the e r is very different from its effect on golgi membranes incubated under identical conditions. in the latter case, buds appearing in replicas are isolated, single structures and are uniformly distributed throughout the entire golgi surface (weidman et al., ). the regional confinement of budding in e r membranes to necklaces or clusters therefore also supports our interpretation that export occurs from areas of luminal and/or membrane specialization. since budding from the e r frequently occurs from the tips of short, coated tubules emanating from e r cisternae, clusters and necklaces could be derived from either sequential or synchronous fission of these tubular elements. in contrast with the fact that export complexes observed in vivo appear to be completely surrounded by budding profiles from topologically distant e r cisternae (fig. a, tier ii), semi-intact cells lacked this feature (fig. b) . thus, permeabilization destroys confinement of several sites to one area and allows them to form in a more random fashion. we have previously noted that er-derived vesicles and downstream compartments are not released from semi-intact cells during incubation in vitro at °c (beckers et al., ) . in contrast, assays that reconstitute vesicle budding from semi-intact yeast cells (baker et al., ) release free -nm copii-coated vesicular carriers (barlowe et al., ) . the inability of mammalian semiintact cells to release vesicles suggests that vesicles are tethered to a scaffold of unknown composition. in yeast, either vesicles are not linked to such a scaffold, or this aspect has not been successfully reconstituted. in either event, while the striking degree of morphological specialization observed in mammalian cells may contribute to the overall efficiency of budding and transport in the early secretory pathway, it is apparently not essential. we have previously suggested that export from the e r is accompanied by concentration of vsv-g . this point was the subject of a recent debate (balch and farquhar, ; griffiths et al., a) . we have now applied an independent approach using quick-freeze, deepetch methodologies in conjunction with immunolabeling of vsv-g to generate three-dimensional replicas that allow us to directly assess the concentration of vsv-g in er-derived vesicles. we found, on average, a value of ~ gold particles per txm in planar projection of clusters accumulated in either the absence or presence of gtp~s, or in the presence of the activated sarl-gtp restricted mutant. the inclusion of the inhibitors prevents further rounds of vesicle budding, ensuring that we are examining concentration associated with export from the er. a planar projection, however, is not a good approximation of the total surface area available on clusters for antibody binding. since only the external surface of vesicles found on the perimeter of clusters is available for antibody binding pind et al., a ) (see fig. d) , a more reasonable estimate of vsv-g density can be determined if we assume that the antibody has access to the outer-half of a shell of -nm vesicles that occupy the perimeter of a . -~m sphere (the size of a typical vtc). compared with the surface area of the planar projection (~ . ~m ), the surface area of such a population of vesicles corresponds to a value of ~ . txm . this is an increase of approximately fivefold over the planar projection. therefore the actual surface density of vsv-g in clusters observed in replicas is gold particles per i~m divided by or gold particles per ixm . this value, when compared to the average surface density of vsv-g before incubation in vitro ( gold particles per ~m ), sug- figure . diagram summarizing the three tiers of organization of er export complexes in vivo (a) and in vitro (b). (a) an individual er cisterna contains a collection of closely opposed buds that define a local transitional region (light zone with buds on the er). this specialized region is dominated by the presence of copii coats and is referred to as tier (box outlined with dotted line). tier ii (cylindrical region outlined by dashed lines) includes buds on distantly connected er strands that face a central vtc consisting of a collection of distinct vesicular-tubular elements that have copi coats. tier iii includes the entire export complex and is outlined by the box with the solid line that encompasses both er buds and a central vtc. the possible elevated concentration of copii and copi coat components within the local cytoplasm of export complexes is depicted by small dots and lines. (b) in semi-intact cells, there appears to be a more limited number of er buds associated with the local transitional region defined by tier i (box outlined with dotted lines). the tier ii level of organization is completely missing in semi-intact cells, as the association of distantly connected er strands appears to be lost during cell permeabilization. however, tier iii (box outlined with solid line) is maintained, highlighting the juxtaposition of vtcs to buds on one er strand. gests that vsv-g is concentrated five-to sixfold during budding from the er. the fold-concentration detected here is very consistent with that observed previously using serial thin-sections pind et al., a) or in the present studies in the presence of the sarl-gtp restricted mutant (table iii) . these results confirm the validity of our previous technical advancements balch and farquhar, ) and now firmly establish that cargo is concentrated during er export . sequential coupling between copii and copi vesicle coats in endoplasmic reticulum to golgi transport estimation of surface area from vertical sections reconstitution of sec gene product-dependent intercompartmental protein transport beyond bulk flow atp-coupled transport of vesicular stomatitis virus g protein between the endoplasmie reticulum and the golgi vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum copii: a membrane coat that forms endoplasmic reticulumderived vesicles copii: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the golgi complex regulation of constitutive exocytic transport by membrane receptors: a biochemical and morphological study localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments transport into and out of the golgi complex studied by transfecting cells with cdnas encoding horseradish peroxidase differential inhibition of multiple vesicular transport steps between the endoplasmic reticulum and trans-golgi network density of newly synthesized plasma membrane proteins in intracellular membranes. i. stereological studies the dynamic nature of the golgi complex the bulk-flow hypothesis: not quite the end immunocytochemical localization of [ -cop to the er-golgi boundary and the tgn disruption of endoplasmic reticulum to golgi transport leads to the accumulation of large aggregates containing -cop in pancreatic acinar cells three-dimensional visualization of coated vesicle formation in fibroblasts microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step sarl promotes vesicle budding from the endoplasmic reticulum but not golgi compartments envelope viruses of vesicular stomatitis virus: effect of temperature-sensitive mutations in complementation groups iii and v coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum bi-directional membrane traffic between the endoplasmic reticulum and golgi apparatus kinesin is the motor for microtubule-mediated golgi-to-er membrane traffic immunocytochemical analysis of the transfer of vesicular stomatitis virus g glycoprotein from the intermediate compartment to the golgi complex mitotic golgi fragments in hela cells and their role in the reassembly pathway the gotgi complex: in vitro veritas? celt the reorganization of the golgi complex in anoxic pancreatic acinar ceils mammalian sec p homologue is restricted to the endoplasmic reticulum transitional cytoplasm coated vesicle assembly in the golgi requires only coatomer and arf proteins from the cytosol bfa bodies: a subcompartment of the endoplasmic reticulum lntracellular aspects of the process of protein transport. science (wash. dc) evidence that luminal er proteins are sorted from secreted proteins in a post-er compartment -cop is essential for biosynthetic membrane transport from the endoplasmic reticulum to the golgi complex in vivo -cop is essential for transport of protein from the endoplasmic reticulum to the golgi in vitro rabl and ca + are required for the fusion of carrier vesicles mediating endoplasmic reticulum to golgi transport participation of the endoplasmic reticulum chaperone calnexin (p , ip ) in the biogenesis of the cystic fibrosis transmembrane conductance regulator morphological analysis of protein transport from the endoplasmic reticulum to golgi membranes in digitonin-permeabilized cells: role of the p containing compartment biosynthesis and intracellular sorting of growth hormone-viral envelope glycoprotein hybrids pre-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the ceil surface distribution of the intermediate elements operating in er to golgi transport identification, by a monoclonal antibody, of a -kd protein associated with a tubulo-vesicular compartment at the cis-side of the golgi apparatus identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to golgi apparatus a threedimensional reconstruction study of the rough er-golgi interface in serial thin sections of the pancreatic acinar cell of the rat human sec rp functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum estimating number, mean sizes and variations in size of particles in -d specimens using disectors anterograde and retrograde traffic between the rough endoplasmic reticulum and the golgi complex segregation of ergic and the mammalian kdel receptor upon exit from the °c compartment replication of coronavirus mhv-a in sac cells: determination of the first site of budding of progeny virions site of addition of n-acetyl-galactosamine to the e glycoprotein of mouse hepatitis virus-a stereoiogical methods. . practical methods for biological morphometry a principle for counting tissue structures on random sections integrated stereological and biochemical studies on hepatocytic membranes. ii. correction of section thickness effect on volume and surface density estimates golgi membrane dynamics imaged by freeze-etch electron microscopy: views of different membrane coatings involved in tubulation versus vesiculation speculations based on the morphology of the golgi system in several types of protein secreting cells we thank dr. g. palade, dr. m.g. farquhar, and michael mccaffery for their many helpful comments concerning the em. this work was supported by grants from the national institutes of health (gm ; ca ) (to w.e. balch), and postdoctoral fellowships from the human frontier science program organization, muscular dystrophy association (to t. rowe), and the cystic fibrosis foundation (to s. bannykh). this study made extensive use of core b (immunoelectron microscopy) in ca .received for publication april and in revised form june . a third tier of organization of export sites was found in the striking relationship between flanking er-connected budding profiles and vtcs to form the functional morphological unit we refer to as export complexes (fig. a, area enclosed by box with solid line). images reconstructed from conventional tem and those observed in replicas yielded identical results. the distinctive morphological characteristics of the export complexes reconstructed in the present studies are consistent with previous qualitative morphological descriptions (saraste and kuismanen, ; schweizer et al., schweizer et al., , saraste and svensson, ) and a recent study in which a hrp-tagged reporter protein was used to characterize the organization of the er/golgi region using immunocytochemistry (stinchcombe et al., ) . the replicas were particularly striking in that they allowed us to visualize for first time the overall compact composition of vtcs and their localization to specific foci found on the er surface.the overall topological organization of export complexes fits well with the proposed function of the er in the sorting and concentration of cargo during budding via copii coats, and the subsequent coupled recycling of proteins from vtcs via copi coats (aridor et al., ) . the close association of these two sorting stations (er and vtcs) may provide for increased efficiency in er to golgi transport and/or promote more rapid exchange of coats within the confines of the complex where a higher local coat concentration may be found. consistent with this proposal, incubation of pancreatic acinar cells in the absence of atp (merisko et al., ) , ) , is also considered to be a site of membrane recycling. it may be an enlarged variation of the more compact vtcs, reflecting the intensity of vesicular traffic in this region of the cell. in general, our studies have provided insight into the fundamental morphological organization of the first steps in the secretory pathway that promote the movement of cargo from the er to the golgi complex. there has been considerable controversy regarding the morphological organization of this stage of secretory pathway, given the complexity of the pre-golgi region and the frequent use of reduced temperature to augment the visibility of intermediates (vtcs). our ability to provide a description of the three-dimensional organization of export complexes at sites distant from the golgi apparatus under normal incubation conditions now illustrates their basic organization in living cells and their essential role in er to golgi transport. key: cord- -f b ygg authors: nan title: site of addition of n-acetyl-galactosamine to the e glycoprotein of mouse hepatitis virus-a date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: f b ygg by pulse-chase labeling with [ s]methionine and long-term labeling with h-sugars, the e glycoprotein of coronavirus mhv-a has been shown to acquire o-linked oligosaccharides in a two-step process. about min after synthesis of the e protein, n-acetyl-galactosamine was added. this was followed approximately min later by the addition of both galactose and sialic acid to give the mature oligosaccharides. this sequence of additions was confirmed by analyzing the h-labeled oligosaccharides bound to each of the e forms using gel filtration on p columns. the intracellular location of the first step was determined by exploiting the temperature sensitivity of virus release. the virus normally buds first into a smooth membrane compartment lying between the rough endoplasmic reticulum and the cis side of the golgi stack (tooze et al., ). at degrees c the virus is assembled but does not appear to enter the golgi stacks. the addition of n-acetyl- galactosamine is unaffected although the addition of galactose and sialic acid is inhibited. these results strongly suggest that addition of n-acetyl-galactosamine occurs in this budding compartment, the morphology of which is similar to that of transitional elements and vesicles. ploiting the temperature sensitivity of virus release. the virus normally buds first into a smooth membrane compartment lying between the rough endoplasmic reticulum and the cis side of the golgi stack (tooze et al., ) . at °c the virus is assembled but does not appear to enter the golgi stacks. the addition of n-acetyl-galactosamine is unaffected although the addition of galactose and sialic acid is inhibited. these results strongly suggest that addition of n-acetyl-galactosamine occurs in this budding compartment, the morphology of which is similar to that of transitional elements and vesicles. p roteins exported from the endoplasmic reticulum (er)' move through each of the cisternae of the golgi stack before they are directed to their correct destination either inside or outside the cell (rothman, ; griffiths and simons, ; pfeffer and rothman, ) . many proteins undergo a series of posttranslational modifications during this transport including the addition of oligosaccharides which are either n-linked to asparagine or o-linked to serine or threonine. the sequence of modifications to these covalently bound oligosaccharides reflects the movement of the protein from one compartment to the next on the transport pathway. the addition and modification of the n-linked oligosaccharides has been extensively investigated (reviewed by kornfeld and kornfeld, ) but much less is known about the addition of o-linked oligosaccharides. for the simplest and most common type there is indirect evidence that construction of the o-linked glycans is a two-step process, the addition of n-acetyl-galactosamine (galnac) preceding that of galactose (gal) and sialic acid (nana). the addition of the last two sugars almost certainly occurs in the trans part of the golgi stack (cummings et al., ; elhammer and kornfeld, ) . proposals for the site of addition of the first sugar, however, range from the rough er (strous, ) , where the proteins are assembled, to the . abbreviations used in this paper: er, endoplasmic reticulum; gal, galactose; galnac, n-acetyl-galactosamine; mem-met, methionine-free modified eagle's medium; mhv-a , mouse hepatitis virus, strain a ; nana, n-acetyl-neuraminic acid (sialic acid); tx- , triton x-ti . cis part of the golgi stack which is the entry point for proteins that have left the er (cummings et al., ; roth, ) , to the medial part (niemann et al., ; elhammer and kornfeld, ) , and finally to the trans-golgi cisternae (johnson and spear, ) . most of the evidence supports the idea that galnac is added either just before or just after the protein leaves the er. there are several reasons why it has proven so difficult to locate the site of this first sugar addition, of which the absence of a combined biochemical and morphological study of the o-glycosylation of a single protein must rank as the most important. the e envelope protein of coronavirus mhv-a (mouse hepatitis virus, strain a ) contains only o-linked oligosaccharides and is, therefore, a good model for studying addition of o-linked sugars. mhv-a buds intracellularly and e is the only viral membrane protein needed for budding (holmes et al., a, b; rottier et al., ) . after synthesis in the rough er this protein is transported to a smooth membrane compartment between the rough er and golgi stack where it accumulates allowing nucleocapsids in the cytoplasm to bind which results in the formation of a virion (holmes et al., a; tooze et al., ) . the virions then follow the secretory pathway out of the cell (holmes et al., b; tooze et al., ) . the virions contain in addition to e a second envelope glycoprotein, e , which is essential for infectivity (sturman, ) and is incorporated at some as yet undefined stage during the budding or transport of the virions through intracellular compartments. e is a fusogen and is also transported in cellular membranes to the plasma membrane where it helps spread the infection by inducing the fusion of infected cells with contiguous uninfected cells (sturman and holmes, ) . reduced temperature has proven to be a very useful tool in analyzing compartments on the exocytic transport pathway. different steps are differentially sensitive so that proteins accumulate at different stages. this has led to the morphological characterization of a compartment similar to the coronavirus budding compartment, but found in baby hamster kidney (bhk) cells infected with semliki forest virus and incubated at °c (saraste and kuismanen, ) . here we exploit reduced temperature to prevent movement of coronavirus and analyze the oligosaccharides bound to e on the arrested virus. we also present evidence showing that the budding compartment, which by morphological criteria is clearly related to transitional elements and transitional vesicles, as classically defined in exocrine pancreatic cells by the work of palade and colleagues (caro and palade, ; jamieson and palade, ) , is not created by the process of infection but exists in the uninfected cell. a physiological function of this compartment appears to be the addition of galnac, implying that it contains the enzyme which adds this sugar to serine and threonine residues. n-acetyl [ , , , , , - c] mhv-a was propagated on sac(-) cells (tooze et al., ) and titered by plaque titration on confluent monolayers of l cells as follows. after infection for h at °c, the monolayers were overlaid with % carboxymethyl cellulose and incubated for h at °c in % air/ % cos incubator. the monolayers were then fixed and stained with mm tris-cl buffer, ph . , containing . % (wt/vol) crystal violet, . % (vol/vol) formaldehyde, % (vol/vol) meoh, and . % (wt/vol) caci (k~ari~inen et al., ) . sac(-) cells grown on coverslips and infected with mhv-a were fixed with % formaldehyde and labeled (tooze et al., ) using either a mab directed against e ( - -m -f -m ; gift of m. koolen, utrecht, and a. osterhus, national institute of public health, bilthoven, holland) or an anti-body directed against an endogenous golgi protein (burke et al., ) . the second antibody (rabbit anti-mouse), kindly provided by dr. b. burke, european molecular biology laboratory, heidelberg, frg, was affinity purified and conjugated to rhodamine. the fixed and labeled coverslips were mounted in moviol and viewed by epifluorescence on a zeiss photomicroscope iii. infected sac(-) cells in -mm dishes were fixed, dehydrated, and embedded in epon as previously detailed (tooze et al., ) . the anti-e antibody ( - -m -f -m ) was used for immunoperoxidase labeling after our standard protocol (tooze et al., ) . infected sac(-) cells, in -mm dishes, were either pulse labeled or continously labeled with [ s]methionine. after washing three times in methionine-free modified eagle's medium (mem-met), cells were labeled with - ~tci [ s]methionine for - min at °c, or for longer periods at , , , , and °c . some cells were chased at or °c for various times by the addition of growth medium containing . mg/ml of methionine, × the normal amount. for quantitation of the number of el molecules labeled [ s]methionine with a defined specific activity was used. the cells were'washed with ice cold pbs and extracted with % triton x- (tx-ll ) in pbs containing ~tg/ml phenylmethylsulfonyl fluoride (pmsf) at °c. the insoluble residue was removed by centrifugation at , g~ for min at °c. the detergent phase, enriched in the el glycoprotein, was isolated from the detergent-soluble extract as described by bordier ( ) . the medium containing labeled virions was collected and layered onto a - % continuous sucrose gradient as described by spaan et al. ( ) . the virions were harvested from the gradients after centrifugation for h at , ga~. the el glycoprotein was extracted from virions using tx-ll as detailed above. the el-enriched detergent extracts from cells or virus were solubilized and reduced in sample buffer, alkylated (green et al., ) , and then fractionated by sds-page using a % running gel and a % stacking gel (maizel, ) . incorporation of radioactive [ s]methionine was determined after tca precipitation (mans and novelli, ) and equal counts were loaded onto each lane unless otherwise indicated. gels were fixed using % (wt/vol) tca, treated with en hance, dried, and exposed to kodak xs film for - d at - °c. to quantitate the [ ~s]methionine incorporated in the e glycoprotein the fluorogram was exposed to kodak x-ar film. the fluorograph was then used to locate the e bands in the fluorogram. the excised pieces were rehydrated and incubated in . ml of % (vol/vol) h in mm hc at °c for h. scintillation fluid ( ml) (rotizant , firma roth, karlsruhe, frg) was then added and the samples were cooled to °c and counted. gel pieces of equivalent sizes from areas containing no samples were treated similarly and used to provide blanks which were typically < % of the experimental values. [ s]methionine-labeled cells were solubilized in ripa buffer ( mm tris-hci, ph . , . m naci, % [wt/vol] triton x- , . % [wt/vol] sds, and % [wt/vol] sodium deoxycholate) containing a cocktail of protease inhibitors ( ~tg/ml pmsf, lag/ml leupeptin, ixg/ml antipain, mm benzamidine, and u/ml trasylol). the extract was centrifuged at , ga~ and the supernatant was treated with of a polyclonal antiserum raised against a triton x- extract of gradient-purified virions (tooze and stanley, ) . after overnight incubation at °c, the immune complexes were immobilized on pansorbin for h at °c and washed three times with ripa buffer. one half of the sample was then digested with mu/ml neuraminidase from clostridium perfringens in mm sodium acetate buffer, ph . , conmining mm cac , for h at °c. the digested immune complexes were then washed well with mm tris-hcl buffer, ph . , containing the protease inhibitor cocktail. these samples, together with the untreated ones, were then prepared for and fractionated by sds-page. the growth medium was changed at h after infection and cultures were either left at °c or shifted to °c for the time period indicated. the cultures were treated at . h after infection with proteinase k to remove any virions from the infecting stock bound to the surface. * temperature of incubation. nd, none detectable in undiluted supernatant. infected sac(-) cells ( × cells) were labeled for h with laci/ml [ , - h]glucosamine in mem containing % the normal concentration of glucose ( i.tg/ml), % (vol/vol) dialyzed fcs, mm hepes buffer, ph z , mm sodium pyruvate, and ixg/ml each of penicillin and streptomycin. the cells were solubilized in ripa buffer and the el glycoprotein was immunoprecipitated as described above. the procedures described below are adapted from cummings et al. ( ) . after sds-page the individual glycosylated forms of e were excised from the dried gel, rehydrated, and digested with pronase (final concentration mg/ml). the glycopeptides were eluted from the gel pieces using hzo. the eluate conmining the glycopeptides was then lyophilized and resuspended in h . glycopeptides were acetylated by treatment with acetic anhydride (baenziger and kornfeld, ) and desalted over an amberlite mixed bed resin before mild alkaline hydrolysis. the eluate, after lyophilization, was resuspended in . m sodium carbonate, ph . , then treated with an equal volume of m nabh in mm naoh, and incubated for h at °c as described by niemann and klenk ( ) . after neutralization at °c with glacial acetic acid, the oligosaccharides were passed over a dowex w-x h + column and eluted with % (vol/vol) acetic acid. the peak fractions containing the oligosaccbarides were pooled and lyophilized. the borate was removed by repeated evaporation with methanol at °c. the oligosaccharides were finally resuspended in ~t distilled h . the final recovery of [ h]-iabeled material was - %. one-half of the material was applied directly to a p ( - mesh) column ( × . cm), equilibrated, and run in a m nahco , ph . . the remainder was first digested with neuraminidase from vibrio cholerae at a final concentration of i u/ml in mm sodium acetate buffer, ph . , containing mm naci and mm cac ,. after incubation under a toluene atmosphere for h at °c (niemann and klenk, ) , the reaction was stopped by boiling, and the samples were lyophilized and then resuspended in i.tl of distilled h.,o before application to the p column. [ h]glucose mannose~/-acetylglucosamine was prepared from chinese hamster ovary (cho) (clone b cells) infected with vesicular stomatitis virus as described by featherstone et al. ( ) . the glycopeptides were prepared and the dolicol-linked oligosaccharide in the tx-ll pellet was released by endoglycosidase h digestion. [ hlgall (l-- )-galnacitol was prepared by reduction of gall ( -' )-galnac (generously supplied by dr. w. gielen, university of cologne, frg), with nab h (li et al., ) . [t c]galnacitol was prepared by acid hydrolysis of udp-n-acetyl-d-[ -t c]galactosamine (cummings et al., ) , followed by reduction with nabh (li et al., ) . at °c, sac(-) cells release progeny virus exponentially, beginning at ~ h after the start of infection and continuing until the cells lyse. the release of e molecules in virions parallels the release of virus particles consistent with the known dependence of budding on the e glycoprotein (tooze, ) . holmes and behnke ( ) observed in a preliminary morphological study that at °c virions were assembled, but accumulated within the infected cell instead of being released. to confirm this observation, infected sac(-) cells were incubated for h at °c and the release of infectious virus was measured by a plaque titration assay over the next h in cultures either kept at °c or shifted to °c. as an added precaution, cells were treated for - min with proteinase k . h after infection to remove any virions from the infecting inoculum still remaining on the cell surface. this ensured that only released progeny particles were measured. infected cells were labeled from to h after infection with [ ~s]methionine in mem-met containing ixg/p.i cold methionine at or °c. at the end of the labeling period the cells were treated with . mg/ml proteinase k at °c for - min. the proteinase k was neutralized with pmsf and the cells were pelleted at °c. the supernatant was then pooled with the medium collected from the cells before the proteinase k treatment and the labeled virions were purified on sucrose gradients. both cells and virions were subjected to tx- extraction followed by sds-page. * counts per minute in all forms of el detected by fluorography, including the lower molecular weight forms of el generated by proteinase k digestion (rottier et al., (rottier et al., ). a rottier et al., ) . to eliminate the possibility that at °c the cells were releasing large numbers of noninfectious particles, which would not be scored in an infectivity assay, el contained in virus particles released from the cells was quantitated. infected cells were labeled from to h after infection with pss]methionine at and °c, the medium was collected, and any labeled virions associated with the plasma membrane were removed by proteinase k treatment at °c and pooled with the virions in the medium. after sds-page and fluorography of virions purified from the medium, the e band was excised and counted. as shown in table ii , the °c drop in temperature during the labeling period inhibited the release of virion-associated e by > %. in marked contrast, however, the total amount of e synthesized was reduced by only ,x, % upon shifting the temperature to °c (table ii) . total protein synthesis, in both infected and uninfected cells, was on average decreased by only % over the same time period. the discrepancy between the observed , -fold decrease in the release of infectious virions (table i ) and the fivefold decrease in the release of virion-associated e (table ii ) may mean that at °c more noninfectious particles are assembled and released. however, we favor an alternative explanation, namely, that proteinase k treatment damages some cells and leads to artifactual release of intracellular virions because, when medium is collected from cells at °c without proteinase k treatment, the amount of methioninelabeled virion-associated e is below the limits of detection by fluorography after sds-page (data not shown). furthermore, electron microscopic studies of thin sections of infected cells incubated at °c from to h after infection reveal very few extracellular virions associated with the plasma membrane (see below and fig. ) . these data show that e synthesis continues almost normally at °c but the release into the medium of virions is dramatically inhibited. to determine the site of accumulation of virions and e at °c a combination of both biochemical and morphological techniques was used. at °c and h after infection, sac(-) cells were pulsed for min with [ s]methionine and then chased in the presence of excess unlabeled methionine for up to min. fig. shows that a proportion of the unglycosylated e protein (e/u, lane ) is converted after min of chase to an intermediate form of higher molecular weight (e/i, lane ) and after ",, min of chase (lane ) to the mature forms (e/m), which in this experiment consisted of three distinct polypeptides differing in molecular mass by ~ kd. three forms of e were always detected (fig. , lane ) , and in some experiments varying but small amounts of two additional higher molecular mass forms were resolved. the observed increases in molecular mass must be due to the addition of o-linked oligosaccharides (holmes et al., a; niemann and klenk, ; rottier et al., ) , which is the only known posttranslational modification of el. the intermediate form ei~ has not, however, been previously detected and the heterogeneity of mature forms resolved in fig. was not seen in previous work with the same virus propagated in a different cell line (e.g., niemann et al., ) . to determine the sugar composition of the oligosaccharides bound to each of the e forms, infected cells were labeled from to . h after infection with h-precursors of each of the sugars expected to be present in the o-linked oligosaccharides, extracted with tx-ll , and subjected to sds-page followed by fluorography. as expected, elu, the unglycosylated form of el, was not labeled by any of the h-sugars (fig. , cf. lane with lanes - ) . [ h] galactose and [ h]n-acetyl-mannosamine are specific metabolic precursors of gal and nana, respectively (neutra and leblond, ; monaco and robbins, ) , and these were found to label only the elm forms (fig. , cf. lane with lanes and ). the presence of nana in elm was also confirmed by treatment of [ s]methionine-labeled e with neuraminidase. the elm polypeptides alone were reduced in molecular mass to a form which migrated slightly slower than ei~ (fig. , lane ) and was assumed to have o-linked oligosaccharides composed of gal-galnac. mature forms of e are known to contain two types of o-linked oligosaccharides: (a) a disialylated form (niemann et al., ) , the intermediate form of e (eli) was labeled by [ h]glucosamine (fig. , lane ) (fig. , lane ) . ei~ was also not labeled by [ h]gal (fig. , lane ) . based on the known structure of the oligosaccharides, these data indicate that galnac is most likely the only sugar present in the ei~ form. the journal of cell biology, volume , were pulse labeled for min with . isci/~l [ ss]methionine or pulse labeled and chased for min at °c. after immunoprecipitation, the samples were divided in half and were either (lanes and ) incubated without neuraminidase in the digestion buffer or (lanes and ) with . mu/ml neuraminidase in the digestion buffer at °c overnight. equal volumes were loaded onto each lane. *, the major digestion product after neuraminidase treatment. to confirm that the intermediate form of e results from the addition of galnac, infected cells were labeled from to h after infection with [ h]glucosamine at °c. the virus was purified from the medium, solubilized in tx- , and pooled with the labeled cells that were also solubilized in tx- . after phase extraction, e was immunoprecipitated and resolved by sds-page followed by fluorography. the polypeptides were excised from the gel in two groups, the lower (elu and eli) and higher (elm) molecular mass forms, and analyzed by p column chromatography after alkaline hydrolysis to release the oligosaccharide side chains. any oligosaccharides released from the lower molecular mass forms of e must have originated from eli since elu is not labeled by [ h]glucosamine (fig. , lane ) . as shown in figure a, a single peak of h-sugar ran at the position of galnacitol (the alcohol derivative of galnac), which was not sensitive to treatment with neuraminidase ( fig. b) . the two predominant h-labeled oligosaccharides from elm, (peaks fig. c, a and b) correspond to the dis!alyated and monosialylated oligosaccharides. after treatment with neuraminidase, both a and b are converted quantitatively to nana (fig. d, peak c) and gall ( --' )galnacitol (fig. d, peak d) . these sugars, as well as galnacitol (fig. d, peak e), are present in the untreated samples (fig. c) , most likely as degradation products of alkaline hydrolysis. taken together, the experiments described so far show that galnac is the first sugar added to the e protein, followed at a later time by gal and nana. to examine the effect of decreased temperature on the glycosylation of el, infected cultures were labeled for rain at °c and then chased at either or °c (fig. ) . after rain of chase at °c, all forms of e were detectable in cell extracts (fig. , lane ) . after rain there was a decrease in the amount of all forms of labeled e (fig. , lane ) , presumably because the labeled e molecules had been incorporated into virions which were then transported out of the cell. when the cultures were shifted to °c for the chase period, the glycosylation of e was blocked in the intermediate form (fig. , lanes and ) . this block was maintained after min of chase, with no detectable decrease in either the unglycosylated or the intermediate form (fig. , compare lanes and with lanes and ). if, after min of chase at °c, the infected cultures were returned to °c for a further min, the amounts of the elu and eli forms decreased (fig. , and with lanes and ) . .~ o forms of el were excised from the gel and quantitated; the results are presented in fig. a. as expected from fig. , a decrease in the incubation temperature reduces the percentage of e m and increases the percentage of e o. surprisingly, the percentage of ei~ remains approximately constant at all the temperatures. in a separate experiment the absolute amounts of each of the e forms were measured at both and °c and, as shown in fig. b, the decrease in the mature forms, elm, was accompanied by an increase in the unglycosylated form, elo, but there was no change in the amount of the intermediate form, ei~. to relate the block in oligosaccharide maturation to the intracellular localization of el, infected cells were labeled for indirect immunofluorescence microscopy, using a mab against el. at h after infection at °c, e was restricted to a perinuclear region (fig. a) and the pattern of labeling was similar to, but more extensive than, that observed with antibodies specific for an endogenous golgi protein (data not shown). between and h after infection at °c syncytia developed, each containing several hundred nuclei, and concomitantly e became detectable in the rough er throughout the cytoplasm (fig. b) . at these late times the mab against e also labeled patches at the cell surface (fig. b) ; this labeling we attribute to released progeny virions, which are known to remain closely associated with the cell surface (dubois-dalcq et al., ) . in contrast, when the cells were shifted at h after infection to °c and incubated until h after infection, the e labeling within the cells remained restricted to the peri-golgi region, although the area and intensity of labeling increased above that seen at h after infection (fig. , a and c) . furthermore, neither the rough er nor the surface of cells incubated from to h after infection at °c labeled for el. these observations imply that e accumulated in the perinuclear region during the incubation at °c and that virions were not transported out of the cell. during the -h incubation at °c, the syncitia also failed to enlarge significantly, the viral envelope glycoprotein e is responsible for viral-induced cell fusion (sturman and holmes, ) and we attribute the lack of growth of the syncytia to the failure in the transport of virions out of the cells. there may also be, at °c, a reduction in the transport to the plasma membrane of e as a membrane glycoprotein, independent of the transport of virions, but this was not investigated. this inhibition of the enlargement of the syncytia was reversible; they expanded rapidly within - h after a return to °c (data not shown). budding of progeny virions, which occurs at membranes with a high concentration of the viral el spike glycoprotein, was first detected in thin sections under the electron microscope between and h after infection. at these early times, the first and only site of budding was a smooth membraned compartment with a complex and irregular vesiculotubular morphology (fig. ) . this compartment, which we refer to here as the budding compartment, was always closely associated with cisternae of the rough er, including transitional elements. usually, but not always, the budding compartment was also juxtaposed to the cis side of the golgi stack (see fig. ). since the membrane of the budding compartment is smooth it cannot be the site of synthesis of e or its insertion into membranes. furthermore, we have reported previously that the budding compartment does not contain a cytochemical marker of the rough er (tooze et al., ) . we conclude that the initial site of accumulation of e to levels that support budding is a distinct smooth- membraned compartment usually found between transitional elements of the rough er and usually lies between them and the golgi complex. examination of single and serial sections showed that this distinct compartment also exists in uninfected cells (fig. ) , where it is also invariably associated with transitional elements of the rough er and golgi stacks. comparison of figs. and shows that the budding compartment is more dilated in infected than in uninfected cells. in part, no doubt, this is a result of the budding of virions, each ~ , /~ in diam, into this compartment in the infected cells. although we have not made extensive morphometric analyses of the budding compartment, it is clear from examination of single and serial sections that its volume and surface area are smaller than those of either the golgi complex or the rough er. beginning between and h after infection at °c, the rough er becomes a site of budding of virions and progressively more viruses bud and accumulate in this compartment (tooze et al., (tooze et al., , . virions can also be seen increasingly in the rims of golgi cisternae and in post-golgi vesicles transporting the virus to the cell surface. the golgi cisternae become distended and eventually vesiculate; by h after infection cell lysis starts (tooze et al., ; and unpublished observations) . by contrast, when cultures are shifted at - h after infection from to °c, the infected cells survive for at least a further h. at h after infection, their golgi stacks have retained their morphological integrity. few virions are seen within the golgi cisternae or in post-golgi transport vesicles (fig. ) , and very few are seen outside the cells associated with the plasma membrane. on the other hand, the rough er contains massive accumulations of virions (fig. ) . these morphological observations indicate that there is little trans-port of virions into the golgi stacks and beyond at °c. we believe that it is this block to virion transport that prevents the disruption of the golgi stacks, which is one of the cytopathic effects of mhv-a infection at °c and is also observed in cells infected at °c with uukuniemi virus, a bunyavirus that buds into intracellular membranes (gahmberg et al., ) . using an immunoperoxidase labeling technique and a mab against an amino-terminal epitope of e (tooze and stanley, ) , we determined at the level of resolution of the electron microscope which membranes contained e at different times after infection at the two temperatures. at - h after infection at °c, the rough er was not labeled but the budding compartment was labeled as expected from indirect immunofluorescence microscopy. there was also distinct labeling of all the stacked cisternal membranes of the golgi complex (fig. ) . the reaction product was found on the lumenal side of these compartments (fig. ) consistent with the fact that the mab recognizes an amino-terminal epitope of el. continued incubation at °c increased the number of labeled compartments to include the rough er (tooze et al. ) . the labeling of golgi cisternae at - h after infection indicates that some e reaches the cisternal membranes as an integral membrane protein. however, the absence of budding of virions directly into the golgi cisternae at these early times after infection (tooze et al., ; figs. and ) indicates that the amount of e in these membranes is below the threshold concentration necessary for budding but clearly is not below the threshold of detection by the immunoperoxidase technique which involves enzymatic amplification. after a shift from to °c at times between and h after infection, with - h further incubation at the lower temperature, the labeling pattern of e using the im- to show budding compartments not juxtaposed to golgi stacks and therefore are comparable to the budding compartment in an uninfected cell shown in fig. . (budding compartments close to golgi stacks are shown in fig. .) note the irregular form of this compartment and its distension by the accumulating virions. frequently parts of the cytoplasmic face of the budding compartment have a "bristle" coat and appear to be sites of fusion of small vesicles (arrowheads in a, b, d, and e). this coat differs in morphology from the coats of typical coated vesicles (open arrows in a and c). orci et al. ( ) have shown by immunocytochemistry that coats of this type associated with golgi membranes lack clathrin. bars, . vtm. the journal of cell biology, volume , figure . serial sections through a budding compartment in an uninfected sac(-) cell. in uninfected cells this compartment (arrowheads) is found closely associated with transitional element regions of the rough er (arrows). often the budding compartment occurs between transitional elements and golgi stacks but sometimes, as this figure shows, it is not adjacent to a golgi stack. in this situation, because of the absence of smooth cis-golgi membranes, it is easier to trace the budding compartment through serial sections. note the complex irregular morphology of the budding compartment and the absence of ribosomes from its membrane. the micrographs shown are of nine sections (si-$ , $ , and $i -s ) from a continuous ribbon of sections, each ,~ - nm thick. in this example, therefore, the budding compartment extended through a depth of ,,, nm. all micrographs are at the same magnification. bar, . lam. munoperoxidase technique was very similar to that seen at h after infection at °c. reaction product was seen in the budding compartment and some in the golgi cisternae (data not shown). there are at least two posttranslational modifications which result in a change in the molecular mass of coronavirus el. the first involves addition of galnac ~ min after synthe-sis; the molecular mass increase of kd would correspond to the addition of three gainac molecules. the sequence of e (armstrong et al., ) shows that there are four potential glycosylation sites for o-linked oligosaccharides at the amino terminus of the protein (nh -ser-ser-thr-thr) of which a maximum of three are used (niemann et al., ) . about min later, gal and nana are added to give a variety of mature e forms which can be explained by variation in the number of galnac residues that are modified and by the heterogeneity of the mature sialylated oligosaccharides. the addition of gal and nana probably occurs in the trans-golgi complex, because the necessary enzymes are found there (carlson et al., ; sadler et al., ; rearick et al., ; elhammer and kornfeld, ) and drugs which inhibit transport into and through the golgi complex (quinn et al., ; tartakoff et al., ) inhibit formation of the mature oligosaccharide structure. we have found that both monensin and cccp inhibit the glycosylation of e in a manner essentially identical to the inhibition at °c (tooze, ) . such results must, however, be interpreted with some caution because with these drugs it is often impossible to distinguish between inhibition of transport and direct inhibition of the enzymes which add the sugars. the pathway taken by newly synthesized e is complicated by the intracellular budding of the virus. at its simplest, one can envisage that e moves to the budding compartment, nucleocapsids bind and bud, and e then moves through the secretory pathway as part of the maturing virion. however, immunoperoxidase labeling shows clearly that e in the golgi complex is not only present in virions but also in every cisternal membrane (see fig. ). the amount of e in golgi membranes cannot be estimated because of the catalytic nature of the peroxidase technique but, until very late in the viral infection, it must be lower than that needed to support budding of the virus. this accumulation of e in the golgi membranes during infection may represent a dead end caused by "leakage" of e from the budding compartment if the nucleocapsids are unable to trap all of the e in budding virions. this idea is supported by the finding that e is not transported beyond the golgi stack in cellular membranes (tooze and stanley, ; tooze et al., ) . alternatively, cisternal e may represent a genuine part of the maturation pathway. el may have to move through golgi cisternae and then back again to the budding compartment before it can participate in the budding reaction. this seems very unlikely, however, because all attempts to show movement of other er proteins to the golgi stack and back again have failed (yamamoto et al., ; brands et al., ) . it is, therefore, more reasonable to assume that e is transported from the membrane of the er to the budding compartment where it reaches a concentration sufficient to allow budding for the first time. thereafter, incorporated into the envelope of progeny virions, it moves through the golgi stack and thence to the cell surface with only a small amount leaking to the golgi stack independent of the virions. as the infection progresses the amount of el being synthesized increases (our unpublished data) and at the same time budding of progeny virions directly into the lumen of the rough er begins and increases. we interpret this to indicate that the rate of insertion of e into the rough er membrane beings to exceed the rate of its transport out of the er to the budding compartment. reducing the temperature to °c profoundly inhibits the release of virions into the medium. all the morphological evidence suggests that at °c it is the entry of virions into the golgi complex that is inhibited; as a result, few virions are seen in the golgi cisternae or in the post golgi transport vesicles and the golgi stack maintains its normal morphology. the inhibition of virion transport is accompanied by an inhibition of terminal glycosylation. this would be expected if (a) terminal glycosylation occurred in the trans-golgi complex and, (b) if the e is delivered there normally incorporated in the envelopes of progeny virions rather than as an integral protein of cellular membranes. but at °c, addition of galnac still occurs to an unchanged extent suggesting that this sugar is added in or just before the budding compartment. interestingly, the amount of e containing galnac (el does not change when the temperature is reduced. the de-crease in elm is accompanied only by an increase in el°. this correlates with the increase in budding into the rough er and further argues that galnac is not added in the rough er but in the budding compartment. if galnac was added either cotranslationally or while the e resided in the rough er membrane, an increase in the amount of the eli form would be expected both at and °c as the concentration of e reaches levels that not only support budding into this compartment but also can readily be detected by immunocytochemistry (tooze et al., ) . however, the constant amount of e containing galnac (eli) implies that the compartment in which the sugar is added has a limited capacity and is smaller than the rough er. morphological evidence indicates that the budding compartment fulfills these criteria. the budding compartment is not a product of infection because it is found in uninfected sac(-) cells and our preliminary observations of serial sections of rat exocrine pancreatic cells and murine att cells shows that it is not peculiar to sac(-) cells. in all three cell types, the budding compartment is found frequently in close association with patches of rough er differentiated into transitional elements in regions juxtaposed to the cis side of the golgi apparatus. however, the budding compartment and transitional elements of the rough er in sac(-) cells are not restricted to this region; they also occur associated with patches of rough er remote from the golgi apparatus. the existence of peripheral transitional elements in pancreatic cells has long been known (jamieson and palade, ) . our observations, together with those of saraste and kuismanen ( ) , tartakoff ( ) , and merisko et al. ( ) indicate that the structure of the transitional element-transitional vesicle region of the cell is considerably more complex than hitherto appreciated. fig. summarizes our interpretation of the results described above which suggest that galnac is added in the budding compartment. this is not to say that the enzyme, n-acetyl-galactosaminyltransferase, is found exclusively in this compartment, although available fractionation data shows that it is restricted to smooth membranes (kim et al., ; ko and raghupathy, ; hanover et al., hanover et al., , elhammer and kornfeld, ) . one must remember that the position of an enzyme can only be inferred from the position of the substrate and product subject to certain strict criteria. obviously, the enzyme cannot be located in the pathway after the point at which the substrate acquires the sugar, but it can exist at any site up to the point at which the substrate appears to be modified. this is because the substate may have to assume a particular conformation before it can be acted upon by the enzyme. the n-acetyl-galactosaminyltransferase could be present earlier in the pathway than the budding compartment but the e may not be a substrate until it reaches the budding compartment. alternatively, the means of detecting the modification may depend on the substrate conformation. the presence of protein-bound galnac in the cis golgi as detected by lectin labeling (roth, ) may reflect addition of the sugar in this compartment or in some prior compartment in which the protein-bound oligosaccharide is inaccessible to the lectin. these possibilities are not normally considered but they could explain the large and otherwise irreconcilable discrepancies in the location that has been assigned to n-acetyl-galactosaminyltransferase (strous, ; niemann et al., ; cummings et al., figure . schematic illustrating the first site of budding of mhv-a into a smooth membrane compartment between the transitional elements of the er and the golgi stack. reduced temperature blocks entry of the virions into the golgi stack and prevents el acquiring galactose and sialic acid but not galnac. this sugar is probably added in the budding compartment which is of limited size and distinct from both the er and golgi apparatus. ; johnson and spear, ) . these differences will only be resolved by locating the enzyme itself using an immunocytochemical technique. the transport to the plasma membrane of the envelope glycoproteins of the influenza virus, sfv, and vsv can be slowed in the trans-most golgi compartment by reducing the incubation temperature from to °c (matlin and simons, ; saraste and kuismanen, ; fuller et al., ) . blocking the transport of these proteins from the rough er to the golgi stacks requires a further reduction in temperature to °c (saraste and kuismanen, ) . by contrast, lowering the incubation temperature by only °c, from to °c, results in at least a , -fold reduction in the release of mhv-a virions and concommitantly blocks the completion of the o-glycosylation of the e viral envelope glycoprotein, as we have shown here. why the transport of mhv-a particles from the budding compartment to the golgi apparatus and thence out of the cell is so very much more cold sensitive than the transport out of the rough er of the envelope glycoproteins of vsv and sfv remains to be explained. possibly the difference stems from the fact that in one case it is whole virions which are being transported and, in the other, viral glycoproteins inserted into cellular membranes. sequence and topology of a model intracellular membrane protein, e glycoprotein, from a coronavirus structure of the carbohydrate units of lga~ immunoglobulins. structure of the o-glycosidically linked oligosaccharide units phase separation of integral membrane proteins in triton x-i solution retention of membrane proteins by the endoplasmic reticulum a monoclonal antibody against a k golgi membrane protein the sialic acids. xvi. isolation of a mucin sialytransferase from sheep submaxillary glands protein synthesis, storage, and discharge in the pancreatic exocrine cell. an autoradiographic study biosynthesis of n-and o-linked oligosaccharides of the low density lipoprotein receptor assembly of enveloped rna viruses two enzymes involved in the synthesis of o-linked oligosaccharides are localized on membranes of different densities in mouse lymphoma bw cells newly synthesized g protein of vesicular stomatitis virus is not transported to the golgi complex in mitotic cells an enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late golgi compartments uukuniemi virus glycoproteins accumulate in and cause morphological changes of the golgi complex in the absence of virus maturation passage of viral membrane proteins through the golgi complex the trans golgi network: sorting at the exit site of the golgi complex synthesis of n-and o-linked glycopeptides in oviduct membrane preparations temporal aspects of the n-and o-linked glycosylation of human chorionic gonadotropin evolution ofa coronavirus during persistent infection in vitro analysis of the functions of coronavirus glycoproteins by differential inhibition of synthesis with tunicamycin tunicamycin resistant glycosylation of a coronavirus glycoprntein: demonstration of a novel type of viral glycoprotein lntracellular transport of secretory proteins in the pancreatic exocrine cell. l role of the peripheral elements of the golgi complex o-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the golgi complex studies on semliki forest virus subviral components glycoprotein biosynthesis in the developing rat brain. i. a study of glycotransferases in microsomal subfractions glycoprotein biosynthesis in the developing rat brain. ii. microsomal galactosaminyltransferase utilizing endogenous and exogenous substrate acceptors structure of glycoproteins and their oligosaccharide units the synthesis of complex-type oligosaccharides. i. structure of the lipid-linked oligosaccharide precursor of the complex-type oligosaccharide of the vesicular stomatitis virus g protein polyacrylamide gel electrophoresis of viral proteins measurement of the incorporation of radioactive amino acids into proteins by a filter-paper disc method reduced temperature prevents transfer of a membrane protein to the cell surface but does not prevent terminal glycosylation the reorganization of the golgi complex in anoxic pancreatic acinar ceils incorporation of n-acetylmannosamine and n-acetylglucosamine in rat thyroid in vitro radiographic comparison of the uptake of galactose-[ h] and glucose-[ h] in the golgi region of various cells secreting glycoproteins or mucopolysaccharides coronavirus glycoprotein el, a new type of viral glycoprotein post-translational glycosylation of coronavirus glycoprotein el : inhibition by monensin the carbohydrates of mhv-a : structures of the o-glycosidically linked oligosaccharides of glycoprotein el a new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the goigi stack biosynthetic protein transport and sorting by the endoplasmic reticulum dissection of the golgi complex. ii. density separation of specific golgi functions in vitally infected cells treated with monensin enzymatic characterization of a i~-v-galactoside ¢t ~ sialytransferase from porcine submaxillary glands cytochemical localization of terminal n-acetylgalactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of o-glycosylation the golgi apparatus: two organelles in tandem viral protein synthesis in mouse hepatitis virus-strain a infected cells: effect of tunicamycin purification to homogeneity and enzymatic characterization of an a-n-acetylgalacostamine a -" sialytransferase from porcine submaxillary glands pre-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface isolation and identification of virus-specific mrna's in cells infected with mhv-a initial glycosylation of proteins with acetylgalactosaminyl serine linkages the structure and behavior of coronavirus a glycoproteins the molecular biology of the coronaviruses the novel glycoproteins of coronaviruses pertubation of vesicular traffic with the carboxylic ionophore monensin temperature and energy dependence of secretory protein transport in the exocrine pancreas sorting of coronavirus from condensed secretory proteins at the exit from the trans golgi network of att cells replication of coronavirus mhv-a in sac(-) cells: determination of the first site of budding of progeny virions laminated cisteruae of the rough endoplasmic reticulum induced by coronavirus mhv-a infection biogenesis of the e glycoprotein of mhv-a identification of two epitopes in the carboxyterminal amino acids of the e glycoprotein of mouse hepatitis virus a by using hybrid proteins is cytochrome p- transported from the endoplasmic reticulum to the golgi apparatus in rat hepatocytes? received for publication october , and in revised form january . key: cord- -awip k authors: nan title: molecular characterization of two human autoantigens: unique cdnas encoding - and -kd proteins of a putative family in the golgi complex date: - - journal: j exp med doi: nan sha: doc_id: cord_uid: awip k serum autoantibodies from a patient with autoantibodies directed against the golgi complex were used to screen clones from a hepg lambda zap cdna library. three related clones, designated sy , sy , and sy , encoding two distinct polypeptides were purified for further analysis. antibodies affinity purified by adsorption to the lambda zap- cloned recombinant proteins and antibodies from nzw rabbits immunized with purified recombinant proteins reproduced golgi staining and bound two different proteins, and kd, from whole cell extracts. the sy protein was provisionally named golgin- and the sy /sy protein was named golgin- . the deduced amino acid sequence of the cdna clone of sy and sy represented . - and -kd proteins of and amino acids. the in vitro translation products of sy and sy cdnas migrated in sds-page at and kd, respectively. the in vitro translated proteins were immunoprecipitated by human anti-golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. features of the cdna suggested that sy was a full- length clone encoding golgin- but sy and sy together encoded a partial sequence of golgin- . analysis of the sy recombinant protein identified a leucine zipper spanning positions - , a glutamic acid-rich tract spanning positions - , and a proline-rich tract spanning positions - . a search of the swissprot data bank indicated sequence similarity of sy to human restin, the heavy chain of kinesin, and the heavy chain of myosin. sy shared sequence similarity with the heavy chain of myosin, the uso transport protein from yeast, and the -kd cytoplasmic dynein-associated polypeptide. sequence analysis demonstrated that golgin- and golgin- share % sequence similarity and, therefore, may be functionally related proteins. h uman autoantibodies have proven to be useful reagents in elucidating the structure and function of eucaryotic organelhs. for example, naturally occurring autoantibodies have been used to identify and clone chromatin, nucleolar, nuclear envelope, and cytoplasmic proteins (reviewed in references - ). antibodies directed against the golgi complex have been reported in the sera of patients with sle ( ), sj gren's syndrome and other systemic rheumatic diseases ( ) ( ) ( ) ( ) ( ) ( ) , idiopathic cerebellar ataxia ( ) , paraneoplastic cerebellar degeneration ( ) , and viral infections, including the ebv ( ) , and hiv ( ) . this study reports the identification and cloning of two golgi autoantigens that react with autoantibodies in the sera of patients with autoimmune diseases. the golgi apparatus is a complex cytoplasmic organelle that has a prominent function in the processing, transporting, and sorting of intracellular proteins ( , ) . structurally, the golgi complex is localized in the perinuclear region of most mammalian cells and is characterized by stacks of membranebound cisternae as well as a functionally distinct trans-golgi network ( , ) . the intraceuular transport of newly synthesized and recycled proteins requires directed movement between the endoplasmic reticulum, the intracdlular vesicles to the c/s, medial, and trans compartments of the golgi complex, and the plasma membrane ( , , ) . the signals and molecular characteristics of the proteins that control this intracellular traffic are poorly understood, but it is known that intraceuular microtubules are important structural and functional components ( , ) . constituent and resident proteins of the golgi complex bdieved to play a role in these processes include families of proteins such as the adaptins ( ) , the "coatomer" proteins (e.g., cz,fl,%/~ cops) ( ) ( ) ( ) , gtp-binding proteins ( ) , including adp ribosylation factors (arfs) ( , ) , and resident enzymes (reviewed in reference ) . although the golgi complex has been identified as a target of autoantibodies for almost a decade, little is known about the antigenic targets or the events that lead to the induction of anti-golgi antibodies. an attractive feature to the study of golgi antigens is the knowledge that viruses, including hiv ( ) , coronaviruses ( ) , rubella ( ) , and various others (reviewed in reference ) , are processed in, and disrupt ( , ) , the golgi complex. observations that certain viruses induce anti-golgi antibodies in mice ( , ) , and that individuals infected with ebv ( ) and the hiv-i virus ( ) bear anti-golgi antibodies, add incentive to the study of the molecular characteristics of the golgi autoantigens. in this study, we have used the antibodies from a patient with sle to clone and characterize two distinct golgi autoantigens. studies of the characteristics of these proteins and the effects of brefeldin a (bfa) on their intracellular distribution suggest that the proteins are related to the coatomer proteins and may belong to a family of myosin-like molecules. the molecular features of these proteins suggest that they have a unique function in the processing and transport of proteins through the golgi complex. human sera. sera from patients with golgi autoantibodies were obtained from serum banks at the university of calgary (calgary, alberta), the scripps research institute (la jolla, ca), and immuno concepts inc. (sacramento, ca). the serum samples were stored at - or - ~ the specificity of the autoantibodies for golgi antigens was first identified on the basis of indirect immunofinorescence (iif) microscopy ( ) . control sera were randomly selected from a bank of , female red cross blood donors ( ) or sera pooled from healthy volunteers. cell lines. hela (atcc ccl . ; american type culture collection, rockville, md); molt- (human t cell lymphobhstic leukemia; atcc crl ); hep- (human epidermoid carcinoma ceils), and hepg (human hepatic carcinoma, atcc hb ) cells were maintained in rpmi supplemented with % fcs, mm t-glutamine, and /~g/ml gentamycin sulfate. indirect immunafluorescence. iif was performed on cells grown on teflon-masked slides or commercially prepared hep- cell substrates (immuno concepts inc.) using a fluorescein-conjugated goat anti-human igg (light and heavy chain) as previously described ( , ) . double-hbeling and colocalization studies used tritcconjugated wheat germ agglutinin (sigma chemical co., st. louis, mo) and rhodamine-conjugated goat anti-rabbit igg (pierce, rockford, il). slides were viewed with a microscope fitted with epifinorescence (carl zeiss, inc., thornwood, ny) or a confocal scanning laser microscope equipped with a krypton-argon light source (mrc ; bio-rad laboratories, richmond, ca). immunoelectron micmscolv. human hep- cells were grown in dmem containing % fcs in lux permanox dishes (electron microscopy sciences, fort washington, pa). after d in culture, monolayer cells were rinsed in pbs, fixed min at room tempera- abbreviations used in thispaper: bfa, brefeldin a; cop, coatomer protein; lie indirect immunofluorescence. ture (rt) with % electron microscopy-grade formaldehyde (polysciences, warrington, pa) buffered with pbs, rinsed with pbs, and then permeabilized with % acetone for min at - ~ after a rinse in pbs, cells were blocked for min at rt with i% bsa/pbs, incubated h at ~ with the serum sy diluted : in % bsa/pbs, rinsed three times for min in pbs, blocked min with % bsa/pbs, and then incubated i h at ~ with affinity-purified goat anti-human igg coupled to peroxidase (cappel laboratories, cochranville, pa) diluted / in % bsa/pbs. cells were then rinsed three times for min in pbs, fixed min at rt with % glutaraldehyde buffered with pbs, rinsed in pbs, rinsed in mm tris-hc (ph . ), and then incubated min at rt in mg/ml diaminobenzamine/ . % hzoz dissolved in tris buffer and distilled water, and then stained for min with % oso in distilled water, rinsed in distilled water, dehydrated with ethanol, and then embedded in polybed (polysciences). embedded whole cells were photographed in the light microscope, thin sectioned as monolayers, and then examined unstained in the electron microscope. sds-page and immunoblotting. proteins or cellular preparations were solubilized in sds sample buffer and separated by discontinuous sds-page according to the method of laemmli ( ) . the separated proteins were transferred to nitrocellulose as described by towbin and gordon ( ) and adapted in our laboratory ( ) . after transfer, nitrocellulose strips were blocked with % nonfat milk in pbs containing . % tween- (pbst), and then overlaid with the primary antibody and washed with pbst to remove any unbound antibody. bound antibody was traced with polyvalent, peroxidase-conjugated goat anti-human ig (calbiochem-behring corp., la jolla, ca) or si-protein a ( - x s cpm/ml (icn radiochemicals, irvine, ca). bound antibodies were visualized by incubating the washed nitrocellulose strips in substrate solution or by exposing the air-dried nitrocellulose to x-omat ar film (eastman kodak co., rochester, ny). edna library screening. clones from a hepg xzap cdna library were initially screened by an immunological method as modified by young and davis ( ) . all screening was performed on duplicate filters and positive bacteriophages were subsequently purified to %. before screening the cdna library, the sera were extensively adsorbed against bacteria and wild-type )~_ap phage to reduce background binding. bound antibodies were detected by immunoblotting as described above. dna subcloning and sequence determination. purified )~ _ap clones were subcloned in vivo into pbluescript plasmids using r helper phage as recommended in the manufacturer's instructions (stratagene inc., la jolla, ca). the nucleotide sequence was determined using the dye primer cycle sequencing kit and dna sequencer ( a; applied biosystems, inc., [abi], foster city, ca). oligonucleotide primers were synthesized with a dna synthesizer ( b; abi). dna sequences were determined in both strands and compiled using the alignment program seqed (abi). pcr. pcr was used to determine the size and orientation of cdna inserts using methods essentially as described in the geneamp dna amplification reagent kit (perkin elmer corp., norwalk, ct). -or -/~ reaction volumes were used and were composed of pcr buffer ( mm tris-hcl, ph . , mm kci, . mm mgc , . % gelatin), datp, dctp, dgtp, and dttp to a final concentration of . /~m. sample dna was added and solutions were heated to ~ for min, followed by addition of . u amplitaq (rtaq dna polymerase; perkin elmer corp.), and one drop of paraffin oil dna amplification was performed using a microcycler (eppendorf inc., freemont, ca) with up to cycles of ~ for s; ~ for s; ~ for s; and a final -rain elongation step at ~ typically, - % of the reaction volumes were separated on % agarose gds and stained with ethidium bromide to visualize pcr products. dna samples for pcr were obtained from phage suspensions or bacterial colonies ( ) . a~inity purification ofautoantibodies. affanity purification of antibody from kzap dones used confluent plates that were induced to produce recombinant protein with isopropyl-thiogalacto-pyranoside (iptg; fisher scientific, springfield, nj)-impregnated nitrocdlulose filters. after incubating the plates overnight, the filters were blocked and probed with primary antibody and washed as described above for immunoblotting. bound antibody was ehted from the filters with ml ofelution buffer ( mm khzpo,, ph . , mm nac , . % bsa) by rocking at room temperature for min. the filter was quickly rinsed with an additional -ml dution buffer and the eluates were collected and neutralized with ~ m "iris, ph . . the eluted antibodies were concentrated with a microconcentrator (centricon ; amicon corp., danvers, ma) and used for iif and immunoblotting analysis. in vitro rna transcription and translation. appropriate clones identified in the screening outlined above were used for in vitro transcription and translation experiments. /zg of linearized plasmid dna was used as template for in vitro transcription with t or t rna polymerase. rna transcripts were analyzed in . % agarose gel containing . m formaldehyde. ~g of the transcribed rna was added in a -/a translation reaction containing rabbit reticulocyte lysate, [ ss]methioniue (trans-~ss label, % methionine, % cysteiue; icn biochemicals, irvine, ca), and rnase block ii (stratagene inc.) as suggested by the manufacturer (promega biotec, madison, wi). translation was carried out at ~ for i h followed by sds-page of a - -/zl aliquot to confirm the presence of translation products. samples were stored at - ~ until required. lmmunoprecipitation. immunoprecipitation of ~ss-labeled in vitro translation products was performed using protein a-sepharose beads ( ) . briefly, # of human serum and - ~ of in vitro translation product were incubated with protein a-sepharose beads - h at ~ after incubation, the sepharose beads were washed five times with buffer and resuspended in sds sample buffer. samples were then analyzed by sds-fage.and autoradiography as described above. recombinant protein production. plasmids were transformed in escherichia coil strain xl- biue (stratageue inc.). the recombinant protein was prepared from -ml cultures of the recombinant ceils grown to od~ = . at ~ and induced with iiri'g added to a final concentration of ram. after overnight incubation, the bacteria were harvested by centrifugation. the recombinant protein was enriched by the method of adams et al. ( ) . the final pellet of iuclusion bodies was extracted with m urea in . m tris (ph . ) on ice for min to remove residual bacterial proteins. the inclusion bodies were then pelleted in an eppendorf microfuge at , rpm and extracted with m urea in . m tr/s buffer for min on ice. in some experiments, the syll inclusion granules were extracted with tris buffer containing m urea, . % -mercaptoethanol. the supernatants from the extractions were stored at - ~ proteins were separated by sds-page and stained with coomassie blue or transferred to nitrocellulose for immunoblotting. rabbit immunization. three new zealand white rabbits were separately immunized by subcutaneous and intramuscular injection of . mg of extracted recombinant proteins in an equal volume of cfa. wk later, the animals were boosted with . mg of the protein in ifa. the appearance and titer of golgi antibodies was monitored by indirect immunofluorescence using goat anti-rabbit igg (h + l chain) antibody (calbiochem-behring corp., lajoua, ca) as described above. bfa. hep- cells growing under standard conditions on teflonmasked microscope slides (cell-line associates inc., newfield, nj) were incubated in . pm bfa (sigma chemical co., st. louis, mo). slides were removed from the bfa media after , , , , and min, washed in ice-cold pbs, then fixed in ice-cold methanol for rain. in cultures growing in paraud, the bfa media was replenished with fresh media without bfa and slides were removed for methanol fixation rain later. changes to the structure and distribution of the golgi complex antigens were followed by indirect immunoperoxidase staining ( ) using the prototype serum sy, rabbit antibodies to the recombinant sy , sy , and syll proteins, and affnity-pufifed antibodies as described above. computer analysis of nucleic acid and protein sequences. nucleic acid and protein sequences were analyzed by the university of wisconsin genetics group sequence analysis software package ( ) . alignment of protein sequences was achieved with the gap program that used a published algorithm ( ) . multiple sequence alignments were performed with the clustal programs ( ). nine sera that demonstrated the typical iif pattern for golgi autoantlbodies ( ) on hep- cells were identified. a serum from a sle patient with pericarditis and central nervous system involvement (sy) was selected for the cloning studies because it demonstrated high titer (~> : , ) and relatively monospecific staining of the golgi complex on methanol-fixed hep- cells (fig. a) . analysis of a variety of cell lines, including hepg .and hela cells, revealed similar patterns of staining. fixation of cells in acetone or % paraformaldehyde did not alter the staining pattern. immunoelectron microscopy of hep- cells demonstrated that antibodies from the serum sy bound to .juxtanudear cisternae of the golgi complex ( fig. , a and b ). further confirmation that the antigens were localized to the golgi complex was demonstrated by colocalization of tritcconjugated wheat germ agghtinin to the corresponding structures stained by the prototype serum (fig. , c and d). immunoblotting. immunoblotting of the prototype serum retained specificity after secondary and tertiary screening to % purity. when antibodies from the prototype serum were affinity purified on nitrocellulose filters containing x phage-expressing sy and syll recombinant proteins, all reproduced the golgi pattern of immunofluorescence on hep- cells (fig. b) . the stereo reconstruction of the staining derived from confocal scanning laser microscopy showed that the antibodies affinity purified from sy clones ( fig. b) bound primarily to the lamellar golgi stacks of hep cells. some reactivity with vesicular structures was also noted. no reactivity with microtubules, intranuclear components, or the plasma membrane was observed with the affinity-purified antibodies. a similar pattern of reactivity was observed with affinity-purified anti-sy (not shown). the cdna inserts were subdoned in vivo into pbluescript plasmid, and both strands were sequenced across the polylinker arms. the nucleotide sequences of the dna and the deduced amino acid sequences of sy and syll are shown in fig. . our data suggested that syll encoded a complete protein sequence because it contained a ' poly(a) tail and an open reading frame of amino acids (fig. a) . the deduced amino acid sequence of the cdna clone of sy and sy represented . -and -kd proteins of and amino acids, and calculated isoelectric points (pls) of . and . , respectively ( table ). the three clones were found to represent two groups of unique sequences with sy and sy having an "~ % overlap (figs. b and a) . a comparison of the length of the cdna clones and the overlap of sy and sy is illustrated in fig. a. analysis of the deduced amino acid sequence of sy predicted that this polypeptide was exclusively an c~-helical structure with an absence of/ sheets (fig. b) . similarly, syll had primarily an o~-helical structure with some sheet regions identified (fig. c) . other special features of syll included a leucine zipper (coiled-coil motif) spanning positions - , a glutamic acid-rich tract spanning positions - , a proline-rich tract spanning positions - , and a single putative glycosylation site (fig. c) . analysis of the sequences of sy or sy did not disclose any transmembrane domains or signal sequences. of interest, the amino acid sequence of sy demonstrated % sequence similarity and % sequence identity with sy (fig. d) . the nudeotide and deduced amino acid sequences were used to search the genebank, embl, and nbkf data banks for homologous sequences (table ). up to % similarity of amino acid sequences was found with several other reported sequences. a search of the swissprot data bank indicated that sy and sy had "~ % sequence similarity with the heavy chain of myosin from a number of species, including humans. the sequence similarity with the heavy chain of myosin is distributed over a -amino acid segment (amino acids - of syll and - of the human heavy chain of myosin). syll also shared '~ % sequence similarity with the human protein restin and the yeast protein num . sy shared ,o % sequence similarity with the yeast intracellular protein transport protein uso and the -kd dynein-associated polypeptide. neither sy nor syll shared significant sequence similarity with/ -adaptin, j -cop, or other known mammalian golgi components. rabbit antibody to recombinant protein. further support for the authenticity of the cloned cdna as coding for golgi complex antigens was obtained by analyzing rabbit antisera raised against syll, sy , and sy recombinant proteins. the resulting antibodies were shown to react in a similar manner to the prototype sera by indirect immunofluorescence (fig. ) and immunoblotting (fig. ) . fig. a illustrates the immunofluorescence staining pattern on hep- cells of the prototype gotgi serum using a fitc-conjugated anti-human igg reagent. fig. b shows the anti-golgi reactivity of rabbit sera after immunization with recombinant protein sy . double staining with fluorescein anti-human and rhodamine anti-rabbit igg showed overlap of the staining patterns. similarly, rabbits immunized with the sy or sy recombinant proteins demonstrated high titer (> : , ) anti-golgi re- whole cell light microscopy, the peraxidase staining is confined to thejuxtanuclear colgi complex (arrowheads); x . the antibody reactivity of the prototype serum colocalized to the same perinuclear and cytoplasmic structures (c) that were stained by tritcconjugated wheat germ agglutinin (d); x . activity by iif (fig. d) in a pattern that overlapped completely with the human antibody (fig. c) . immunoblotting of hepg cellular extract with rabbit antisera showed reactivities similar to that produced by human serum (fig. ) . the antibodies from the rabbit immunized with sy reacted specifically with the -kd antigen in hepg extract (fig. , lane i) . this band has the same molecular mass as the one identified by the prototype serum in molt cell extracts (fig. , lane ) and in hepg extracts (fig. , lane ) . of interest, the serum from the rabbit immunized with the sy recombinant protein also reacted with the -kd protein (fig. , lane ) and several other proteins of , , , and kd. the rabbits immunized with recombinant sy protein produced strong reactivity with a -kd protein but also weaker reactivity with > -, -, and -kd proteins (fig. , lane ) . the > -, -, and -kd proteins in hep-g cell extracts were also identified by the prototype serum (fig. , lane ) . rabbit sera wk after immunization with sy (fig. , lane ) showed weak reactivity with some of the same proteins, as did the rabbitimmune serum (fig. , lane ) . preimmune rabbit serum (fig. , lane ) and normal human serum (fig. , lane ) did not react with any proteins. in vitro translation and immunolmcipitation. the s-labeled in vitro translation product of syll cdna migrated in sds-page at kd ( fig. b, lane i) and was immunoprecipitated by the original human anti-golgi serum (fig. b, lane ) . the in vitro translation product of syll was also immunoprecipitated by rabbit antisera raised against the sy recombinant protein (fig. b, lane ) . another anti-golgi serum (fig. , lane ) that reacted with the -and -kd but not the -kd proteins did not precipitate the -kd in vitro translated product (fig. b, lane ) . on the other hand, a serum that reacted with the -and -kd proteins (fig. , lane ) immunoprecipitated the -kd in vitro translated products (fig. b, lane ) . these data supported the immunoblotting results in which sera that reacted with the cellular -kd antigen also tmmunopredpitated the in vitro translated sy protein. these observations also support the conclusion that syll encodes a full-length protein. evidence that the rabbit antiserum raised by immunization with recombinant sy recognized the same protein as the prototype human serum was shown when the rabbit antiserum immunoprecipitated the in vitro translation product in an identical manner to human golgi sera (fig. b, compare lanes and with lane ). by comparison, the in vitro translation product of sy migrated in sds-page at kd (fig. a, lane ) and was immunoprecipitated by serum from rabbits immunized with sy (fig. a, lanes and ) and sy (fig. a, lanes and ) , but not by preimmune rabbit serum (fig. a, lanes and ) . the prototype serum (fig. a, lane ) and another anti-golgi serum (fig. a, lane ) also did not precipitate the sy in vitro translation product. the lower molecular mass translation products of sy also appeared to be specifically immunopredpitated by the immune rabbit sera, indicating the presence of at least one epitope in these smaller fragments. the observation that rabbits immunized with sy or sy immunoprecipitated the in vitro translation product of sy is in agreement with the conclusion that there was ",~ % overlap of the sy and sy clones. the reason why the prototype human serum or other anti-golgi sera did not immunoprecipitate the in vitro translated sy product is not clear. for example, the recombinant proteins produced by sy and sy in e. coli were reactive with anti-golgi sera previously shown to recognize golgin- and golgin- in whole cell extracts (fig. , c and d, lane i). the prototype serum ( fig. d and c, lane ), affinity-purified antibodies to sy (fig. c, lane ) and sy (fig. d, lane ) , and rabbit serum raised against sy (fig. c, lane ) and sy (fig. , d, lane ) also reacted with the recombinant protein. normal human serum (fig. , c and d, lane ) and preimmune rabbit serum (data not shown) did not react with the purified recombinant proteins. the data suggest that the epitopes critical for human antibody binding are present on the recombinant protein derived from the original x phage and the subcloned pbluescript plasmid but that they are not present on the in vitro transcribed and translated protein. bfa. our attention turned to possible relationships of the cloned proteins to the coatomer protein fl-cop. bfa, an isoprenoid fungal antibiotic, is known to have an early and characteristic effect on fl-cop and other coatomer proteins of the golgi complex ( , , ) . intermediate concentrations ( . /~m) of the drug were used to study the effects on the golgi antigens identified by the prototype serum and the rabbit antisera raised against recombinant proteins. at and min, the golgi staining remained typical of untreated cells (fig. , a and b) . however, and min after exposure of hep cells to the drug, there is a remarkable reduction of staining in the perinuclear golgi complex and the appearance of vesicular and elongated microtubular structures (fig. , c and d) . at min, there is complete loss of golgi staining (e) that is restored min after removal of the drug (~. this work is aimed at characterizing and identifying the golgi complex autoantigens that are the targets of autoantibodies in the sera of patients with autoimmune diseases. to begin analysis of the golgi complex, we have cloned the cdna encoding the complete protein sequence of a -kd autoantigen identified by a sle serum, and have named this golgin- . we also obtained a cdna encoding a portion of a second a i ta__._~atcaggggctggtaatggagtcggttagacaactacaaatggagagagataaatat g c a t gt~t~a~t g~a~g ( ) i gold# complex antigen, named gold#n- , that reacted with the same sera and has a molecular mass of kd. the clinical relevance of anti-gold# antibodies is uncertain. it is interesting that two of the sera with antibodies to golgin- , including the serum used to clone the protein, were from patients who had cerebellar disease. likewise, the report of anti-gold# antibodies in patients with cerebellar ataxia ( ), paraneoplastic cerebellar degeneration ( ), and patients with sj gren's syndrome ( ) who can develop central nervous system disease ( , ) may be clues to the clinical importance and pathogenic role of gold# antibodies. v aagagggagctggga~gaagct gggcgagctgcaggagaagctgagcgagctgaagga~ggtggagctgaa~tc~t~a~a~a~ca~g k r e l g k k l g e l q e k l s e l k g t v e l k s q e a q s l q q q r d q y l ggacacctgcagcagtatgtggccgcctatcagcagctgacctct gagaaggaggtgctc-cataatcagctactgctgcagacccagc ~gt~a~a~a~ag g h l q q y v a a y q q l t s e k e v l h n q l l l q t q l v d q l q q q e a q ggcaaagcggtggccgagatggcccgccaagagttgcaggaaacccaggagcgcct ggaagctgccacccagcagaa~ ~agtt~at~tc~ g k a v a e h a r q s q e t q e r l e a a t q q n q q l r a q l s l m a h p g gaaggagatggact ggaccgggaggaggaggaggatgaggaggaggaggaggaggaggcggtggcagt~a~t~c~a~~~tg e g d g l d r e e e s d e e e s s s s a v a v p q p m p s i p s d l e s r s a h gtggcatttttcaactcagctgtagccagtgcc gaggaggagcaggcaaggctacgt gggcagctgaaggagc~t~t~t~a~t ~t~ tc~ag v a f f n s a v a s a g g e q a r l r g q l k g q r v r c r r l a h l l a s a q aaggagcctgaggcagcag~agccccagggacc gggggtgattctgtgtgtggggagacccaccgggccctgcaggg~t~a~ ~tat~atg k e p s a a a p a p g t g g d s v c g s t h r a l q g a m e k t. q s r f h e l m caggagaaggcagacctgaaggagagggtagaggaactggaacatcgctgcatccagctttctggagagacagacaccatt gga~gtaca~t~a~aga~a~a~ g q e k a d v. k e r v e e l e h r c i q l s g e t d t i g e y i a l y q s q r a v ctgaaggagcggcaccgggagaac-gaggagta~atcagcaggctggc~aagacaaggaggagatgaa~t~t~t~~~~ l k s r h r e ) e e y i s r l a q d k g e m k v k l l e l q e l v l r l v g d r we considered the possibility that the gold# antigens that we have cloned bear resemblance to gold# autoantigens described by others. the best characterized gold# autoantigen was recently reported by kooy dynein-associated polypeptide lo lo figure . immunofluorescence localization of golgin- and gdgin- . anti-golgi antibodies from the prototype serum (sy) at~nity purified with xzap recombinant protein sy (a) or syll (c) colocalized with rabbit antibodies raised against the golgin- (b) or golgin- recombinant proteins (d), respectively. the binding of afl:lnity-pudfied anti-syll was traced with fluorescein-conjugated goat anti-human antibodies and the binding of rabbit antibodies was traced with rhodamine-conjugated goat anti-rabbit antibodies; x . ( ) . although proteins with leucine zippers are able to bind dna and are thought to be predominantly regulatory in their function ( ) , this motif also mediates the dimerization of certain transactivators ( , ) , and is essential for recombination of certain viral proteins ( ) and oligomerization of viral epitope proteins ( , , ) . unlike golgin- , some proteins with leucine zipper motifs contain a domain of - amino acids with a high ratio of the basic amino acids arginine and lysine, which are immediately adjacent to the putative zipper structure ( ) . golgin- has a stretch of acidic residues from amino acids to . consecutive stretches of acidic amino acid residues have been described in other autoantigens, including nor- (human upstream binding factor [hubf]) ( ), high mobility group protein i (hmg- ), centromere protein b (cenp-b), and nucleolin (reference and references therein). although the role of these sequences is not clear, it is likely that these stretches are responsible for binding to cationic molecules. the golgi complex, localized in the perinudear region of most mammalian cells, consists of stacks of membranous cisternae with functional and topological polarity (for reviews see references and ). the processing, maturation, and sorting of secretory and membranous proteins take place in this organelle. many of the specific enzymes responsible for the sequential modification of proteins passing through the golgi complex have been characterized. although considerable detail about these enzymes is known, the mechanisms by which proteins are transported and sorted in the golgi complex remain unclear. figure . immunoblot analysis of hepg whole cell extracts separated on % get sds-page and probed using rabbit antisera raised against golgin- and golgin- . the rabbit antisera diluted : show reactivity with proteins of similar molecular mass as the prototype serum (sy) diluted : . rabbit antibodies raised against sy (lane i) react with a -kd protein that has the same molecular mass as the fainter upper band reacting with the prototype serum (lane ). rabbit antibodies raised against syll (lane ) react strongly with a -kd protein that has the same molecular mass as a band recognized by the prototype serum (lane ). lane represents the reactivity of a rabbit serum ( : ) wk after immunization with sy . lane was probed with an immune rabbit serum wk after a boost with the recombinant sy protein. this serum reacted with the -kd protein and several other lower molecular mass proteins. lanes and are preimmune rabbit serum and normal human serum, respectively. recent evidence suggests that important molecules in this process are four high molecular mass coat proteins (cops: or-cop, kd; b-cop, kd; "y-cop, kd; and fi-cop, kd) that are major constituents of the nondathrincoated vesicles ( ) . these proteins are associated with several other lower molecular mass ( - -kd) proteins in the cytosol ( ) , bind reversibly to membranes of the golgi complex, and exist as a high molecular mass complex in the cytosol referred to as the "coatomer" ( , , ) . b-cop displays homology with the human clathrin-coated vesicle protein b-adaptin ( , ) . b-cop does not bear sequence similarity to kinesin or other microtubule-associated proteins (maps), nor does it have a transmembrane or other obvious target sequences ( ) . bfa, a drug that blocks intracellular transport, blocks the binding of b-cop to golgi membranes producing a characteristic pattern of immunofluorescence ( ) . in our study, we have noted similar effects of bfa on the intracellular distribution of golgin-g and golgin- . taken together, these observations suggest that golgin- and golgin- may be structurally and functionally related to the cops. however, they do not have sequence similarity to b-cop or the adaptins, and do not bear the nonapeptide slgeipive described by serafini et al. ( ) or the putative microtubule-binding motif kkex motif in b-cop suggested by duden et al. ( ) . a notable feature of golgin- is the sequence similarity to microtubule proteins and, like b-cop, the absence of a transmembrane or signal sequence. it is possible that golgin- and golgin- are functionally and structurally related because they share > % sequence similarity and . % sequence identity over a stretch of amino acids. in this regard, it is interesting that golgin- and golgin- showed significant sequence similarity to several cytoskeleton-related proteins, including kinesin ( ), the -kd dynein-associated protein ( ) , the myosin family proteins (heavy chain myosin, tropomyosin), and desmin. kinesin is a microtubule-stimulated atpase ( , ) and a likely motor protein for organdie transport along microtubules. the sequence similarity of the golgins with these proteins is only ~ %, but the aligned sequences span > amino acids in or-helical domains of these proteins. this suggests that the golgi complex has unique motor proteins that are part of the myosin family. another observation that supports this conclusion is that golgin- has sequence similarity to the yeast cytoskeleton-related protein uso . it has been demonstrated that uso is required for protein transport from the endoplasmic reticulum to the golgi apparatus ( ) . in summary, autoantibodies from a patient with sle have been used to identify, done, and sequence two apparently related golgi-associated proteins having molecular masses of and kd. we have designated these proteins golgin- and golgin- . the function(s) of these two proteins is unknown. based on the analysis of the sequences of these proteins and the effects of bfa on their distribution, several possibilities can be considered. first, these proteins may be kinesin-like motor proteins that have a role in the transport of vesicles from the endoplasmic reticulum to the golgi complex or within the golgi stack. second, both proteins may form a cytoskeletal structure that is the framework for transport of golgi vesicles. sequence similarity of the cloned proteins to cytoskeleton-related proteins supports this possibility. the deduced secondary structure suggests a predominantly coiled-coil structure. third, golgin- may be a structural protein within the golgi complex responsible for protein transport. the presence of a leucine zipper, and stretches of acidic residues, is consistent with features of a coat protein. this is further substantiated by observations that the behavior of both proteins is similar to b-cop after bfa exposure. studies using a number of cell biology and molecular tools can help determine the more precise roles of these proteins in the future. in the meantime, sera from patients with sle continue to provide valuable reagents in the study of immune responses and the structure and function of key cellular organelles. figure . immunoprecipitation of in vitro translation and transcription products of syll and sy . in vitro translation products derived from the syll or sy plasmid templates linearized with saci restriction enzyme and transcribed with t or t rna polymerase. the [ ss]methionine-labeled in vitro translated products were incubated with rabbit or human serum and the antigen-antibody complexes adsorbed to protein a-sepharose. the complexes were separated by sds-page and the dried gel was processed and exposed to x-ray film to trace the bound antigen. (a) the translation product [ sslmethionine-hbeled sy (lane i) migrated at kd with smaller translation products also observed. the -kd [ ss]methionine-hbeled in vitro translated product was not immunoprecipitated by preimmune rabbit serum (lanes and ) but it was by antibodies from rabbits immunized with the sy recombinant protein (hnes and ) and rabbits immunized with sy (lanes and ) . . effects of bfa on the goigi apparatus. bfa at . /~m was added to logarithmic-growing hep- cells in culture. at selected time intervals, slides were removed, fixed, and processed for indirect immunofluorescence using affinity-purified anti-syll. (a) time , demonstrating clearly defined golgi organization; (b) min after bfa, the golgi antigen shows early signs ofredisttibution; (c) at min, there is loss of staining in the golgi region; (d) at min, there is more loss of the lamellar golgi antigen with apparent redistribution to the cytoplasm; (e) at min, there is virtual loss of golgi staining; (/) min after bfa media was removed and replaced with fresh media, there is a restoration of the staining in vesicular and lamellar structures consistent with the golgi staining. antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology recombinant autoantigens. ann. rheum. d/s the diversity and origin of rheumatologic autoantibodies antibodies from patients with autoimmune disease react with a cytoplasmic antigen in the golgi apparatus anti-golgi complex autoantibodies in a patient with sjrgren's syndrome and lymphoma anti-golgi complex autoantibodies in patients with primary sjrgren's syndrome a monoclonal antibody recognizing golgi apparatus produced using affinity purified material from a patient with connective tissue disease human autoantibodies as reagents to conserved golgi components anti-golgi antibody in rheumatoid arthritis patients recognizes a novel antigen of kda (doublet) by western blot presence of an autoantibody against a golgi cisternal membrane protein in the serum and cerebrospinal fluid from a patient with idiopathic late onset cerebeuar ataxia immunoperoxidase labeling of rat brain sections with sera from patients with paraneoplastic cerebellar degeneration and systemic neoplasia anti-organelle and anticytoskeletal autoantibodies in the serum of epstein-barr virusinfected patients nonorgan-specific autoantibodies in individuals infected with type human immunodeficiency virus progress in unraveling pathways of golgi traffic the golgi complex: in vitro veritas? compartmental organization of the golgi stack the trans golgi network: sorting at the exit site of the golgi complex intracellular aspects of the process of protein synthesis movement of proteins through the golgi stack: a molecular dissection of vesicular transport role of microtubules in the organization of the golgi apparatus the directed migration of eukaryotic cells coatomer": a cytosolic protein complex containing subunits of non-clathrincoated golgi transport vesicles a coat subunit of golgiderived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein b-adaptin /~-cop, a kd protein associated with non-clathrincoated vesicles and the golgi complex, shows homology to b-adaption trimeric g proteins in golgi transport purification of a protein cofactor required for adp-ribosylation of the stimuhtory regulatory component of adenylate cyclase by cholera toxin adp-ribosylation factor is functionally and physically associated with the golgi complex dual infection of the nervous system by aids viruses with distinct cellular tropisms induction of demyelination by a temperature sensitive mutant of the coronavirus mhv-a; is associated with restriction of viral replication in the brain the rubellar virus e glycoprotein is arrested in the novel post-er, pre-golgi compartment protein localization and virus assembly at intracellular membranes the assembly of hiv within the golgi apparatus and golgi-derived vesicles of jm cell syncytia cytomegalovirus prevents antigen presentation by blocking the transport ofpeptideloaded major histocompatibility complex class i molecules into the medial-golgi compartment autoimmunity induced by lactate dehydrogenase-elevating virus: monoclonal autoantibodies against golgi antigens and other subcellular elements lactate dehydrogenase-elevating virus induces anti-golgi apparatus antibodies antinuclear, anticytoplasmic, and anti-sj gren's syndrome antigen a (ss-a/ro) antibodies in female blood donors antinuclear antibodies and the connective tissue diseases autoantibody testing: procedures and significance in systemic rheumatic diseases cleavage of structural proteins during the assembly of the head of bacteriophage t immunoblotting and dot immunobinding-current status and outlook antibodies in procainamideinduced and systemic lupus erythematosus bind the c-terminus of histone (h ) eilident isolation of genes using antibody probes the polymerase chain reaction colony miniprep human autoantibodyreactive epitopes of ss-b/la are highly conserved in comparison with epitopes recognized by murine monoclonal antibodies mrna polyadenylate binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence the detection of autoantibodies on hep- cells using an indirect immunoperoxidase kit (colorzyme). i~'agn a comprehensive set of sequence analysis for the vax a general method applicable to the search for similarities in the amino acid sequence of two proteins clustal: a package for performing multiple sequence alignment on a microcomputer multiple targets for brefeldin a brefeldin a: insights into the control of membrane traffic and organelh structure sj gren syndrome: central nervous system manifestations primary sj gren's syndrome with central nervous system disease mimicking multiple sclerosis the leucine zipper: a hypothetical structure common to a new class of dna binding proteins evidence that the leucine zipper is a coiled-coil molecular definition and sequence motifs of the -kd component of human ss-a/ro autoantigen cdnaderived amino acid sequence of the -kda subunit of the ku antigen leucine repeats and an adjacent dna binding domain mediate the formation of functional cfos-cjun heterodimers coronavirus motif leucine zipper extends. nature monensin action on the golgi complex in perfused rat liver: evidence against bile salt vesicular transport action ofleucine zippers. nature isolation of a recombinant copy of the gene encoding c/ebp a leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion retroviral envelope glycoproteins contain a leucine zipper-like repeat molecular domains involved in oligomerization of the friend leukemia virus envelope protein human autoantibody to rna polymerase i transcription factor hubf. molecular identity of nucleolus organizer region autoantigen nor- and ribosomal rna transcription upstream binding factor anionic regions in nuclear proteins purification of a novel class of coated vesicles mediating biosynthetic protein transport through the golgi stack brefeldin a, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on golgi cisternae a novel -kda protein associates with the golgi and microtubules homology of a k cytoplasmic dynein-associated polypeptide with the drosophila gene glued bovine brain kinesin is a microtubule-activated atp-ase native structure and physical properties of bovine brain kinesin, and identification of the atp-binding subunit polypeptide a cytoskeleton-related gene, uso , is required for intraceuular protein transport in saccharomyces cerevisiae key: cord- -og sg qw authors: howell, gareth j.; holloway, zoe g.; cobbold, christian; monaco, anthony p.; ponnambalam, sreenivasan title: cell biology of membrane trafficking in human disease date: - - journal: int rev cytol doi: . /s - ( ) - sha: doc_id: cord_uid: og sg qw understanding the molecular and cellular mechanisms underlying membrane traffic pathways is crucial to the treatment and cure of human disease. various human diseases caused by changes in cellular homeostasis arise through a single gene mutation(s) resulting in compromised membrane trafficking. many pathogenic agents such as viruses, bacteria, or parasites have evolved mechanisms to subvert the host cell response to infection, or have hijacked cellular mechanisms to proliferate and ensure pathogen survival. understanding the consequence of genetic mutations or pathogenic infection on membrane traffic has also enabled greater understanding of the interactions between organisms and the surrounding environment. this review focuses on human genetic defects and molecular mechanisms that underlie eukaryote exocytosis and endocytosis and current and future prospects for alleviation of a variety of human diseases. the human cell is a complex network of membranes and protein enclosed in a membrane lipid bilayer. the interactions within and associated with such biomembrane bilayers have profound consequences for the organism as a whole; a single defect in just of the potential - , gene products made by each cell can cause devastating, if not fatal, evects for the whole organism. in addition to this, humans pass genetic information onto their ovspring and, with it, any genetic mutations or polymorphisms. it is believed that at least in people have, or will eventually develop, a disease caused by mutation or variation at the gene level. understanding how genetic mutations increase risk for human disease is critical in our understanding and treatment of the majority of human ailments that are caused by interactions between the organism and the environment. this review focuses on the research undertaken in the past years relating to the molecular mechanisms that underlie membrane traycking within eukaryotic cells. we address mechanisms and factors that control protein progression through the secretory and internalization pathways and highlight key human diseases that illuminate mechanisms of membrane traycking. in addition, current and future strategies for therapeutic intervention in such genetic disorders are considered. common to all eukaryotic cells is the presence of multiple biomembrane lipid bilayer compartments, or organelles, which are maintained by specific protein-protein and protein-lipid interactions. such interactions are maintained within each compartment in spite of continuous traycking of membrane-bound and soluble components to diverent intracellular locations, and for secretion from the cell. in the majority of cases, this transfer of material occurs through vesicular movement: fission, docking, and fusion of membrane bilayer-enclosed intermediates occurs between donor and acceptor compartments (palade, ) . proteins, including membrane-bound receptors, secreted enzymes, and antibodies, begin their journey by entering the early secretory pathway at the endoplasmic reticulum (er). from here they are transported through the golgi apparatus and finally distributed to their final destination such as other intracellular organelles, the plasma membrane, or the extracellular environment. but how does a specific protein ''know'' how to reach a specific cellular destination when hundreds of newly synthesized, diverent molecules require specific transport and targeting? many of these transport intermediates or vesicles, whether derived from the er, other internal organelles, or the plasma membrane, are ''coated'' with unique protein complexes, tethering factors, and regulatory factors that ensure correct targeting to an acceptor compartment. vesicle coat proteins, such as the clathrin or coat protein (cop) complexes, are relatively well studied. such complexes are assembled onto the cytoplasmic face of donor compartments to facilitate the fission of transport intermediates. allied with these coat proteins are diverent molecules that mediate recognition of cytoplasmic motifs in cargo proteins either directly (e.g., transmembrane proteins) or indirectly (e.g., soluble secreted enzymes). the snare hypothesis is central to our understanding of vesicular targeting to intracellular compartments (rothman, ; sollner et al., ) . initially uncovered in a screen for intra-golgi transport docking and fusion regulators, the snare (soluble n-ethylmaleimide-sensitive fusion attachment protein receptor) proteins have been found to regulate diverent membrane interactions in all eukaryotes via a highly conserved mechanism for membrane traycking based on accessory docking and fusion regulators. snare proteins are present on both the vesicle (vesicular or v-snare) and the acceptor (target or t-snare) and comprise coiled-coil domains that assemble to facilitate vesicle docking and membrane fusion (bennett, ; pelham, ) . in conjunction with snare proteins, small ras-related rab gtpases are implicated in further ensuring the fidelity of vesicle docking and fusion (olkkonen and stenmark, ) . these -to -kda proteins are gtphydrolyzing enzymes that act to recruit diverent proteins or evectors to membranes in a gtp/gdp-regulated manner (collins, ) . rab gtpase activity and protein conformation are regulated by interaction with soluble and membrane-bound proteins; such regulators can also tether vesicles to acceptor membranes and mediate intracellular signaling. a. early secretory pathway the endoplasmic reticulum (er) is the first stage of quality control along the secretory pathway. proteins destined for secretion (e.g., hormones), the plasma membrane (e.g., membrane-bound receptors), or other intracellular membrane compartments such as the lysosome (e.g., lysosomal proteases) emphysema and liver cirrhosis (perlmutter, ) endophillin ii clathrin-coated pit formation leukemia (dreyling et al., ; jones et al., ; narita et al., ; tebar et al., ) alzheimer's disease presenilin presenilin -involved in cleavage and trafficking of amyloid precursor protein to plasma membrane neurodegenerative disorder (uemura et al., ) tau tau -microtubular stability through formation of aggregates autosomal dominant polycystic kidney disease (adpkd) polycystin- or causes a defect in e-cadherin assembly and basolateral trafficking renal cysts in kidney and other tissues leading to endstage renal failure (charron et al., ) (continued ) autosomal dominant retinitis pigmentosa rhodopsin inhibited interaction of rhodopsin and arf , leading to inhibited post-golgi delivery to rod outer segment narrowing of visual fields, night blindness (deretic et al., ) autosomal dominant ventricular tachycardia cardiac arrhythmia, hyperthermia (yano et al., ) autosomal recessive primary hyperoxaluria mistargeting of peroxisomal proteins to mitochondria kidney disease (danpure, ) ab-lipoproteinaemia mtp er retention thus preventing apob secretion vascular disease (sharp et al., ) batten's disease cln -cln group of gene products implicated in regulating the processing and targeting of lysosomal and synaptic proteins neurological disease (pearce, ) breast cancer caveolin- deletion or dominant negative mutation of caveolin- promotes tumor progression breast cancer (bouras et al., ; williams and lisanti, ) (hayasaka et al., ; matsuyama et al., ) chediak-higashi syndrome (chs) chs /lyst lyst involved in regulation of protein secretion from lysosomes -enlarged lysosomes partial albinism, recurrent bacterial infections, impaired chemotaxis and abnormal natural killer cell function (shiflett et al., ; ward et al., ) choroideremia (chm) rab escort protein (rep ) rab a remains cytosolic due to defective geranylgeranyl modification in chm lymphoblasts x-linked form of retinal degeneration combined factors v and viii deficiency ergic- /p c-type lectin er retention and defective secretion of factors v and viii blood disease congenital finnish nephritic syndrome nephrin (nphs ), podocin (nphs ) er retention kidney inflammation (kestila et al., ; kramer -zucker et al., ) pancreatic atp-sensitive potassium channel (k-atp) er or golgi retention of k-atp due to mutations in its sulfonylurea- (sur ) subunit excess insulin leading to hypoglycaemia (dunne et al., ; yan et al., ) congenital hypothyroid goiter thyroglobulin er storage disease. thyroglobulin is misfolded and accumulates in er constipation, large tongue, swelling around the eyes, failure to suckle, mental retardation (hishinuma et al., ; kim and arvan, ) (fan et al., ; garman and garboczi, ) familial hemophagocytic lymphoschistiocytosis (fhl) perforin -defective ctl (cytotoxic t lymphocytes) mediated killing immunodeficiency (feldmann et al., ; stepp et al., ) munc (hogg et al ., ; mathew et al., ) three forms of menkes disease can arise from diverent mutations in the atp a gene: premature stop codons, deletions, or splicing defects. these can prevent atp a function and/or traycking. classical menkes disease is the most common and fatality usually results by the age of years. in two other nonfatal forms of menkes, mild and occipital horn syndrome, atp a maintains the ability to transport copper ions across intracellular membranes, although traycking to the plasma membrane can be compromised (la fontaine et al., ) . atp a is ubiquitously expressed and is the major copper transporter in cells of the intestine, kidney, and brain. in the liver, however, the major copper transporter is the wilson's disease protein, atp b (bull et al., ) . this second p-type atpase shares strong similarity with atp a and also translocates copper ions across membranes. although these gene products share functional similarities, mutations in atp b result in copper accumulation in the liver and brain. familial hypercholesterolemia is an autosomal dominantly inherited disease caused by mutations in the low-density lipoprotein receptor (ldlr), leading to premature atherosclerosis and coronary heart disease. in healthy individuals, the ldlr is expressed on the surface of cells, where it binds circulating ldl particles and promotes uptake and cellular metabolism of its constituents, which includes cholesterol. in these patients, ldlr alleles display amino acid substitutions (cassanelli et al., ; jensen et al., ) , truncations (lehrm an et al ., ) , or mis sense mutat ions ( leiter sdorf et al ., ) , which can result in er retention and degradation. the point mutation at residue of the insulin receptor compromises the ability of the receptor to dimerize correctly within the er, therefore leading to er retention. decreased plasma membrane levels of insulin receptor cause inhibited insulin binding after stimulus by a meal, and subsequent elevations in plasma glucose levels. this then leads to type ii diabetes mellitus (kadowaki et al., ) . a number of human diseases can induce the er stress response. here, the mutant protein is retained within the er, resulting in either dilation of the organelle, such as in congenital hyperthyroidism (medeiros-neto et al., ) and hypofibrinogenemia (callea et al., ) , or chronic er stress as is the case for hereditary emphysema (perlmutter, ) . in pelizaeus-merzbacher disease, an x-linked leukodystrophy disease, er accumulation of proteolipid protein (plp) results in oligodendrocyte apoptosis (gow et al., ) and the subsequent disruption of white matter formation in the brain observed in humans and mouse models. plp is a central nervous system protein that is the major component of myelin and, when expressed in cultured fibroblasts, is localized to the plasma membrane (gow et al., ) . the link between plp and the er stress response provides a tool for elucidating the cellular response to misfolded protein accumulation . accumulation of proteins within the er, leading to blockage of protein secretion, is an unwanted cellular property and mechanisms have evolved to overcome such events. this disposal of unwanted proteins is termed er-associated degradation (erad) (fig. ) . as recently as the early s, it was still believed that aberrant proteins were degraded within the er (fra and sitia, ) ; however, current models suggest that aberrant er-retained proteins actually undergo retrotranslocation and subsequent degradation in the cytoplasm. retrotranslocation has been proposed to occur through the same ''pore'' used to translocate nascent proteins into the er lumen during translation, namely the sec translocon (biederer et al., ; rö misch, ) . various yeast and mammalian proteins have been shown to be retrotranslocated from the er and degraded within the cytoplasm in a proteasomedependent manner, including the budding yeast proteins carboxypeptidase y, and a mutant pro-a-factor. when a mammalian protein such as cftr is expressed in budding yeast, it matures relatively slowly within the yeast er, leading to retrotranslocation to the cytoplasm and degradation (ward et al., ) . a further example is that of a -antitrypsin deficiency. a -antitrypsin is responsible for inactivating the enzyme elastase produced by lung neutrophils. in this inherited disease, a mutated form of a -antitrypsin is retrotranslocated and degraded in proteasomes, leading to retention of active elastase in lung tissues and thus is a cause of lung emphysema (rutishauser and spiess, ) . however, retrotranslocation and proteasomal degradation may not be functionally coupled processes. pharmacological inhibitors that cause proteasome inactivation lead to egress of molecules such as mhc class i (wiertz et al., a,b) , ribophorin (de virgilio et al., ) , and carboxypeptidase y (biederer et al., ) from the er to the cell cytoplasm. in contrast, inhibition of protein ubiquitination results in the retention of such molecules within the er. schmitz et al. ( ) suggest that two distinct proteasome-regulated pathways mediate degradation of retrotranslocated b-amyloid precursor protein. interestingly, endocytosed toxins that target key cytosolic factors appear to use the erad pathway to move out of the er and into the cytosol (deeks et al., ; hazes and read, ) . cholera and ricin toxins are routed from the cell surface through the golgi apparatus and to the er before being retrotranslocated into the cell cytosol. it is believed that the unusually low lysine content of these protein toxins prevents subsequent er-associated ubiquitination for degradation by the cytosolic proteasome. protein cargo is shuttled between the er and golgi within vesicular intermediates or -nm-diameter spherical vesicles containing coat protein complexes fig. quality control of protein assembly within the endoplasmic reticulum. proteins destined for the secretory pathway (this example shows a transmembrane protein) are cotranslationally translocated from the ribosome into the lumen of the endoplasmic reticulum (er) through a portal referred to as the sec translocon. as the newly synthesized protein enters the er, quality control mechanisms in the form of protein chaperones bind to it and fold it to its correct conformation. further processing occurs through interactions with other chaperones before the successfully folded protein is loaded into copii-coated vesicles and shuttled from the er to the golgi apparatus. however, if the protein carries a mutation that causes it to take on an aberrant conformation the er chaperones will trigger a misfolded protein response. this has two outcomes: either the chaperones will remain bound to the misfolded protein, preventing its escape from the organelle (er retention), or the protein will be ubiquitinated and retrotranslocated through the sec complex for proteasomal degradation in the cell cytoplasm. a number of human genetic diseases are a result of key proteins failing to trayc through the secretory pathway and as a consequence are retained or degraded in this manner. such as copi or copii. initially discovered in mammals and yeast (kaiser and schekman, ; malhotra et al., ; novick et al., ; rothman and wieland, ) , cop complexes are required for the formation of vesicles at the er, er-golgi intermediate compartment (ergic), and golgi apparatus. cop recruitment to membranes facilitates the specific capture, packaging, transport, and delivery of membrane-bound and soluble protein cargo to an acceptor compartment. copii recruitment to sites on the smooth er initiates the formation of anterograde (forward) transport vesicles. these copii vesicles move from the er to the ergic, or vesicular tubular clusters (vtcs). from here, copi-coated vesicles are thought to mediate the continued anterograde movement from the ergic to the cis face of the golgi apparatus (scales et al., ) . the sar p gtpase regulates copii vesicle formation via interaction with the sec p guanine exchange factor (gef). sec p-mediated activation of sar p to a gtp-bound form leads to recruitment of the sec p-sec p heterodimer to membranes; this also initiates protein cargo selection within the er and recruitment of v-snares such as bet p and bos p. binding of sec p-sec p mediates further recruitment of the sec p-sec p complex. this copii complex then acts as a protein scavold that causes deformation of the membrane, resulting in vesicular fission, with anterograde movement of protein cargo-containing copii vesicles to the ergic. copii docking at an acceptor compartment is thought to trigger sec p function, causing a conformational change in sar p and gtp hydrolysis and dissociation or uncoating of the copii complex. thus copii vesicle docking and fusion with an acceptor compartment are mediated by cognate v-snare/ t-snare interactions (kirchhausen, ; kuehn et al., ; matsuoka et al., ; tang et al., ) . a severe hereditary bleeding disorder called combined deficiency of factor v factor viii (f f d) highlights the functional importance of traycking between the er and ergic. some f f d patients are deficient in the ergic-localized ergic- (lman ) protein and display defective secretion of the factor v and viii clotting factors. ergic- is a mannosebinding lectin that acts as a ''cargo receptor'' and recycles between the er and ergic (neerman-arbez et al., ; . however, % of f f d patients show normal levels of ergic- /lman , but are deficient in an associated protein, mcfd , another ergic resident that interacts with ergic- /lman in a calcium-dependent manner . small intestinal cells called enterocytes absorb fats and fat-soluble vitamins from food in the form of fatty acids and monoglycerides. the fats enter the lumenal surface of absorptive enterocytes by free divusion across their membranes, and emerge from the basolateral surface as particulate structures referred to as chylomicrons. formation of chylomicrons occurs within the er and golgi apparatus by vesicular transport before being traycked from the golgi to the plasma membrane. chylomicron retention disease (cmrd), anderson disease, and a neuromuscular disorder, cmrd associated with marinesco-sjö gren syndrome (cmrd-mss) , are examples of inherited diseases that result in compromised fat absorption, low blood cholesterol, and severely depleted blood chylomicron levels. jones et al. ( ) identified eight mutations in the sar p gene product and copii component associated with these lipid absorption diseases, thus strongly implicating a role for the copii vesicular transport system in the movement of dietary fats from the intestine to the circulating bloodstream. copii mediates anterograde trayc from the er to the golgi apparatus; however, copi vesicles appear to function primarily in the retrograde (backward) transfer of proteins from the golgi and ergic back to the er. this retrograde trayc is necessary for recovering escaped er resident proteins, coat and snare proteins that have arrived at the ergic and golgi from copii vesicles, or glycosylation enzymes that have been incorrectly modified (duden, ; lee et al., ) . the golgi-associated copi coatomer is a complex of seven polypeptides: a-, b-, b -, g-, d-, e-, and z-cop gene products, which interact with the donor membrane to form copi vesicles. vesicle formation is triggered by the gtpase adp-ribosylation factor (arf ), which recruits copi coatomer to the donor membrane. transmembrane proteins containing cytoplasmic lysine-based motifs such as kkxx or kxkxx, or soluble proteins containing the c-terminal kdel motif, are recycled by copi-coated vesicles from the golgi apparatus back to the er. the kdel motif, present in soluble er chaperones such as bip and protein disulfide isomerase, is recognized by the membrane-bound kdel receptor (majoul et al., ) . in both cases, cytoplasmic motifs in these transmembrane proteins are recognized and bound by copi coatomer, promoting inclusion into vesicles destined for the er. actin microfilaments are also involved in this retrograde transport step (valderrama et al., ) . this golgi-er step is regulated by the gtpase cdc and n-wasp protein (luna et al., ) , factors previously implicated in actin-linked processes at the plasma membrane. live imaging of cells expressing an engineered fluorescent and temperaturesensitive vesicular stomatitis virus g-glycoprotein (ts vsvg) demonstrated sequential action of copii-and copi-coated vesicles (scales et al., ) . vsvg accumulated in structures close to the er that contained intermediate compartment resident proteins. these structures then matured into vesicles that contained copi proteins. stephens et al. ( ) showed that this ''segregation'' between copii and copi vesicles occurred at a location in close proximity to exit sites on er membranes. a cop-independent mechanism has also been implicated in retrograde trayc between the golgi apparatus and the er. the rab gtpase is implicated in regulating the movement of bacterial shiga toxin b fragment (stb) via a retrograde step from the golgi apparatus to the er. expression of a dominant-negative gdp-bound form of rab inhibited stb retrograde movement, whereas copi transport was unavected (white et al., ) . the golgi apparatus is composed of flattened cisternae and membrane compartments that are closely juxtaposed in a stack-like appearance. in mammalian cells these stacks are positioned end-to-end, forming a ribbonlike structure near the nucleus (barr and warren, ) . the golgi apparatus is a highly dynamic organelle sited at the hub of the secretory pathway with key processing and sorting functions. the golgi is a polarized structure with proteins and lipids from the er received at the cis side, followed by the medial and trans subcompartments, where further glycosylation modifications occur; the trans-golgi network (tgn) is the final subcompartment where sorting and packaging events take place. the golgi apparatus also sorts proteins and lipids bound on a retrograde pathway from the cis-golgi back to the er. in addition, proteins can also return to the tgn from the endomembrane/lysosomal system (fig. ) . controversy exists regarding the mechanism for anterograde movement of cargo proteins within the golgi apparatus. the golgi apparatus contains secretory proteins that can vary in physical size, from relatively small polypeptides to large, bulky multisubunit complexes; all need to reach the tgn for final sorting into transport intermediates. there are also resident glycosylation enzymes that have spatially restricted functions within the golgi, that is, enzymes that function within specific subcompartments to ensure the correct addition or trimming of n-and o-linked sugars on secreted proteins as they progress through the pathway. this raises a key question: how do protein and lipid cargo move through the golgi apparatus while resident enzymes retain their localization? we know that many golgi enzymes contain transmembrane golgi localization signals that mediate targeting to a specific compartment (munro, ) . two models have been proposed: the cisternal maturation model and the vesicular transport model (elsner et al., ; storrie et al., ) . briefly, the cisternal maturation model suggests that large proteins or aggregates remain within a single golgi cisterna, which matures through the retrograde transfer of resident enzymes via copi vesicles. in contrast, the vesicular transport model proposes that newly synthesized protein is traycked from cisterna to cisterna via copi-coated vesicles that sequentially bud ov membranes and fuse with the next subcompartment. in either case, copi-coated vesicles play a central role in intra-golgi the secretory pathway and vesicular traycking. protein enters the secretory pathway at the endoplasmic reticulum (er) and is traycked in copii-coated vesicular structures to the intermediate compartment (ergic/vtc), from which copi-coated vesicles carry it to the cis face of the golgi. cargo protein (c) continues along the secretory pathway through the golgi apparatus to the trans-golgi network (tgn). retention signals in er resident proteins (r) ensure they undergo retrograde traycking from the golgi in copi vesicles. retrograde transport of transport. a number of snare proteins, such as membrin, rbet , gs , and syntaxin- , have also been localized to the golgi apparatus and are required for intra-golgi transport and homeostasis (nichols and pelham, ) . golgi-tethering molecules called golgins and golgi reassembly stacking proteins (grasps) belong to a family of regulatory factors involved in golgi maintenance and vesicular transport. the reader is pointed to an indepth review that covers golgins in more detail (short et al., ) . in brief, the golgins can be anchored to golgi membranes through various mechanisms and contain characteristic coiled-coil domains that extend from the membranes as a rod-like structure (burkhard et al., ) . golgins such as giantin and golgin- are securely anchored to the membrane via a transmembrane domain near their c terminus. electrostatic or ionic interactions mediate the attachment of other golgins to membranes. for example, proteins of the grasp family (grasp and grasp ) bind to gm and golgin- to recruit these factors to the cis and medial golgi membranes, respectively. moreover, a large number of golgins are recruited to membranes via interactions with the rab, arf, and arl (arf-like) gtpases. vesicular and cis-golgi membrane recruitment of golgin p is regulated by rab , whereas membrane attachment of yeast golgin rud p is regulated by arf p. golgin- binds to membranes by interaction with arl p, a member of a new class of arf-like gtpases termed arls (short et al., ) . interestingly, autoantibodies directed against giantin, golgin- , golgin- , gm , and golgin- golgins and grasps are present in patients with autoimmune conditions such as sjö gren's syndrome and systemic lupus erythematosus. in sjö gren's syndrome, moisture-producing glands are targeted by the autoimmune response, resulting in dry eyes and mouth (lichtenfeld et al., ) . systemic lupus erythematosus is a chronic rheumatic condition that avects joints and muscles, causing skin rash and kidney problems. sjö gren's syndrome patients can also simultaneously display both rheumatoid arthritis and systemic lupus erythematosus. golgi biogenesis requires golgin function at diverent stages during cell division. mammalian p is crucial for maintenance of the stacked nature of the golgi cisternae (puthenveedu and linstedt, ) . during mitosis, the golgi stack disperses into clustered vesicles. these vesicles then fuse in the daughter cells to form new cisternae, alignment and stacking of which result in the formation of a fully functional organelle. grasp tethers have been proposed to hold cisternae in close proximity through interactions with p and gm (shorter and warren, ) . the golgin p is also involved in tethering copi vesicles to golgi membranes (sonnichsen et al., ) and may be needed for snare complex assembly (shorter et al., ) . the budding yeast p homolog (uso p) tethers copii-coated vesicles to golgi membranes during anterograde transport from er exit sites to the cis-golgi (barlowe, ; cao et al., ; sapperstein et al., ) . mammalian p is also essential for the tethering of transport vesicles to the cis-golgi (alvarez et al., ) and during intra-golgi transport (seemann et al., ; waters et al., ) . golgins such as golgin- are implicated in the regulation of golgi structure and the formation of the golgi ribbon (diao et al., ) . golgin- may function as a tethering molecule in retrograde trayc from the endosome to the tgn (lu et al., ) . moreover golgins are also implicated as tethering components between the cytoskeleton and the golgi apparatus (short et al., ) . the trans-golgi network (tgn) is the final golgi subcompartment where secreted proteins are sorted, packaged, and directed to their final destination. traycking from the tgn can occur in either a constitutive or regulated manner. constitutive transport is the continuous release of protein from the trans-golgi network. regulated secretion occurs in response to extracellular stimuli such as secretagogues, metal ions, hormones, or growth factors, which trigger the docking and fusion of secretory granules or vesicles with the plasma membrane. various mechanisms control the traycking of proteins from the tgn by the formation and delivery of membrane-derived transport vesicles to the plasma membrane, endosomes, or lysosomal structures (ponnambalam and baldwin, ) . the expression of inactive (dominant-negative) protein kinase d isoforms in tumor lines (liljedahl et al., ) , polarized canine kidney cells (yeaman et al., ) , and mouse fibroblasts (prigozhina and waterman-storer, ) has been shown to inhibit vesicle fission (release) from the tgn. vesicle release is modulated by this family of kinases in response to cellular diacylglycerol (baron and malhotra, ) and binding to an as yet unknown evector protein on the cytoplasmic face of the tgn (van lint et al., ) . in addition, the cdc gtpase is linked to actin remodeling and has been shown to inhibit the exit of basolateral targeted proteins in polarized cells (kroschewski et al., ; musch et al., ) and copper-regulated protein transport (cobbold et al., ) . copper is an essential element and cofactor required for functionality of many secreted enzymes (cuproenzymes). at steady state, atp a (menkes howell et al. disease copper transporter; section iii.a. ) resides in the tgn, where it provides newly synthesized cuproenzymes such as lysyl oxidase with copper ions as they traverse the secretory pathway. when intracellular copper ion levels rise, atp a responds to this environmental danger by redistributing to the plasma membrane in a cdc -regulated manner (cobbold et al., ) . here, atp a acts as a copper ezux pump to remove copper ions from the cytoplasm to maintain homeostatic function and prevent toxicity. when copper levels are reduced, atp a recycles back to the tgn. this endocytic internalization and sorting event is independent of both clathrin and caveolae (cobbold et al., ) , although relying on a cytoplasmic dileucine motif present in the atp a c-terminus petris and mercer, ) . dent's disease, an x-linked kidney disorder that presents with hypercalciuria, nephrocalcinosis (kidney stone formation), and progressive renal failure, is caused by missense, nonsense, and deletion mutations within the endosomal clc- voltage-gated chloride channel. clc- is a member of a large family of voltage-gated chloride channels that have a diverse array of cellular functions including membrane excitability, transepithelial ion transport, and cell volume regulation (thakker, ) . when expressed in xenopus oocytes, a number of missense mutations in the clc- gene localized the channel to the golgi apparatus and showed reduced conductance and significantly reduced plasma membrane (pm) localization (ludwig et al., ) . similarly, expression of mutant clc- alleles in cultured cells revealed an approximate -fold increase in golgi retention (carr et al., ) . a. receptor-mediated endocytosis clathrin-coated vesicles (ccvs) are a route for protein internalization conserved from yeast to humans. roth and porter ( ) first observed this process in mosquito oocytes and these vesicles have subsequently become one of the best characterized membrane transport steps in eukaryotes. clathrin is one of the principal proteins involved in this transport step and, in combination with more than clathrin-associated factors, this unique structural component forms transport vesicles on the cytoplasmic face of the tgn, endosomes, and the plasma membrane. clathrin-coated vesicles bud from their donor membranes and are directed to target membranes by associated proteins and factors. this highly conserved -kda clathrin complex comprises heavy ( kda) and light ( kda) chain proteins that are assembled into a three-legged structure called a triskelion. triskelions can be polymerized by accessory factors into striking lattice-like ''cages'' comprising pentagons and hexagons, resembling a soccer ball structure or buckminsterfullerene. clathrin cages are - nm in diameter; significantly larger than copi or copii vesicles (ccvs). clathrin-coated vesicles are believed to assemble through a sequence of events that can be designated as activation, cargo capture, coat assembly, scission, movement, and vesicle uncoating (kirchhausen, ) . members of a class of clathrin-associated factor termed adaptor protein (ap) complexes are recruited to donor membranes through interactions with a docking complex, which then further interacts with motifs within the cytoplasmic tail of cargo proteins, resulting in ''cargo capture.'' this leads to clathrin cage assembly and the concomitant polymerization of the clathrin triskelion and resultant deformation of the donor membrane. scission, or vesicle release from the plasma membrane, is believed to occur through the action of the gtpase dynamin and other accessory proteins, such as amphiphysin (wigge et al., ) . in the fruit fly drosophila melanogaster, a dynamin gene mutation (shibire) causes temperature-sensitive paralysis. this is likely due to a block in the endocytic uptake of synaptic vesicle proteins at the plasma membrane, leading to a block in recycling and reformation of competent synaptic vesicles at nerve terminals (chen et al., ; koenig and ikeda, ; kosaka and ikeda, ; van der bliek and meyerowitz, ) . the expression of a dominant-negative gdp-bound dynamin mutant, k a, results in compromised ccv formation (herskovits et al., ; van der bliek et al., ) and inhibition of clathrin-mediated internalization of the glucose transporter glut (al-hasani et al., ) , human immunodeficiency virus (hiv) (daecke et al., ) , and influenza virus (roy et al., ) . the scission function of dynamin is assisted by specific lipid-modifying enzymes such as endophilin, synaptojanin, and phospholipase d (bi et al., ; havner et al., ; ringstad et al., ; schmidt et al., ; woscholski et al., ) . finally, ccv uncoating at the target membrane occurs through the actions of the heat shock protein hsc (schlossman et al., ) and auxilin (ungewickell et al., ) . sorting of proteins from donor to target membranes involves the recognition of cytoplasmic sequences in membrane proteins by clathrin-associated ap complexes. four adaptor protein complexes (ap -ap ), each comprising four diverent subunits, have been identified (robinson, ) . the ap complex is involved in clathrin-coated vesicle formation at the tgn for transport to late endosomes; evidence has also implicated a role for this complex in a tgn-to-plasma membrane step (folsch et al., ) . ap is the best-studied of the four complexes and mediates internalization of transmembrane howell et al. receptors at the plasma membrane via clathrin-coated vesicles. the ap complex is involved in traycking from early endosomes to either late endosomes or lysosome-related organelles such as melanosomes, platelet-dense bodies, and antigen-processing compartments. finally, the ap complex was the last to be cloned (dell'angelica et al., a; hirst et al., ; ) . in contrast to ap -ap , ap does not possess the b ''ear'' domain (see below), which allows interaction with clathrin and other cytosolic factors such as eps and auxilin (lundmark and carlsson, ) . by electron microscopy, ap has been localized to vesicles at the tgn, plasma membrane, and early endosomes, although there is debate as to whether these vesicles are clathrin-coated (barois and bakke, ; hirst et al., ) . interestingly, ap and ap may function independently of clathrin (hirst et al., ; vowels and payne, ) , suggesting the existence of another, as yet unidentified, coat protein that is analogous to clathrin. all four ap complexes comprise two large -kda subunits: a b subunit (b -b ) plus a g (ap ), a (ap ), d (ap ), or e (ap ) subunit. in addition, each ap complex contains a -kda subunit (m -m ) and a small -kda subunit (s -s ). ap , - , and - contain two carboxyl ''ear'' domains connected to the head of each large -kda subunit by a flexible hinge of approximately - residues. importantly, the ear domain of the b subunit and the hinge domains of the g and a subunits have been shown to bind clathrin (goodman and keen, ; morgan et al., ; owen et al., ) , and consensus sequences in the hinge domains of b and b have clathrin-binding properties (dell'angelica et al., ) . the b and m subunits of the ap complex interact with motifs present in the cytoplasmic domains of transmembrane proteins to mediate cargo recruitment into clathrin-coated vesicles. such motifs include npxy, yxxØ, and dileucine-based sequences (Ø represents a bulky hydrophobic amino acid). one such motif, npxy, is present in key cellular receptors such as low density lipoprotein receptor (ldlr), epidermal growth factor receptor (egfr or erb ), and insulin receptor, and mediates endocytosis and sorting. importantly, the jd mutation (y c) in ldlr lies within this key motif and causes familial hypercholesterolemia (knoblauch et al., ) . the second tyrosinebased motif, yxxØ, mediates plasma membrane internalization, lysosomal targeting, and basolateral targeting of cargo. this motif is found in lysosomal residents such as lamp- and - , cd , the recycling transferrin receptor (tfr), and tgn-associated recycling membrane proteins, furin and tgn . di-leucine motifs present on transmembrane transporters such as glut (glucose transporter), atp a, and mannose- -phosphate receptors (m pr) can fall into two categories: [de]xxx[li] and dxxll related motifs. the [de]xxx[li] motif is associated with proteins internalized from the plasma membrane and targeted to lysosomes, while dxxll motif is found in transmembrane proteins that shuttle between the tgn and endosomal system (bonifacino and traub, ) . another class of clathrin-associated factor is the golgi-localized, g-earcontaining, arf-binding proteins (ggas) found on the tgn and postulated to interact with ap to mediate transport of m pr (section v.b) to endosomes (doray et al., ) . ggas can act as multifunctional adaptors that link transmembrane proteins, arf gtpases, clathrin and accessory proteins at sites of ccv formation (robinson and bonifacino, ) . the disease oculocerebrorenal syndrome of lowe (ocrl) is an x-linked disorder caused by mutations in the ocrl gene (lowe, ) . the gene product is an inositol -phosphatase that catalyzes the removal of the phosphate from this position on the inositol moiety. the preferred ocrl substrate is pi( , )p , a phosphoinositide shown to be important in endocytosis because of its central role in recruiting accessory proteins to ccvs (padron et al., ) . ocrl has been localized to clathrin-coated vesicles associated with endosomal and tgn membranes (choudhury et al., ) . this is not surprising as ocrl interacts with clathrin and promotes its assembly into clathrin lattices and cages (choudhury et al., ; ungewickell et al., ) . ocrl also interacts with the rac gtpase that regulates actin dynamics, possibly via a gtpase activation domain to accelerate gtp hydrolysis (faucherre et al., ) . although the exact function of ocrl is still unclear, the disease phenotype hints to ocrl function in membrane traycking. ocrl mutations can cause loss of protein expression and phosphatase activity. rnai-mediated inhibition of ocrl expression in cultured human cells results in partial redistribution of a cation-independent mannose- -phosphate receptor and a tgn recycling protein (tgn ) to early endosomes (choudhury et al., ) . this suggests that loss of ocrl perturbs endosome-to-tgn vesicle transport, suggesting a functional requirement for this membrane trayc step. it is possible that ocrl plays a role in anterograde traycking from the tgn-to-endosomes as well, since ocrl is abundantly present on tgn-associated clathrin buds destined for the endocytic pathway. ocrl disease symptoms include congenital cataracts, mental retardation, and renal tubular dysfunction (lowe et al., ) . renal failure in ocrl patients is probably partly caused by defects in solute and protein readsorption in kidney proximal tubules. this is likely due to missorting of megalin and cubilin, cell surface receptors involved in kidney solute uptake. in ocrl patients plasma membrane shedding of these receptors is reduced (norden et al., ) , indicating ocrl regulation of either receptor traycking from the tgn-to-plasma membrane or recycling from plasma membrane-to-tgn. paraneoplastic stiv-person syndrome (sps) is a neurological autoimmune disease characterized by severe muscle stivness and spasms, and often has secondary symptoms including diabetes, epilepsy, and breast cancer. autoantibodies are produced against the clathrin-associated regulator, amphiphysin i (de camilli et al., ) , a protein shown to bind dynamin in nerve terminals (david et al., ) and which is implicated in regulating the endocytosis of neuronal synaptic vesicle components (burns, ) . in support of this hypothesis, sommer et al. ( ) showed that sps-like symptoms could be triggered in rats injected with anti-amphiphysin antibodies from a human sps patient. genetic translocations leading to the formation of hybrid clathrin-accessory proteins can lead to other forms of acute myeloid leukemia, lymphoblastic leukemia and acute megakaryoblastic leukemia (dreyling et al., ; jones et al., ; narita et al., ; tebar et al., ) . in these diseases, an aberrant hybrid protein consisting of the putative transcription factor af and the clathrin accessory protein calm (clathrin assembly lymphoid myeloid leukemia protein) is formed because of a partial inversion of the af gene on chromosome (salmon-nguyen et al., ) . finally, in hermansky-pudlak syndrome (hps) type , a condition that results in partial albinism and prolonged bleeding, mutations have been found in the b a gene that encodes a subunit of the ap adaptor complex (dell'angelica et al., b) . hps is discussed in more detail in section v.c. originally identified more than years ago (palade, ; yamada, ) , caveolae are flask-shaped invaginations of approximately - nm in diameter at the plasma membrane. these plasma membrane profiles are related to lipid rafts and contain unique mixtures of gpi-anchored proteins, transmembrane proteins, signaling factors and lipids, such as cholesterol. caveolae are believed to mediate the uptake of small solutes, regulate protein traycking (hommelgaard et al., ; tagawa et al., ) , transcytosis (transport across endothelial cells) (simionescu et al., ) , signal transduction (insel et al., ; lisanti et al., ; ostrom and insel, ) and cholesterol homeostasis (fielding and fielding, ) . however, their exact role in the internalization of membrane proteins and soluble protein ligands is controversial. caveolin- , also known as vip , is a structural component essential for the formation and stability of caveolae (kurzchalia et al., ; rothberg et al., ) . of the three members of the caveolin gene family (caveolin- , - , and - ) tang et al., ) , caveolin- and - are abundant in a wide variety of cell types including endothelial cells, adipocytes, alveolar type i pneumocytes, and smooth muscle cells (williams and lisanti, ) , whereas caveolin- is a muscle-specific isoform expressed in striated muscle cells such as cardiac and skeletal myocytes (cohen et al., ; tang et al., ) . caveolin- and - are both able to induce formation of caveolae at the plasma membrane (galbiati et al., ; li et al., ) . however, caveolin- requires the presence of caveolin- for expression, membrane localization, and formation of caveolae (razani et al., ) . caveolae are absent from cells that lack caveolin- but can be induced by ectopic expression of the gene (fra et al., ) . caveolins adopt a hairpinlike structure that inserts into the membrane such that the n and c termini are cytoplasmic. caveolins can polymerize to form a striated coat surrounding an invagination site . caveolin- can bind cholesterol (murata et al., ) , which is enriched within both caveolae and lipid rafts (sargiacomo et al., ) ; this may explain why caveolae have been considered a subset of lipid rafts. however, caveolae and lipid rafts are considered to be independent entities as some proteins can be found in one but not the other (liu et al., ) . certain ligands can internalize via a lipid raftdependent but clathrin-independent mechanism in cells that lack caveolae (lamaze et al., ) . a large pool of the plasma membrane caveolar vesicles cluster into dense grape-like structures where individual caveolae appear stacked on top of each another (thomsen et al., ) . these structures are intimately associated with the actin cytoskeleton (stahlhut and van deurs, ) ; caveolaassociated proteins are also implicated in regulating plasma membrane dynamics and cellular movement. a small pool of ''transport-competent'' caveolar vesicles may undergo short-range constitutive fusion and budding cycles just under the plasma membrane . caveolae and caveolins can also be detected at the tgn (dupree et al., ; kurzchalia et al., ) and may form stable ''platforms'' for the movement of proteins and lipids from the tgn to the plasma membrane (tagawa et al., ) . the caveolar pathway can be hijacked and used by pathogens or toxins to gain entry into the cell. viruses such as polyomavirus, echovirus , and simian virus (sv ) use caveolae to internalize viral particles. these viruses cluster lipid rafts and sequester them into caveolae through interactions with raft components such as integrins and glycosphingolipids ; in the case of sv , the virus binds to the raft component ganglioside gm (tsai et al., ) . tagawa et al. ( ) have shown that sv can trigger the long-range movement of transport-competent caveolar vesicles. moreover, cell infection with sv more than doubles the number of caveolae capable of undergoing viral internalization and long-range traycking. caveolae contain much of the molecular machinery required for ''classical'' vesicle fission, docking, and fusion, for example, snare proteins, monomeric and trimeric gtpases, annexins ii and vi, n-ethylmaleimide (nem)sensitive fusion protein (nsf), and atpases (schnitzer et al., ) . caveolae also contain the dynamin gtpases, which can be transiently recruited to sv -loaded caveolae and implicated in membrane scission (henley et al., ; oh et al., ; pelkmans and helenius, ) . internalized caveoladerived vesicles move to an endocytic compartment termed the ''caveosome'' and eventually arrive at the early endosome. after fusion with the target compartment, caveolae do not disassemble but maintain their integrity in the membrane, preserving their compartmentalization and retaining their lipid and protein components (pelkmans et al., ) . the fate of internalized sv viruses after reaching the caveosome eventually results in arrival at the smooth er (pelkmans et al., ) . interestingly, mutations in caveolin have been implicated in muscular dystrophy and cardiovascular disease, and mutations causing the downregulation of caveolin have been linked to the progression of various human carcinomas; it is therefore possible that caveolins may have a tumor suppressor role. the caveolin- and caveolin- genes are located on human q . near the microsatellite repeat marker d s . this region is commonly deleted in various cancers (engelman et al., ) , hinting that caveolin gene deletion may be advantageous for tumor progression. in one report, the caveolin- p l mutation was present in % of breast cancer patients studied (hayashi et al., ) . the p l mutation was also linked to the metastatic potential of tumors and disease prognosis. the caveolin- p l mutation also conferred increased cell migration and altered morphology. caveolin- protein levels can be reduced or absent from a number of human breast cancer cell lines compared with normal mammary cells (lee et al., ) . similarly, silent and missense mutations in caveolin- have also been associated with oral carcinomas (han et al., ) . caveolin- , and to a lesser extent caveolin- , gene expression is downregulated in some cases of thyroid carcinoma (aldred et al., ) . although it remains unclear as to why the loss of caveolin causes cell proliferation diseases such as cancer, one can speculate on the role of caveolin in regulating signaling pathways. in endothelial cells, which have a high abundance of caveolin, the key vascular endothelial growth factor receptor (vegfr ) has been shown to be inactive when localized to caveolae (labrecque et al., ) . this receptor tyrosine kinase modulates the endothelial response to the key vegf-a cytokine and controls angiogenesis and new blood vessel formation, thus regulating neovascularization and tumor growth (neufeld et al., ) . similarly, platelet-derived growth factor (pdgf) receptor tyrosine kinase activity is reduced when associated with caveolae (yamamoto et al., ) . in addition to vegfr and pdgfr, a number of g protein-coupled receptors (gpcrs) have been shown to interact with caveola-associated factors (insel et al., ) . gpcrs are a large family of transmembrane receptors involved in a variety of signal transduction events. these receptors are activated by a range of ligands, including hormones and peptides, and have been linked to a number of cancers such as cell biology of membrane trafficking in human disease thyroid, lung, and gastric. the presence of a number of gpcrs in caveolae suggests that these plasma membrane structures may interact with gpcrs and modulate their signaling potential. lisanti and others (li et al., a) have shown that caveolin- interacts solely with inactive forms of g-protein a subunits, lending credence to the negative regulation hypothesis caused by the association of caveolae with transmembrane signaling receptors. a number of mutations in muscle-specific caveolin- have been associated with four distinct but related autosomal dominant muscle disease phenotypes (woodman et al., ) : limb girdle muscular dystrophy type c (minetti et al., ) , rippling muscle disease, hyperckemia (persistently elevated levels of serum creatine kinase), and distal myopathy. some mutations cause aberrant retention of caveolin- in the golgi and subsequent degradation; other mutations may cause mutant caveolin- to act in a dominant-negative manner by forming unstable aggregates with wild-type caveolin- (galbiati et al., ; sotgia et al., a,b) . hypertrophic cardiomyopathy (hcm) patients have a caveolin- t s mutation that reduces plasma membrane levels (hayashi et al., ) . caveolin gene knockout mice are providing insights into protein function in diverent human diseases. for example, lack of caveolins can cause diabetes, atherosclerosis, and cardiomyopathies in mouse models (cohen et al., ; williams and lisanti, ) . however, such phenotypes have yet to be linked to caveolin dysfunction in humans. phagocytosis is a process used by white blood cells such as macrophages, neutrophils, and dendrites to ingest large particulate material into specialized vesicles called phagosomes. these professional phagocytes are paramount in the defense against infection as they engulf and ingest whole microorganisms such as bacteria. they also use this route for ''mopping up'' apoptotic debris or senescent cells from tissues. in contrast to constitutive pinocytic transport, phagocytosis is regulated by cell surface-localized fc receptor (fcr) contact or interaction with complement-or antibody-coated particles which results in clustering of fcr on the cell surface, a step important for subsequent intracellular signaling and cellular activation (daeron, ) . polymorphisms in leukocyte-specific fcg receptors may contribute to autoimmune diseases such as guillain-barré syndrome or rheumatoid arthritis, and enhanced susceptibility to infection (van sorge et al., ) . fc-mediated binding can trigger a complex signaling response involving extrusion of fine plasma membrane projections (pseudopodia) from the macrophage to surround and engulf the pathogen, forming a phagosome. the signaling response is reviewed in greater detail elsewhere (bokoch, ; chimini and chavrier, ; niedergang and chavrier, ) . in brief, the activation of tyrosine kinases and rho gtpases is triggered through fcr signaling. the rac and cdc gtpases, in conjunction with the downstream evector wasp, mediate remodeling of the actin cytoskeleton, leading to pseudopodium formation and phagosome closure (castellano et al., ; chimini and chavrier, ) . in contrast, the complement mediated-uptake of opsonized particles divers such that they appear to ''fall'' into the cell in a process that requires rho, but not rac or cdc (bokoch, ) . phagocytosis, although designed to destroy pathogens, can paradoxically be used as a route of entry by pathogens such as mycobacterium (m. leprae and m. tuberculosis) or leishmania (nguyen and pieters, ; scott et al., ) . normally, internalized pathogens are destroyed successfully through phagosome maturation into lysosomes and subsequent degradation. mycobacterium can evade host degradation by secreting a soluble serine/threonine protein kinase g molecule into the phagosome. this molecule initiates a signaling response that interferes with phagosome-lysosome fusion, and promotes intracellular pathogen survival (walburger et al., ) . furthermore, phagosome maturation is compromised by a pathogen induced block of p map (mitogen-activated protein) kinase recruitment to the tethering molecule early endosome antigen (fratti et al., ) . the leishmania protozoan parasite, which is transmitted to humans by sand flies, produces a membrane molecule called a lipophosphoglycan, which is inserted into the lipid bilayer of the phagosome in infected macrophages. this lipophosphoglycan is thought to modulate intracellular signaling pathways, resulting in a less fusogenic phagosome and preventing maturation; this would facilitate pathogen replication and disease progression (lodge and descoteaux, ) . molecules internalized from the cell surface by receptor-mediated endocytosis and clathrin-coated vesicles are delivered to the early endosome for sorting. molecules such as low-density lipoprotein receptor (ldlr) and transferrin receptor (tfr) are eyciently recycled between the early endosome and the plasma membrane. however, after ligand-mediated activation (fig. ) , receptor tyrosine kinases such as epidermal growth factor receptor (egfr) are sorted along the endocytic pathway for degradation. early endosomes are thought to be formed through the fusion of internalized vesicles and recruitment of specific proteins and lipids. one key protein traycking through the endosomal-lysosomal system. cell surface receptors are internalized through clathrin-coated vesicles (ccvs) at the plasma membrane. in the cell cytoplasm, ccvs shed their coat components and fuse to produce endosomes. internalized receptors are either recycled from sorting endosomes (housekeeping receptors, e.g., transferrin receptor) or targeted for degradation within the lysosome (signaling receptors, e.g., growth factor receptors) after movement through the late endosome and multivesicular body (mvb) compartments. endosomal regulator is the ubiquitously expressed rab a gtpase. rab a is present on the cytosolic face of the plasma membrane, vesicles, and tubular endosomal profiles (chavrier et al., ) . a number of rab a-associated evector proteins regulate endosomal fusion and mediate protein cargo movement and endosomal sorting (zerial and mcbride, ) . such evector proteins, including , are clustered on the cytosolic face of the early endosome and stabilize the gtp-bound rab a in an activated state (horiuchi et al., ) . gtp-bound rab a directly binds to early endosome antigen eea to regulate vesicular and endosomal tethering. eea contains a c-terminal rab a-binding domain, and a phosphatidylinositol -phosphate-binding zinc finger domain referred to as an fyve (conserved in fab , yotb, vac , and eea ) domain (gaullier et al., ; stenmark et al., ) . overexpression of wild-type rab a, or a constitutively active rab a mutant, causes endosome enlargement and defective traycking through this compartment, whereas expression of a constitutively inactive rab a mutant leads to formation of small endosomes and decreased endocytosis (bucci et al., ) . a family of evector proteins that accelerate gtpase hydrolysis (rabgaps) have been identified: rabgap- binds to rab a and regulates traycking through the endocytic pathway . the importance of rab a activity is further illustrated in the genetic disorder tuberous sclerosis (ts), a disease that causes tumors in the brain, eyes, heart, kidneys, lungs, and skin. ts arises when the tumor suppressor gene, tuberous sclerosis complex (tsc), is absent; introduction of the wild-type tsc gene into an animal model or cultured cells results in tumor suppression and reduced cellular proliferation (kobayashi et al., ; yeung et al., ) . interestingly, the tsc gene product (tuberin) is implicated in regulating gtp/gdp exchange on rab a, thus regulating traycking through this endosome system (xiao et al., ) . in chronic myelomonocytic leukemia (cmml) a genetic translocation causes fusion of rab a evector rabaptin- and the pdgfbr (magnusson et al., ) . this chromosomal translocation results in enhanced cellular proliferation by compromising endosomal fusion and traycking, and thus regulation of growth factor degradation. it is likely that this aberrant gene product is not degraded and triggers sustained intracellular signaling, leading to cell proliferation and tumor progression in a subset of lymphoid cells. recycling from endosomes back to the cell surface is often used by receptors that internalize nutrients such as lipoproteins and ions. receptor recycling rather than degradation conserves receptor functionality and nutrient uptake and reduces energy expenditure in the synthesis of new receptors (mukherjee et al., ) . genetic screens in the nematode caenorhabditis elegans identified rme- and delineated a new family of conserved class of eps homology (eh) domain proteins . both the worm and mouse homologs of rme- are associated with the endosomal compartment: a dominant-negative rme- g r mutant had little evect on receptor-mediated endocytosis but had a substantial evect on endosomal recycling, suggesting a functional role in this step . although information is currently limited, a number of neurological diseases are associated with dysfunction of early endosomal proteins. in some cases of demyelinating polyneuropathy, characterized by progressive weakening and sensory dysfunction of the legs and arms, eea autoantibodies have been detected (selak et al., ) . a number of disorders, from muscular dystrophy to rheumatoid arthritis, reveal the presence of circulating anti-eea antibodies. interestingly, eea epitopes recognized by such autoantibodies varied from patient to patient (selak et al., ) . autoantibodies against eea have also been detected in cases of subacute cutaneous systemic lupus erythematosus (scle), characterized by the appearance of an unsightly red rash, often occurring after sun exposure (mu et al., ) . lysosomes are terminal, membrane-enclosed degradative compartments that interact with other organelles through vesicular transport originating from the secretory, endocytic, and autophagic pathways. this organelle stores various proteases, lipases, hydrolases, and degradative enzymes within an acidic environment that maximizes enzymatic activity and degradation. resident lysosomal membrane proteins, integral proteins, and glycoproteins are targeted to the organelle via the endosome. lysosomal proteases such as cathepsin d are processed in the golgi apparatus to add a mannose -phosphate (m p) moiety to n-linked sugars. the m p moiety is recognized by plasma membrane or tgn-resident mannose -phosphate receptors (m prs) and sorted to the late endosome and eventually the lysosome. here, the acidic ph (ph < . ) results in receptor-ligand disassociation and recycling of the m pr to the tgn. fusion between the endosome and preexisting primary lysosomes allows the delivery of lysosomal resident proteins. the importance of m p-mediated targeting of lysosomal proteins is highlighted in the human neurological disorder, i-cell disease (mucolipidosis ii), where lysosomal enzymes are secreted from cells rather than targeted to the lysosome. the defect in i-cell disease involves lack of m p moiety addition as a result of mutations to the n-acetylglucosamine- -phosphotransferase enzyme usually present within the golgi apparatus (ben-yoseph et al., ) . how lysosomes are formed is still unclear (luzio et al., ) . three mechanisms have been proposed to explain lysosomal biogenesis: vesicular transport between late endosomes and preformed primary lysosomes (griyths and gruenberg, ), early endosomal ''maturation'' to lysosomes (murphy, ) , or the current favored model of ''kiss-and-run,'' in which transient interactions between endosomes and lysosomes transfer endosomal contents to the latter compartment (duclos et al., ; storrie and desjardins, ) . late endosome and lysosome interactions in the kiss-and-run model are thought to be regulated by the rab gtpase, which is present on late endosomes; a vps complex, homologous to budding yeast vacuole fusion regulators, is also implicated in sorting and delivery to lysosomes (seals et al., ) . the mammalian form of the vps complex interacts with syntaxin- , a t-snare that is concerned in regulating membrane dynamics along this route (kim et al., ) . danon disease is caused by point mutations in, or complete absence of lysosome-associated membrane protein (lamp ) or complete absence of this protein: changes which result in cardiomyopathy, myopathy, and mental retardation. in danon disease patients and lamp -deficient mice, autophagic vacuoles accumulate within the cytoplasm; these vacuoles arise via intracellular engulfment of old membranes to form an autophagosome, thus sequestering membranes and proteins for eventual degradation (shintani and klionsky, ) . autophagosomes fuse with lysosomes, leading to degradation for provision of molecules for cellular homeostasis. the accumulation of autophagic vacuoles in lamp -deficient cells suggests that lamp mediates interactions between autophagosomes and lysosomes. this pathway is commonly activated during conditions of cellular stress such as starvation or pathogenic infection (kirkegaard et al., ) . lysosomal storage diseases are caused through insufficient degradation of targeted components within lysosomes, leading to substrate accumulation and lysosome enlargement. more than lysosomal storage diseases have been documented and generally manifest themselves as neurological disorders; disease severity correlates with the levels of lysosomal enzyme activity. niemann-pick disease is a neurodegenerative condition caused by sphingomyelin accumulation in reticuloendothelial cells and ganglion neurons, leading to cell death. it is classified into five types (a-e), each distinguished by either clinical severity or age-related disease phenotype. niemann-pick type a (npa) is most common, with death occurring before years of age. npa patients have point mutations in the smpd gene that encodes a lysosomal sphingomyelinase (levran et al., ; takahashi et al., ) . interestingly, in npc patients, endocytosed ldl particles are not fully degraded in lysosomes, leading to defects in cholesterol metabolism (li et al., a) . the npc disease is caused by mutations in the npc gene, which encodes a lysosomal resident protein with similarity to sterol-sensing enzymes and proteins (scott and ioannou, ) . fabry disease is an x-linked condition caused by changes in lysosomal a-galactosidase activity resulting in glycosphingolipid accumulation within vascular endothelial lysosomes. this leads to angiokeratomas (a wart-like thickening of the skin), progressive renal impairment, cardiomyopathy, and cerebrovascular disease. mutations in the a-galactosidase a gene can also show reduced enzymatic activity of the encoded protein and retention within the endoplasmic reticulum (yasuda et al., ) . receptor tyrosine kinases such as epidermal growth factor receptor (egfr) are degraded by lysosomes after ligand binding and receptor activation. egfr lysosomal targeting is dependent on ligand-stimulated ubiquitination of the cytoplasmic domain. binding of egf to egfr causes downstream signaling, clathrin-mediated internalization, and traycking to endosomes. internalized receptor-ligand complexes are sorted to the late endosome or multivesicular bodies (mvbs), which eventually deliver their contents to the lysosome (katzmann et al., ) . whereas other receptors such as tfr are recycled to the plasma membrane, egfr is moved through the endosome-lysosome system by a ubiquitin-dependent sorting and recognition system. these include the hrs/stam heterodimer and tsg (bilodeau et al., ) present on endosomal membranes. the tsg tumor suppressor gene is mutated in nearly % of breast cancer patients and encodes a membrane-associated protein (lee and feinberg, ) . this factor participates in the sorting of ubiquitinated proteins on the endosome, but its exact function is not clear. in some specialized cells, such as cytotoxic t lymphocytes (ctls), platelets, and melanocytes, regulated secretion can be routed through compartments other than the tgn. such cells have evolved mechanisms whereby modified or secretory lysosomes release their contents at the plasma membrane in response to extracellular stimuli. these secretory lysosomes (sls) share lysosomal characteristics such as acid ph and lamp (lysosome-associated membrane proteins) residents but also contain unique markers such as tyrosinase, present in melanosomes. the ctl secretory lysosomes contain unique components such as perforin and granzymes required for triggering apoptosis in target cells. on ctl contact with a target cell, sls trayc toward the immunological synapse formed between the ctl and target cell. a signal then causes sl fusion with the ctl plasma membrane (stinchcombe et al., ) , and release of sl contents and subsequent target cell death. a number of autosomal genetic diseases causing immunodeficiency and albinism involve defects in regulated lysosomal secretion (stinchcombe et al., ) . in the rare, fatal disease familial hemophagocytic lymphohistiocytosis (fhl) sls congregate at the plasma membrane in ctls, where they can dock but cannot fuse with the membrane. in one group of fhl patients, this disease is due to a mutation in the gene encoding munc - ; this is closely related to the neuronal munc - gene product that is involved in snare complex formation in neuronal cells (feldmann et al., ) . assembly of this neuronal syntaxin- , snap- , and synaptobrevin complex is regulated by munc - , which binds and locks syntaxin- (t-snare) in a closed, inactive conformation, thus preventing it from interacting with snap- (yang et al., ) . however, munc - (sassa et al., ) and rim (rab a-interacting protein evector) (koushika et al., ) can compete with munc - and displace it from syntaxin- . this reinforces the syntaxin- open conformation and allows snare complex formation to occur. munc - may act as a conformational switch to promote t-snare into an ''open'' state, thus allowing formation of the snare complex that mediates synaptic vesicle docking and fusion. from observations of fhl patients, one speculation is that munc - has a role similar to that of munc - in regulating snare complex formation for sl docking and fusion in ctls (yang et al., ) . chediak-higashi syndrome (chs) is a key example of a disease avecting sl function in ctls, with patients displaying hypopigmentation (stinchcombe et al., ) . chs patients have genetic mutations in the lyst or chs gene (barbosa et al., ; perou et al., ) and produce ctls containing strikingly enlarged sls that are able to polarize to the immunological synapse but are unable to fuse with the pm. this suggests a role for the chs gene product in regulating membrane docking and fusion . overexpression of chs leads to the presence of small lysosomes, indicating increased lysosomal fission (ward et al., ) . in addition, chs interacts with snare proteins, further indicating a role in sl fusion (tchernev et al., ) . griscelli syndrome patients also display defects in sl dynamics within ctls and exhibit hypopigmentation and silvery hair. in melanocytes, cells responsible for pigment storage and production, rab a is required to recruit melanophilin to pigment granules called melanosomes (sls). melanophilin binds the myosin motor protein myosin va and regulates melanosome movement along actin cables to the plasma membrane strom et al., ; wu et al., ) . in type griscelli syndrome patients, rab a gtpase is missing or defective, whereas in type griscelli syndrome patients the myosin va motor protein is absent. these defects are also evident in mouse models such as ashen (rab a defective), dilate (myosin va defective), and leaden (melanophilin defective). in both the human griscelli syndromes and the mouse models, melanosomes are clustered in a perinuclear location, a defect attributed to rab a dysfunction (wilson et al., ; wu et al., ) . interestingly, ctls isolated from type griscelli syndrome patients and ashen mice (rab a deficient) are unable to kill target cells, whereas type griscelli syndrome patient and dilate mouse ctls are functional. this suggests that rab a interacts with diverent evectors to induce sl fusion with the plasma membrane in diverent cell types (haddad et al., ) . hermansky-pudlak syndrome (hps) is a fourth example of sl dysfunction and is characterized by oculocutaneous albinism, ceroid deposition, and excessive prolonged bleeding (hermansky and pudlak, ; swank et al., ) . however, hps cannot be viewed as a single disease but a group of at least seven autosomal genetic disorders. each of the seven subgroups (hps - ) is due to mutations in individual genes, most of which encode components of multisubunit protein complexes involved in vesicle traycking , whereas the function of others remains unclear. three of these complexes, termed blocs (biogenesis of lysosome-related organelle complexes), play a role in regulating traycking involved in platelet and melanosome secretion, but their exact functions are unclear (di pietro and dell'angelica, ) . in hps patients a nonsense mutation in the gene encoding the b a subunit of the ap adaptor protein prevents expression of this subunit (huizing et al., ) . as previously mentioned, ap is involved in the recruitment of transmembrane proteins into vesicles at the early endosome for delivery to the lysosome (peden et al., ) . in melanocytes derived from hps patients, the tyrosinase that catalyzes the formation of melanin is not transported to maturing melanosomes (huizing et al., ) . this leads to the characteristic pattern of albinism seen in patients with this condition. furthermore, patients with hps display an impaired ctl response and immune response. ctls from hps patients have lytic granules that cannot move in an oriented fashion toward the microtubule-organizing center; therefore, when ctls are stimulated by contact with target cells, the lytic granules are not targeted to the immunological synapse for cell killing . studies on the cell biology of hiv infection have suggested the existence of a viral secretory compartment. work by marsh and others pelchen-matthews et al., ) has localized viral envelope (gp ) and matrix proteins (p ) to tetraspanin-positive endosome-related organelles in infected macrophages and dendritic cells. these viral secretory compartments move from an intracellular localization to an infectious synapse when infected macrophages or dendritic cells form an immunological synapse with activated t cells. this may be one mechanism for subsequent viral infection of cd -positive t cells, thus causing the impaired immune response seen in patients with acquired immunodeficiency syndrome (aids). cells require a highly organized framework or cytoskeleton to station and move membrane organelles within three-dimensional space. components of the cytoskeleton can guide organelles or vesicles to specific destinations within the cell. the microtubule cytoskeleton is commonly associated with the directional movement of intracellular transport vesicles or intermediates. in contrast, actin has been envisioned to have a structural role in determining cell shape, plasma membrane dynamics, and cell locomotion. however, evidence points to a role for actin in regulated traycking from the tgn (allan et al., ; badizadegan et al., ; cobbold et al., ) and endocytosis (ascough, ; engqvist-goldstein and drubin, ) . the cytoskeleton is a dynamic structure likened to a collapsible scavold that can be rapidly disassembled and reconstituted depending on cellular requirements. actin or tubulin polymerization (elongation) and depolymerization (breakdown) rely on the controlled addition or removal of monomers in a polarized and energy-dependent manner. protofilaments in either structure are both polarized, with the plus end growing at a faster rate. actin cables are each composed of two parallel protofilaments that twist around each other, whereas microtubules are composed of a hollow cylindrical structure comprising parallel protofilaments. actin nucleation is an initial step required for elongation involving formation of a stable trimer subunit base for protofilament elongation. a heptameric complex termed arp / (actin-related protein) binds to the ends and sides of actin filaments to nucleate and further accelerate the growth of the actin network (millard et al., ) . the function of the arp / complex can be regulated by membrane-associated rho gtpases. these regulators, which include cdc and various rac isoforms, act as molecular switches that cycle between an active gtp-bound state and an inactive gdp-bound state. cdc regulates arp / indirectly through its downstream target wiskott-aldrich syndrome protein (wasp), which binds directly to the arp / complex (jave and hall, ) . patients with x-linked wiskott-aldrich syndrome display mutations in the wasp gene and have thrombocytopenia (reduced platelet count), eczema, recurrent infections, hematologic malignancy, and autoimmune disorders (lemahieu et al., ) . approximately disease mutations in wasp have been reported, which lead to defective control of wasp in actin polymerization and severe disease phenotypes (burns et al., ) . wasp expression is restricted to hematopoietic cells, although the ubiquitously expressed n-wasp is present in various cells and tissues (burns et al., ) . the actin network is important for the formation of immunological synapses between cytotoxic t lymphocytes (ctls) and their targets, as well as t lymphocytes and antigen-presenting cells such as macrophages. the formation of the immunological synapse in ctls is essential for the transport, docking, and fusion of sls and subsequent destruction of the target cell as described above. defective wasp inhibits the formation of the immunological synapse and t cell activation (notarangelo and ochs, ) , probably causing the immunological deficiencies observed in wiskott-aldrich syndrome patients. wasp deficiency in t lymphocytes also avects the regulation and composition of lipid rafts (dupre et al., ) , indicating that the cell biology of membrane trafficking in human disease formation of the immunological synapse is dependent on lipid rafts, wasp, and actin dynamics. motor proteins provide the physical force to move membrane vesicles along the polymerized cytoskeletal filaments via atp-dependent hydrolysis. actinbased motor proteins belong to the myosin superfamily. the myosin va gene is mutated in a small number of patients with griscelli syndrome (bahadoran et al., ; pastural et al., ) (other traycking mutations contributing to griscelli syndrome are discussed in section v.c). mutations in the myosin viia gene can cause usher's syndrome, resulting in blindness and deafness. intracellular transport is probably compromised in usher syndrome patients; the mouse shaker model has a mutant myosin viia gene, displaying defective melanosome transport in retinal pigment epithelial cells (liu et al., ) and altered distribution in photoreceptor cells (richardson et al., ) . microtubule motor proteins, which actively move vesicles along the microtubules, and microtubule-associated proteins (maps), serve as docking molecules to bind cargo to motor proteins (gerdes and katsanis, ) . microtubule motor proteins belong to either the kinesin or dynein families. with the exception of the c-terminal kinesins, kinesin-based motors generally transport cargo toward the plus end of the microtubule, whereas the dyneins are minus end-directed motors. long-range vesicular transport is particularly important in neurons, where axons can reach up to m in length. newly synthesized lipids, and secreted or membrane-associated proteins, are made in the cell body; long-range and directional transport is crucial for replenishing the constituents of the presynaptic cleft (at the terminal end of the axon) with synaptic vesicles and plasma membrane receptors (holzbaur, ) . a number of human neurological diseases are linked to mutations in microtubule motors and associated proteins. lissencephaly, a greek term meaning ''smooth brain,'' causes severe brain malformation resulting in mental retardation and epilepsy. one of the genes mutated in the disease is lis (originally identified in miller-dieker syndrome patients with lissencephaly) (reiner et al., ) . the lis protein regulates microtubule motor function by binding dynein and p glued , a component of the dynactin complex that binds to and activates dynein (smith et al., ) . it is proposed that lis regulates retrograde axonal transport. another gene mutated in some patients with lissencephaly is doublecortin, a microtubule-associated protein that binds tubulin and stabilizes microtubules (horesh et al., ; moores et al., ) . the kif b kinesin regulates transport of synaptic vesicle precursors along the neuronal axon. patients with charcot-marie-tooth disease type a display neuronal axonal degeneration due to a loss-of-function mutation in the motor domain of kif b . in alzheimer's disease, a classical sign is hyperphosphorylated aggregates of the microtubule-associated protein, tau, in neuronal cells and tissues. tau protein can influence vesicular transport (ebneth et al., ) by regulating the attachment of motors to microtubules (trinczek et al., ) . one theory is that the tau protein can interfere with kinesin-dependent transport by blocking motor access to microtubules, thus slowing or preventing vesicle movement along axons (mandelkow et al., ) . moreover, an early sign of alzheimer's disease is the loss of synapses and retrograde degeneration of neurons, complemented by a breakdown in intracellular transport. the disruption of microtubule-mediated vesicular traycking may also be a causative factor of the neurodegenerative phenotype of huntington's disease. this disease is caused by expansion of polyglutamine repeats occurring in the brain-enriched protein huntingtin (htt). it has been demonstrated that htt enhances vesicular transport of brain-derived neurotrophic factor (bdnf) along microtubules (gauthier et al., ) . htt is localized in the cytoplasm and is associated with vesicular and microtubule-based trayc through its ability to bind huntingtin-associated protein (hap ) (li et al., b) , a protein that has aynity for the dynactin p glued subunit (engelender et al., ) . dysfunctional polyq-htt associated with the disease state may disrupt the transport of bdnf by binding and blocking the hap /dynactinmediated delivery of bdnf vesicles along microtubules (gauthier et al., ) . this is further supported by the finding that bdnf levels are decreased in brains of huntington's disease patients (ferrer et al., ) . the current treatment of genetic disorders involves addressing the symptoms rather than the cause. to that end, many mild forms of disorders such as niemann-pick disease, and familial hypercholesterolemia, can be controlled by diet regimens and lifestyle changes. in contrast, a life-threatening disease such as cystic fibrosis requires extensive physiotherapy and pulmonary exercise to loosen and prevent mucus accumulation within the lungs. new antidementia drugs are increasingly successful in treating neurological disorders such as alzheimer's disease. drugs such as galantamine, donepezil, rivastigmine, and memantine target the posttranslational processing of bapp to reduce amyloid deposits (prasher, ) . in familial hypercholesterolemia, statin treatment is a common strategy for reducing plasma ldl and cholesterol levels by targeting hmg-coa reductase, the rate-limiting enzyme in cellular cholesterol biosynthesis. furthermore, less commonly used ldl-lowering drugs such as probucol have shown some success in (buckley et al., ) . the administration of adrenalin receptor antagonists (b-blockers) to patients with the cardiac condition long-qt syndrome reduces arrhythmia risk. enzyme replacement therapy (ert) has been carried out in patients with fabry's disease, a lysosomal storage disease. patients are given recombinant lysosomal a-galactosidase (mignani and cagnoli, ) to reduce the risks of strokes and kidney failure associated with the condition. finally, organ transplantation is occasionally carried out for some disease states: for example, bone marrow transplants for chediak-higashi syndrome patients (liang et al., ) and rab a-defective patients with griscelli syndrome (schuster et al., ) and wiskott-aldrich syndrome (filipovich et al., ) . however, although transplant operations can be successful in alleviating the immunological issues associated with these diseases, it does not address problems associated with the nervous system or pigmentation. b. gene therapy: the next generation of medical treatment? completion of the human genome sequencing project has given science the ability to track gene(s) responsible for potentially any genetic disorder and, as a consequence, to allow these genes to be corrected in patients. this is the goal of gene therapy research. of course, gene therapy has a fundamental limitation: it is only really suitable for single-gene defect diseases, and multigenic or chromosomal defects will be beyond the ability of the technique because of the complex nature of the disease. however, there are more than single gene defects that cause human disease, so there are many diseases requiring such approaches. the history of gene therapy is discussed in more detail by russell ( ) and scollay ( ) . much evort has been made in developing techniques that allow successful replacement or augmentation of defective genes. gene therapy is performed by introducing a gene vehicle directly into the patient (in vivo) or by removing cells from a patient, introducing the gene into these cells in culture, and replacing the cells back in the patient (ex vivo). most studies have focused on the use of viral vectors as delivery vehicles. retroviruses are potentially the best gene delivery system (kurian et al., ) . these rna viruses are able to infect a great many cell types and replicate by inserting their viral genes into the genome of the host. the host cellular machinery is then modulated to produce and assemble viral particles. in gene therapy, retroviruses could be used to express the target gene to be replaced but be modified to prevent viral disease (hiv, which is the causative agent of aids, is also a retrovirus). the principal drawback of a retrovirus vector is the possibility that genomic integration could elevate oncogene expression, thus causing cancer. therefore, the majority of clinical trials using retrovirus vectors have been performed ex vivo. a ''successful'' gene therapy experiment was exemplified in the case of a -year-old female patient lacking adenosine deaminase (ada), which results in severely compromised immunodeficiency (ada-scid) and dysfunctional t cells (blaese et al., ) . in this case, a retroviral vector was used to deliver the coding sequence for ada into cells, resulting in successful expression of this enzyme in hitherto defective cells. although successful, it is uncertain whether enzyme replacement treatment (recombinant ada injections) also influenced the patient outcome. adenovirus (adv) (mcconnell and imperiale, ) is a dna virus and key gene therapy vehicle that maintains the viral genome as a separate transmissible episome within the nuclei of infected host cells. the use of attenuated or inactivated adv for human gene delivery has attracted much interest. the advantages of adv gene transfer are that its genome can easily be manipulated and recombinant virus can be grown to high titers in vitro with eycient transduction of target cells in vitro or in vivo. as adv can evectively infect nondividing cells such as lung pulmonary tissues it is a popular vector of choice for gene therapy to treat cystic fibrosis (cf) patients. although there are promising studies (zabner et al., ) , failures have also been noted (knowles et al., ) . a major disadvantage of an adv-based approach is the triggering of a strong host immune response to the virus, which becomes a serious problem in subsequent long-term delivery of recombinant virus for disease alleviation. one approach to circumventing such an issue is to use a viral delivery system that produces a low host immune response such as the adeno-associated virus (aav) (flotte, ) . aav is a nonpathogenic virus that requires coinfection with a helper virus to replicate. however, aav has broad host cell specificity and is diycult to grow in large quantities, probably because of its reliance on a helper virus. finally, nonviral methods are increasingly available for the delivery of dna constructs directly into cells and tissues. these are often lipid-based reagents (e.g., liposomes) that bind to the plasmid dna and fuse with the plasma membrane, thus enabling cytosolic delivery of the gene. the plasmid dna would then be transported into the host nucleus by endogenous cellular machinery. this type of gene delivery can only be performed ex vivo and can be limited by the poor dna transfection eyciency of primary cells or tissues. this type of method, however, is a potentially useful method for delivering genes into progenitors or precursors (e.g., stem cells) before cellular cell biology of membrane trafficking in human disease diverentiation and tissue formation within a particular microenvironment in the body (mendell et al., ) . numerous disease states are caused by protein misfolding within the er, leading to degradat ion (table i ; section s iii. a. an d iii.a. ). one strategy would be to promote the correct protein conformation in a mutant gene product either chemically or pharmacologically. a number of membrane-divusible chemical and pharmacological ''chaperones'' have been identified that could avect protein folding in cells. chemical chaperones such as glycerol and trimethylamine n-oxide (tmao) can restore the wild-type traycking and activity of cftrÁf in cultured epithelial cells (brown et al., ) , and porcine kidney epithelial cells expressing cftrÁf and treated with dimethyl sulfoxide (dmso) increased plasma membrane levels of the channel protein (bebok et al., ) . loo et al. ( ) have demonstrated that a novel quinazoline derivative specific for cftr will rescue the defective traycking of cftrÁf in cultured cells. cell surface levels of a water channel, aquaporin- , can be enhanced with dmso (tamarappoo and verkman, ) . defects in this gene can result in x-linked nephrogenic diabetes insipidus, a condition in which patients are unable to concentrate their urine because of an inability to reabsorb water from the kidneys into the blood. although chemical chaperones are somewhat nonspecific in their action (the protein folding of the whole cell is avected and not just the target protein), pharmacological chaperones can be tailored to individual proteins. for example, the compound sr a is a nonpeptide vasopressin v receptor antagonist (morello et al., ; robert et al., ) . patients with a mutant vasopressin v receptor can also display nephrogenic diabetes insipidus. on treatment, the cell-permeant sr a compound would act as a chaperone and accompany the mutant v receptor to the cell surface to rescue correct functionality. geldanamycin, a naturally occurring antifungal agent, has potential as an anticancer drug (beliakov and whitesell, ; miyata, ) . geldanamycin interacts with and inhibits activity of the heat shock protein and chaperone hsp , a cytosolic cellular stress protein that supports the correct folding, stability, and function of ''client'' proteins. many hsp client proteins are implicated in cell cycle progression, proliferation, and angiogenesis (whitesell and lindquist, ) . the erbb tyrosine kinase complex is implicated in regulation and development of epithelial breast tumors and is an hsp client. inhibition of hsp action by geldanamycin results in degradation of both erbb and downstream signaling evectors, resulting in reduced cellular growth and tumor formation (citri et al., ) . a number of cell-permeable peptide sequences found in viruses and host proteins have been discovered that mediate the delivery of cargo (proteins, drugs, plasmid dna, oligonucleotides) directly into cells (brooks et al., ; gupta et al., ; schwartz and zhang, ) . such peptide sequences could be fused or attached to recombinant or engineered proteins and administered to patients to complement defects of a particular gene product. for example, the drosophila melanogaster antennapedia homeodomain (antp) transcription factor contains a short -residue sequence that mediates protein translocation across biological membrane bilayers in an energy-independent manner (derossi et al., ; joliot et al., ) . other sources of cell membrane-permeable proteins have been uncovered in viruses. the hiv- replication protein tat contains a basic, arginine-and lysine-rich peptide sequence (residues - ) that modulates the translocation of exogenous tat across the plasma membrane in a number of cell types, and is able to activate intracellular genes controlled by an hiv promoter (frankel and pabo, ; mann and frankel, ) . this basic -residue sequence can internalize conjugated b-galactosidase and horseradish peroxidase (fawell et al., ) as well as a fab antibody fragment (anderson et al., ) . the major structural protein of herpesvirus (hsv- ), vp , can trayc between cells (elliott and o'hare, ) , whereas the pres- domain of hepatitis b virus surface antigen acts as a shuttle for peptides and functional proteins (such as egfp) in hepatocytes and other cells (oess and hildt, ) , suggesting further the existence of naturally occurring peptide sequences that may act as drug delivery vectors. finally, a ''synthetic'' amphipathic peptide, fluos-klalklalkalkaalkla-nh , has been shown to be internalized in mast and endothelial cells (oehlke et al., ) . the employment of small molecular inhibitors as a method of treating human disease has moved at exponential pace. a number of compounds have been synthesized or isolated from nonhuman organisms that directly avect cellular function and have been used in research on a variety of diseases. plant-and microorganism-derived polyhydroxylated alkaloids referred to as iminosugars have been used in the treatment of patients with gaucher disease (cox et al., ) . gaucher disease type i and type ii is a lysosomal storage disorder caused by a mutation in the gene encoding the cell biology of membrane trafficking in human disease acid b-glucosidase (gba) enzyme and results in the accumulation of toxic glucosylceramide in a patient's spleen, liver, and bones; manifesting itself in enlargement of these organs, as well as heart and lung disease. iminosugars act on glycosylating enzymes present within the er and golgi and inhibit their ability to transfer sugar moieties onto proteins. one member of the iminosugar family, n-butyldeoxynojirimycin (nb-dnj; also called miglustat or zavesca) inhibits the enzyme important in the maturation of the gba substrate glucocerebroside, namely ceramide glucosyltransferase (cgt) (butters et al., ) . inhibition of cgt has resulted in significantly reduced levels of glucocerebroside in the liver and spleen of patient in clinical trials (cox et al., ) . however, nearly % of patients in the trials displayed osmotic diarrhea as a side evect of the treatment. in a mouse model for human tay-sachs disease, which is caused by a mutation in the gene encoding hexosamidase a, levels of the harmful glycophospholipid gm were significantly reduced on treatment with nb-dnj (platt et al., ) . in addition to the treatment of lysosomal storage diseases, an nb-dnj derivative called miglitol has been used in the treatment of diabetes mellitus, resulting in reduced activity of the sucrose-isomaltase enzyme complex and reduction of carbohydrate digestion (mitrakou et al., ) . a major aspect of human disease is the production and subsequent degradation of misfolded proteins, either by the proteasome or within the lysosome. lysosomotropic agents such as chloroquine cause an increase in the intralumenal ph of endosomes and lysosomes, reducing lysosomal protease activities and the traycking of proteins through the endosome-lysosome system. a number of proteasome inhibitors such as mg , lactacystin, and alln can specifically inhibit the activity of a range of serine and cysteine proteases and chymotrypsin-like enzymes (kisselev and goldberg, ) . proteasome inhibition has been linked with a number of aspects of human disease. treatment of endothelial cells with proteasome inhibitors resulted in apoptosis of proliferating cells (drexler et al., ) and inhibition of plasminogen activator levels; this factor promotes angiogenesis and new blood vessel sprouting (oikawa et al., ) . however, inhibiting proteasome function has broad cytotoxic and apoptotic evects in cells and tissues. chemotherapeutic agents targeting signaling pathways are currently of much interest in relation to cancer therapeutics. cellular proliferation can be regulated by growth factor binding to a cell surface receptor and signaling through either the mitogen-activated protein kinase (mapk) or phosphoinositide- -kinase (pi k) cascades. activation of these pathways induces the expression of oncogenes such as c-jun and c-fos and inhibits apoptosis through a sequence of protein phosphorylation events. shelton et al. ( ) demonstrated that inhibition of the mapk pathway with small molecule inhibitors specific for raf- or mek reduced cell proliferation and induced apoptosis in conditionally transformed hematopoietic cells. however, such pathways also regulate other cellular functions besides proliferation or apoptosis and there are likely to be consequences for cellular homeostasis. structural studies are important in the development of new small molecule inhibitors that target specific enzymes and regulators. c-akt (pkb) is a serine/ threonine protein kinase required for survival and proliferation in many human cancers and its structure has been elucidated (kumar and madison, ; yang et al ., ; and refer ences therei n). chem otherape utic agents have been consequently designed that inhibit c-akt activity; molecules such as h- target the atp-binding pocket in c-akt (kumar and madison, ) . compounds that bind specifically to c-akt isoforms or target specific domains within the kinase have been reported (barnett et al., ) , but there are no reports of clinical trials with such compounds (kumar and madison, ) . finally, small molecule inhibitors are being developed to target the posttranslational processing of proteins or peptides implicated in human disease. the enzyme that catalyzes the initial steps in b-amyloid synthesis, g-secretase, is an attractive target for prevention of amyloid deposits in alzheimer's disease patients (churcher and beher, ) . such small molecule inhibitors could also be used to treat pathogenic infections such as those caused by severe acute respiratory syndrome (sars), influenza, hiv, or hepatitis c viruses. attractive targets are virus-encoded or host proteases required for processing of viral proteins to generate infectious virus particles from the host cell. in the case of the sars virus, a viral chymotrypsin-like cysteine protease is responsible for processing sars viral proteins required for viral replication. inhibition of this protease would evectively inhibit viral replication. a molecule referred to as cs was found to inhibit the replication of human sars with no toxic evect on normal cells (dooley et al., ) . much work is also being carried out in targeting host proteases required for the processing of hiv envelope glycoproteins by the biosynthetic secretory pathway to generate viral gp and gp polypeptides. the completion of the human genome sequencing project has led to the prediction that a large number of diseases will be identified and understood at the gene level . as noted in this review, a number of examples exist in which a single gene mutation can have devastating evects on human function. at present, the symptoms of some mild forms of genetic diseases can be modulated through diet or drug regimens, and some success has been achieved with organ transplantation. gene therapy has attracted much attention but has suvered setbacks due to viral toxicity issues. an alternative strategy is the use of small molecule therapeutics, which cell biology of membrane trafficking in human disease may override specific defects or target specific pathways to compensate for gene defect(s). in addition, our understanding of how we respond at a genetic level to pathological infection will enable us to design evective drug strategies to presently chronic infections. in essence, understanding the cell biological basis for human diseases will enable us to design evective methods to deliver therapeutic strategies to patients. caveolin- and caveolin- , together with three bone morphogenetic protein-related genes, may encode novel tumor suppressors down-regulated in sporadic follicular thyroid carcinogenesis endocytosis of the glucose transporter glut is mediated by the gtpase dynamin motoring around the golgi the p -interactive proteins gm and giantin participate in endoplasmic reticulum-golgi trayc cftr and chaperones: processing and degradation tumor cell retention of antibody fab fragments is enhanced by an attached hiv tat protein-derived peptide endocytosis: actin in the driving seat traycking of cholera toxin-ganglioside gm complex into golgi and induction of toxicity depend on actin cytoskeleton comment on elejalde syndrome and relationship with griscelli syndrome identification of the homologous beige and chediak-higashi syndrome genes coupled er to golgi transport reconstituted with purified cytosolic proteins the akt/pkb family of protein kinases: a review of small molecule inhibitors and progress towards target validation the adaptor protein ap- as a component of the clathrin coat machinery: a morphological study role of diacylglycerol in pkd recruitment to the tgn and protein transport to the plasma membrane loss of function associated with novel mutations of the scn a gene in patients with brugada syndrome disassembly and reassembly of the golgi apparatus activation of deltaf cftr in an epithelial monolayer hsp : an emerging target for breast cancer therapy snares and the specificity of transport vesicle targeting mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase altered molecular size of n-acetylglucosamine -phosphotransferase in i-cell disease and pseudo-hurler polydystrophy congenital and acquired neutropenia the role of regulated cftr traycking in epithelial secretion phosphatidic acid formation by phospholipase d is required for transport from the endoplasmic reticulum to the golgi complex degradation of subunits of the sec p complex, an integral component of the er membrane, by the ubiquitin-proteasome pathway role of cue p in ubiquitination and degradation at the er surface the vps p hse p complex binds ubiquitin and mediates endosomal protein sorting t lymphocyte-directed gene therapy for ada-scid: initial trial results after years regulation of innate immunity by rho gtpases signals for sorting of transmembrane proteins to endosomes and lysosomes caveolin- in breast cancer tat peptide-mediated cellular delivery: back to basics chemical chaperones correct the mutant phenotype of the delta f cystic fibrosis transmembrane conductance regulator protein the small gtpase rab functions as a regulatory factor in the early endocytic pathway probucol. a reappraisal of its pharmacological properties and therapeutic use in hypercholesterolaemia the wilson disease gene is a putative copper transporting p-type atpase similar to the menkes gene coiled coils: a highly versatile protein folding motif mechanisms of waspmediated hematologic and immunologic disease a step forward for stiv-person syndrome small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders hepatic endoplasmic reticulum storage diseases initial docking of er-derived vesicles requires uso p and ypt p but is independent of snare proteins a role for cbs domain in traycking of chloride channel clc- a ''de novo'' point mutation of the low-density lipoprotein receptor gene in an italian subject with primary hypercholesterolemia actin dynamics during phagocytosis adpkd: a human disease altering golgi function and basolateral exocytosis in renal epithelia localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments multiple forms of dynamin are encoded by shibire, a drosophila gene involved in endocytosis function of rho family proteins in actin dynamics during phagocytosis and engulfment lowe syndrome protein ocrl interacts with clathrin and regulates protein traycking between endosomes and the trans-golgi network gamma-secretase as a therapeutic target for the treatment of alzheimer's disease the achilles heel of erbb- /her : regulation by the hsp chaperone machine and potential for pharmacological intervention lytic granules, secretory lysosomes and disease adaptor protein -dependent microtubule-mediated movement of lytic granules to the immunological synapse novel membrane trayc steps regulate the exocytosis of the menkes disease atpase the menkes disease atpase (atp a) is internalized via a rac -regulated, clathrin-and caveolae-independent pathway actin and microtubule regulation of trans-golgi network architecture, and copper-dependent protein transport to the cell surface role of caveolae and caveolins in health and disease the human genome project: lessons from large-scale biology rab and arf gtpase regulation of exocytosis novel oral treatment of gaucher's disease with n-butyldeoxynojirimycin (ogt ) to decrease substrate biosynthesis copper transporting p-type atpases and human disease defective intracellular transport and processing of oa is a major cause of ocular albinism type involvement of clathrin-mediated endocytosis in human immunodeficiency virus type entry fc receptor biology the molecular basis of alanine: glyoxylate aminotransferase mistargeting: the most common single cause of primary hyperoxaluria type a role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals the synaptic vesicle-associated protein amphiphysin is the -kd autoantigen of stiv-man syndrome with breast cancer a novel form of hereditary myeloperoxidase deficiency linked to endoplasmic reticulum/proteasome degradation the low lysine content of ricin a chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol low-density lipoprotein receptor-its structure, function, and mutations association of the ap- adaptor complex with clathrin ap- , a novel protein complex related to clathrin adaptors altered traycking of lysosomal proteins in hermansky-pudlak syndrome due to mutations in the beta a subunit of the ap- adaptor rhodopsin c terminus, the site of mutations causing retinal disease, regulates traycking by binding to adp-ribosylation factor (arf ) the third helix of the antennapedia homeodomain translocates through biological membranes rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces rab prenylation and platelet synthesis ubiquitination is required for the retrotranslocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome mutations in the mdr gene cause progressive familial intrahepatic cholestasis the coiled-coil membrane protein golgin- is a novel rab evector required for golgi ribbon formation the cell biology of hermansky-pudlak syndrome: recent advances from genome to drug lead: identification of a small-molecule inhibitor of the sars virus cooperation of ggas and ap- in packaging mprs at the trans-golgi network inhibition of proteasome function induces programmed cell death in proliferating endothelial cells the t( ; )(p ;q ) in the u cell line results in the fusion of the af gene and calm, encoding a new member of the ap- clathrin assembly protein family remodeling of endosomes during lysosome biogenesis involves ''kiss and run'' fusion events regulated by rab er-to-golgi transport: cop i and cop ii function (review) hyperinsulinism in infancy: from basic science to clinical disease wiskott-aldrich syndrome protein regulates lipid raft dynamics during immunological synapse formation caveolae and sorting in the trans-golgi network of epithelial cells overexpression of tau protein inhibits kinesin-dependent traycking of vesicles, mitochondria, and endoplasmic reticulum: implications for alzheimer's disease a novel gene for autosomal dominant stargardt-like macular dystrophy with homology to the sur protein family intercellular traycking and protein delivery by a herpesvirus structural protein cisternal maturation and vesicle transport: join the band wagon! (review) huntingtin-associated protein (hap ) interacts with the p glued subunit of dynactin genes encoding human caveolin- and - are co-localized to the d s locus ( q . ), a known fragile site (fra g) that is frequently deleted in human cancers actin assembly and endocytosis: from yeast to mammals accelerated transport and maturation of lysosomal alpha-galactosidase a in fabry lymphoblasts by an enzyme inhibitor lowe syndrome protein ocrl interacts with rac gtpase in the trans-golgi network tat-mediated delivery of heterologous proteins into cells munc - is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (fhl ) brain-derived neurotrophic factor in huntington disease caveolae and intracellular traycking of cholesterol impact of donor type on outcome of bone marrow transplantation for wiskott-aldrich syndrome: collaborative study of the international bone marrow transplant registry and the national marrow donor program adeno-associated virus-mediated gene transfer for lung diseases the ap- a and ap- b clathrin adaptor complexes define biochemically and functionally distinct membrane domains the endoplasmic reticulum as a site of protein degradation de novo formation of caveolae in lymphocytes by expression of vip -caveolin identification of a di-leucine motif within the c terminus domain of the menkes disease protein that mediates endocytosis from the plasma membrane cellular uptake of the tat protein from human immunodeficiency virus induction of p mitogen-activated protein kinase reduces early endosome autoantigen (eea ) recruitment to phagosomal membranes caveolin- null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities phenotypic behavior of caveolin- mutations that cause autosomal dominant limb girdle muscular dystrophy (lgmd- c). retention of lgmd- c caveolin- mutants within the golgi complex hiv- traycking to the dendritic cell-t-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse structural basis of fabry disease fyve fingers bind ptdins huntingtin controls neurotrophic support and survival of neurons by enhancing bdnf vesicular transport along microtubules microtubule transport defects in neurological and ciliary disease wilson disease the alpha chain of the ap- adaptor is a clathrin binding subunit intracellular transport and sorting of the oligodendrocyte transmembrane proteolipid protein disrupted proteolipid protein traycking results in oligodendrocyte apoptosis in an animal model of pelizaeus-merzbacher disease evidence that rme- , a conserved c. elegans eh-domain protein, functions in endocytic recycling the arguments for pre-existing early and late endosomes intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides a gtpase-activating protein controls rab function in endocytic traycking defective granule exocytosis in rab a-deficient lymphocytes from ashen mice synaptojanin : localization on coated endocytic intermediates in nerve terminals and interaction of its kda isoform with eps mutation and aberrant expression of caveolin- in human oral squamous cell carcinomas and oral cancer cell lines traycking, turnover and membrane topology of prp charcot-marie-tooth neuropathy type b is associated with mutations of the myelin p gene invasion activating caveolin- mutation in human scirrhous breast cancers identification and functional analysis of a caveolin- mutation associated with familial hypertrophic cardiomyopathy accumulating evidence suggests that several ab-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells the delta f mutation shortens the biochemical half-life of plasma membrane cftr in polarized epithelial cells dynaminmediated internalization of caveolae albinism associated with hemorrhagic diathesis and unusual pigmented reticular cells in the bone marrow: report of two cases with histochemical studies evects of mutant rat dynamin on endocytosis characterization of a fourth adaptor-related protein complex missense mutation (c r) in the thyroglobulin gene causes congenital goiter with mild hypothyroidism by impaired intracellular transport a novel leukocyte adhesion deficiency caused by expressed but nonfunctional beta integrins mac- and lfa- caveolae: stable membrane domains with a potential for internalization doublecortin, a stabilizer of microtubules a novel rab gdp/gtp exchange factor complexed to rabaptin- links nucleotide exchange to evector recruitment and function hereditary neutropenia: dogs explain human neutrophil elastase mutations ap- mediates tyrosinase but not trp- traycking in human melanocytes nonsense mutations in adtb a cause complete deficiency of the {beta} a subunit of adaptor complex- and severe hermansky-pudlak syndrome type the leaden gene product is required with rab a to recruit myosin va to melanosomes in melanocytes caveolae and lipid rafts: g protein-coupled receptor signaling microdomains in cardiac myocytes rho gtpases: biochemistry and biology a common w s mutation in the ldl receptor gene of danish patients with familial hypercholesterolemia encodes a transport-defective protein antennapedia homeobox peptide regulates neural morphogenesis identification and molecular characterisation of a calm-af fusion in acute megakaryoblastic leukaemia mutations in a sar gtpase of copii vesicles are associated with lipid absorption disorders substitution of arginine for histidine at position in the alpha-subunit of the human insulin receptor. a mutation that impairs receptor dimerization and transport of receptors to the cell surface distinct sets of sec genes govern transport vesicle formation and fusion early in the secretory pathway metabolic and molecular bases of menkes disease and occipital horn syndrome receptor downregulation and multivesicular-body sorting stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls positionally cloned gene for a novel glomerular protein-nephrin-is mutated in congenital nephrotic syndrome endocrinopathies in the family of endoplasmic reticulum (er) storage diseases: disorders of protein traycking and the role of er molecular chaperones a conditional mutation avecting localization of the menkes disease copper atpase. suppression by copper supplementation molecular characterization of mammalian homologues of class c vps proteins that interact with syntaxin- three ways to make a vesicle cellular autophagy: surrender, avoidance and subversion by microorganisms proteasome inhibitors: from research tools to drug candidates a cholesterol-lowering gene maps to chromosome q a controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis a germline insertion in the tuberous sclerosis (tsc ) gene gives rise to the eker rat model of dominantly inherited cancer disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval possible temperature-dependent blockage of synaptic vesicle recycling induced by a single gene mutation in drosophila a post-docking role for active zone protein rim hiv interaction with endosomes in macrophages and dendritic cells organization of the pronephric filtration apparatus in zebrafish requires nephrin, podocin and the ferm domain protein mosaic eyes cdc controls secretory and endocytic transport to the basolateral plasma membrane of mdck cells copii-cargo interactions direct protein sorting into er-derived transport vesicles akt crystal structure and akt-specific inhibitors defective human ether-a-go-go-related gene traycking linked to an endoplasmic reticulum retention signal in the c terminus retroviral vectors vip , a -kd membrane protein is an integral component of trans-golginetwork-derived transport vesicles regulation of vascular endothelial growth factor receptor- activity by caveolin- and plasma membrane cholesterol intracellular localization and loss of copper responsiveness of mnk, the murine homologue of the menkes protein, in cells from blotchy (mo blo) and brindled (mo br) mouse mutants interleukin receptors and detergent-resistant membrane domains define a clathrinindependent endocytic pathway aberrant splicing but not mutations of tsg in human breast cancer tumor cell growth inhibition by caveolin re-expression in human breast cancer cells bi-directional protein transport between the er and golgi a missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in sephardic jews novel mutations in the wiskott-aldrich syndrome protein gene and their evects on transcriptional, translational, and clinical phenotypes niemann-pick disease: a frequent missense mutation in the acid sphingomyelinase gene of ashkenazi jewish type a and b patients evidence for a regulated interaction between heterotrimeric g proteins and caveolin a huntingtin-associated protein enriched in brain with implications for pathology expression and characterization of recombinant caveolin hermansky-pudlak syndrome type (hps- ) results from mutant dysbindin molecular, anatomical, and biochemical events associated with neurodegeneration in mice with niemann-pick type c disease the listeria protein internalin b mimics hepatocyte growth factor-induced receptor traycking bone marrow transplantation from an hla-matched unrelated donor for treatment of chediak-higashi syndrome familial sjogren's syndrome with associated primary salivary gland lymphoma protein kinase d regulates the fission of cell surface destined transport carriers from the trans-golgi network rme- regulates the distribution and function of the endocytic recycling compartment in mammalian cells caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains mutant myosin viia causes defective melanosome distribution in the rpe of shaker- mice rescue of deltaf and other misprocessed cftr mutants by a novel quinazoline compound organic-aciduria, decreased renal ammonia production, hydrophthalmos, and mental retardation; a clinical entity structure and function of the lowe syndrome protein ocrl autoantigen golgin- , an evector of arl gtpase, participates in trayc from the endosome to the trans-golgi network functional evaluation of dent's disease-causing mutations: implications for clc- channel traycking and internalization regulation of protein transport from the golgi complex to the endoplasmic reticulum by cdc and n-wasp the beta-appendages of the four adaptor-protein (ap) complexes: structure and binding properties, and identification of sorting nexin as an accessory protein to ap- function and regulation of the mammalian coppertransporting atpases: insights from biochemical and cell biological approaches membrane dynamics and the biogenesis of lysosomes rabaptin- is a novel fusion partner to platelet-derived growth factor beta receptor in chronic myelomonocytic leukemia kdel-cargo regulates interactions between proteins involved in copi vesicle trayc: measurements in living cells using fret purification of a novel class of coated vesicles mediating biosynthetic protein transport through the golgi stack clogging of axons by tau, inhibition of axonal trayc and starvation of synapses endocytosis and targeting of exogenous hiv- tat protein a novel point mutation in cd causing the expression of dysfunctional cd /cd leucocyte integrins in a patient with leucocyte adhesion deficiency (lad) copii-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes altered traycking and adhesion function of mpz mutations and phenotypes of charcot-marie-tooth disease b biology of adenovirus and its use as a vector for gene therapy the putative tumor suppressors ext and ext form a stable complex that accumulates in the golgi apparatus and catalyzes the synthesis of heparan sulfate congenital hypothyroid goiter with deficient thyroglobulin. identification of an endoplasmic reticulum storage disease with induction of molecular chaperones mutations in rab a cause griscelli syndrome associated with haemophagocytic syndrome myoblast transfer in the treatment of duchenne's muscular dystrophy enzyme replacement therapy in fabry's disease: recent advances and clinical applications signalling to actin assembly via the wasp (wiskott-aldrich syndrome protein)-family proteins and the arp / complex mutations in the caveolin- gene cause autosomal dominant limb-girdle muscular dystrophy long-term evectiveness of a new alpha-glucosidase inhibitor (bay m -miglitol) in insulin-treated type diabetes mellitus genetic disorders avecting proteins of iron and copper metabolism: clinical implications hsp inhibitor geldanamycin and its derivatives as novel cancer chemotherapeutic agents mechanism of microtubule stabilization by doublecortin identification and functional analysis of two novel mutations in the multidrug resistance protein gene in israeli patients with dubin-johnson syndrome pharmacological chaperones: a new twist on receptor folding a conserved clathrin assembly motif essential for synaptic vesicle endocytosis eea , an early endosome-associated protein. eea is a conserved alpha-helical peripheral membrane protein flanked by cysteine ''fingers'' and contains a calmodulin-binding iq motif localization of proteins to the golgi apparatus vip /caveolin is a cholesterol-binding protein maturation models for endosome and lysosome biogenesis cdc regulates the exit of apical and basolateral proteins from the trans-golgi network consistent detection of calm-af chimaeric transcripts in haematological malignancies with t( ; )(p ;q ) and identification of novel transcripts molecular analysis of the ergic- gene in families with combined factor v-factor viii deficiency vascular endothelial growth factor (vegf) and its receptors the trojan horse: survival tactics of pathogenic mycobacteria in macrophages snares and membrane fusion in the golgi apparatus mutations in the er-golgi intermediate compartment protein ergic- cause combined deficiency of coagulation factors v and viii regulation of phagocytosis by rho gtpases urinary megalin deficiency implicates abnormal tubular endocytic function in fanconi syndrome wiskott-aldrich syndrome: a model for defective actin reorganization, cell traycking and synapse formation identification of complementation groups required for post-translational events in the yeast secretory pathway cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically novel cell permeable motif derived from the pres -domain of hepatitis-b virus surface antigens dynamin at the neck of caveolae mediates their budding to form transport vesicles by gtp-driven fission from the plasma membrane of endothelium the proteasome is involved in angiogenesis role of rab gtpases in membrane trayc the evolving role of lipid rafts and caveolae in g proteincoupled receptor signaling: implications for molecular pharmacology the structure and function of the beta -adaptin appendage domain phosphatidylinositol phosphate -kinase i{beta} recruits ap- to the plasma membrane and regulates rates of constitutive endocytosis fine structure of blood capillaries intracellular aspects of the process of protein synthesis two genes are responsible for griscelli syndrome at the same q locus localization and processing of cln , the protein associated to batten disease: where is it and what does it do? localization of the ap- adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins infectious hiv- assembles in late endosomes in primary macrophages snares and the specificity of transport vesicle targeting caveolar endocytosis of simian virus reveals a new two-step vesicular-transport pathway to the er caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane trayc alpha -antitrypsin deficiency: liver disease associated with retention of a mutant secretory glycoprotein in the endoplasmic reticulum alpha- -antitrypsin deficiency: diagnosis and treatment identification of the murine beige gene by yac complementation and positional cloning the menkes protein (atp a; mnk) cycles via the plasma membrane both in basal and elevated extracellular copper using a c-terminal dileucine endocytic signal participation of the endoplasmic reticulum chaperone calnexin (p , ip ) in the biogenesis of the cystic fibrosis transmembrane conductance regulator prevention of lysosomal storage in tay-sachs mice treated with n-butyldeoxynojirimycin correction of a mineralization defect by overexpression of a wild-type cdna for col a in marrow stromal cells (mscs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects constitutive protein secretion from the trans-golgi network to the plasma membrane review of donepezil, rivastigmine, galantamine and memantine for the treatment of dementia in alzheimer's disease in adults with down syndrome: implications for the intellectual disability population protein kinase d-mediated anterograde membrane traycking is required for fibroblast motility gene replacement reveals that p /snare interactions are essential for golgi biogenesis constitutive skipping of alternatively spliced exon in the atp a gene abolishes golgi localization of the menkes protein and produces the occipital horn syndrome traycking and folding defects in hereditary spherocytosis mutants of the human red cell anion exchanger caveolin- -deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae isolation of a miller-dicker lissencephaly gene containing g protein [beta]-subunit-like repeats myosin viia is required for aminoglycoside accumulation in cochlear hair cells endophilin/sh p is required for the transition from early to late stages in clathrinmediated synaptic vesicle endocytosis mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin v b/v receptor by a pharmacological chaperone adaptor-related proteins adaptable adaptors for coated vesicles surfing the sec channel: bidirectional protein translocation across the er membrane yolk protein uptake in the oocyte of the mosquito aedes aegypti caveolin, a protein component of caveolae membrane coats mechanisms of intracellular protein transport protein sorting by transport vesicles early stages of influenza virus entry into mv- lung cells: involvement of dynamin science medicine, and the future. gene therapy endoplasmic reticulum storage diseases calm-af fusion gene in leukemias: simple and inversion-associated translocation ( ; ) assembly of the er to golgi snare complex requires uso p signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in mdck cells regulation of the unc- -caenorhabditis elegans syntaxin complex by unc- visualization of er-to-golgi transport in living cells reveals a sequential mode of action for copii and copi identification, sequence, and expression of caveolin- defines a caveolin gene family an enzyme that removes clathrin coats: purification of an uncoating atpase endophilin i mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid endoplasmic reticulumlocalized amyloid beta-peptide is degraded in the cytosol by two distinct degradation pathways endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including vamp, nsf, snap, annexins, and gtpases griscelli syndrome: report of the first peripheral blood stem cell transplant and the role of mutations in the rab a gene as an indication for bmt peptide-mediated cellular delivery gene therapy: a brief overview of the past, present, and future phagosome maturation: a few bugs in the system the npc protein: structure implies function rab gtpases, intracellular trayc and disease a ypt/rab evector complex containing the sec homolog vps p is required for homotypic vacuole fusion the role of the tethering proteins p and gm in transport through the golgi apparatus in vivo early endosome antigen. : an autoantigen associated with neurological diseases identification of the b-cell epitopes of the early endosome antigen (eea ) cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia diverential evects of kinase cascade inhibitors on neoplastic and cytokine-mediated cell proliferation chediak-higashi syndrome: a rare disorder of lysosomes and lysosome related organelles autophagy in health and disease: a double-edged sword golgins and gtpases, giving identity and structure to the golgi apparatus a role for the vesicle tethering protein, p , in the postmitotic stacking of reassembling golgi cisternae in a cell-free system sequential tethering of golgins and catalysis of snarepin assembly by the vesicle-tethering protein p transcytosis of plasma macromolecules in endothelial cells: a cell biological survey regulation of cytoplasmic dynein behaviour and microtubule organization by mammalian lis snap receptors implicated in vesicle targeting and fusion paraneoplastic stiv-person syndrome: passive transfer to rats by means of igg antibodies to amphiphysin a role for giantin in docking copi vesicles to golgi membranes phenotypic behavior of caveolin- r q, a mutant associated with hyperckemia, distal myopathy, and rippling muscle disease phosphofructokinase muscle-specific isoform requires caveolin- expression for plasma membrane recruitment and caveolar targeting: implications for the pathogenesis of caveolin-related muscle diseases identification of filamin as a novel ligand for caveolin- : evidence for the organization of caveolin- -associated membrane domains by the actin cytoskeleton endosomal localization of the autoantigen eea is mediated by a zinc-binding fyve finger perforin gene defects in familial hemophagocytic lymphohistiocytosis copicoated er-to-golgi transport complexes segregate from copii in close proximity to er exit sites the immunological synapse of ctl contains a secretory domain and membrane bridges linking albinism and immunity: the secrets of secretory lysosomes the biogenesis of lysosomes: is it a kiss and run, continuous fusion and fission process? breaking the copi monopoly on golgi recycling a family of rab -binding proteins. melanophilin links rab a and myosin va function in melanosome transport retinal stimulates atp hydrolysis by purified and reconstituted abcr, the photoreceptor-specific atp-binding cassette transporter responsible for stargardt disease abnormal vesicular traycking in mouse models of hermansky-pudlak syndrome protein folding and translocation across the endoplasmic reticulum membrane role of calnexin in the glycan-independent quality control of proteolipid protein assembly and traycking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters identification and expression of five mutations in the human acid sphingomyelinase gene causing types a and b niemann-pick disease. molecular evidence for genetic heterogeneity in the neuronopathic and non-neuronopathic forms defective aquaporin- traycking in nephrogenic diabetes insipidus and correction by chemical chaperones misfolding of mutant aquaporin- water channels in nephrogenic diabetes insipidus molecular cloning of caveolin- , a novel member of the caveolin gene family expressed predominantly in muscle copii and exit from the endoplasmic reticulum identification of a familial hyperinsulinism-causing mutation in the sulfonylurea receptor that prevents normal traycking and function of katp channels mutations of the pds gene, encoding pendrin, are associated with protein mislocalization and loss of iodide ezux: implications for thyroid dysfunction in pendred syndrome the chediak-higashi protein interacts with snare complex and signal transduction proteins clathrin assembly lymphoid myeloid leukemia (calm) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated trayc how peroxisomes arise chloride channels cough up caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic traycking tau regulates the attachment/detachment but not the speed of motors in microtubule-dependent transport of single vesicles and organelles gangliosides are receptors for murine polyoma virus and sv protein traycking and alzheimer's disease role of auxilin in uncoating clathrincoated vesicles the inositol polyphosphate -phosphatase ocrl associates with endosomes that are partially coated with clathrin actin microfilaments facilitate the retrograde transport from the golgi complex to the endoplasmic reticulum in mammalian cells dynamin-like protein encoded by the drosophila shibire gene associated with vesicular trayc protein kinase d: an intracellular trayc regulator on the move fcgammar polymorphisms: implications for function, disease susceptibility and immunotherapy a dileucine-like sorting signal directs transport into an ap- -dependent, clathrin-independent pathway to the yeast vacuole protein kinase g from pathogenic mycobacteria promotes survival within macrophages degradation of cftr by the ubiquitinproteasome pathway use of expression constructs to dissect the functional domains of the chs/beige protein: identification of multiple phenotypes a novel -kd peripheral membrane protein is required for intercisternal transport in the golgi stack rab coordinates a novel golgi to er retrograde transport pathway in live cells hsp and the chaperoning of cancer sec -mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction the human cytomegalovirus us gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol amphiphysin heterodimers: potential role in clathrin-mediated endocytosis the caveolin genes: from cell biology to medicine a mutation in rab a causes the vesicle transport defects observed in ashen mice caveolinopathies: mutations in caveolin- cause four distinct autosomal dominant muscle diseases four contiguous amino acid substitutions, identified in patients with laron syndrome, diverently avect the binding aynity and intracellular traycking of the growth hormone receptor synaptojanin is the major constitutively active phosphatidylinositol- , , -trisphosphate -phosphatase in rodent brain rab a enables myosin va-dependent melanosome capture by recruiting the myosin to the organelle identification of an organelle receptor for myosin-va the tuberous sclerosis gene product, tuberin, functions as a rab gtpase activating protein (gap) in modulating endocytosis the fine structure of the gall bladder epithelium of the mouse caveolin is an inhibitor of platelet-derived growth factor receptor signaling sulfonylureas correct traycking defects of atp-sensitive potassium channels caused by mutations in the sulfonylurea receptor nsec binds a closed conformation of syntaxin a abnormal ryanodine receptor function in heart failure fabry disease: characterization of alpha-galactosidase a double mutations and the d y plasma enzyme pseudodeficiency allele protein kinase d regulates basolateral membrane protein exit from trans-golgi network predisposition to renal carcinoma in the eker rat is determined by germ-line mutation of the tuberous sclerosis (tsc ) gene adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis rab proteins as membrane organizers bleeding due to disruption of a cargo-specific er-to-golgi transport complex charcot-marie-tooth disease type a caused by mutation in a microtubule motor kif b the lebanese allele at the low density lipoprotein receptor locus. nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum sucrase-isomaltase deficiency in humans. diverent mutations disrupt intracellular transport, processing, and function of an intestinal brush border enzyme surfing the sec channel: bidirectional protein translocation across the er membrane crystal structure of an activated akt/protein kinase b ternary complex with gsk -peptide and amp-pnp key: cord- -w zvfkyl authors: tooze, sharon a title: biogenesis of secretory granules in the trans-golgi network of neuroendocrine and endocrine cells date: - - journal: biochim biophys acta mol cell res doi: . /s - ( ) - sha: doc_id: cord_uid: w zvfkyl secretory granule formation requires selection of soluble and membrane proteins into nascent secretory granules, and exclusion of proteins not required for the function of secretory granules. both selection and exclusion presumably can occur in the compartment where assembly of the secretory granule begins, the trans most cisternae of the golgi complex. current research focused on the initial stages of secretory granule formation includes a search for the ‘signals’ which may mediate active sorting of components into secretory granules, and the role of aggregation of regulated secretory proteins in sorting. in addition, the temporal sequence of the sorting events in the golgi, and post-golgi compartments has gained much attention, as summarized by the alternative but not mutually exclusive ‘sorting for entry’ vs. ‘sorting by retention’ models. ‘sorting for entry’ which encompasses the most popular models requires selection of cargo and membrane and exclusion of non-secretory granule proteins in the tgn prior to secretory granule formation. ‘sorting by retention’ stipulates that protein selection or exclusion may occur after secretory granule formation: secretory granule specific components are retained during maturation of the granule while non-secretory granule molecules are removed in vesicles which bud from maturing secretory granules. finally, some progress has been made in the identification of cytosolic components involved in the budding of nascent secretory granules from the tgn. this review will focus on the recent data concerning the events in secretory granule formation which occur, in the trans-golgi network. dense core secretory granules are found in all exocrine, endocrine, neuroendocrine, and neuronal cells. vesicles which are similar to secretory granules are also found in cells of the haemopoetic and immune systems. formation of these vesicles is initiated in the trans-golgi network (tgn) and results in the secretory proteins being packaged in a concentrated form in the secretory granules. unstimulated exocytosis of these granules occurs at low levels, but ampli¢ed secretion from the cell of proteins within secretory granules is regulated exclusively via external stimuli which triggers the fusion of the secretory granule membrane with the plasma membrane. secretory granules in endocrine and neuroendocrine cells have a very similar composition. the limiting membrane contains proteins required for secretory granule acidi¢cation, transport, targeting and fusion, while the dense core content is composed of a set of soluble proteins, some of which are substrates, some of which are enzymes, others of which have an as yet unidenti¢ed function. it is known that there is variation in the content of the secretory granule, depending on the type of tissue the cell is derived from, however, very little is known about the membrane composition and the variation which might be present in the molecules involved in secretory granule biogenesis, targeting and fusion. research into the formation of the secretory granules from the tgn is mainly concerned with how the soluble regulated secretory proteins are targeted into the secretory granule, how soluble non-secretory granule proteins are excluded, if at all, what factors determine the membrane composition, and how secretory granule budding occurs from the tgn. this research is complicated by the fact that secretory granule formation could occur in parallel with formation of vesicles directed to endosomes or the plasma membrane. this review will explore the recent progress in these areas and attempt to compare the current models of secretory granule biogenesis from the tgn in endocrine cells and neuroendocrine cells. regulated secretory proteins are packaged into nascent secretory granules (which are referred to as immature secretory granules (isgs)) in a process that is thought to be initiated by selective aggregation of the secretory proteins in the tgn, followed by interaction of the aggregate with the tgn membrane which may involve receptor molecules. it is also possible that selection may be achieved by interaction of individual regulated secretory proteins with a putative membrane receptor prior to aggregation. proteins which are not present in regulated secretory protein aggregates, or do not interact with the recep-tor for the regulated secretory proteins, are thought to exit the tgn in constitutive secretory vesicles (csvs). an alternative hypothesis is that there is very little constitutive secretion originating from the tgn of regulated cells, and that most molecules in the biosynthetic pathway exit into isgs. in the isg, the non-secretory granule proteins would presumably be sorted from the regulated secretory proteins via interaction with a receptor and removed from the isg in constitutive-like vesicles. aggregation of the regulated secretory proteins would still occur in the isg and would function to allow retention in the isg by preventing any interaction with receptors for non-secretory granule proteins. in both models, one could make predictions about the stoichiometry of the receptor relative to the ligand, depending on whether it is a single secretory protein interacting with a single receptor, or an aggregate of regulated secretory proteins interacting with a single receptor. in both models, interaction with a receptor would allow removal of non-secretory granule proteins from the forming or maturing secretory granule. an important point of clari¢cation is that there may well be multiple classes of receptors for both regulated secretory proteins and constitutive secretory proteins in the tgn and the isg. in addition, interaction of any ligand with its receptor is most probably transient and disrupted, for example, by changes in the ph of the maturing isg. one mechanism for sorting soluble secretory proteins into secretory granules is via a selective aggregation of a subset of soluble proteins to the exclusion of other soluble, non-aggregating secretory proteins. for e¤cient aggregation, the local concentration of the secretory proteins should be high in the sorting compartment. soluble regulated secretory proteins are for the most part very abundant in cells with a regulated secretory pathway. in particular, a family of proteins known as the granins [ ] are highly expressed in most endocrine and neuroendocrine cells. the members of this family of proteins include chromogranin a (cga), chromogranin b (cgb), secretogranin ii (sgii), secretogranin iii, and secretogranin iv (also known as b ). based on the primary sequence of the family members, it is thought they have similar structural features which may enable them to e¤ciently aggregate in the tgn [ ] . although the exact function of the granins is not known, they might be acting as`helper proteins' [ ] . if sorting only occurs via aggregation and does not rely on a speci¢c and stoichiometric receptor-ligand interaction, then e¤cient sorting of prohormones which either have a less than optimal ability to condense, or are present at very low concentration, might be enhanced by`helper proteins' that aid aggregate formation. recently, natori and huttner [ ] have demonstrated that increasing the amount of cgb in att cells (a cell line derived from the pituitary corticotroph) indeed promoted e¤cient sorting of the kda pomc (pro-opiomelanocortin) fragment into secretory granules, resulting in increased storage of the kda pomc fragment and acth. aggregation of sgii has been shown in vitro as well as in permeabilized tgn membranes, under conditions (low ph, high ca ) believed to be present in the lumenal environment of the tgn [ , ] . other soluble secretory proteins, other members of the granin family and other proteins found in secretory granules, have also been shown to aggregate under these low ph, high ca conditions (see e.g. [ , ] ). indeed, morphological evidence demonstrating the presence of aggregates of regulated secretory proteins in the tgn [ , ] , but not in earlier compartments under normal conditions supports both the location of the initiation of aggregation and its role in sorting. experiments performed several years ago with weak bases, such as chloroquine and ammonium chloride argued that perturbation of the ph in the tgn caused missorting of regulated secretory proteins into the constitutive pathway (see for example [ ] ). although the data did not gain complete support [ ] these experiments were the ¢rst to suggest that ph may have a role in the sorting of soluble proteins. more recent results obtained with streptolysin o-permeabilized pc cells support the idea in that ph may be crucial for sorting of regulated secretory proteins in the tgn [ ] . concerning the exact ph and ca requirements in vivo, values for the ph in the tgn of cells lacking a regulated secretory pathway have been proposed in two recent studies ( . þ . and . þ . ) [ , ] supporting the notion that the tgn is slightly more acidic than previous golgi compartments, but there are still no data available on the free ca concentration in the lumen of the tgn. interpretation of all these results to determine the precise role of ph and ca in the tgn requires accurate measurements for ph and ca in the lumen of the tgn in cells with a regulated secretory pathway. some controversy exists concerning the role of the tgn-localized aggregation event in sorting which may relate to di¡erences between the cellular systems studied. l-cells of the endocrine pancreas secrete large quantities of insulin [ ] . insulin forms insoluble, close packed crystalline arrays in the presence of zinc, whereas proinsulin has distinct physical properties which prevent its condensation into insoluble crystals. processing of proinsulin to insulin, which requires two processing enzymes, prohormone converting enzymes (pcs) and , acting at two distinct sites (for review see [ ] ), is crucial for the condensation of insulin. however, the intracellular location where processing of proinsulin and other prohormones begins is unresolved. there are reports which demonstrate that processing of prohormones by pc enzymes can begin in the tgn [ , , ] whereas other studies indicate that processing is initiated in the isg [ ^ ] . proinsulin conversion has been demonstrated to occur only in secretory granules ([ ^ ] and references included therein). therefore, if sorting of regulated secretory proteins into dense cores relies on exclusion of soluble non-granule material through aggregation, or condensation, insulin can only be sorted in a post-tgn compartment. this result also implies that proinsulin cannot be sorted by aggregation in the tgn or any post-tgn compartment. these results have provided the basis for the`sorting by retention' hypothesis [ ] (fig. ). this hypothesis suggests that regulated secretory proteins are sorted largely by retention in a post-golgi isg. non-secretory granule proteins, or processing remnants of regulated secretory proteins, such as the c-peptide (which is derived from proinsulin and not found in mature insulin), can be in part removed by constitutive-like vesicle budding from the isg, although the e¤ciency of this removal is likely to be related to whether a selection process (i.e. receptor-mediated) process is employed. thus, the e¤ciency for removal from isgs might be expected to vary for di¡erent proteins (see below). an interesting example which reinforces the importance of the precise location of prohormone processing on sorting comes from experiments done on aplysia californica [ ] and lymnaea stagnalis [ ] . these two model systems have been used to demonstrate that sorting of proelh (egg-laying hormone) can be in£uenced by the location of cleavage, which in turn presumably a¡ects the hormones ability to aggregate or condense. in aplysia, the proelh is cleaved in the golgi and the two n-terminal and c-terminal intermediates are sorted into two distinct populations of large dense core vesicles (ldcvs) [ ] . lymnaea presents a more complicated scenario in that there are two di¡erent types of neurones which produce elh: in one type (type i neurones) processing occurs in the tgn and results in the appearance of two morphologically distinguishable condensed protein cores, containing either the n-terminal or c-terminal region of elh, which are sorted to di¡erent populations of secretory vesicles, leg (large electrondense granule) and ldcvs [ ] . in the second type of neurone (type ii), the elh is not cleaved in the tgn and is sorted into the ldcvs as a prohormone, after which it undergoes cleavage to mature elh. this raises an interesting question concerning how proteins exit from the golgi in type i and type ii neurones. it would be of great interest to compare the sorting of true`constitutive' proteins in type i and type ii neurones to see if in type i neurones constitutive secretory proteins are secreted constitutively, implying that csvs are formed directly from the golgi, and likewise if in type ii neurones if these proteins are secreted in a constitutive-like fashion, implying secretion originates from isgs. interestingly, the ldcvs from the two types of neurones di¡er in size, the ldcv from the type i neurone having a size of approximately nm whereas the ldcvs in the type ii neurone was nm [ ] suggesting that the composition of the content has an in£uence fig. . depicted in a very simple fashion is a scheme of the sorting mechanisms in the secretory pathway of neuroendocrine and endocrine cells proposed by arvan and colleagues [ ] to explain the di¡erent models for formation of regulated secretory vesicles from the tgn, i.e. sorting for entry vs. sorting by retention. also included are other vesicle classes, such as csvs and constitutive-like secretory vesicles, as well as a pathway for lysosomal enzymes (blue circle) and mpr (black cup-shaped object). the low amount of missorted proteins, such as furin [ ] , and mpr found in the isg in the top panel have not been illustrated for simplicity. the regulated secretory proteins are shown as yellow circles, and the constitutive secretory proteins are shown as red circles. for simplicity, all coats and coat proteins have been omitted, as well as any putative interactions of the regulated secretory proteins with the membrane. on the ¢nal size. in support of this idea, a size difference has also been seen in the secretory granules in endocrine cells of the pituitary (for review see [ ] ). while the data to support`sorting by retention' [ ] is compelling, the data to support the`sorting for entry' hypothesis, where the bulk of sorting has been demonstrated to occur in the tgn prior to isg formation, is equally compelling (see above). to reconcile these two hypotheses, it has been suggested [ , ] that whether sorting is predominantly tgnbased (sorting for entry) or isg-based (sorting by retention) might depend both on the propensity of the major prohormone species to aggregate, or not, and the relative rates of prohormone synthesis and vesicle formation in the tgn. the paradigm for sorting of soluble proteins into vesicles is receptor-mediated endocytosis at the plasma membrane: a receptor binds a ligand and sequesters the ligand for transport to a di¡erent compartment in a coat-dependent fashion. this model has been extended to a variety of intracellular transport events including sorting of regulated secretory proteins in the tgn [ ] . regardless of whether a single molecule or a molecular aggregate is sorted, this paradigm has remained attractive enough for investigators to continue to pursue the putative`sorting receptor'. the putative sorting receptor could then interact directly or indirectly with the cytosolic machinery involved in the vesicle formation. the search for a sorting receptor has been approached from both the ligand side, i.e. identi¢cation of sorting signals in the ligand, and the receptor side, i.e. identi-¢cation of membrane proteins which bind regulated secretory proteins. recent experiments have focused on an n-terminal disulphide loop structure found in pomc, provasopressin, pro-oxytocin, prodynorphin, proenkephalin, cga and cgb [ , ] . the n-terminal amino acids of pomc have been shown to contain a signal which sorts a reporter molecule into secretory granules in att cells [ ] . it has been further demonstrated that a disulphide bond, within this sequence of amino acids, forms an amphipathic loop structure which may play a role in sorting pomc to isgs [ ] . this data is supported by experiments in which the analogous disulphide bond of cgb was disrupted by incubation of pc cells with dtt, leading to constitutive secretion of cgb [ ] . in pursuit of a receptor which would recognize the sorting motif researchers have used assays based on the binding of solubilized secretory granule membrane components to immobilized regulated secretory proteins. as some granule`matrix' proteins such as cgb exist as membrane-bound isoforms [ ] , it has been suggested that the membrane bound forms of such proteins would act as nucleating receptors to recruit soluble regulated secretory proteins [ ] . indeed, recent experiments have demonstrated that the membrane associated form of carboxypeptidase e, a granule speci¢c carboxypeptidase [ ] , can function as a sorting receptor [ ] . these observations are the subject of some debate [ ] ; in particular, in light of a recent report by halban [ ] where the absence of carboxypeptidase e in a mouse mutant cpe fatafat [ ] does not result in abnormal insulin processing. however, the principle behind the observations follows a common theme: soluble regulated secretory proteins appear to interact in a homo-or heterophillic manner with membrane associated forms of regulated secretory proteins. at this stage, however, it could also be that other unrelated bone ¢de membrane proteins act as receptors for the aggregate, such as the ip receptor [ , ] identi¢ed using the same approach with immobilized, soluble cga. a variety of exogenous soluble proteins, such as renin, normally expressed in the kidney [ ] to trypsinogen, normally expressed in pancreatic exocrine cells [ ] , have been expressed by transfection in endocrine and neuroendocrine cell lines. these exogenous molecules have been found to be secreted in a regulated fashion from the transfected cells lines, suggesting that these proteins have been stored in secretory granules. from these data it was hypothesized that a putative sorting signal, a common sequence or structural feature, appears to be conserved across a variety of systems. this signal is not, however, dominant as fusion of proinsulin with the trans-membrane and cytoplasmic domain of cd resulted in targeting of proinsulin to the constitutive pathway and appearance on the cell surface [ ] . these experiments demonstrate that a diverse set of soluble molecules can be targeted to secretory granules and cast doubts on the existence of a singlè sorting receptor' because it is di¤cult to reconcile the accurate sorting of such a variety of molecules with a single receptor. gerdes and colleagues [ ] have argued that e¤cient sorting of these molecules was obtained because the exogenous molecules were sorted by co-aggregation with the endogenous regulated secretory proteins. therefore identi¢cation of a sorting sequence could only be achieved in essentially a null background of endogenous regulated secretory proteins. they have achieved this experimentally using a vaccinia virus expression system which shuts o¡ host protein synthesis, and have shown that exogenous cgb when expressed in vaccinia virus infected pc cells can be e¤ciently sorted to secretory granules but that cgb without the n-terminal disulphide loop (vcys-cgb) was not correctly sorted. furthermore, in the same set of experiments the authors demonstrated that vcys-cgb was correctly and e¤ciently sorted after conventional transfection protocols providing additional support for their hypothesis. using this powerful expression system the authors should be able to begin to identify the requirements for sorting of soluble proteins to the secretory granule. lastly, supplementary to the postulated role of receptors in sorting regulated secretory proteins or aggregates, sorting receptors may also serve the function of selecting an appropriate membrane for the nascent secretory granule. indeed, there must be a recognition mechanism between soluble granule proteins and granule membrane proteins or components to ensure that the membrane which ¢nally envelops the dense core contains all the proteins necessary for secretory granule maturation, storage and exocytosis. again, it is possible that this selection only occurs later in the pathway, in the isg, and that the proteins not required are removed from the isg instead of being left behind in the tgn. at present we have very little knowledge about the membrane composition of the nascent isg which would allow one to speculate on how and where the ¢nal selection is achieved. several years ago it was ¢rst shown that att cells, which have a regulated secretory pathway, have a distinct pathway by which they transport proteins to the plasma membrane, the constitutive secretory pathway [ ] . more recently, the vesicles involved in constitutive secretion of proteins from pc cells, which also have a regulated secretory pathway, have been identi¢ed [ ] . it was shown in pc cells, using two sulphated proteins (a hspg and sgii), as markers for constitutive and regulated pathways, respectively, that formation of csvs occurs concomitantly with formation of isgs from the tgn. these budding reactions have very similar characteristics to those described below for secretory granules and those identi¢ed using other systems which reconstitute vesicle formation from cells without a regulated pathway (see for example [ ] ). formation of csvs appears to be regulated by heterotrimeric gtp-binding proteins [ , ] , and be dependent upon arf [ , ] . although the ¢delity of sorting into the constitutive and regulated pathways appears to vary in di¡erent pc cells clones (compare [ ] and [ ] ) the principle of sorting di¡erent populations of proteins in the tgn into distinct vesicle populations remains intact in neuroendocrine cells. in contrast, no one has to date identi¢ed a constitutive secretory pathway in endocrine l-cells using a bone ¢de constitutive secretory protein. the c-peptide, derived from processing of proinsulin, has been shown to be secreted constitutively, however, this might be through a constitutive-like pathway originating from the isg [ ] . it may be that indeed that no constitutive secretory proteins in csvs are secreted directly from the tgn in l-cells, however, using immunogold labelling techniques csvs have been identi¢ed in l-cells using antibodies to the viral-spike glycoprotein ha of in£uenza virus [ ] . at the same time, experiments were done which demonstrated that corona virus virions, which are released constitutively, were found segregated from dense-cores in att cells, a neuroendocrine cell line [ ] . in retrospect, the ideal experiment would have been to study the transport of coronavirus particles, which have the advantage of being morphologically identi¢able marker for constitutive secretion, in l-cells. however, in the absence of experiments to prove whether secretory proteins are also conveyed by this pathway, the question remains an open one. transport of lysosomal enzymes from the tgn to endosomes occurs via clathrin-coated vesicles (ccvs), and is mediated by binding of the clathrincoat proteins to the mannose- -phosphate receptor (mpr, see review by ho£ack in this issue). it has been assumed that this pathway exists in all cells. clathrin coats assemble on the tgn via the ap- adaptor complex (robinson, this issue) through an interaction with the cytoplasmic domain of receptors, such as mpr, and other proteins such as furin which tra¤c to and from the plasma membrane [ ^ ]. while the distribution of lysosomal enzymes has not been studied in pc cells, recent data using both gh c cells [ ] and a l-cell line [ ] have demonstrated that some lysosomal enzymes are present in isgs. the appearance of lysosomal enzymes in the regulated secretory pathway provides additional support for the`sorting by retention' hypothesis. these authors [ , ] were able to demonstrate that lysosomal enzymes can be released from the cells by agents used to stimulate regulated secretion. some of the lysosomal enzymes (cathepsin b) were absent from the msg, while others (cathepsin l) were detected in msgs [ ] . these results suggest that in the tgn newly synthesized lysosomal enzymes are incorporated into the nascent secretory granule. this could be due to a missorting of the receptors for lysosomal enzymes, mpr, into secretory granules. however, newly synthesized lysosomal enzymes that have diminished or abolished a¤nity for mprs continue to enter isgs in equal or even greater abundance [ , ] . as an alternative explanation, there may be a decrease in the e¤ciency of sorting of lysosomal enzymes by the mprs in the tgn of cells with a regulated secretory pathway which allows them to escape from the tgn in other exit pathways, such as the regulated or constitutive secretory pathway, and be secreted or recaptured at a post-golgi location such as the endosome (see review [ ] ). a variety of experiments, including those cited above, have con¢rmed the transient appearance of lysosomal enzymes in the regulated pathway, suggesting that lysosomal enzymes are being removed from the maturing isgs. it is assumed that this is occurs via the mpr and ccvs. indeed, this may explain the transient presence of clathrin coats on the isgs. further support for this idea comes from experiments which show that the ap- is used on the isg to assemble the clathrin coat [ ] , and the mpr has been found in isgs but not msgs in isolated secretory granule fractions derived from pc cells (a.s. dittië, submitted). a second protein which is involved in recruiting ap- and therefore clathrin onto isgs is furin, an endopeptidase present in tgn membranes. furin, like mpr, is found in isgs but not msgs [ ] . these results suggest that furin, like mpr, is removed from the isg via ccvs. the destination of the ccvs, containing either furin, mpr, or both, could be any one of several possibilities, for example, the tgn, early endosomes, or plasma membrane. there is no data at present regarding the content, composition and destination of ccv budding from the isg, and indeed the ccvs themselves have not been identi¢ed. a related question concerns the constitutive-like secretion of proteins which could be considered true markers of the constitutive pathway, and those which are unwanted products from the processing of prohormones, such as c-peptide. it has been proposed that ccvs might be a vesicular carrier for these population of molecules [ ] . this raises a very intriguing set of questions: are there multiple classes of ccvs forming from the isg all with a di¡erent cargo?, would these ccvs all have a di¡erent destination? or is there a single species of ccvs forming with a mixed cargo? would this ccv deliver its cargo to one destination, for example, the endosome, from where further sorting would occur? while the mechanism for selection and transport via ccvs is clear when one assumes the cargo associates with a transmembrane receptor, it is more complex for other cargo proteins such as the c-peptide, or those proteins which follow a bulk-£ow pathway [ ] which has not been thought to be signal-mediated. how does this signal-less type of cargo enter the ccvs allowing it to be removed? if it is simply a case of exclusion, as formulated by arvan et al. in their`sorting by retention' hypothesis, one would predict it could not be % e¤cient and some c-peptide would be found in the medium after stimulation of msg. this has been demonstrated for c-peptide (see [ ] and the references therein). alternatively, it may be that multiple vesicular carriers bud from the isg with di¡erent cargo. depending on the cargo, distinct mechanisms could be used for selection to increase the e¤ciency of removal of nonsecretory granule proteins from the maturing isgs. while the mechanisms involved in the formation of both constitutive and regulated secretory vesicles from the tgn is largely unknown there have been recent reports which suggest that both these budding reactions maybe unique in that they function independently of known coat proteins. morphological data based on high voltage electron microscopy suggests that the formation of constitutive secretory vesicles from the tgn involves a`lace-like' coat [ ] . the components of this coat have yet to be identi¢ed but may be associated with the p complex identi¢ed by howell and colleagues [ ] and include p [ ] . conventional electron microscopy and immunogold labelling has revealed that patches of clathrin are present on the tgn in the vicinity of forming isgs and are also found on isgs [ , ] . however, there is no biochemical data available to date which supports the role of clathrin in secretory granule formation. recent results concerning the formation of secretory granules have been obtained primarily from two in vitro systems which reconstitute budding from tgn membranes; one using a post-nuclear supernatant derived from pc cells [ ] and the second semi-intact gh c cells [ ] . while in the former assay it is possible to identify two classes of vesicles budding from the tgn, in the latter this has not been possible. however, because there is no data which so far suggests the requirements for formation of an isg are di¡erent from that for a csv, they will be considered as congruent assays and the data ob-tained from both, and other assays monitoring constitutive secretory vesicle formation will be compared. the ¢rst observations made using the in vitro budding assays described above, both of which are atp and cytosol dependent, concerned the requirement for gtp. the formation of secretory vesicles from the tgn requires gtp and is inhibited by non-hydrolysable gtp analogs [ , ] . in the pc cell assay, the same e¡ect was observed with [alf ] À and several other lines of evidence demonstrated that heterotrimeric gtp-binding proteins are involved in the regulation of post-golgi vesicle formation (see [ ] for review). the precise function of gtp is not yet clear as there maybe several di¡erent gtp-binding proteins involved in vesicle formation from the golgi, including arf (see below). an intriguing related observation concerns the role of an as yet unidenti¢ed cytoplasmic phosphoprotein as a modulator of the heterotrimeric g protein(s) that regulate vesicle formation from the tgn [ ] . a similar requirement has been seen in the semi-intact gh c in vitro assay [ ] in experiments using inhibitors of tyrosine kinases and phosphatases. this observation is supported by several other reports from cell-free budding assays which indicate that phosphorylation regulates vesicle formation [ , , ] . a role for tyrosine-phosphorylation in secretory granule biogenesis and/or function has also been suggested by two recent reports that have iden-ti¢ed two highly homologous proteins in the membrane of secretory granules which appear to be nonfunctional protein-tyrosine phosphatases, called phogrin and the ica autoantigen [ , ] . most of the well characterized transport steps in the cell (for example from the endoplasmic reticulum to the cis-golgi, intra-golgi, plasma membrane to endosome) have been shown to be mediated by vesicles which form by coat-mediated budding reactions from donor compartments. in contrast no known coat, or coat protein, has been demonstrated by morphological or biochemical techniques to be involved in secretory granule formation. as mentioned above, if a coat were required, the most likely coat protein candidate would be clathrin. formation of clathrin-coats on the tgn requires ap- (see above) and the monomeric gtpase adp-ribosylation factor, or arf [ , ] and these components might therefore be thought to function in the formation of secretory granules. however, to date there is no evidence which implicates clathrin in the formation of either constitutive secretory vesicles or secretory granules from the tgn. on the other hand, reports from the two cell-free assays described above have demonstrated that arf is required for the budding of secretory granules and post-tgn vesicles. myristoylated arf peptide stimulated both the formation of secretory granules and constitutive secretory vesicles from isolated tgn membranes [ ] , while in a semi-intact cell system [ ] arf peptide was su¤cient for stimulation of budding of gh-and prl-containing vesicles. importantly, although arf is known to be involved in the recruitment of cop i coats to membranes (see article by emr in this issue), barr and huttner [ ] could demonstrate that depletion of cop i from cytosol did not inhibit post-golgi vesicle formation. several reports have recently demonstrated that arf may function by activating phospholipase d ([ ^ ] and see [ ] for review). interestingly, addition of phospholipase d (pld) has been shown to stimulate budding from the tgn in the semi-intact assay using gh c cells [ ] . the stimulatory e¡ect of pld might be attributed to its ability to catalyse the hydrolysis of pc to pa and alter the lipid composition of the membrane. this local increase in charged lipids has been proposed to function to stabilize ap- association with endosomal membranes [ ] . alternatively, the production of pa may have several consequences including the production of pip (phosphatidylinositol , -bisphosphate) in the membrane [ ] . pip has been implicated in a variety of membrane tra¤c events [ ] and has been shown to regulate the nucleotide bound state of arf [ , ] and to stimulate pld activity [ ] leading to a feedback cycle of interactions between arf, pld, pa and pip . it has been proposed [ ] that the e¡ect of arf and pld on budding might alter-natively be to recruit ap- to regions of the membrane which are high in pa. however, from more recent experiments we now know that pld does not play a role in ap- recruitment [ ] to tgn membranes, it would thus seem most likely that arf and pld are functioning to promote budding from the tgn via a signalling cascade. consistent with this scenario [ ] shields and colleagues had previously shown that pkc can stimulate budding, and suggest that pa could function as a second messenger to trigger intracellular signalling events such as activation of pkc. further exciting results have been obtained with regard to cytoplasmic proteins required for vesicle formation from the tgn. a phosphatidylinositol transfer protein (pitp) has been implicated in the formation of both regulated and constitutive secretory vesicles [ ] . the mammalian pitp activity can be substituted for by sec p, a yeast protein from saccharomyces cerevisiae (which is, however, not homologous to the mammalian pitp) that is required for normal golgi function and shares with pitp the ability to exchange phosphatidylinositol for phosphatidylcholine ( [ ] ; see also contribution in this issue by bankaitis). additional evidence recently published related to the function of sec p, obtained from the study of sac p, a sec -bypass suppressor [ ] would suggest that pitp can also function to cause a local production of diacylglycerol which may be involved in coat recruitment in much the same way the pa has been postulated to be involved in arf and ap- recruitment (see review [ ] ). ptdins is also the substrate for the yeast kinase vps which has pi -kinase activity [ ] . the synthesis of pdtins( )p by vps has been implicated in the transport from the golgi to the vacuole in yeast and it has been suggested that vps activity is required for the formation of a speci¢c vesicle type from the golgi [ ] . a wortmannin-sensitive pi kinase activity is also required for the sorting of lysosomal enzymes from the golgi to the endosome in mammalian cells (see [ ] for review). in addition, a recent report has shown that a pi -kinase activity is required for formation of constitutive secretory vesicles from isolated rat liver golgi membranes [ ] . interestingly, this pi -kinase regulatory subunit associates with the cytoplasmic domain of a tgn-resident protein tgn in a complex with a small molecular weight gtpase, previously identi¢ed as rab [ ] . the authors hypothesis is that the activity of the pi -kinase is stimulated by the activation of the gtpase [ ] . so far there has been no report implicating a pi -kinase in the budding of regulated secretory granules. in fact, incubation of pc cells with wortmannin had no e¡ect on either constitutive or regulated secretory vesicle formation, transport or exocytosis of secretory proteins (s. urbë and s.a. tooze, unpublished observations). it maybe that the activation of the pi -kinase is dependent upon the presence of particular receptors in the forming bud, for example, the presence of tgn . data from experiments performed to examine the composition of the nascent isg demonstrates that there is no tgn present in isgs [ ] . with regard to the role of ptdins-metabolites in vesicle-formation, it is of interest that a kda protein, oculocerebrorenal syndrome of lowe (ocrl) [ ] , belonging to the inositol (ins) -phosphatase family has recently been localized to the golgi complex [ ] . ocrl dephosphorylates ins-( , , , )p , ins( , , )p and most importantly pip in vitro and seems to act preferentially as a lipidphosphatase in vivo, and could therefore regulate the levels of pip in the golgi-membrane [ ] . another inositol- -phosphatase, synaptojanin, has been implicated in the recycling of synaptic vesicles, a process that involves the formation of clathrincoated vesicles [ ] . synaptojanin interacts through amphiphysin [ ] with the small gtpase dynamin which appears to play an important role in the budding of clathrin-coated vesicles from the plasmamembrane (for reviews see [ ^ ]). although the function of dynamin has predominantly, but not exclusively [ ] , been linked to the function of clathrin, it has been speculated that there might be a dynamin for every budding event in the cell [ ] . a protein with immunoreactivity to antibodies raised against conserved regions of the dynamin-family has been localized to the golgi complex in ¢broblasts and melanocytes [ ] . as gtp-hydrolysis is required for dynamin function it is possible that the inhibitory e¡ect of gtpqs on the budding of secre-tory vesicles from the tgn re£ects not only an involvement of heterotrimeric g proteins and small gtpase, but also of dynamin. research on secretory granule biogenesis has revolved to a large extent around the granule content selection mechanisms. the only coat-proteins identi-¢ed so far on the secretory granule, which are present only on isgs are clathrin and adaptor-complex ap- , and they function to mediate vesicle budding from the isg rather than formation of the isg from the tgn [ ] . it has been suggested that budding of nascent secretory granules does not require a coat protein, but that the aggregated core of the granule is able to`drive' vesicle formation in a way analogous to the budding of a viral particle. this hypothesis predicts that there have to be speci¢c interactions between the granule core and the granule membrane that mirror the binding of viral nucleocapsid proteins to the membrane-bound envelope proteins. in this way the secretory granule core could mould its own membrane, driven only by a thermodynamically favourable binding of granule membrane proteins with the aggregating content. this hypothesis becomes less attractive if the nascent isg contains largely uncondensed, unaggregated secretory proteins which, along with the membrane proteins, are not sorted prior to budding (see the sorting by retention model above). work on this sorting mechanism is in progress and new results will no doubt be forthcoming to clarify the inconsistencies present. to address these questions, it would be most interesting to have an in vitro system for formation of isgs using the cells, or cell lines (i.e. l-cells), from which the sorting by retention hypothesis has been developed. the development of such a system would allow a direct test for common structural and regulatory molecules involved in formation of secretory granules in neuroendocrine and endocrine cells. network (erb-fmrxct ) for stimulating discussions on which a large part of this review is founded. in particular, i thank my network colleagues, hans-hermann gerdes and philippe halban for copies of manuscripts before publication. finally, i wish to thank peter arvan for taking the time to read this review and for his valuable comments. the granin (chromogranin/secretogranin) family what the granins tell us about the formation of secretory granules in neuroendocrine cells regulated secretion. helper proteins for neuroendocrine secretion chromogranin b (secretogranin i) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor the primary structure of human secretogranin ii, a widespread tyrosine-sulfated secretory granule protein that exhibits low ph-and calcium-induced aggregation milieu-induced, selective aggregation of regulated secretory proteins in the trans-golgi network calcium-and phdependent aggregation and membrane association of the precursor of the prohormone convertase pc secretory granule content proteins and the luminal domains of granule membrane proteins aggregate in vitro at mildly acidic ph conversion of proinsulin to insulin occurs coordinately with acidi¢cation of maturing secretory vesicles clathrin-coated vesicular transport of secretory proteins during the formation of acth-containing secretory granules in att cells chloroquine diverts acth from a regulated to a constitutive secretory pathway in att- cells the role of a low ph intracellular compartment in the processing, storage, and secretion of atch and endorphin transport via the regulated secretory pathway in semi-intact pc cells: role of intra-cisternal calcium and ph in the transport and sorting of secretogranin ii direct measurement of trans-golgi ph in living cells and regulation by second messengers the insulin factory: a tour of the plant surroundings and a visit to the assembly line sorting and processing of secretory proteins prohormone processing in the trans-golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells proteolytic processing of pro-acth/endorphin begins in the golgi complex of pituitary corticotropes and att- cells proteolytic maturation of insulin is a post-golgi event which occurs in acidifying clathrin-coated secretory vesicles an antibody speci¢c for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in att cells before its cleavage ph-dependent processing of secretogranin ii by the endopeptidase pc in isolated immature secretory granules formation of the insulin-containing secretory granule core occurs within immature beta-granules ph-independent and -dependent cleavage of proinsulin in the same secretory vesicle distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic l-cells sorting within the regulated secretory pathway occurs in the trans-golgi network cell type-speci¢c sorting of neuropeptides: a mechanism to modulate peptide composition of large dense-core vesicles constitutive and regulated secretion of proteins di¡er-ential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic l-cells protein secretion : puzzling receptors pathways of protein secretion in eukaryotes identi¢cation of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway the disul¢de bond in chromogranin b, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans-golgi network chromogranin b (secretogranin i), a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in pc cells carboxypeptidase e is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in cpe fat mice proinsulin targeting to the regulated pathway is not impaired in carboxypeptidase e-de¢cient cpe fat /cpe fat mice hyperinsulinaemia in obese fat/fat mice associated with a carboxypeptidase e mutation which reduces enzyme activity ph-dependent interaction of an intraluminal loop of inositol , , -triphosphate receptor with chromogranin a ph-dependent interaction of chromogranin a with integral membrane proteins of secretory vesicle including -kda protein reactive to inositol , , -triphosphate receptor antibody a targeting sequence for dense secretory granules resides in the active renin protein moiety of human preprorenin the exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer a chimeric proinsulin-cd protein expressed in att cells is directed to the cell surface via the constitutive pathway two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells requirement for gtp hydrolysis in the formation of secretory vesicles the in vitro generation of post-golgi vesicles carrying viral envelope glycoproteins requires an arflike gtp-binding protein and a protein kinase c associated with the golgi apparatus multiple trimeric g-proteins on the trans-golgi network exert stimulatory and inhibitory e¡ects on secretory vesicle formation an elevation of cytosolic protein phosphorylation modulates trimeric g-protein regulation of secretory vesicle formation from the trans-golgi network a role for adp-ribosylation factor , but not cop i, in secretory vesicle biogenesis from the trans-golgi network adp-ribosylation factor- stimulates formation of nascent secretory vesicles from the trans-golgi network of endocrine cells intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells protein discharge from immature secretory granules displays both regulated and constitutive characteristics the trans-most cisternae of the golgi complex: a compartment for sorting of secretory and plasma membrane proteins sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells intracellular tra¤cking of furin is modulated by the phosphorylation state of a casein kinase ii site in its cytoplasmic tail two independent targeting signals in the cytoplasmic domain determine trans-golgi network localization and endosomal tra¤cking of the proprotein convertase furin an acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-golgi network localization and internalization from the cell surface cysteine proteinases in gh c cells, a rat pituitary tumor cell line, are secreted by the constitutive and regulated secretory pathways mannose -phosphate receptors in sorting and transport of lysosomal enzymes the ap- adaptor complex binds to immature secretory granules from pc cells, and is regulated by adp-ribosylation factor interaction of furin in immature secretory granules from neuroendocrine cells with the ap- adaptor complex is modulated by casein kinase ii phosphorylation protein targeting via the`constitutive-like' secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment biosynthetic protein transport and sorting by endoplasmic reticulum hvem tomography of the trans-golgi network: structural insights and identi¢cation of a lace-like vesicle coat a cytosolic complex of p and rab associates with tgn / and is involved in budding of exocytic vesicles from the trans-golgi network distinct coated vesicles labeled for p bud from trans-golgi network membranes perrelet, clathrin-immunoreactive sites in the golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells cell-free protein sorting to the regulated and constitutive secretory pathways trimeric g proteins and vesicle formation formation of nascent secretory vesicles from the trans-golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors regulated formation of golgi secretory vesicles containing alzheimer beta-amyloid precursor protein metabolism of alzheimer beta-amyloid precursor protein: regulation by protein kinase a in intact cells and in a cell-free system ica , an autoantigen of type i diabetes, is an intrinsic membrane protein of neurosecretory granules molecular cloning of phogrin, a protein-tyrosine phosphatase homologue localized to insulin secretory granule membranes the binding of ap- clathrin adaptor particles to golgi membranes requires adp-ribosylation factor, a small gtp-binding protein mannose -phosphate receptors and adp-ribosylation factors cooperate for high a¤nity interaction of the ap- golgi assembly proteins with membranes adp-ribosylation factor, a small gtp-dependent regulatory protein, stimulates phospholipase d activity phospholipase d: a downstream e¡ector of arf in granulocytes human adp-ribosylation factor-activated phosphatidylcholine-speci¢c phospholipase d de¢nes a new and highly conserved gene family arf proteins: the membrane tra¤c police? phospholipase d stimulates release of nascent secretory vesicles from the trans-golgi network the role of adpribosylation factor and phospholipase d in adaptor recruitment type i phosphatidylinositol -phosphate -kinase isoforms are speci¢cally stimulated by phosphatidic acid phosphoinositides as regulators in membrane tra¤c e¡ects of acid phospholipids on nucleotide exchange properties of adp-ribosylation factor . evidence for speci¢c interaction with phosphatidylinositol , -bisphosphate gtp hydrolysis by adp-ribosylation factor is dependent on both an adp-ribosylation factor gtpase-activating protein and acid phospholipids cantley, novel function of phosphatidylinositol , -bisphosphate as a cofactor for brain membrane phospholipase d a role for phosphatidylinositol transfer protein in secretory vesicle formation an essential role for a phospholipid transfer protein in yeast golgi function essential role for diacylglycerol in protein transport from the yeast golgi complex greasing the golgi budding machine vps p required for yeast vacuolar protein sorting is a multiple speci¢city kinase that exhibits both protein kinase and phosphatidylinositol-speci¢c pi -kinase activities receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and gtp-binding proteins phosphoinositide -kinases and membrane tra¤c phospatidylinositol -kinase is required for the formation of constitutive transport vesicles from the tgn the lowe's oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate- -phosphatase the oculocerebrorenal syndrome gene product is a -kd protein localized to the golgi complex the protein de¢cient in lowe syndrome is a phosphatidylinositol- , -bisphosphate -phosphatase a role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals the role of clathrin, adaptors and dynamin in endocytosis dynamin and receptor-mediated endocytosis molecular mechanisms in synaptic vesicle endocytosis and recycling rapid endocytosis coupled to exocytosis in adrenal chro-ma¤n cells involves ca +, gtp, and dynamin but not clathrin ringing necks with dynamin association of a dynamin-like protein with the golgi apparatus in mammalian cells the ap- adaptor complex binds to immature secretory granules from pc cells, and is regulated by adp-ribosylation factor essential role of the disul¢de-bonded loop of chromogranin b for sorting to secretory granules is revealed by expression of a deletion mutant in the absence of endogenous granin synthesis key: cord- -p sgaypq authors: west, christopher m. title: current ideas on the significance of protein glycosylation date: journal: mol cell biochem doi: . /bf sha: doc_id: cord_uid: p sgaypq carbohydrate has been removed from a number of glycoproteins without major effect on the structure or enzyme activity of the protein. thus carbohydrate has been suggested to underly a non-primary function for proteins, such as in relatively non-specific interactions with other carbohydrates or macromolecules, stabilization of protein conformation, or protection from proteolysis. this non-specific concept is consistent with both the general similarity in carbohydrate structure on very diverse glycoproteins and the frequent structural microheterogeneity of carbohydrate chains at given sites. the concept is supported in a general sense by the viability of cells whose glycosylation processes have been globally disrupted by mutation or pharmacological inhibitors. in contrast to the above observations, other studies have revealed the existence of specific, selective receptors for discrete oligosaccharide structures on glycoproteins which seem to be important for compartmentalization of the glycoprotein, or the positioning of cells on which the glycoprotein is concentrated. sometimes multivalency in the carbohydrate-receptor interaction is crucial. there are additional possible roles for carbohydrate in the transduction of information upon binding to a receptor. the possibility of specific roles for carbohydrate is supported by the existence of numerous unique carbohydrate structures, many of which have been detected as glycoantigens by monoclonal antibodies, with unique distributions in developing and differentiated cells. this article attempts to summarize and rationalize the contradictory results. it appears that in general carbohydrate does in fact underlie only roles secondary to a protein's primary function. these secondary roles are simple non-specific ones of protection and stabilization, but often also satisfy the more sophisticated needs of spatial position control and compartmentalization in multicellular eukaryotic organisms. it is suggested that there are advantages, evolutionarily speaking, for the shared use of carbohydrate for non-specific roles and for specific roles primarily as luxury functions to be executed during the processes of cell differentiation and morphogenesis. new information about the significance of protein glycosylation is rapidly accumulating. this can be attributed to advancements in methods to detect and isolate new structures, to determine structure, and to modify carbohydrate structures experimentally. nevertheless, the field continues to experience profound unrest. many studies have failed to un-cover significant cellular or molecular consequences for dramatic changes in carbohydrate structures. other studies have pointed to isolated specific roles but no sweeping generalizations of function have been possible. interpreters of the former studies would attribute apparent specific effects to secondary consequences of changes in carbohydrate on protein structure. interpreters of the latter studies would argue that the former studies simply failed to ask the right questions of function. it is clear that there is much to be done to extend our knowledge for this particular type of posttranslational modification. the purpose of this article is to outline the scope of investigations presently being carried out in this subject area and to generalize the possible significance of these new findings. in particular, this article will attempt to compare and contrast the two prevailing views in the field. as intimated above, one view is that glycosylation plays a relatively non-specific role in maintaining and preserving polypeptide function. the alternate view is that carbohydrate structures participate in numerous specific interactions with discrete protein receptors, and that these interactions lead to predictable modifications in the localization or activity of the glycoprotein. these opposing views will be simply referred to as the non-specific and specific models. the importance of this issue is dramatized by the fact that nearly all secretory, extracellular matrix, cell surface and lysosomal proteins are glycosylated. there are many published comprehensive reviews on aspects of this subject ( - , , , , , , , , ) and the reader is referred to these for more detailed summaries and documentation. since this article will in part serve as an introduction and a background to the others in this volume, an overview of general features of the glycosylation process has been provided to precede the summary of existing and new ideas in the field which follows. oligosaccharides linked to asparagine occur in cellular slime molds, yeast, higher plants, insects, and humans, and several essential features of these structures are highly conserved throughout this phylogenetic range ( ) . typically though not invariably, a glc man glcnac structure is synthesized on a polyisoprenoid derivative (dolichol-p-p) and transferred en bloc to asparagine residues on polypeptide acceptors forming an n-glycosidic linkage (hence n-linked). the three glcs are usually trimmed by a tandem pair of a-glucosidases to yield a high mannose n-linked structure. the highmannose structure is variably modified, beginning with removal of mannoses in a typical sequence by a tandem pair of o~-mannosidases. one pathway retains most mannoses but permits additional substitutions of e.g., sulfate and/or phosphate. a second pathway trims down to mansglcnac and makes limited additions such as glcnac, and sometimes gal and core-linked fuc. these are referred to as hybrid structures. the third pathway continues the second pathway to build complex structures usually terminated by sialic acid. there can be two, three or four chains emanating from the branching mannose (and secondary branching mans) and these structures are accordingly referred to as biantennary, triantennary or tetraantennary complex nlinked oligosaccharides. the sialic acids, which appear not to precede echinoderms in their evolutionary appearance, comprise a diverse family of sugars ( ) . numerous additional substituents including sulfate, phosphate, methyl groups and others have been documented ( , , ) . each step in the glycosylation pathway is catalyzed enzymatically; there is usually a unique enzyme for each linkage position formed as monosaccharides are accreted or deleted ( ) . n-linked glycosylation is not template driven. only the first step might be considered so, inasmuch there is a necessary but not sufficient requirement of an xasn-x-ser/thr-x sequence for the oligosaccharide acceptor. protein structure or corfformation has also been shown to influence whether a site becomes glycosylated as well as whether it becomes high mannose, hybrid, or complex ( , ) or whether it receives a phosphodiester-linked glcnac-po ( ) or galnac-so ( ). a relationship between position in the polypeptide chain (distance from n-to c-terminus) and whether an oligosaccharide becomes high mannose or complex has been observed ( ) . finally these relations are not absolutely determinate, for spontaneous heterogeneity of carbohydrate structures at particular positions has been documented ( , , ) . changes in structure also result from extracellular influences (see below). the other major structural class of oligosaccharides is o-linked on ser on thr ( , ) . a largely distinct set of enzymes catalyzes the synthesis of these structures. these oligosaccharides are built by monomer addition from sugar-nucleotide donors directly on the polypeptide rather than on a lipid precursor. trimming does not appear to play a major role. gal and galnac are typical proximal sugars and man is rarely found. o-linked structures are found both in branched and straight chain forms. o-linked structures often resemble the oligosaccharides of glycolipids ( ) , and in fact these may be built from shared enzymes. an experimental consideration of the role of these oligosaccharides on protein must inevitably involve an examination of homologous glycolipids. although the n-linked and o-linked structures discussed above account for - % of incorporation of labelled sugar precursors into cellular glycoproteins, numerous additional small classes of oligosaccharides are also found. for example, galglc is found on hydroxylysine and hydroxyproline of collagens. glcnac-pon-ser is found on a lysosomal enzyme in d. discoideum ( ) and glcnac-asn is found on certain glycoproteins of what is probably the nuclear envelope ( ) . archeobacterial proteins are also glycosylated. a major class of these glycosylations shares some homology with n-glycosylation and may be evolutionarily related ( , , ) . a polyisoprenoid lipid carrier is utilized prior to en bloc transfer. the ancient character of the mammalian n-linked glycan suggests important functional attributes for his posttranslational modification. several physical methods have been applied to ascertain the conformation of oligosaccharides on proteins ( , ) . studies in model systems have implied that there is a preferred conformation at least for the core sugars of n-linked oligosaccharide chains. this conformation is stabilized by hydrogen bonding with the polypeptide chain, which orients the structure, and limited intrachain hydrogenbonding. the existence of preferred conformations implies that shape may be important for specific interactions of protein-linked carbohydrate with receptors. n-linked glycosylation appears to occur only in the rough endoplasmic reticulum (rer) and hence only to proteins which possess a signal sequence ( ) . the process is co-or post-translational ( ) . mannose trimming begins in the rer but can be concluded in golgi. modification of high-mannose forms or conversion to hybrid or complex forms occurs in the golgi. nearly all glycosylation is believed to be restricted to these two compartments. certain steps in glycosylation have, however, been postulated to occur in the ser, secretory vesicles, nuclear envelope and cell surface (e.g., , , ) . of course, deglycosylation occurs in lysosomes as a part of protein turnover ( ) . o-linked glycosylation appears to be initiated and concluded in the golgi ( , , but see ). as for n-linked glycosylation, there has been some resolution of processes between the cis, medial and trans elements of the golgi apparatus. nearly all proteins which pass through the rer and golgi become glycosylated in some form. one exception is the secretory protein serum albumin ( ) , which is synthesized in hepatocytes. there is no evidence for a glycosylated precursor. it should be recognized that at least some glycoproteins, e.g., cell surface transferrin receptor, can probably be reexposed to the golgi compartment subsequent to primary passage through this organelle ( ) . this provides new opportunities for glycosylation subsequent to initial synthesis. occasional reports have appeared in the literature regarding the presence of glycoproteins in cytoplasmic compartments topologically discontinuous with the lumen of the rer and golgi. histones and other nucleoplasmic proteins ( , , ) , a ribosomal protein ( ) and certain mitochondrial proteins ( , ) have been suggested to be glycoproteins. these findings contradict the dogma that glycosylation is strictly an rer and golgi dependent event unless it is postulated that modified proteins subsequently translocate across the membrane back to the cytoplasmic space. further work remains to be performed to confirm the existence of oligosaccharides on these proteins and to characterize their structure as a means for understanding their enzymatic basis. it must be appreciated that the description of the glycosylation pathway as we now understand it suggests many interesting kinds of regulatory mechanisms. for example, each step of the pathway can be influenced by enzyme synthesis and turnover, acceptor accessibility, as well as the supply of donor sugar, cofactors such as lipids, divalent cations, etc. the question of accessibility is particularly interesting because sequentially acting enzymes tend not to be mixed but are distributed vectorially through the rer and the multiple compartments of the golgi. movement of polypeptides through these compartments appears to be mediated by atp-dependent dissociative movement of vesicles ( ) . thus glycosylation may conceivably be regulated by the pathway of movement of the acceptor protein. there is also an indication that some glycosyltransferases form supramolecular complexes. thus, it is possible that certain carbohydrate structures are dictated not only by vesicle movement, but also by which enzyme cluster initially captures the acceptor protein ( ) . ideas about the role of protein glycosylation may be divided into two general groups: ) those which imply specificity associated with particular carbohydrate structures, and ) those which stress the general similarities of oligosaccharide structures, and the presence of microheterogeneity in the structure of particular oligosaccharides, in suggesting that combined carbohydrate bulk creates a common microenvironment for resident polypeptides much as the lipid bilayer provides an environment for integral membrane proteins. most effort has been devoted to the former category of ideas because of the greater ease of formulating hypotheses of this type. a non-specific role for oligosaccharides that is dependent on their generalized chemical properties has been suggested in several contexts. it was appreciated early on that carbohydrate afforded a stabilizing effect on protein solubility and against heat denaturation (see, e.g. , and ) . it was also realized that oligosaccharides afford resistance to proteolytic degradation ( ) , presumably by a mechanism of steric hindrance, and these ideas have been reinforced in numerous studies up to the present (e.g., , , ) . there are in addition several instances where carbohydrate is known to interfere sterically with protein-protein interaction not involving proteolysis ( , ) . predating this understanding, it was known that carbohydrate is a major constituent of the extracellular matrix in the form of polysaccharides known as glycosaminoglycans (gags) ( ). in the past years, it has been discovered that most gags are covalently attached to polypeptides, resulting in the formation of glycoproteins which, due to the specialized nature of the attached carbohydrate, are known as proteoglycans. gags are anionic polymers which hydrate to form enlarged domains which can interact with one another, as well as with polycationic polymers such as collagen and with other glycoproteins ( ) . a three dimensional space is thus defined by polysaccharide, protein, and the bound water which provides an environment for cells. convection and diffusion are modified, and fluctuations in ionic strength and ph are minimized. these structures depend on the polyanionic nature of the carbohydrate, and the general chemistry rather than the specific monosaccharide identities and sequences may be dominant in governing the properties of the system. carbohydrate oligomers are also known to selfassociate in the formation of mucous gels and in yeast and plant cell walls ( ) ( ) ( ) ) . in the former, the anionic nature of the carbohydrate appears to be important although in the algal and yeast gels the interactions are non-coulombic. the collective oligosaccharide density on cell surface proteins is sufficiently great so as to invite comparison with the carbohydrate density of the matrix space. this observation has led to the suggestion that cell surface carbohydrate might also establish a convection and diffusionqimited region around the cell in an area thick enough to be named, in certain cell types, the glycocalyx or fuzz. the polyanionic nature of the carbohydrate found on some cell surfaces probably results in marked cation and ph differences in this region relative to the surrounding medium ( ). it has recently been proposed that the heterogeneity of carbohydrate structure found on proteins of this region might result in minor differences in protein function, leading to a broader range in the ionic strength and ph values optimal for protein function ( , ) . as such, protein oligosaccharides might subserve a homeostatic process used by cells to maintain their viability in potentially changing milieus. it has also been suggested that a layer of non-immunogenic protein-associated carbohydrate may play an immuno-protective role for underlying cell surface antigens, especially on neoplastic cells ( ) . plasma membrane carbohydrate, owing to intercarbohydrate associations, might also influence the lateral diffusion of certain glycoproteins. inter-molecular association at the cell surface has been suggested to be so extensive in an archeobacterial system that it can influence the shape of the included cell ( ) . an extension of intermolecular association to contact between two cells in close mutual proximity leads to a possible mechanism for cell adhesion. interaction of oligosaccharides might be due to an apposition of the hydrophobic faces of two monosaccharides such as mannose ( ) . implicit in this model is the idea of a large number of weak interactions equalling in strength a smaller number of strong interactions. this mechanism is feasible in polymeric systems with repreating similar subunits. cell surface oligosaccharides could simulate a polymer inasmuch as they are coanchored in the same membrane. the interactions involved in these phenomena may be relatively nonspecific. the notion of a 'non-specific' role for carbohydrate has received perhaps its strongest support from studies on cells whose glycosylation processes have been globally altered by mutation or drugs. the most dramatic studies have involved mutant cells selected on the basis of resistance to the toxic effects of certain lectins. many mutations leading to loss of lectin recognition have been identified in cho and other cells ( ) . some have been characterized and in fact most steps in the pathway to complex, n-linked glycan formation are mutable. the striking finding is that mutant cells are largely viable in culture. most mutant cho cells remain adherent under appropriate conditions, proliferate, and are tumorigenic. mutants defective in early steps leading to the formation of high-mannose glycans are also viable ( ) . this result was not, however, found in a yeast mutant ( ) . selected activities of cells have also been investigated in some mutant strains. for example, the half-life of cell surface proteins was found not to be dramatically affected ( ) . similar results were found following treatment of cells with sialidase ( ) . a considerable literature details the consequences of carbohydrate modification on the secretory process. though several examples of apparently specific effects will be enumerated in a later section, the number of cases where little or no effect is found is noteworthy ( , , , ) . in some cases detailed kinetic analysis has found only small effects on secretory rates ( ) . similar results have been found regarding accxumulation of receptors in the plasma membrane and in their function at that site ( , , , ) . genetic modification of the gene for the ldl receptor has deleted a sequence required for a cluster of o-linked glycans and even for this poorlystudied glycan type no consequence on function could be detected ( ) . mutants in the enzymatic pathway of o-glycosylation still place the ldl receptor, albeit in a less stable form, on the cell surface ( ) . these negative results have suggested that carbohydrate cannot underlie essential housekeeping functions in cells. this implies a non-specificity of function in the sense that obvious functions cannot specifically be attributed to the carbohydrate when it is present. this represents a shift in meaning from the above definition of nonspecificity inasmuch as specific receptors may be involved in functions non-essential in the tissue culture environment. thus it has alternatively been suggested that carbohydrate may be more important for particular differentiated functions, which are not expressed in cell culture systems such as the cho cell line. there are but few investigations into this possibility. for example, in the cellular slime mold d. discoideum, mutations have eliminated two developmentally regulated glycoantigens ( , ) . nevertheless, the cells develop reasonably normally to produce fruiting bodies and viable spores. the result may imply that carbohydrate is either nonspecific or subserves subtle modulatory roles or that their roles are redundant with alternate (or back-up) mechanisms. alternatively, the value of these carbohydrate structures may only become evident under the competitive conditions of natural survival outside of the laboratory. although the above ideas have been grouped together under 'non-specific mechanisms', this is not meant to imply that carbohydrate structure per se is completely irrelevant. 'non-specific' should be interpreted relative to the types to be discussed below, were specific, saturable recognition mechanisms related in principle to enzyme-substrate or hormone-receptor interactions are apperently involved. in any case, the non-specific mechanisms tend to rely on the bulk chemical properties of the participating oligo(poly)saccharides. this phrase implies interactions between receptors and ligands (usually the oligosaccharide) of saturability and high selectivity and avidity. selectivity can be achieved by high affinity monovalent interactions or multiple weaker interactions. selective interaction is often equated with molecular or, on a more complex level, cellular recognition. the phrase also implies that a specific function can be attributed to the carbohydrate structure. perhaps the strongest indication for specific roles are the variety of structures which have been detected on protein carbohydrate. the following sections outline how several characterized structures are thought to function in specialized processes. in some cases, a receptor protein capable of recognizing a given structure is known. in eylar ( ) presented the idea that proteil' glycans constitute a 'chemical passport' signalling export of a protein to the cell surface or for secretion. though this idea has since been supplanted by the 'signal hypothesis' of blobel and sabbatini ( ) , it has served as a precursor to more refined ideas of chemical signals leading to the accumulation of glycoproteins in specific compartments ( ) . for example, a mannose- -phosphate moiety has been suggested to be responsible for associating proteins with receptors which in turn lead to protein accumulation in lysosomes ( ) . the key to this understanding was the finding of mutant cells which appeared to secrete constitutively certain lysosomal enzymes that were distinct in their oligosaccharide structures: namely that normal enzymes possessed multiple phosphate residues - inked to non-peripheral mans in high-mannose structures, and that mutant enzymes lacked these phosphate moieties. this suggested that there was a man- -po receptor in membranes of the golgi, and plasma membrane (since lysosomal enzymes can also be absorbed from the medium and translocated to lysosomes), which was responsible for binding the man- -po recognition marker on certain glycoproteins. two such receptors, one of kd and the other of kd, have been discovered ( , ) . these are located in the golgi and/or on the cell surface in concordance with their proposed functions. from studies on receptor movement and the effects of inhibitors there is fairly good evidence that the man- -po moiety, together with one or both of these receptors, plays a recognition role in the intracellular routing. however, not all lysosomal enzymes seem to be involved because some copies of the altered enzymes still accumulate in mutant cells ( ) , and some lysosomal enzymes are not affected. it has been suggested that there is lysosomal heterogeneity with only one class of lysosome employing this recognition marker. it is nevertheless apparent that for some lysosomal enzymes in some cell types the man- -po receptor does not function alone, even as an intermediate signal, for lysosomal routing. there must be other recognition signals on the enzyme polypeptide which lead to its potential lysosomal accumulation. it is interesting to note that the distinctive carbohydrate structure plays a compartmentalization role separate from the enzyme's function in catalytic degradation. furthermore, carbohydrate is employed to perform a common role shared by multiple proteins. a posttranslational modification such as glycosylation is well-suited to the demands of a mechanism for compartmentalization. the rer, golgi, smooth endoplasmic reticulum, and nuclear envelope are also intracellular destinations for proteins which traverse the rer cotranslationally. these compartments contain numerous characteristic and uniquely distributed glycoproteins, but there is not enough information to evaluate the possibility that carbohydrate structure is involved in routing or maintaining the positions of the glycoproteins. several cases of rer-specific glycoproteins have been considered. cells infected with rotavirus synthesize, under control of the virus, several proteins including vp , an integral membrane glycoprotein whose distribution is restricted to the rer ( ). this protein together with the membrane of the rer form a temporary coat for the virus. in a genetically modified form of vp , a hydrophobic region, which presumably serves as a transmembrane anchor defining the proteins as an integral membrane glycoprotein, was deleted. the truncated gene encoding vp was introduced to cells by transfection. truncated vp was synthesized, but was then secreted despite this fact that glycosylation at its single site toward the c-terminus retained its high-mannose character. these results show that the key factor which retained the glycoprotein in the rer was the transmembrane sequence and was not related to the glycosylation site. the results would also imply that glycosylation is not essential to the process of secretion. several nuclear envelope glycoproteins have been found to express an unusual carbohydrate structure consisting of glcnac-asn ( ) , which is an expected product of endo-glycosidic degradation. it is at present unknown where this structure is synthesized and thus whether glycosylation precedes or follows arrival into the nuclear envelope. the secretion of protein appears to involve at least two pathways: a constitutive pathway and a pathway which includes a secretory vesicle which is stored cytoplasmically ( ) . a range of high mannose and complex bi-, tri-and tetra-antennary nlinked structures has been observed on both secretory and plasma membrane associated glycoproteins. most studies have been directed, however, toward n-linked glycans since these structures are easier to modify and detect than are olinked and other, unknown structures. one approach to evaluate the potential role of nlinked glycans has involved the use of pharmacological inhibitors of several steps, namely the initial steps of synthesis, and deglucosylation and demannosylation. tunicamycin blocks n-linked glycosylation completely (as well, possibly, as other types of glycosylation ( )), whereas deoxynojirimycin and swainsonine block certain steps leading from high mannose structures to the complex type. though the effects are not simple it tentatively appears that lack of n-linked glycosylation has a measureable affect on the rate of secretion of some proteins from hepatocytes or hepatoma cells ( , , ) . secretion from these cells appears not to involve a stored secretory vesicle. modification of glycosylation by deoxynojirimycin or swainsonine appears in some cases to affect the rate of movement from the rer to the golgi and, in other cases, from the golgi to the extracellular space ( , , ) . some glycoproteins and albumin are not affected at all. this result has led to speculation that there is a receptor which can recognize and retain glycoproteins bearing the high mannose marker. this mechanism would not be universal for all proteins. alternatively, these differences may reflect spurious differences in rates of dissociation from glycosylation enzymes. it seems reasonable to consider that glycans may be designed to play a modulatory role in the kinetics of secretion for only selected proteins. since secretion of these proteins is not regulated by storage in a secretory vesicle, this mechanism may be used by the cell as a finetuning mechanism for controlling release of protein. it is plausible that the passive constitutive pathway for secretion ( , ) does not involve carbohydrate per se, but that modifications of this pathway or its kinetics do. further work remains to be done to compare carbohydrate structure on different glycoproteins and to consider the possible involvement of neighboring polypeptide regions in the interaction with a hypothetical receptor. entry of protein into stored secretory vesicles requires a specific signal as shown by the observation that some hormone polypeptides produced by pituitary cells are stored whereas others are constitutively secreted. it has been observed that peptide hormones which are stored in pituitary gonadotropes and thyrotropes are sulfated whereas related peptide hormones released from placental cells, but not stored there, are not sulfated. this sulfate has subsequently been shown to reside at one or more non-reducing termini of n-linked complex oligosaccharides in the position usually occupied by sialic acid. this substitution may be directed by a substitution of the galnac for underlying gal ( ) . the functional significance of the relationship between galnac-so and delivery to a vesicle which is stored cytoplasmically prior to release by a secretagogue remains to be explored. an exception to this structure-function correlation is posed by human fsh, which is stored in human pituitary gonadotropes but, unlike the lh stored in these cells, is not sulfated. possibilities consistent with a role for galnac-so are that fsh may segregate to a discrete vesicle population relative to lh, or that the two homones colocalize and that some intermolecular association substitutes for the lack of this carbohydrate modification. there is no concensus oligosaccharide structure discovered thus far which distinguishes plasma membrane from secreted glycoproteins. although this may suggest the possibility that plasma membrane and secretory glycoproteins may in fact share a common signal, on account of their topologically equivalent destinations, inhibitor experiments have shown that an absence of n-linked glycans does not consistently block the appearance of glycoproteins on the cell surface ( , ) . there are, however, some instances of inhibition ( , , , ( ) ( ) ( ) , which allow for the possibility that nlinked glycans may act as a signal or modulator for the export of specific proteins to the plasma membrane. alternatively, the failure of a few altered glycoproteins to be exported may be explained by enhanced proteolysis, by a secondary effect on conformation, or by the display of another signal elsewhere on the polypeptide ( ) . a special class of extrinsic plasma membrane glycoproteins is known, however, for which a glycan structure appears to be significant for localization. this includes glycoproteins which are recognized by the cell surface protein ligatin, which recognizes a man- -po - -glc, linked to a highmannose backbone ( ) . this structure appears to be important for the cell surface association of these glycoproteins since glc-i-po can elute them from cells and a high concentration of ca + +, which can elute ligatin, also elutes the associated glycoproteins. glycoproteins associated by this mechanism were first recognized ultrastructurally on the surface of the intestinal mucosa of the suckling rat, but have since been discovered on the surfaces of a variety of cell types in sea urchin, chicken and mouse. it is unclear whether the gic-i-po marker serves as a routing signal for export to the cell surface, using ligatin as an intermediate carrier, or whether it serves a recognition role for secondary association with the cell surface once secretion in soluble form has already occurred. though the identity of many of the ligatin associated glycoproteins is unknown, the first to be discovered in the intestinal mucosa was j -nacetylhexosaminidase. in this case, the primary function of the glycoprotein can be viewed as an enzymatic one. the appended carbohydrate appears to serve a role in localization, contributing to the spatial organization of the system. consideration of the possible role of carbohydrate in other molecular associations with the plasma membrane will be considered below in sections on hormone-receptor interactions and cellsubstratum (e.g., extracellular matrix) and cell-cell adhesion. it has been proposed that specific carbohydrate/protein interactions might designate the localization of secreted glycoproteins to specific sites in the extracellular matrix ( ). this idea has received support recently from the detection of animal lectins in matrices, basement membranes, and mucous secretions ( ) . lectins, which are multivalent carbohydrate binding proteins with oligosaccharide specificity, are believed to crosslink other molecules to contribute to, e.g., the viscosity of certain mucous structures ( ) . binding of matrix proteins to carbohydrate of gags has been reported ( , ) . a recent report suggests that the neural adhesion protein n-cam has a binding site for heparan sulfate-like gags ( ) . in the early s, ashwell and morrell demonstrated that desialylation of serum glycoproteins, resulting in the exposure of a penultimate galactose in mammalian species, caused rapid removal of proteins by hepatocytes once these altered proteins were restored to the blood (reviewed in , ) . this suggested that normal turnover of serum proteins is regulated by the structure of their glycans, which would be regulated by interaction with sialidases. though a carbohydrate binding receptor for (non-reducing) terminal gal/galnac has been characterized in hepatocytes, it has not been possible to prove this hypothesis directly. this mechanism is not universal because not all serum proteins, albumin being a notable example, are enzymatically glycosylated. further work must be done to determine whether this system actually functions in serum protein turnover. it is interesting to note that a family of serum proteins (iga ) binds this receptor by way of o-linked oligosaccharides, but only upon denaturation ( ). it has also been recently shown that liver endothelial cells can desialate a serum protein, thus rendering it susceptible to uptake by hepatocytes via the gal/galnac receptor ( ) . additional studies have revealed the presence of a distinct carbohydrate binding protein in kupffer and other macrophage-like cells ( , , , ) . following recognition, these and other carbo-hs~drate binding proteins become internalized together with their ligands, which carry a man/glcnac/fuc specificity, but later return to the cell surface free of ligand. in addition, cell surface localized man- -po receptors have been suggested to be responsible for hepatic uptake of enzymes supplied to the fetal blood from placental fluids ( ). it has been suggested that receptors on the kupffer cell and other types of macrophages may also recognize cells which present the appropriate configuration of carbohydrate residues ( ) . binding in these circumstances may lead to phagocytosis. this kind of binding has been detected in model systems ( ) . several examples of a role for protein glycan in other forms of intermolecular recognition have been offered. these include glycans on the fc portion of igg being involved in the recognition by complement proteins and cell surface receptors ( , ) , glycans of human chorionic gonadotropin being involved in activation of its receptor subsequent to the actual binding event ( ) ( ) ( ) , and interaction between anti-thrombin iii and heparin leading to activation of inhibition of thrombin protease activity ( ). as early as , burnett ( ) and his colleagues observed the importance of cell surface proteinbound sialic acid in virus binding and infection. this phenomenon is sufficiently complex, however, that it is necessary to discuss the kinds of evidence which are involved in concluding that carbohydrate plays a direct, rather than a regulatory, role in this and other examples of adhesion. any demonstration that carbohydrate comprises part of a physical bridge in cell adhesion (or any molecular binding) requires that four general criteria be satisfied: ) removal or alteration of the carbohydrate diminishes cell adhesion; ) reconstitution of the carbohydrate either on the cell or on an artificial surface re- stores cell adhesion; ) a specific receptor for the carbohydrate is identified on the cell (complementary) surface; ) inactivation of the receptor diminishes cell adhesion. satisfaction of these criteria can be complicated because cells appear to have multiple adhesion systems, rendering it difficult to assay one independently of the others. removal of carbohydrate can have other effects on cells. for example, cell viability can be reduced by drugs or mutations affecting glycosylation, and transport of polypeptides to the cell surface and other mechanisms of turnover and placement of glycoproteins can be altered. specific receptors may be difficult to detect because individual receptor-ligand interactions may have low affinity (e.g., ). a rationale for this generalization is that, because of the large area of cell surfaces, cells can theoretically interact with other surfaces using a large number of low affinity associations. this may in fact be desirable for cell interactions which would need to be reversible. the results of several approaches where cell reassociation has been competed with model sugars have shown that the competing sugars must be crosslinked to form multivalent structures in order to be competitive ( ) . a multivalency requirement has also been implicated in the man- -po receptor system ( ) . examination of cell-ceu and cell-substratum adhesion in several systems has identified candidate molecules which participate in the formation of the physical bridge ( , ) . in some cases independent approaches have converged on the same candidate molecules. the candidate molecules are usually glycoproteins and in some cases the relative roles of carbohydrate and protein have yet to be resolved. in other studies, candidates have not yet been identified. the simplest adhesion systems involve parasite/host interaction. considerable evidence has been adduced that various parasites such as viruses, bacteria, protozoa, etc., possess carbohydrate binding proteins which can interact with sugar structures on the surfaces of cells with which the parasites associate ( , , ( ) ( ) ( ) ( ) . in some cases it is suspected that this sugar is associated with glycolipid but in other cases man is believed to be involved on the basis of competition studies (e.g., ). this would imply the involvement of glycoprotein because this sugar is rare in glycolipid. the use of carbohydrate as attachment points on cells presumably reflects the stability of these structures on cell surfaces and their specific associations with target cells suitable for the parasite. these would of course be useful features for any mechanism of specific cell-cell interaction. species-specific cell-cell adhesion (the so-called secondary type) in the phylogenetically-primitive marine sponge is suspected to involve carbohydrate ligands ( ) . ca-free sea water elutes a fraction from cells of microcionaprolifera which is required for their reaggregation. this fraction is the source of a purified proteoglycan which is referred to as aggregation factor. hypotonic shock elutes another molecular species (the so-caued baseplate or aggregation factor receptor) which is also required for reaggregation and may recognize the proteoglycan by a mechanism involving glucuronic acid on the proteoglycan. these preliminary findings were ascertained by competition studies with model sugars and enzymatic treatments, and it will be interesting to confirm these findings using purified molecules ( ) . results of comparable studies in another marine sponge, geodia cydonium, are even more intriguing ( ) . bound t -glucuronic acid is also believed to be important in aggregation factor/baseplate recognition but its position is reversed so that the key sugar is on the baseplate glycoprotein. beta-glucuronidase and j -glucuronyltransferase can be eluted from extracellular material suggesting a mechanism for the dynamic regulation of cell recognition. an investigation into the tissue specificity of cell recognition showed that ~-galactosidase treatment of nonaggregating cells permitted their aggregation in the presence of aggregation factor. a protein was then isolated which could inhibit aggregation of aggregation-competent cells in a fl-galactosidase sensitive fashion. finally, a t -galactoside binding protein was isolated from extracellular material which rendered an effect similar to that of the glycosidase. though the significance of these observations is as yet unclear, it has been suggested that carbohydrate structures may underlie specific mechanisms of cell recognition in multiple ways in these species. cell-substratum adhesion in another primitive multicellular system, aggregating cellular slime mold cells, appears to involve a multivalent carbohydrate binding protein termed discoidin i. the involvement of discoidin i was initially recognized based on sequence homology between this protein and an oligopeptide sequence of vertebrate fibronectin which has been shown to contain its cell binding site. this oligopeptide, which blocks fibronectin binding to cells, also affects association of dictyosteliurn cells with their substrata ( ) . the ceils become incapable of migrating properly on a surface. the effect can be mimicked by antidiscoidin i. the effect is also replicated if the cells are transformed with a gene which encodes an mrna which hybridizes with authentic discoidin i mrna and blocks its translation ( ) . the evidence is very strong that this carbohydrate binding protein is involved in cell-substratum association. what is unclear, however, is the role of the carbohydrate binding activity. there is evidence that it does not associate with the cell via this site ( ) . it is unclear whether the carbohydrate-binding site might associate the protein with elements of certain substrata, but it is apparent that this is not necessary. there is recent evidence that food bacteria are recognized by this lectin and hence discoidin i might assist in the adhesion of ceils to their food source ( ) . perhaps the best documented instance of a direct role for carbohydrate in cell-cell adhesion occurs in higher organisms and involves the protein galactosyltransferase and bound glcnac, sometimes associated with oligolactosamine ( , , , ) . this pair of molecules has been implicated in adhesion between mouse sperm and egg, neural crest cells and substrated, and embryonal carcinoma ceils. each of the four criteria cited above has been addressed by experimental approaches using antibodies, enzymes and drugs to inactivate the receptor and the ligand. a genetic approach to modify receptor or ligand has yet to be applied. though the latter approach with its inherent specificity is very important, the evidence using the approaches already employed is strong. the system is interesting because the receptor-ligand interaction can be readily reversed by supplying substrate for the galactosyltransferase enzyme activity. an independent approach to the problem of mouse sperm-egg recognition has resulted in the isolation, from the jelly coat of the egg, of a glycoprotein, zp , which can, upon presentation to the sperm, block its ability to recognize eggs ( ) . it was found that careful alkaline borohydride treatment to strip o-linked glycans without altering the polypeptide chain neutralized the inhibitory activity of zp . finally, it was shown that the released glycans possessed inhibitory activity and in addition bound to sperm, suggesting that the carbohydrate itself served as the recognition site in the jelly coat for sperm. further work remains to be done to identify the sperm receptor ( ) as well as to relate the findings to the above results implicating the galactosyltransferase. carbohydrate of a different type has also been implicated in cell-cell adhesion between mature mammalian cells. for example, it has been suggested that cell surface carbohydrate may be important for recognition between certain lymphocytes and certain high endothelial venule surfaces (hev) in lymphatic tissues ( ) . pretreatment of lymphocytes with specific sugars renders them incapable of binding to hevs in an in vitro assay, and glycosidase treatment of hevs renders them incapable of being recognized by lymphocytes. there is conflicting evidence from these studies regarding the nature of the carbohydrate structure involved. additional results have indicated that one lymphocyte subclass recognition site involves a previously studied homing receptor, the ubiquitin-conjugated glycoprotein defined by the mel- antigen. recent work has found carbohydrate-derivatized polyacrylamide surfaces to which lymphocytes will specifically bind ( ) . an effort is underway to compare the specificity of this interaction with that to hevs. the exciting prospect is that the manipulability of the artificial surface ( ) will allow dissection of multiple adhesion mechanisms and permit more rapid investigation of the nature of the carbohydrate recognition system believed to be responsible for lymphocyte recognition preparatory to extravasation. carbohydrate has been suggested to perform an opposite kind of role in the function of the protein n-cam in associating neural cells ( ) . fab fragments from anti-n-cam inhibit reassociation of dissociated neural cells and isolated n-cam can self associate. there are several forms of n-cam which differ in their degree of sialylation. the more heavily sialylated forms show less affinity for one another in the self association assays. thus it has been postulated that sialylation is a posttranslational modification which modulates the strength of adhesion mediated by this glycoprotein and in vivo evidence has now been provided ( ) . recent-ly it has alo been suggested that n-cam can bind carbohydrate in the heparan sulfate proteoglycan ( ) and that this recognition is involved in intercellular ( ) and cell-substratum adhesion. it is too soon to speculate whether binding to a carbohydrate ligand may have secondary effects on cells as as been suggested for, e.g., binding of human chorionic gonadotropin ( ) ( ) ( ) . this hormone does not depend upon its carbohydrate for binding to its receptor but the carbohydrate is important for activating the cellular response. the selected examples discussed here indicate that protein-linked (or possibly lipid-linked) carbohydrate may play a fundamental role in multicellular organization in a wide phylogenetic range of organisms. though in all of the examples cited there is a specificity in the adhesion, in some cases this is due to multiple possibly low-affinity binding interactions. in the extreme, each single binding event may approach non-specificity, with specificity accruing through multivalency. perhaps this implies that the formalization of non-specific carbohydrate-mediated events obscures an actual continuum of specificity in these events between these two extremes. it is unclear whether the involvement of carbohydrate in adhesion is universal. several other adhesion molecules, including glycoproteins such as gp in d. discoideurn ( ) , cam / in mammalian cells, etc. and glycolipids ( ) , are glycosylated but the role of carbohydrate is not known. if the use of carbohydrate is universal, then the specificity inherent in the examples discussed would suggest an underlying structural code which may be important for the affiliated processes of cell sorting and morphogenesis, to which adhesion makes a very important contribution. this may explain some of the interesting morphogenetic effects circumstantially suggested by many studies involving carbohydrate structure perturbations on developing cells. a role for carbohydrate in cell adhesion choice or sorting correlates with its postulated role in intracellular or extracellular compartmentalization of proteins. cell sorting and molecular compartmentalization can be regarded as sorting at different levels. it will be interesting to learn whether the proteins on which these carbohydrates are located exist only to place the carbohydrate or whether the carbohydrate piggybacks on another essential function of the protein. were the latter true, this would strengthen the analogy with the role of carbohydrate in routing or molecular compartmentalization. it may then become feasible to generalize carbohydrate's role as important for the three-dimensional compartmentalization of proteins, and the cells, with which they are associated. as such, carbohydrate may be the chemical structure cells used to address and organize space as they evolved from prokaryotes to unicellular eukaryotes, and continued to exploit as they evolved to form multicellular tissues and organs. numerous studies have implicated protein-linked carbohydrate in more complicated cellular phenomena ( , ) . glycosylation is modulated coincidentally with numerous developmental processes ( , - , , , ) . carbohydrate interactions have been implicated in mammalian morula compaction ( , , ) . proliferation has been reported to require glycosylation ( ) or to be inhibited by glycopeptides and a defined fragment of heparan sulfate ( , ) . oligosaccharides released from plant cell walls can stimulate cell proliferation and alter cell differentiation in plant cells ( ) . myogenesis and chondrogenesis have been speculated to be affected by gags linked to proteoglycans (e.g., ), but conclusions in this field are very controversial. attachment of cells to substratum-associated carbohydrate can result in its covalent modification ( , ). neuritogenesis has been shown to be inhibited in vitro by tunicamycin ( ) , though the specificity of the effect is difficult to assess, as tunicamycin-sensitivity does not convincingly demonstrate the involvement of protein-linked carbohydrate ( , ) . a recent report suggests that alteration of processing of nlinked oligosaccharides with swainsonine, without affecting cell viablility, inhibits the ability of a line of melanoma cells to metastasize to the lung ( ). this finding is consistent with another report documenting carbohydrate differences between metastatic and non-metastatic cell lines ( ) . there is much new territory to explore in the field. the use of monoclonal antibodies and lectins which recognize carbohydrate-dependent epitopes continues to reveal an unexpected diversity of carbohydrate structures with surprisingly intricate distributions in embryonic and adult tissues. this has recently been appreciated in amphibian ( ) and mouse ( , ) embryos and in the simpler model system dictyostelium discoideum ( , ) . tumor cells also differ in many cases in specific carbohydrate structures on both lipid and protein ( - , - , ) . several of the antibodies employed recognize carbohydrate epitopes whose corresponding structures have been determined. the antibodies provide unrivaled sensitivity and specificity relative to standard methods for detection of unique carbohydrate structures. caution must be applied, however, because ) it is theoretically possible that unique structures at the monosaccharide level may present structures similar enough at the atomic level to be immunologically cross-reactive and ) identical or similar structures may be immunologically distinguished by conformational restrictions imposed by the surrounding polypeptide environment. thus inference of structure based on antibody data must be confirmed directly by structural studies. in any case, antibodies and lectins may model potential receptors in cells which might specifically recognize carbohydrate-associated structures. the significance of the association of distinct carbohydrate structures with distinct embryonic or tumor cell types is absolutely unclear at present. ultrastructural localization has placed some epitopes intracellularly in specific compartments and others on the cell surface or extracellularly in the surrounding matrix. a cell surface localization is a necessary condition for a direct role in cell recognition and, on the strength of suggestions from other systems, this has been promiscuously interpreted to imply a role in cell recognition. this reflects the primitive status of our general understanding of the significance of protein glycosylation. thus the potential developmental roles of protein-linked carbohydrate in morphogenesis, differentiation, adherence and proliferation will likely be a primary target of investigation in this field in the future. interest in developmental involvement of carbohydrate is based not only on a large bank of indirect data but also on an intrigue in the special kinds of epigenetic regulatory mechanisms with which carbohydrate might be associated. for example, glycosylation offers the cell a mechanism to regulate the structure of multiple proteins without altering gene expression. in addition, monosaccharide residues may be processed independently of the associated polypeptide ( , ) either by recycling through the golgi ( ) or at locations near the site of funtion ( , ) , far from the point of origin in the rer and golgi. it can be envisaged that the cell responded to the evolutionary requirements for cell differentiation by using epigenetic pathways such as glycosylation and other posttranslational modifications ( ) . consistent with these speculations, there are, as cited above, numerous examples of glycosylation steps modified by differentiation ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ) , as well as by the anchorage state of the cell and the ph and composition of the surrounding medium ( , ) . an example of what the future may bear is exemplified by a group of developmental arrest mutants recently characterized in d. discoideum ( ) . isolated on the basis of failure to accumulate an early regulated enzyme, many of these mutants appear to be altered in posttranslational modifications, including glycosylation. thus many potentially necessary roles played by glycosylation in development remain unexplored. been shown that carbohydrate interaction is multivalent, composed of multiple weak associations which only in aggregate are strong ( , , ) . it would appear that most protein-linked carbohydrate is not solely responsible for the essential housekeeping functions of cells required for life in the tissue culture environment. perhaps this is because specification of its structure, which is nontemplate-directed, is not precise enough. glycosylation presumably has instead afforded the cell, during the evolution of developmental mechanisms, an economy in allowing the structural modification of many preexisting proteins using the same enzymatic mechanism. it provides the cell with another control point over structure which can be exerted on many proteins at the site of function (away from the site of synthesis) without necessitating a change in gene expression. change in structure by glycosylation may be very suitable for the regulation of the 'luxury' functions of cell differentiation and morphogenesis. an economy of shared non-specifc and specific functions of carbohydrate would thus offer an unifying explanation for the often contradictory conclusions yielded from the varied experimental approaches summarized in this article. the nearly ubiquitous glycosylation of extracellular and cell surface proteins and proteins from certain organelles may reflect a primitive, nonspecific function supporting protein structure and protection. this carbohydrate carries out a shared function, however, when it becomes modified in subtle ways without affecting non-specific functions, to allow discrimination by receptors. recognition by discriminate receptors allows the carbohydrate to subserve specific functions. carbohydrate would appear to be used by cells to carry out secondary functions (both specific and non-specific) for proteins. these include specialized instances of compartmentalization, rate of transport through the cell, intermolecular association, conformation control and general protection. interaction of the cell with the environment, which is in a sense secondary to the life of the cell, would also appear to involve protein-linked carbohydrate. this might include compartmentalization or sorting of cells into tissues. in several instances it has structure, biosynthesis and functions of glycoprotein glycans carbohydrate moieties of glycoproteins complex carbohydrates of the extracellnlar matrix. structures, interactions and biological roles glycoprotein biosynthesis in yeast control of asparagine-linked oligosaccharide chain processing: studies on bovine pancreatic ribonuclease b correlation of glycosylation forms with position in amino acid sequence lysosomal enzyme phosphorylation occurrence of n-acetylglucosamine -phosphate in complex carbohydrates - -methylation of mannose residues transient metbylation of dolichyl oligosaccharides is an obligatory step in halobacterial sulfated glycoprotein biosynthesis glycoproteins as ceil-surface components either high-mannose-type or hybrid type oligosaccharide is linked to the same asparagine residue in ovalbumin posttranslational covalent modification of proteins assembly of asparagine-linked oligosaccharides tunicamycin inhibits ganglioside biosynthesis in neuronal cells tunicamycin blocks neuritogenesis and glucosamine labeling of gangliosides in developing cerebral neuron cultures suppression of placental alkaline phosphatase biosynthesis by tunicamycin occurrence of nacetylglucosamine-l-phosphate in proteinase i from dictyostelium discoideum glycosylation mutants of animal cells laminin, proteoglycan, nidogen and collagen iv: structural models and molecular interactions purification, composition, molecular weight and subunit structure of ovine submaxillary mucin effect of altered oligosaccharide structure onithe cell surface number, distribution and turnover of the high molecular weight acidic glycoproteins of cho cells preliminary characterization of a chinese hamster ovary cell glycosylation mutant isolated by screening for low intracellular lysosomal enzyme activity temporal aspects of the n-and o-glycosylation of human chorionic gonadotropin o-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the golgi apparatus structural and functional aspects of tumor cell sialomucins antigenic determinants shared by lysosomal proteins of dictyostelium discoideum adhesion mutants of dictyostelium disclideum lacking the saccharide determinant recognized by two adhesion blocking monoclonal antibodies conformation of the glycopeptide linkage in asparagine-linked glycoproteins conformation of the complex oligosaccharides of glycoproteins multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites effects of deglycosylation on the architecture of ovine submaxillary mucin glycoprotein preparation and properties of serum and plasma proteins. xxix. separation from human plasma of polysaccharides, peptides and proteins of low molecular weight. crystallization of an acid glycoprotein studies on fetuin, a glycoprotein of fetal serum. i. isolation, chemical composition, and physicochemical properties variation of the carbohydrates of glycoproteins of cells growing on different surfaces the function of protein-bound carbohydrates in normal and pathological cells asparaginyl-nacetylgalactosamine olden k: retinoic acid alters the proportion of high mannose to complex type oligosaccharides on fibronectin secreted by cultured chondrocytes a temperature-sensitive n-glycosylation mutant of s. cerevisiae that behaves like a ceil-cycle mutant nglycosylation of nascent proteins early in the prereplicative phase constitutes a process for controlling animal cell proliferation studies on the regulation of the biosynthesis of glucose-containing oligosaccharide-lipids carbohydrate structure, biological recognition, and immune function. in: ivatt rj (ed). the biology of glycoproteins glybopeptide changes and malignant transformation. a possible role for carbohydrate in maligant behavior demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens glycosphingolipids as differentiationdependent, tumor-associated markers and as regulators of cell proliferation biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides aggregation-deficient embryonal carcinoma cells: defects in peanut agglutinin (pna) receptors asparagine-linked oligosaccharides in murine tumor cells: comparison of a wga-resistant (wga r) nonmetastatic mutant and a related wga-sensitive (wga s) metastatic line wp: carbohydrates of the tumor cell surface oligosaccharide modification by swainsonine treatment inhibits pulmonary colonization by b -f murine melanoma cells regional specificity of glycoconjugates in xenopus and axolotl embryos saccharide structures of the mouse embryo during the first eight days of development reactivity of five nacetylgalactosamine-recognizing lectins with preimplantation embryos, early postimplantation embryos, and teratocarcinoma cells of the mouse inhibition of glycoprotein synthesis in saccharomyces cerevisiae by mating pheromones ssea- , a stagespecific antigen of the mouse, is carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells trypanosoma cruzi cells undergo an alteration in protein n-glycosylation upon differentiation appearance of a class of cell-surface fucosyl-glyeopeptides in differentiated muscle cells in culture changes in protein glycosylation during chick embryo development changes in the expression of blood-group carbohydrates during oral mucosal development in human fetuses cell surface glycoconjugates as oncodifferentiation markers in hematopoietic cells glycoprotein synthesis and embryonic development decreased transfer of oligosaccharide from oligosaccharideqipid to protein acceptors in regenerating rat liver stimulation of lipid-linked oligosaccharide assembly duing oviduct differentiation cell-type-related segregation of surface galactosyl-containing components at an early developmental stage in hemopoietic bone marrow cells in the rabbit changes in asparagine-linked sugar chains of human promyelocytic leukemic cells (hl- ) during monocytoid differentiation and myeloid differentiation are glycoproteins and glycosaminoglycans components of the eukaryotic genome? n-linked glycoprotein biosynthesis in the developing mouse embryo biological significance of carbohydrate chains on monoclonal antibodies a role for glycosylation of the c~-subunit in transduction of biological signal in glycoprotein hormones role of carbohydrate in human chorionic gonadotropin antibodies against human chorionic gonadotropin convert the deglycosylated hormone from an antagonist to an agonist cell surface galactosyltransferase as a recognition molecule during development nelsestuen gl: deglycosylated prothrombin fragment polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin investigations of the possible functions for glycosylation in the high mobility group proteins preferential association of glycoproteins to the euchromatin regions of crossfractured nuclei is revealed by fracture-label eukaryotic ribosomes possess a binding site for concanavalin a on the biological role of glycoproteins the significance of glycosylated proteins effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes differing requirements for glycosylation in the secretion of related glycoproteins is determined neither by the producing cell nor by the relative number of oligosaccharide units effect of the threonine analog/ -hydroxynorvaline on the glycosylation and secretion of c~l-acid glycoprotein by rat hepatocytes differential effects of -deoxynojirimycin on the intracellular transport of secretory glycoproteins of human hepatoma cells in culture the effects of processing inhibitors of nlinked oligosaccharides on the intracellular migration of glycoprotein e of mouse hepatitis virus and the maturation of coronavirus particles role of glycosylation in processing of newly translated insulin proreceptor in t -l adipocytes influence of the nlinked oligosaccharides on the biosynthesis, intracellular routing, and function of the human asialoglycoprotein receptor glycosylation of the epidermal growth factor receptor in a- cells inhibition of n-linked igosaccharide trimming does not interfere with surface expression of certain integral membrane proteins evidence for a glycoprotein 'signal' involved in transport between subcellular organelles a single nlinked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface the role of protein glycosylation in the compartmentalization and processing of mouse mammary tumor virus glycoproteins in mouse mammary tumor virus-infected rat hepatoma cells the formation of vesicular stomatitis virus (san juan strain) becomes temperature-sensitive when glucose residues are retained on the oligosaccharides of the glycoprotein deletions into a ni-i -terrninal hydrophobic domain result in secretion of rotavirus vp , a resident endoplasmic reticulum membrane glycoprotein glycoantigen expression is regulated both temporally and spatially during development in the cellular slime molds dictyostelium discoideum and d. mucoroides the identification of n-linked oligosaccharides on the human cr /epstein-barr virus receptor and their function in receptor metabolism, plasma membrane expression, and ligand binding glycosylation and secretion of surfactant-associated glycoprotein a complete glycosylation of the insulin and insulin-like growth factor receptors is not necessary for their biosynthesis and function chemistry, metabolism, and biological functions of sialic acids effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease swainsonine inhibits glycoprotein degradation by isolated rat liver lysosomes slime mold lectins bacterial glycoconjugates are natural ligands for the carbohydrate binding site of discoidin i and influence its cellular compartmentalization carbohydrate-specific receptors of the liver hepatic clearance of serum glycoproteins the repair of the surface structure of animal cells carbohydrate-mediated clearance of immune complexes from the circulation carbohydrate-mediated clearance of antibody-antigen complexes from the circulation surface carbohydrates and surface lectins are recognition determinants in phagocytosis the carbohydrate structure of porcine uteroferrin and the role of the high mannose chains in promoting uptake by the reticuloendothelial cells of the fetal liver intracellular movement of cell surface receptors after endocytosis: resialylation of asialotransferrin receptor in human erythroleukemia cells hyaluronic acid bonded to cell culture surfaces stimulated chondrogenesis in stage limb mesenchyme cell cultures lectin activation in giardia lamblia by host protease: a novel hostparasite interaction carbohydrates as recognition determinants in phagocytosis and in lectin-mediated killing of target cells specificity of binding of a strain of uropathogenic escherichia coli to galcd- gal-containing glycosphingolipids evidence for a malarial parasite interaction site on the major transmembrane protein of the human erythrocyte adherent bacterial colonization in the pathogenesis of osteomyelitis mucoproteins in relation to virus action the synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion the receptor function of galactosyltransferase during cellular interactions the biology of glycoproteins a multivalent lacto-n-fucopentaose iii-lysyllysine conjugate decompacts preimplantation mouse embryos, while the free oligosaccharide is ineffective receptor function of mouse sperm surface galactosyltransferase during fertilization o-linked oligosaccharides of mouse egg zp account for its sperm receptor activity differential cell adhesion may result from nonspecific interactions between cell surface glycoproteins specific alteration of n-cam-mediated cell adhesion by an endoneuraminidase a heparin-bindifig domain from n-cam is involved in neural cell-substratum adhesion recognition of igg by fc receptor and complement: effects of glycosidase digestion cell surface carbohydrates in cell recognition and response feizi t: cell interactions in preimplantation embryos: evidence for involvement of saccharides of the poly-nacetyllactosamine series mitochondrial synthesis of glycoproteins and surface properties of mitochondrial membranes cell membranes in sponges the phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes cell-cell recognition in yeast, characterization of the sexual agglutination factors from saccharomyces kluyveri lymphocyte attachement to high endothelial venules during recirculation: a possible role for carbohydrates as recognition determinants lymphocyte adhesion to immobolized polysaccharides suggests multiple carbohydrate receptors for recirculation spontaneous glycosylation of glycosaminoglycan substrates by adherent fibroblasts immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells soluble lectins: a new class of extraeellular proteins effect of choroquine on the degradation of l-fucose and the polypeptide moiety of plasma membrane glycoproteins different half-lives of the carbohydrate and protein moieties of a dalton glycoprotein isolated from plasma membraned of rat liver structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. ii. evidence for a pentasaccharide sequence that contains a - -sulfate group pathways of protein secretion in eukaryotes structure and function of plant cell wall polysaccharides gangliosides support neural retina cell adhesion ~-mannosidase- mutants of dictyostelium discoideum: early aggregationessential genes regulate enzyme precursor synthesis, modification, and processing discoidin i is implicated in cell-substratum attachment and ordered cell migration of dictyostelium discoideum phenocopy of discoidin i-minus mutants by antisense transformation in dictyostelium mechanisms for the incorporation of proteins in membranes and organelles glycopeptides prepared from mouse cerebrum inhibit protein synthesis and cell division in baby hamster kidney ceils, but not in their polyoma virus-transformed analogs stereochemistry and binding behavior of carbohydrate chains associations of like and unlike polysaccharides: mechanism and specificity in galactomannans, intreacting bacterial polysaccharides, and related systems model for the structure of the gastric mucous gel freeze hh: interaction of dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor differential processing of asn-linked oligisaccharides on pituitary glycoprotein hormones: implications for biologic function lysosomal enzyme bind!ng to mouse p d l macrophage membranes lacking the -kda mannose -phosphate receptor: evidence for the existence of a second mannose -phosphate receptor identification and characterization of cells deficient in the mannose -phosphate receptor: evidence for an alternate pathway for lysosomal enzyme targetting variability in transport rates of secretory glycoproteins through the endoplasmic reticulum and golgi in human hepatoma cells glucose phosphotransferase and intracellular protein trafficking freeze hh: modifications of lysosomal enzymes in dictyostelium discoideum glucose removal from n-linked oligosaccharides is required for efficient maturation of cerain secretory glycoproteins from the rough endoplasmic reticulum to the golgi complex deletion of clustered o-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts absence of a carbohydrate modification does not affect the level or the subcellular . . . localization of three membrane glycoproteins in modb mutants of dictyostelium discoideum reversible defects in o-linked glycosylation and ldl receptor expression in a udp-gal/udp-galnac -epimerase deficient mutant groth sf: studies on mucoproteins. iii. the accesibility to trypsin of the susceptible bonds in ovine submaxillary land mucoproteins influenza c virus uses - -acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells carbohydrate-binding sites of the mannose-specific fimbrial lectins of enterobacteria the mouse egg's receptor for sperm; what is it and how does it work? cell adhesion molecules neuronal cell-cell adhesion depends on interactions of n-cam with heparin-like molecules reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer localization of galactosyl-and sialyltransferase by immunofluorescence: evidence for different sites outer membrane terminal saccharide of bovine liver nuclei and mitochondria hendil kb: ion exchange properties of the glycocalyx of the amoeba chaos chaos and its relation to pinocytosis liver endothelium mediates the hepatocyte's uptake of ceruloplasmin the molecular basis of species specific cell-cell recognition in marine sponges, and a study on organogenesis during metamorphosis rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri-and tetraantennary glycopeptides containing [glcnac- ( , )man-a( , )man] and poly-nacetyllactosamine sequences than baby hamster kidney cells i am grateful to drs. c. feldherr and g. erdos for their critisms of the manuscript during preparation. key: cord- -l cdllh authors: saraste, j.; marie, m. title: intermediate compartment: a sorting station between the endoplasmic reticulum and the golgi apparatus date: - - journal: encyclopedia of cell biology doi: . /b - - - - . - sha: doc_id: cord_uid: l cdllh the intermediate compartment (ic) is a pleiomorphic membrane system that mediates two-way trafficing in the early biosynthetic-secretory pathway of mammalian cells. the ic associates with the cytoskeleton, binds copi (coat protein i) coats and generates vesicular, tubular and saccular transport carriers. it recieves newly made proteins and lipids from the endoplasmic reticulum (er) and sorts them for transport to the golgi apparatus or recycling back to the er. although the ic appears to be functionally complex and resides at the crossroads of multiple transport routes, it is still disputed whether it represents a transient or stable structure. newly synthesized proteins and lipids leave the endoplasmic reticulum (er) at specialized transitional regions called er exit sites (eres) (jamieson and palade, ; sesso et al., ; bannykh et al., ; hammond and glick, ; tang et al., ) and enter the intermediate compartment (ic) that has been shown to operate as an obligatory a post-er sorting station in the early biosynthetic-secretory trafficking of mammalian cells. from the ic they are typically transported to the cisternal stacks of the golgi apparatus, prior to their delivery to the different organelles of the endomembrane system or secretion to the extracellular space. bidirectional er-golgi trafficking involves the sequential operation of membrane-bound coat protein ii (copii) and copi coats (aridor and balch, ; scales et al., ; stephens et al., ) . er-derived copii vesicles mediate forward (anterograde) transport, while ic-and golgi-derived copi vesicles are thought to act in the opposite (retrograde) direction (lee et al., ; rabouille and klumperman, ) . retrograde transport also involves copiindependent routes (kano et al., ) . despite the conservation of the transport machineries (such as the cop coats), the organization of the er-golgi interface varies in different eukaryotic cells. for example, in plants, certain yeasts, and the fruit fly drosophila melanogaster, the individual golgi stacks lie next to the widespread eres, establishing units for short range er-golgi communication. by contrast, in animal cells the golgi stacks are linked together into a continuous ribbon around the microtubule-organizing center (mtoc)/centrosome, whereas the er extends throughout the cytoplasm. hence, a large proportion of the eres reside at the cell periphery and er-golgi trafficking depends on the long distance movements of the ic elements along mt tracks (saraste and svensson, ; presley et al., ; scales et al., ; brandizzi and barlowe, ; day et al., ) . thus, it has been proposed that the ic represents a late evolutionary invention, which developed to meet the special sorting, transport and recycling requirements of the large-sized animal cells, but lacks lower eukaryotes. however, results showing that the ic constitutes an extensive membrane system that can be compared with the endosomal network in complexity, are questioning this view. electron microscopic (em) studies using a temperature-sensitive mutant of semliki forest virus (sfv ts- ) to synchronize the transport of viral membrane glycoproteins from er to the plasma membrane (pm) showed that when the cells are shifted from c to c the proteins exit the er, but accumulate in vacuoles/saccules (up to . mm in diameter), tubules, and vesicles in the cis-golgi region and more peripheral locations (saraste and kuismanen, ) . by light microscopy (lm) the proteins were localized at c to scattered globular structures in the cytoplasm, a pattern distinct from that of er or golgi (kuismanen and saraste, ) . the transport block was readily reversible, and the proteins entered the golgi stacks and reached the pm following the transfer of cells from c to c, showing that these structures are normal transport intermediates. studies employing a similar mutant of vesicular stomatitis virus (vsv tso ) showed that the ' c compartment' also acts as a way station during the transport of the vsv g glycoprotein (balch et al., ; bonatti et al., ; schweizer et al., ; duden et al., ; lotti et al., ) . like the sfv proteins, the g-protein was found to maintain its er-type high-mannose glycans at c, indicating lack of processing by golgi enzymes. furthermore, cell fractionation experiments showed that newly synthesized secretory proteins are arrested in a post-er location when pancreatic exocrine cells are kept at c (saraste et al., ) . live cell imaging of green fluorescent protein (gfp) equipped with an er targeting signal (ssgfp) and em studies of procollagen and growth hormone constructs verified that the transport of membrane and secretory proteins is similarly affected at - c (blum et al., ; volchuk et al., ; trucco et al., ) . in addition, the transport of virus glycoproteins and cholesterol appears to be blocked at the same site at this temperature (urbani and simoni, ; heino et al. ) . em of mouse hepatitis virus (mhv)-infected cells showed that the budding of progeny virus initially takes place at tubulovesicular membranes located in the transitional region between the er and the golgi apparatus (tooze et al., ) . the first step of o-glycosylation, the attachment of nacetyl-galactosamine (galnac) to the viral membrane (m) glycoprotein, was suggested to occur in this compartment, causing its reactivity with the lectin helix pomatia, which specifically binds galnac (tooze et al., ; krijnse-locker et al., ) . subsequent studies showed that the intracellular maturation of various coronaviruses occurs at the same budding site, which corresponds to the ic (klumperman et al., ) . the discovery of the lys-asp-glu-leu tetra-peptide (kdel)motif in abundant, lumenal er proteins lead to the proposal that the c/budding compartment is the post-er site from which these proteins are retrieved to the er (munro and pelham, ; warren, ; pelham, ). the first mammalian kdel-receptor, a multispanning membrane protein, was identified and shown to predominantly localize to the ic and cis-golgi (lewis and pelham, ; tang et al., ; griffiths et al., ; orci et al., ) . also, kdel proteins, such as the immunoglobulin binding protein (bip/grp ), glucoseregulated protein of kda (grp ), protein disulphide isomerase (pdi) and calreticulin (cr), are present in the ic connolly et al., ; zuber et al., ; ying et al., ; breuza et al., ) . following binding to their receptor, the proteins are retrieved to the er in copi vesicles (orci et al., ; martínez-menárguez et al., ; majoul et al., ) . attachment of the kdel-motif to lysosomal enzymes and the use of the c block suggested that the enzyme that initiates the formation of their lysosomal targeting signal (mannose- -phosphate) resides in the ic (pelham, ; lazzarino and gabel, ; dittmer and von figura, ) . the cytoplasmic tails of certain type i integral membrane proteins were shown to contain dilysine (kkxx)motifs, which by directly interacting with copi coats result in their retrieval from the ic/cis-golgi to the er (nilsson et al., ; jackson et al., jackson et al., , cosson and letourneur, ) . the first endogenous ic markers rat p and human er-golgi intermediate compartment (ergic)- ( % homology) were identified by the generation of antibodies against the c post-er fraction isolated from pancreatic acinar cells (saraste et al., ) and a golgi fraction derived from epithelial caco- cells (schweizer et al., ) , respectively. the cytoplasmic c-terminal tails of these non-glycosylated, hexameric, type- integral membrane proteins (schindler et al., ; lahtinen et al., lahtinen et al., , neve et al., ) contain a kkff-motif, which interacts with copii and copi coats and gives rise to their continuous cycling between er, ic, and cis-golgi (kappeler et al., ; tisdale et al., ) . at c the recycling of p /ergic- to the er is inhibited and the proteins pile up in the same pre-golgi structures that contain the sfv or vsv membrane proteins saraste and svensson, ; plutner et al., ) , verifying that the p /ergic- compartment (figures and ) is equivalent to the site where cargo is arrested at low temperature. ergic- and the related vip were shown to share homology with leguminous plant lectins (fiedler and simons, ) and to be identical with a mannose-binding protein mr of human myelomonocytic (hl ) cells (arar et al., ) . the n-terminal domain of p /ergic- binds mannose in a calcium-dependent manner (itin et al., ; velloso et al., ; ; hence the name lectin mannose-binding protein (lman ). it is the best characterized mammalian cargo receptor, facilitating the copii vesicle-mediated export of a subset of soluble glycoproteins from the er (nichols et al., ; appenzeller et al., ; hauri et al., ) . most well-characterized ic proteins are components of the transport machinery. many of them cycle at the er-golgi boundary and are also found in cis-golgi, supporting the view of the ic as a transient structure (see below). in fact, em studies showing the specific metal (osmium) staining of the ic and cis-golgi provided the first indication of their compositional similarity (friend and murray, ; rambourg et al., ) . however, the fungal compound brefeldin a (bfa) helps to discriminate between ic and golgi components. it releases copi coats, disassembles the golgi stacks and redistributes golgi components to the er, whereas cycling proteins (such as p /ergic- /lman and the kdelreceptor) are arrested in the drug-resistant ic elements (lippincott-schwartz et al., ; saraste and svensson, ; tang et al., b; füllekrug et al., ; ward et al., ; marie et al., ) . the bfa resistance of the ic has been utilized for its proteomics analysis (breuza et al., ) , and indicates its stability. like p /ergic- , the p family proteins contain motifs for copii and copi binding, resulting in their cycling between the er, ic, and cis-golgi (rojo et al., ; dominguez et al., ; blum et al., ; gommel et al., ; strating and martens, ). as abundant type- transmembrane proteins they participate in the biogenesis of copi vesicles (stamnes et al., ; majoul et al., ; beck et al., ) , but have also been implicated in tubulation and the formation of membrane domains (lavoie et al., ; emery et al., ) . studies in yeast first suggested their function as receptors for the exit of glycosylphosphatidylinositol (gpi)-anchored proteins from the er (schimmöller et al., ) . the copi coats are mainly recruited to the cytoplasmic surface of ic and cis-golgi membranes, but also associate with the lateral rims of the golgi stacks (duden et al., ; oprins et al., ; griffiths et al., ; orci et al., ; klumperman et al., ) . copi vesicle budding depends on the activation of small gtpases of the adp-ribosylation factor (arf) family (popoff et al., ) . the guanine nucleotide exchange factor (gef) of these arfs, gbf , is the master regulator of copi recruitment and the target of bfa (niu et al., ) . it localizes to the ic and cis-golgi and plays a key role in er-to-golgi trafficking (kawamoto et al., ; garcia-mata et al., ; zhao et al., ; szul et al., ) . gbf knock down or specific inhibition by golgicide a releases copi coats and arrests the vsv g-protein in the ic, indicating its involvement in anterograde ic-to-golgi transport (manolea et al., ; sáenz et al., ) . however, the prevailing view is that the main function of copi vesicles is to recycle membrane and selected proteins to the er. in pancreatic exocrine cells approximately % of the coats associate with the vtcs, suggesting that the ic represents the main point for recycling (martínez-menárguez et al., ) . the roles of two gtpases of the large rab family, rab and rab , in er-golgi trafficking are relatively well understood (plutner et al., ; tisdale et al., ) . both interact with multiple effectors suggesting that they coordinate successive transport steps (barnekow et al., ) . the association of rab with the cytoplasmic surface of ic and cis-golgi membranes, and its absence from the er, has been demonstrated by em saraste et al., ; satoh et al., ; marie et al., ; figure ). the two rab isoforms, rab a and rab b ( % homology), are recruited to ic membranes at eres, show similar localizations by lm (mochizuki et al., ; figure (a)), but seem to play distinct roles in tubular and vesicular (long and short range) trafficking within the ic. accordingly, live cell imaging and cell fractionation have shown that rab a mainly associates with ic tubules (sannerud et al., ; marie et al., ) , while rab b interacts with gbf and evidently modulates copi recruitment to globular ic domains monetta et al., ; see below) . also, rab a is specifically nu * * * * figure (a) immunofluorescence lm localization of p /ergic- in baby hamster kidney, (b) mouse myeloma, (c) rat neuroendocrine pc , and (d) human hela cells. the protein marks the ic elements accumulating in the perinuclear golgi region (asterisks), scattered throughout the cytoplasm, and located close to the pm (arrows). the reticular staining indicates the er pool of p /ergic- , which varies in the different cell types. nu, nucleus. bar: mm. phosphorylated at mitosis (bailly et al., ) , correlating with the cessation of tubular ic dynamics, whereas copi-mediated vesicular transport continues (marie et al., ) . rab a can functionally replace ypt , which coordinates two-way er-golgi trafficking in the yeast saccharomyces cerevisiae (haubruck et al. ; segev, ; kamena et al., ) . rab has been localized to ic and cis-golgi membranes (chavrier et al., ; lotti et al., ) and proposed to regulate the formation of retrograde copi vesicles that contain p /ergic- (tisdale, ) . it has also been implicated in the recruitment of the motor protein dynein to ic membranes and the association of ic elements with mts (tisdale et al., ; see below) . most of the known rab and rab effectors are cytoplasmically oriented, long coiled-coil proteins, which function in the tethering of vesicle and organelle membranes preceding their soluble nsf attachment protein receptor (snare)-mediated fusion (barnekow et al., ; sztul and lupashin, ). besides the ic/cis-golgi tethers gm and gmap- (rios et al., ) , rab has been shown to interact with medialand trans-golgi tethers, suggesting a more widespread function (short et al., ; sinka et al., ) . the rab effector p is functional already at the peripheral eres (alvarez et al., ) , while gm (bound to its membrane anchor grasp ) cycles between central ic and cis-golgi (marra et al., ) and regulates membrane tethering at a later transport step (alvarez et al., ; marra et al., ) . rab also interacts with the transmembrane tethers golgin- and giantin, which appear to act in copi vesicle trafficking at the lateral rims of the golgi stacks (diao et al., ; malsam et al., ; barnekow et al., ; munro, ) . the membrane fusion machinery (snare proteins) operating in er-golgi trafficking in the yeast saccharomyces cerevisiae has been well characterized (cai et al., ; barlowe and miller, ) , whereas the fusion events that take place in mammalian cells remain enigmatic. the determination of the exact fusion steps is complicated by the continuous cycling of the snares in copi vesicles (hay et al., ; martinez-menárguez et al., ) . in analogy to yeast, rab (ypt ) has been suggested to recruit p (uso p) to copii vesicles at eres (allan et al., ) , followed by the formation of a snare complex (sec b, membrin, bet and syntaxin ), which mediates either homotypic fusion of copii vesicles or their heterotypic fusion with the stationary ic (zhang et al., ; xu et al., ; see below) . another snare complex (syntaxin , gs , bet and ykt ) is involved in a later cis-golgi transport step (zhang and hong, ) . by em the ic elements can be readily distinquished from the er and golgi, but share structural similarity with endosomes. they reside close to peripheral and central eres as clusters of vesicles and tubules (vtcs; figure (f)) that frequently contain copi coats (balch et al., ; bannykh et al., ; martínez-menárguez et al., ) . however, they display considerable size heterogeneity (ying et al., ) and also include large saccules that are found within the membrane clusters, free in the cytoplasm, or at the cis-face of the golgi stacks (saraste and svensson, ; lahtinen et al., , connolly et al., stinchcombe et al., ; ladinsky et al., ; fan et al., ; figures (a)- (e)). these pleiomorphic saccules can accommodate large-sized cargo, such as procollagen or lumenal protein aggregates (volchuk et al., ; trucco et al., ; zuber et al., ) , and based on correlative microscopy (lm/em) correspond to many of the mobile carriers that are visible in living cells (mironov et al., ) . like endosomes, they extend narrow tubules and also bind copi coats, indicating that they represent sites for vesicle budding (saraste and kuismanen, ; volchuk et al., ; horstman et al., ; figures (c) and (d)). the hypertrophy of the saccular domains most likely gives rise to the pre-golgi vacuoles seen in cells kept at c (saraste and kuismanen, ; trucco et al., ) . lm shows the division of the ic into globular and tubular domains (presley et al., ) . the former contain anterograde cargo, cargo receptors and copi coats, most likely corresponding to individual vtcs or free saccules, while the latter are highlighted by rab a (sannerud et al., ) . the tubules extend from the globular domains ( figure ) . some of them contain recycling proteins, but lack anterograde cargo, indicating that they function in retrograde transport (palokangas et al., ; simpson et al., ) . however, under synchronized conditions cargo is also detected in the tubular domain, due to overload or the existence of different types of ic tubules ( figure ). for example, incubation of cells at - c inhibits the formation of ic tubules, but causes the expansion of the globular domain, while the shift of cells to c generates tubular networks containing both antero-and retrograde markers (blum et al., ; ben-takaya et al., ; simpson et al., ) . proliferation of the tubules is induced when copi function is compromised (szul et al., ; marie et al., ; ben-takaya et al., ; tomás et al., ; hamlin et al., ) , but also occurs under physiological conditions. in differentiating neuroendocrine pc cells the rab a-positive tubular ic domain expands and the tubules move from the cell body to the forming neurites accumulating in their growth cones (sannerud et al., ; figure ). an analogous pathway connects the ic with the leading edge of fibroblasts ( figure ) . live imaging of various fluorescent ic markers indicates that the tubules are highly dynamic, while the globular domains are typically more stationary. due to their differential localization within these domains, the constructs highlight different aspects of ic dynamics (see below). long distance er-to-golgi transport involves the movement of ic elements from the cell periphery to the central cis-golgi region, resulting in the division of the ic into spatially distinct early (eresadjacent) and late (cis-golgi-adjacent) domains (saraste and svensson, ; presley et al., ; scales et al., ; marra et al., ) . two types of anterogradely moving ic elements can be resolved by lm, narrow tubules and large pleiomorphic structures. some of the latter appear to represent saccular elements that transform into elongated structures ('thick tubules') (presley et al., ; marie et al., ). in addition, narrow tubules establish dynamic connections between the globular ic domains (ben-tekaya et al., ; sannerud et al., ) . the long distance movements of the ic elements depend on mts (murshid and presley, ; palmer et al., (b) and ). the plus-and minus-end directed motor proteins kinesin and dynein associate with the ic elements (lippincott-schwartz et al., ; roghi and allan, ; stauber et al., ) , explaining their bidirectional movements even along the same mt tracks (sannerud et al., ) . when the mts in mammalian cells are depolymerized by nocodazole the ic elements become immobile and accumulate close to eres (saraste and svensson, ; hammond and glick, ; ben-tekaya et al., ; sannerud et al., ) . although the central golgi ribbon breaks down, the formation of golgi ministacks in the vicinity of eres re-establishes er-golgi communication as a short range process (as in plants), explaining the ongoing golgi modification and secretion of proteins (saraste and svensson, ; cole et al., ; storrie et al., ) . the rho family gtpase cdc , which regulates actin dynamics, interacts with copi coats and affects dynein function, suggesting functional coupling between the actin filament system and mt-dependent motility of ic/cis-golgi carriers (luna et al., ; chen et al., ) . similarly, whamm, which promotes actin nucleation and interacts with mts, has been localized to the ic and implicated in the formation and transport of pre-golgi tubules (campellone et al., ) . based on imaging of fluorescent cargo, such as the vsv g-protein and procollagen, in living cells (presley et al., ; scales et al., ; stephens and pepperkok, ) , it has been proposed that the ic represents a collection of large, pleiomorphic transport complexes (tcs) (bannykh and balch, ; stephens and pepperkok, ) , which form via homotypic fusion of copii vesicles, or protrude directly from the er (mironov et al., ; xu and hay, ; yu et al., ) . thereafter, they move in a mt-and dynein-dependent (burkhardt et al., ) 'stop-and-go' fashion to the golgi region, where they either fuse with or transform into cis-golgi cisternae (figure (a) ). in other words, the ic elements are transient structures which are first formed de novo at eres and then consumed at cis-golgi. these mobile structures (speed of about mm/sec) corresponding to saccules or more complex, pleiomorphic elements (mironov et al., ) appear to consist of subdomains enriched in antero-or retrograde cargo, or copi (shima et al., ; stephens et al., ) . they have also been shown to contain other machinery proteins, such as p / , p , vip , membrin and rab a (blum et al., ; golgi nu figure immunofluorescence lm localization of rab in differentiating pc cells, illustrating the interconnected globular (arrowheads) and tubular (arrows) domains of the ic. two confocal sections of the same cell are shown. the ic has expanded in response to a h treatment with the nerve growth factor (ngf) and the tubules are found both in the cell body and the neurite-like extensions (right panel). nu, nucleus; golgi, perinuclear golgi region. bar: mm. time projection figure bidirectional movements of gfp-rab a-positive tubules in a living nrk cell. selected confocal images from a movie obtained by timelapse microscopy are shown. tubules extending from the same relatively stationary globular ic element (black arrowhead) pinch off and move either in the direction of the cell's leading edge (upper panels; yellow arrowheads), or toward the golgi indicated by an asterisk (lower panels; red arrowheads). the paths taken by the tubules are highlighted in the time projection on the right. adapted from sannerud, r., marie, m., nizak, c., et al., chao et al., ; dahm et al., ; sannerud et al., ; monetta et al., ; tomás et al., ) . another model of the ic (figure (b) ) is largely based on the imaging of gfp-ergic- dynamics in living cells (ben-tekaya et al., ) . it proposes that the ic consists of stationary, long-lived membrane clusters, located close to the eres, which communicate with the er and golgi via distinct transport carriers (appenzeller-herzog and hauri, ) . thus, in a two-step transport process cargo is first transfered from er to the ic via heterotypic fusion of copii vesicles. the ic then generates novel (possibly copi-coated) anterograde carriers (acs) that move along mts to the cis-golgi. the recycling of gfp-ergic- to the er evidently occurs mainly from the eres-associated ic, since it was not detected in the acs containing soluble, golgi-destined cargo. the identification of a motif in the neuronal gaba transporter, that seems to be required for its exit from the ic, supports the stable nature of the ic (farhan et al., ) . visualization of ic dynamics using gfp-rab a showed that the pleiomorphic transport carriers arriving along mts from the cell periphery do not move directly to cis-golgi, but are targeted to the mtoc/centrosome that is normally positioned next to the golgi ribbon. in cells displaying centrosome motility, for example, cells that are migrating or entering mitosis, the centrosome-targeted membranes can be resolved as a separate compartment, called the pericentrosomal ic (pcic), which is distinct from the cis-golgi-adjacent ic domain (marie et al., , mochizuki et al., ; figure (a)). live imaging further showed that the separated pcic and the golgi communicate via tubular and globular carriers. the pcic contains its own pool of copi coats, and mediates the bfainduced, copi-independent backflow of golgi enzymes to the er, suggesting that forward pcic-to-golgi transport depends on copi coats, and thus is blocked by bfa . both the peripheral ic elements and the pcic persist upon golgi disassembly by bfa and maintain their communication with via dynamic tubules . accordingly, the ic has been proposed to constitute a dynamic, but stable membrane network due to its anchoring next to the eres and the centrosome figure (c) ). this model is supported by studies of mitotic cells, showing that the ic persists despite golgi breakdown and the rearrangement of the mts, and maintains its spatial organization due to its ongoing association with the spindle mts and the centrosomes at the spindle poles (marie et al., ; figure (a) ). in the endocytic pathway, soluble proteins and particles bound to lysosomes accumulate in the lumen of vacuolar endosomes, while many membrane proteins are sorted into narrow tubules for recycling to the pm. the observed major the ic consists of stationary membrane clusters located close to eres. in this case er-to-golgi trafficking involves two distinct transport steps, since the ic recieves cargo from the er via heterotypic fusion of copii vesicles and forms special anterograde carriers (ac) that move along mts to cis-golgi. (c) the ic represents a stable interconnected network that is anchored both next to eres and the centrosome. bidirectional transport between these sites and between the central ic elements and the golgi stacks involves vesicular, tubular or saccular carriers. eres-ic communication via copii vesicles occurs as in model b. copi, copii and clathrin coats are shown in gray, orange, and blue, respectively, and the centrosome in red. concentration of soluble secretory proteins within the ic (oprins et al., ) could be explained by similar geometrybased sorting, namely, their exclusion from vesicles and tubules, which recycle lipids and membrane-bound proteins back to the er (martínez-menárguez et al., ) . membrane recycling is a major function of both the ic and endosomes in mammalian cells. due to the similarity of the tubular domain of the ic with the endocytic recycling compartment (erc), its 'mirror compartment' next to the centrosome, it has been designated as the biosynthetic recycling compartment (brc; saraste and goud, ; marie et al., ) . lumenal conditions: receptor-mediated retrieval of kdel proteins from the ic requires that it is discontinuous with the er and maintains special lumenal conditions (pelham, ) . the finding of low ph-dependent binding of kdel-ligands to their receptor in vitro suggested the existence of a ph gradient between the er and cis-golgi (wilson et al., ) . the phsensitive interactions of the low density lipoprotein (ldl)receptor-related protein (lrp) and procollagen with the chaperones rap and hsp , respectively (bu et al., ; satoh et al., ) , and partial co-localization of damp (a marker for acidic compartments) and p /ergic- in central ic elements (palokangas et al., ) , are in accordance with this idea. the ph of the er and cis-golgi has been estimated to be about . and . - . , respectively (paroutis et al., ; vavassori et al., ) . although the effect ph on the binding of mannose-containing cargo to p /ergic- /lman remains unclear, it appears that cargo release is caused by a drop in free calcium concentration between the er and ic lumen (appenzeller-herzog et al., ; bentley et al., ; . on the other hand, the depletion of lumenal calcium stores affects the morphology of the ic and the recycling of cargo receptors at the er-golgi boundary, and the ic has been reported to contain the calcium-atpase serca, as well as the calciumbinding proteins bip, grp , cr, and calnuc (ying et al., ; breuza et al., ; howe et al., ; bentley et al., ) , suggesting its role in intracellular calcium storage. role of copi coats: there are at least three subtypes of copi coats (beck et al., ) , and four arf gtpases that regulate their membrane binding (popoff et al., ) . three arfs appear to act at the er-golgi boundary and two of these associate with membranes in a bfa-resistant manner (volpicelli-daley et al., ; chun et al., ; duijsings et al., ; ben-tekaya et al., ; hamlin et al., ) , suggesting that different types of copi vesicles mediate two-way trafficking at the level of the ic. the role of copi in anterograde transport has been considered for some time (hosobuchi et al., ; pepperkok et al., ; peter et al., ; orci et al., ; malsam et al., ) . in addition, although both p /ergic- and kdel-receptor employ copi vesicles in their recycling, their transport itineraries differ (tang et al., a; marie et al., marie et al., , . in addition to acting in vesicle budding the copi coats may form membrane domains (presley et al., ) . golgi bypass: besides mediating bidirectional er-golgi trafficking, the ic has been suggested to be involved in golgiindependent pathway(s) (marie et al., ; saraste et al., ) . the route between the ic and the leading edge or growth cone of motile fibroblasts or neurons, respectively ( figures and ) , could participate in cholesterol and integrin trafficking (urbani and simoni, ; sannerud et al., ; wang et al., ) or correspond to the bfa-resistant er-ic-pm route that supports phagocytosis (becker et al., ; saraste and goud, ; see below) . direct pericentrosomal communication between the ic and the endosomal system seems to be used by the cystic fibrosis transmembrane conductance regulator (cftr) during its golgi-independent transport to the cell surface (yoo et al., ; marie et al., ; gee et al., ) . further, the presence of the ic in neuronal dendrites may provide a golgi-independent satellite pathway for local dendritic trafficking (krijnse-locker et al., ; sannerud et al., ; ehlers, ) . questioning the studies on coronavirus maturation, which suggested that o-glycosylation is initiated in the ic (see above), subsequent em studies showed that the galnactransferases are predominantly found in the golgi. however, the activation of cells (by epidermal growth factor) causes their incorporation into copi vesicles and relocation to the ic and er, which consequently become positive for the lectin helix pomatia (gill et al., ) . the cycling of cis-golgi proteins to the ic could be a constitutive event (lin et al., ; marra et al., ; jarvela and linstedt, ) . moreover, the construction of the sugar chains on proteoglycans has been suggested to begin in the ic (jönsson et al., ) . the kdel-containing molecular chaperones present in the ic (bip, pdi, and grp ; see above) could be cycling while still bound to their unfolded client proteins, opening for a post-er level of protein folding and quality control. the presence of quality control machinery in the ic and the finding that the er-associated degradation of certain proteins requires their er export is in accordance with this idea (zuber et al., ; anelli and sitia, ) . the pdi family member erp and p / ergic- cooperate in the ic/cis-golgi in sequential assembly of igm polymers (anelli and sitia, ) . unassembled igm or t-cell antigen receptor subunits bound to erp or bip, respectively, can be retrieved to the er by the kdel-receptor in a ph-dependent manner (yamamoto et al., ; vavassori et al., ) , similarly as the overexpressed, misfolded vsv g-protein (hammond and helenius, ) and mutant v vasopressin receptors (hermosilla et al., ) . additional proof for a post-er checkpoint is provided by studies showing the accumulation of the deletion mutant df of cftr (gilbert et al., ) , misfolded mhc class i proteins (hsu et al., ; raposo et al., ) , and proinsulin (zuber et al., ) in expanded ic elements. the level of p /ergic- mrna is up-regulated by the unfolded protein response (upr), which also requires yeast ypt function, supporting a link between this signaling pathway and er-golgi trafficking (nyfeler et al., ; tsvetanova et al., ) . protein kinases src, apkc, and scyl have been implicated in copi function at the level of the ic (tisdale and artalejo, ; hamlin et al., ) and pkc and its downstream effectors appear to control ic morphology (ben-tekaya et al., ; sugawara et al., ) . the activation of neuronal trk receptor tyrosine kinases in the ic initiates signaling via the mek pathway leading to golgi fragmentation (schecterson et al., ) . membranes enriched in ic markers (p /ergic- /lman , kdel-receptor, and sec b) play a role in the biogenesis of autophagosomes by representing the primary membrane source for the lipidation of lc , which triggers this process (ge et al., ) . moreover, ypt /rab is a key regulator of autophagy in yeast and mammals (lynch- day et al. ; winslow et al., ; zoppino et al., ; huang et al., ; lipatova et al., ) . a phosphatidylinositol -phosphatase (mtmr ) acting in vesicle transport and autophagy is regulated by rab b and localizes predominantly to the pcic (mochizuki et al., ) , further supporting the role of the ic in autophagy. supporting the existence of an unconventional pathway that connects the er with the pm-derived phagosome (gagnon et al., ) , the ic-enriched snare sec b/ers- (zhang et al., ) was shown to influence phagocytosis independently of its role in er-golgi trafficking (becker et al., ; hatsuzawa et al., ) . in dendritic cells the delivery of certain proteinssuch as the peptide transporter (tap), cr and tapasinfrom er to the phagosome is required for antigen cross-presentation. this pathway depends on the interaction between sec b and the pm snare syntaxin and involves efficient recruitment of the integral ic components sec b and p /ergic- to the phagosomes (cebrian et al., ) , in accordance with the idea that the ic provides an important membrane source for their formation. the presence of cr, tapasin, and functional tap in the ic/cis-golgi support this possibility (kleijmeer et al., ; howe et al., ; ghanem et al., ) . the ic plays a role in the golgi-independent trafficking of cftr (see above). mutations in p /ergic- /lman , or its partner mcfd (multiple coagulation factor deficiency protein ) result in an autosomal recessive bleeding disorder called combined deficiency of coagulation factors v and viii. they disrupt the lectin mannose-binding protein (lman )-mcfd receptor complex, thereby inhibiting the secretion of these factors and reducing their serum levels (nichols et al., ; zheng and zhang, ) . parkinson's disease-related cytotoxic protein, α-synuclein, has been suggested to interfere with er-golgi trafficking and to arrest autophagy by inhibiting rab function (cooper et al., ; winslow et al., ) . many phagocytosed bacterial pathogens, such as legionella, hijack the er-to-ic-to-phagosome pathway during their intracellular replication (isberg et al., ; arasaki et al., ; see above) . in addition to coronaviruses, the ic has been implicated in the replication of bunya-, entero-, flavi-, picorna, and vacciniaviruses (jäntti et al., ; risco et al., ; miller and krijnse-locker, ; hsu et al., ) . surprisingly, p / ergic- interacts in a lectin-independent manner with fusion proteins of a number of membrane viruses and participates in different stages of their life cycle (klaus et al., ) . rab recruitment of p into a cis-snare complex: programming budding copii vesicles for fusion er to golgi transport: requirement for p at a pre-golgi vtc stage copi recruitment is modulated by a rab b-dependent mechanism the p -interactive proteins gm and giantin participate in endoplasmic reticulum-golgi traffic protein quality control in the early secretory pathway the lectin ergic- is a cargo transport receptor for glycoproteins the er-golgi intermediate compartment (ergic): in search of its identity and function ph-induced conversion of the transport lectin ergic- triggers glycoprotein release ergic- , a membrane protein of the endoplasmic reticulum-golgi intermediate compartment, is identical to mr , an intracellular mannose-specific lectin of myelomonocytic cells the legionella pneumophila effector drra is sufficient to stimulate snare-dependent membrane fusion principles of selective transport: coat complexes hold the key phosphorylation of two small gtp-binding proteins of the rab family by p cdc atp-coupled transport of vesicular stomatitis virus g protein between the endoplasmic reticulum and golgi vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum membrane dynamics at the endoplasmic reticulum-golgi interface the organization of endoplasmic reticulum export complexes secretory protein biogenesis and traffic in the early secretory pathway rab proteins and their interaction partners the copi system: molecular mechanisms and function differential use of endoplasmic reticulum membrane for phagocytosis in j macrophages adp ribosylation factors and and group via phospholipase a regulate morphology and intraorganellar traffic in the endoplasmic reticulum-golgi intermediate compartment live imaging of bidirectional traffic from the ergic vesicular calcium regulates coat retention, fusogenicity, and size of pre-golgi intermediates intracellular localization and in vivo trafficking of p a and p lumenal targeted gfp, used as a marker of soluble cargo, visualises rapid ergic to golgi traffic by a tubulo-vesicular network palmitylation of viral membrane glycoproteins takes place after exit from the endoplasmic reticulum organization of the er-golgi interface for membrane traffic control proteomics of endoplasmic reticulum-golgi intermediate compartment (ergic) membranes from brefeldin a-treated hepg cells identifies ergic- , a new cycling protein that interacts with human erv binding and endocytosis of kda protein by mdbk cells overexpression of the dynamitin (p ) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution coats, tethers, rabs, and snares work together to mediate the intracellular destination of a transport vesicle whamm is an arp / complex activator that binds microtubules and functions in er to golgi transport sec b regulates phagosomal maturation and antigen crosspresentation by dendritic cells snare membrane trafficking dynamics in vivo localization of low molecular weight gtp binding proteins to exocytic and endocytic compartments coatomer-bound cdc regulates dynein recruitment to copi vesicles characterization of class i and class ii adp-ribosylation factors in live cells: gdp-bound class ii arfs associate with the er-golgi intermediate compartment independently of gbf golgi dispersal during microtubule disruption: regeneration of golgi stacks at peripheral endoplasmic reticulum exit sites transport into and out of the golgi complex studied by transfecting cells with cdnas encoding horseradish peroxidase alpha-synuclein blocks er-golgi traffic and rab rescues neuron loss in parkinson's models coatomer (copi)-coated vesicles: role in intracellular transport and protein sorting quantitative er golgi transport kinetics and protein separation upon golgi exit revealed by vesicular integral membrane protein dynamics in live cells a three-stage model of golgi structure and function the coiled-coil membrane protein golgin- is a novel rab effector required for golgi ribbon formation phosphorylation of arylsulphatase a occurs through multiple interactions with the udp-n-acetylglucosamine- -phosphotransferase proximal and distal to its retrieval site by the kdel receptor gp l/emp /p protein family members of the cis-golgi network bind both cop i and ii coatomer beta-cop, a kd protein associated with non-clathrin-coated vesicles and the golgi complex, shows homology to beta-adaptin differential membrane association properties and regulation of class i and class ii arfs dendritic trafficking for neuronal growth and plasticity the transmembrane protein p forms highly specialized domains that regulate membrane composition and dynamics ultrastructural analysis of transitional endoplasmic reticulum and pre-golgi intermediates: a highway for cars and trucks signal-dependent export of gaba transporter from the er-golgi intermediate compartment is specified by a c-terminal motif a putative novel class of animal lectins in the secretory pathway homologous to leguminous lectins osmium impregnation of the golgi apparatus characterization of brefeldin a-induced vesicular structures containing cycling proteins of the intermediate compartment/cis-golgi network endoplasmic reticulummediated phagocytosis is a mechanism of entry into macrophages adp-ribosylation factor/copidependent events at the endoplasmic reticulum-golgi interface are regulated by the guanine nucleotide exchange factor gbf the er-golgi intermediate compartment is a key membrane source for the lc lipidation step of autophagosome biogenesis rescue of Δf -cftr trafficking via a grasp-dependent unconventional secretion pathway the transporter associated with antigen processing (tap) is active in a post-er compartment delta f cftr localizes in the endoplasmic reticulum-golgi intermediate compartment in cystic fibrosis cells regulation of o-glycosylation through golgi-to-er relocation of initiation enzymes p and p , the major transmembrane proteins of copi-coated transport vesicles, form heterooligomeric complexes and cycle between the organelles of the early secretory pathway localization of the lys, asp, glu, leu tetrapeptide receptor to the golgi complex and the intermediate compartment in mammalian cells immunocytochemical localization of beta-cop to the er-golgi boundary and the tgn scyl scaffolds class ii arfs to specific subcomplexes of coatomer through the γ-cop appendage domain dynamics of transitional endoplasmic reticulum sites in vertebrate cells quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the er, intermediate compartment, and golgi apparatus sec b is a negative regulator of phagocytosis in macrophages the ras-related mouse ypt protein can functionally replace the ypt gene product in yeast ergic- and traffic in the secretory pathway localization, dynamics, and protein interactions reveal distinct roles for er and golgi snares dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface disease-causing v( ) vasopressin receptors are retained in different compartments of the early secretory pathway ultrastructural characterization of endoplasmic reticulum-golgi transport containers (egtc) sec is a gene required for er to golgi protein transport that encodes a subunit of a yeast coatomer calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of mhc class i molecules viral reorganization of the secretory pathway generates distinct organelles for rna replication a recycling pathway between the endoplasmic reticulum and the golgi apparatus for retention of unassembled mhc class i molecules antibacterial autophagy occurs at pi( )p-enriched domains of the endoplasmic reticulum and requires rab gtpase the legionella pneumophila replication vacuole: making a cosy niche inside host cells ergic- is a functional mannose-selective and calcium-dependent human homologue of leguminous lectins identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum retrieval of transmembrane proteins to the endoplasmic reticulum intracellular transport of secretory proteins in the pancreatic exocrine cell. i. role of the peripheral elements of the golgi complex immunocytochemical analysis of uukuniemi virus budding compartments: role of the intermediate compartment and the golgi stack in virus maturation irradiation-induced protein inactivation reveals golgi enzyme cycling to cell periphery initiation of the decorin glycosaminoglycan chain in the endoplasmic reticulum-golgi intermediate compartment ypt p is essential for retrograde golgi-er transport and for golgi maintenance in s. cerevisiae yip a regulates the copiindependent retrograde transport from the golgi complex to the er the recycling of ergic- in the early secretory pathway. ergic- carries a cytosolic endoplasmic reticulum-exit determinant interacting with copii gbf , a guanine nucleotide exchange factor for adp-ribosylation factors, is localized to the cis-golgi and involved in membrane association of the copi coat the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles location of mhc-encoded transporters in the endoplasmic reticulum and cis-golgi coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding the recycling pathway of protein ergic- and dynamics of the er-golgi intermediate compartment characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step the organization of the endoplasmic reticulum and the intermediate compartment in cultured rat hippocampal neurons low temperature-induced transport blocks as tools to manipulate membrane traffic golgi structure in three dimensions: functional insights from the normal rat kidney cell characterization of a kda cis-golgi protein in pancreatic exocrine cells molecular cloning and expression of a -kda cis-golgi and intermediate compartment protein roles for alpha( )p and copi in endoplasmic reticulum cargo exit site formation biosynthesis of the mannose- -phosphate recognition marker in transport-impaired mouse lymphoma cells. demonstration of a two-step phosphorylation bi-directional protein transport between the er and golgi a human homologue of the yeast hdel receptor er/golgi intermediates acquire golgi enzymes by brefeldin a-sensitive retrograde transport in vitro regulation of selective autophagy onset by a ypt/rab gtpase module kinesin is the motor for microtubule-mediated golgi-to-er membrane traffic microtubuledependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway immunocytochemical analysis of the transfer of vesicular stomatitis virus g glycoprotein from the intermediate compartment to the golgi complex regulation of protein transport from the golgi complex to the endoplasmic reticulum by cdc and n-wasp trs directs a ypt gef, trappiii, to the phagophore to promote autophagy kdel-cargo regulates interactions between proteins involved in copi vesicle traffic: measurements in living cells using fret golgin tethers define subpopulations of copi vesicles distinct functions for arf nucleotide exchange factors at the golgi complex: gbf and bigs are required for assembly and maintenance of the golgi stack and tgn, respectively division of the intermediate compartment at the onset of mitosis provides a mechanism for golgi inheritance the function of the intermediate compartment in pre-golgi trafficking involves its stable connection with the centrosome take the 'a' train: on fast tracks to the cell surface the gm and grasp golgi proteins cycle through and define a subdomain of the intermediate compartment the biogenesis of the golgi ribbon: the roles of membrane input from the er and of gm vesicular tubular clusters between the er and golgi mediate concentration of soluble secretory proteins by exclusion from copi-coated vesicles peri-golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model of intra-golgi transport modification of intracellular membrane structures for virus replication er-to-golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized er exit domains phosphatidylinositol -phosphatase myotubularin-related protein (mtmr ) is regulated by small gtpase rab b in the early secretory and autophagic pathways rab b interacts with gbf and modulates both arf dynamics and copi association the golgin coiled-coil proteins of the golgi apparatus a c-terminal signal prevents secretion of luminal er proteins er-to-golgi transport and cytoskeletal interactions in animal cells oligomerization and interacellular localization of the glycoprotein receptor ergic- is independent of disulfide bonds mutations in the er-golgi intermediate compartment protein ergic- cause combined deficiency of coagulation factors v and viii short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum dynamics of gbf , a brefeldin a-sensitive arf exchange factor at the golgi the cargo receptor ergic- is a target of the unfolded protein response beta-cop localizes mainly to the cis-golgi side in exocrine pancreas the er-to-golgi interface is the major concentration site of secretory proteins in the exocrine pancreatic cell bidirectional transport by distinct populations of copi-coated vesicles the role of microtubules in transport between the endoplasmic reticulum and golgi apparatus in mammalian cells retrograde transport from the pre-golgi intermediate compartment and the golgi complex is affected by the vacuolar h þ -atpase inhibitor bafilomycin a the ph of the secretory pathway: measurement, determinants, and regulation evidence that luminal er proteins are sorted from secreted proteins in a post-er compartment control of protein exit from the endoplasmic reticulum beta-cop is essential for biosynthetic membrane transport from the endoplasmic reticulum to the golgi complex in vivo β-cop is essential for transport of protein from the endoplasmic reticulum to the golgi in vitro rab b regulates vesicular transport between the endoplasmic reticulum and successive golgi compartments morphological analysis of protein transport from the er to golgi membranes in digitonin-permeabilized cells: role of the p -containing compartment several adp-ribosylation factor (arf) isoforms support copi vesicle formation dissection of copi and arf dynamics in vivo and role in golgi membrane transport er-to-golgi transport visualized in living cells golgi membrane dynamics the maturing role of copi vesicles in intra-golgi transport three-dimensional structure of the osmium-impregnated golgi apparatus as seen in the high voltage electron microscope misfolded major histocompatibility complex class i molecules accumulate in an expanded er-golgi intermediate compartment gmap- recruits γ-tubulin complexes to cis-golgi membranes and is required for golgi ribbon formation endoplasmic reticulum-golgi intermediate compartment membranes and vimentin filaments participate in vaccinia virus assembly dynamic association of cytoplasmic dynein heavy chain a with the golgi apparatus and intermediate compartment involvement of the transmembrane protein p in biosynthetic protein transport golgicide a reveals essential roles for gbf in golgi assembly and function rab defines a novel pathway connecting the pre-golgi intermediate compartment with the cell periphery emerging new roles of the pre-golgi intermediate compartment in biosynthetic-secretory trafficking functional symmetry of endomembranes pre-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the golgi complex localization of the small gtp-binding protein rab to early compartments of the secretory pathway temperature-sensitive steps in the transport of secretory proteins through the golgi complex in exocrine pancreatic cells antibodies to rat pancreas golgi subfractions: identification of a kda cis-golgi protein distribution of the intermediate elements operating in er to golgi transport golgin- is a rab binding partner involved in golgi structure intracellular interaction of collagen-specific stress protein hsp with newly synthesized procollagen visualization of er-to-golgi transport in living cells reveals a sequential mode of action for copii and copi trk activation in the secretory pathway promotes golgi fragmentation the absence of emp p, a component of er-derived copii-coated vesicles, causes a defect in transport of selected proteins to the golgi ergic- , a membrane protein of the er-golgi intermediate compartment, carries an er retention motif identification, by a monoclonal antibody, of a -kd protein associated with a tubulo-vesicular compartment at the cis-side of the golgi apparatus identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to golgi apparatus ypt and rab gtpases: insight into functions through novel interactions a three-dimensional reconstruction study of the rough er-golgi interface in serial thin sections of the pancreatic acinar cell of the rat segregation of copi-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-golgi transport complexes a grasp -rab effector complex linking golgi structure to membrane traffic biogenesis of tubular er-to-golgi transport intermediates golgi coiled-coil proteins contain multiple binding sites for rab family g proteins an integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding a role for kinesin- in copi-dependent recycling between the er and the golgi complex copi-coated er-to-golgi transport complexes segregate from copii in close proximity to er exit sites illuminating the secretory pathway: when do we need vesicles imaging of procollagen transport reveals copidependent cargo sorting during er-to-golgi transport in mammalian cells anterograde and retrograde traffic between the rough endoplasmic reticulum and the golgi complex recycling of golgi-resident glycosyltransferases through the er reveals a novel pathway and provides an explanation for nocodazole-induced golgi scattering the p family and selective transport processes at the er-golgi interface pkcδ and ε regulate the morphological integrity of the er-golgi intermediate compartment (ergic) but not the anterograde and retrograde transports via the golgi apparatus role of vesicle tethering factors in er-golgi membrane traffic dissecting the role of the arf guanine nucleotide exchange factor gbf in golgi biogenesis and protein trafficking segregation of ergic- and the mammalian kdel-receptor upon exit from the c compartment differential response of resident proteins and cycling proteins of the golgi to brefeldin a copii and exit from the endoplasmic reticulum molecular cloning, characterization, subcellular localization and dynamics of p , the mammalian kdel receptor a rab mutant with impaired gtpase activity stimulates vesicle formation from pre-golgi intermediates src-dependent atypical protein kinase c iota/lambda (apkciota/lambda) tyrosine phosphorylation is required for apkciota/lambda association with rab and glyceraldehyde- -phosphate dehydrogenase on pre-golgi intermediates rab utilizes glyceraldehyde- -phosphate dehydrogenase and protein kinase c{iota} to associate with microtubules and to recruit dynein gtpbinding mutants of rab and rab are potent inhibitors of vesicular transport from the endoplasmic reticulum to the golgi complex p / binds copi and is required for selective transport through the early secretory pathway regulation of er-golgi intermediate compartment tubulation and mobility by copi coats, motor proteins and microtubules replication of coronavirus mhv-a in saccells: determination of the first site of budding of progeny virions site of addition of n-acetyl-galactosamine to the e glycoprotein of mouse hepatitis virus-a secretory traffic triggers the formation of tubular continuities across golgi subcompartments the yeast rab gtpase ypt modulates unfolded protein response dynamics by regulating the stability of hac rna cholesterol and vesicular stomatitis virus g protein take separate routes from the endoplasmic reticulum to the plasma membrane a ph-regulated quality control cycle for surveillance of secretory protein assembly the crystal structure of the carbohydrate-recognition domain of the glycoprotein sorting receptor p /ergic- reveals an unpredicted metal-binding site and conformational changes associated with calcium ion binding megavesicles implicated in the rapid transport of intracisternal aggregates across the golgi stack isoform-selective effects of the depletion of adp-ribosylation factors - on membrane traffic regulation of integrin β recycling to lipid rafts by rab a to promote cell migration maintenance of golgi structure and function depends on the integrity of er export signals and salvage sequences ph-dependent binding of kdel to its receptor in vitro α-synuclein impairs macroautophagy: implications for parkinson's disease reconstitution of copii vesicle fusion to generate a pre-golgi intermediate compartment subunit structure of a mammalian er/golgi snare complex the kdel receptor mediates a retrieval mechanism that contributes to quality control at the endoplasmic reticulum the p -positive pre-golgi intermediates consist of distinct subpopulations of particles that show differential binding of copi and copii coats and contain vacuolar h þ -atpase colocalization of ca þ -atpase and grp with p and the effects of thapsigargin on protein recycling suggest the participation of the pre-golgi intermediate compartment in intracellular ca þ -storage non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway mbet p is required for homotypic copii vesicle tethering in mammalian cells ykt forms a snare complex with syntaxin , gs , and bet and participates in a late stage in endoplasmic reticulum-golgi transport the mammalian protein (rbet ) homologous to yeast bet p is primarily associated with the pre-golgi intermediate compartment and is involved in vesicular transport from the endoplasmic reticulum to the golgi apparatus morphological and functional association of sec b/ers- with the pre-golgi intermediate compartment gbf , a cis-golgi and vtcs-localized arf-gef, is implicated in er-to-golgi protein traffic structural characterization of carbohydrate binding by lman protein provides new insight into the endoplasmic reticulum export of factors v (fv) and viii (fviii) combined deficiency of coagulation factors v and viii: an update autophagosome formation depends on the small gtpase rab and functional er exit sites immunolocalization of udp-glucose: glycoprotein glucosyltransferase indicates involvement of pre-golgi intermediates in protein quality control misfolded proinsulin accumulates in expanded pre-golgi intermediates and endoplasmic reticulum subdomains in pancreatic beta cells of akita mice further reading getting into the golgi molecular motors and the golgi complex: staying put and moving through the kdel-receptor: new functions for an old protein protein sorting receptors in the early secretory pathway the golgi apparatus: years of progress and controversy architecture of the mammalian golgi secretory protein trafficking and organelle dynamics in living cells copii and the regulation of protein sorting in mammals key: cord- -aios h s authors: sicari, daria; chatziioannou, aristotelis; koutsandreas, theodoros; sitia, roberto; chevet, eric title: role of the early secretory pathway in sars-cov- infection date: - - journal: j cell biol doi: . /jcb. sha: doc_id: cord_uid: aios h s similar to other rna viruses, sars-cov- must ( ) enter a target/host cell, ( ) reprogram it to ensure its replication, ( ) exit the host cell, and ( ) repeat this cycle for exponential growth. during the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. therefore, secretory pathway–mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of sars-cov- and their relevance for potential antiviral targeting. the fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for sars-cov- biology that could also be targeted with therapeutic objectives. finally, we provide an overview of recent evidence implicating proteins of the early secretory pathway as potential antiviral targets with effective therapeutic applications. as obligatory parasites, viruses must find a suitable host cell, deliver into it their genome (either dna or rna), and hijack the endogenous molecular machineries for their own replication (su et al., ) . thus, some scientists exploit viruses as trojan horses to deliver genes into target cells, whereas others search for the achilles heels of those causing diseases. to find a suitable host, viruses bind to receptors expressed on the cell surface. selective binding activates membrane fusion, allowing the viral genome to enter the target cell. fusion can occur directly at the plasma membrane (e.g., human immunodeficiency virus) or within endocytic compartments (e.g., influenza). in the cytoplasm, the viral genome is replicated while enough structural and nonstructural proteins are produced. all the essential components must then be assembled and surrounded with an envelope composed of viral proteins in combination with host cell-derived membranes. viruses exploit the ability of human cells to produce, assemble, and transport to the cell surface receptors, transporters, and other complex proteins (su et al., ) . for severe acute respiratory syndrome coronavirus (sars-cov- ) and other coronaviruses (covs), these steps take place in organelles of the early secretory pathway, the er, the er-golgi intermediate compartment (ergic) , and the golgi apparatus. sars-cov- , a member of the sars-cov family, is responsible for a global pandemic that started in (lake, ; myint, ; andersen et al., ) . because sars-cov- shares % of the genome with sars-cov, one can use knowledge obtained on the viruses that caused prior pandemics, such as sars and middle east respiratory syndrome (lake, ; lim et al., ) . cov- infection starts when its spike (s) protein binds to angiotensin i-converting enzyme (ace ) receptors on the host cell membrane (lake, ; letko et al., ) . cov- does so with higher affinity than sars-cov (wan et al., ; wu et al., ) . in the cytosol, the virus is uncoated, and its rna genome is used to produce replicases and nonstructural proteins (nsps). replicases generate subgenomic rna capable of producing the structural proteins spike (s), nucleocapside (n), membrane (m), and envelope (e) proteins. the group of proteins helping the assembly and transport of infective viral particles are referred to as accessory proteins (orfs). a large number of these proteins are cotranslationally translocated into the er to be glycosylated, folded, and assembled in preparation for virus budding and release via the secretory pathway. thus, virion spread critically depends on recruiting the most efficient secretory machineries of host cells (su et al., ; proteins of the early secretory pathway bound by sars-cov- as the entire world asks for ways to stop cov- , many laboratories are investigating the virus's achilles heel(s). because the virus is an obligatory parasite, a powerful strategy to identify endogenous molecules essential for its replication starts with the expression of suitably tagged cov- proteins in human cells. the host interactors are identified by biochemical methods and grouped on the basis of their known functions. experiments along these lines have been performed in a cooperative effort led by the krogan laboratory in a study aiming to reposition approved drugs against the virus (gordon et al., ) . taking advantage of affinity purification mass spectrometry assays, the authors, using hek t cells as a model, highlighted putative targetable cov- -host protein-protein interactions among the identified (gordon et al., ) . many (∼ %) involved proteins from the host secretory pathway (fig. , a and b ). these observations are in line with those of previous analyses of other viruses but also revealed a number of proteins specific for cov- , the most representative being orf ( %), orf c ( %), nsp ( %), nsp ( %), and m ( %; fig. c and table ) . hereafter, we comment on the interactions involving elements of the secretory pathway and how these could be targeted to combat cov- . this approach has great potential but also some limitations. perhaps the most relevant caveat is that each viral protein was expressed independently of the other sars-cov- proteins in human cells. on the one hand, this is key to identifying specific interactors in sufficient quantities. on the other hand, however, it might yield artifacts due to the absence of other physiological partner(s) or accessories, nonstructural proteins encoded in the viral genome or produced by the host cell upon viral infection. overexpression per se might elicit er stress responses that do not necessarily reflect what happens in an infected cell. in addition, considering that sars-cov- can infect different human cell types, the hek -based interactome might miss tissue-specific factors and introduce biases. for instance, similar experiments performed in a cells produced slightly different results in terms of interactors (stukalov et al., ) , because protein-protein interaction assays depend on the gene expression patterns specific for each cell line (table ) . nonetheless, the interactome data obtained in hek and a cells clearly identify commonly enriched pathways, including er-to-cytosol trafficking, protein quality control, inflammatory response, and dna damage (stukalov et al., ) . the role of glycosylation and protein quality control in sars-cov- infections most covs bud at the ergic level ( fig. ) and are then transported along the exocytic pathway (klumperman et al., ; stertz et al., ) . the domains facing the er lumen figure . the journey of sars-cov- in the host cell. coronavirus binds to cognate receptors on target cells via the spike proteins (dark red). this drives conformational changes that promote fusion of the virus with the host cell's plasma membrane (entry by endocytosis and green membrane-containing virion, bottom left). in the cytoplasm, viral capsids are uncoated, and the viral rna genome is translated, producing two poly-proteins (pp a and pp ab). these polypeptides are then proteolytically processed by both host and viral proteases, thereby generating nonstructural proteins (nsps) and leading to the formation of the replicase-polymerase complex (rtc). the latter is responsible for the replication of the viral genome and for the production of subgenomic rnas, which are translated into the structural proteins nucleocapsid (n), spike (s), membrane (m), and envelope (e). in addition to these genomic elements shared by other covs, the sars-cov- genome also contains eight open reading frames (orfs) that drive the production of accessory proteins. s, m, and e structural proteins and some accessory proteins are co-translationally translocated into the er, where they undergo diverse post-translational modifications, including disulfide bond formation and n-linked glycosylation. structural proteins concentrate in the ergic, where they assemble around the newly formed genome-nucleocapsid complexes. mature virions are further modified (e.g., o-glycosylated) as they proceed through the golgi complex and later stations of the secretory pathway before being released in the extracellular milieu (release by exocytosis and pink membrane-containing virions, top left). the membrane of the virus derives from the host cell, which synthetizes it in the er. undergo n-glycosylation, disulfide bond formation, and other posttranslational modifications necessary for viral tropism and host cell specificity. addition of n-glycans occurs cotranslationally in the er, and their processing continues in downstream compartments (fung and liu, ; liang et al., ; oostra et al., ) . the glycosylation profiles of the cov- s protein revealed % conservation of the n-sites with sars-cov strains (watanabe et al., ) . it has been proposed that conservation of the n-glycans in the receptor-binding domain is important for recognition by a neutralizing antibody (cr ) raised against sars-cov. indeed, the lower affinity of cr for cov- than for other sars strains might reflect the absence of a specific glycan in the n residue (yuan et al., ) . interestingly, two o-glycosylation sites are instead present only in cov- s protein. these o-glycans are thought to hinder s protein epitopes from host immune system responses and to improve ace receptor binding (watanabe et al., ) . cov- protein orf might support o-glycosylation by binding poglut , poglut , two o-glucosyltransferases, and pofut , an o-fucosyltransferase (table ). these enzymes have been shown to be pivotal for the proper glycosylation and presentation on the plasma membrane of the notch receptor (takeuchi et al., ; yang et al., ; okamura and saga, ) , confirming once more the importance of glycan processing in the maturation and transport of cov- s and other proteins. accordingly, many viral accessory proteins interact with components of the host cell's secretory pathway. we still have a limited understanding of how the assembly of viral structural proteins is coordinated. in sars-cov, copi binds the kxhxx motif found in the cytosolic domain of the s protein. this motif sicari et al. journal of cell biology is necessary for s to accumulate in ergic and interact with m and is hence crucial for assembly (mcbride et al., ) . cov- s protein presents the same motif. in both hek and a cells, it interacts with two golgi-localized proteins, zdhhc and golga (gordon et al., ; stukalov et al., ) , which form a complex localized to the plasma membrane (ko and dixon, ; ohno et al., ) . zdhhc is an acyltransferase that can localize to the er, golgi apparatus, and plasma membrane and controls golgi trafficking and palmitoylation of anthrax toxin (sergeeva and van der goot, ). golga is a golgin subfamily member that assists zdhhc activity on the plasma membrane (ko et al., ) . the zdhhc-golga complex may help s protein to reach the ergic compartment and favor assembly. interestingly, erp , slc a , and slc a were found to interact with orf , orf c, and m proteins, respectively. erp cycles between the er and the golgi to retrieve select clients back to the er (anelli et al., ; otsu et al., ) . this process is regulated by the ph and zinc gradients between the two compartments (vavassori et al., ; sannino et al., ; watanabe et al., ) . two golgi-resident transporters (slc a /znt and slc a /znt ) provide sufficient zinc to activate erp (watanabe et al., ) . moreover, erp binds the lectin ergic /lman , which promotes the production of infective covs (klaus et al., ) . lman (vip ) and ergic , two ergic like lectins, also interact with orf and nsp , respectively (table ) . vip has been shown to control transport of high-mannose proteins (gupta, ; mages et al., ) . thus, it could be that cov- takes advantage of erp -and lectin-dependent er-to-golgi transport processes to accumulate in the ergic for budding. the formation of proper disulfide bonds is key for the folding, assembly, and recognition of ace receptors by spike (oostra et al., ) . cov- orf protein binds to the er-resident oxidase ero β (table ) . orf -mediated ero β interaction may be important for disulfide bond formation in protein s or for the formation of orf multimers. considering that ero β is a powerful oxidase induced by the unfolded protein response (upr), this interaction might also reflect a response to er stress. cov- orf protein was also found to bind fkbp and fkbp (table ) , two er-resident peptidyl-prolyl cis-trans isomerases, and uggt (table ) , a key element in the glycoprotein quality table . proteins of the early secretory pathway found to interact with sars-cov- list of host proteins (columns) related to the secretory pathway that interact with different viral proteins (first line). proteins derived from the input signature (gordon et al., ) have been divided based on the cellular compartment localization (fig. a) . in fig. b, candidates related to er and golgi compartments have been sorted based on the interacting viral protein. sicari et al. journal of cell biology of a closer look at a sars-cov- achilles heel https://doi.org/ . /jcb. table . conserved features in sars-cov- and sars-cov interactomes (continued) hek (gordon et al., ) a ( stukalov et al., ) secretory pathway components proteins listed in table were matched with human protein-protein interactions obtained in a cells expressing sars-cov or sars-cov- proteins (stukalov et al., ) . the comparison highlights cell type-and virus-specific differences within an overall similarity of the interactome. control machinery and part of a large multichaperone in the er lumen. other cov- protein interactors are part of the sophisticated machineries that guarantee protein homeostasis within the exocytic compartment; that is, the balance between protein production, secretion, and degradation, a process generally referred to as "proteostasis" (klaips et al., ) . for instance, orf binds numerous proteins involved in erassociated degradation (erad), a key pathway for limiting protein condensation and aggregation in the early secretory compartment (sun and brodsky, ) . orf binds edem (er degradation enhancing α-mannosidase like protein ) and os (table ) , two erad-related proteins that are abundant in coatomer complex ii-independent vesicles. these structures are referred to as "edemosomes" and are key elements in proteostasis adjustments because they normally control the levels of erad components (araki and nagata, ) . interestingly, during sars-cov infection, edemosomes are used for forming double-membrane vesicles (dmvs) that recruit the replicationtranscription complexes (reggiori et al., ) . dmvs are also important to protecting the viral genome from sensors in the cytosol and from ifns (zinzula and tramontano, ) . finally, emc , a subunit of the er membrane protein complex, was found to interact with orf (table ) . with emc and emc , emc is thought to engage the rab gtpase in late endosomes (bagchi et al., ) . bridging late endosomes and the er, this interaction might enable efficient entry and/or excretion of viral particles. emc proteins also associate with sec (chitwood and hegde, ), a component of the translocon mediating the entry of nascent polypeptides into the er, and have been presented by gordon et al. ( ) as a sars-cov- modulator. the above lines of evidence suggest that the biogenesis of effective viral proteins requires efficient assembly and transport lines in the host's secretory protein factory. how can cov- divert them to its advantage? in general, viral infection induces er stress via the strain imposed by the massive flux of viral proteins on the cell's folding and transport machineries (zhang and wang, ) . er stress induces activation of three er resident receptors, inositol-requiring enzyme α (ire ), protein kinase r-like er kinase (perk), and activating transcription factor (atf ), which in turn promote a transcriptional program named the "upr" (hetz et al., ) . these receptors have all been implied in cov infection. for instance, s protein from sars-cov presents a upr-activating domain mapped in the central region of the s subunit (between and aa) that acts in an n-glycosylation-independent manner (siu et al., ) and is highly conserved among covs. sars-cov orf a activates atf and chop promoter activities, suggesting the involvement of the perk branch during the infection (minakshi et al., ) . perk activation induces phosphorylation of the α-subunit of the eukaryotic initiation factor α (eif α), leading to translation suppression and activation of a downstream signaling pathway known as the "integrated stress response." under the integrated stress response, translation of a majority of cellular mrnas is suppressed while mrnas containing small upstream orfs in their utr are translated, among them the proapoptotic transcription factor atf . during cov infection, eif α phosphorylation, global host mrna translation inhibition, and atf translation were detected (bechill et al., ) . however, it remains unknown how virus mrnas can still be translated normally when eif α is phosphorylated. an attractive hypothesis, based on recently published data, is that sars-cov- infection impacts protein phosphorylation profiles. for instance, larp , which interacts with the n viral protein, has been found to be less phosphorylated in cov- -infected cells. this modification increases the affinity of larp for the utrs of mrnas encoding ribosomal proteins, thus attenuating protein synthesis stukalov et al., ) . er stress induces the cytosolic kinase and rnase activities of ire α. this leads to the nonconventional splicing of x-box binding protein (xbp ) mrna, rna degradation via ire αdependent rna decay (ridd), and jnk phosphorylation. xbp spliced (xbp s) is a transcription factor that promotes transcription of genes encoding proteins involved in entry into the er, folding, glycosylation, erad, lipid biogenesis, and vesicular trafficking (huh et al., ) . ridd activity has been described as either adaptive or terminal (maurel et al., ) . it has been shown to degrade er-associated mrna to facilitate er homeostasis (adaptive) or mrna encoding prosurvival proteins, thereby contributing to er stress-induced cell death (terminal). sars-cov infection failed to induce xbp splicing, ridd, or jnk phosphorylation (dediego et al., ; versteeg et al., ) , suggesting that the ire branch of the upr remains inactive during sars-cov infection. however, cov- nsp protein binds sigma receptor . this er-resident integral membrane protein, together with sr (tmem ), which interacts with cov- orf c, modulates calcium fluxes (table ) and controls ire activation (mori et al., ; rosen et al., ) . thus, cov- may affect ire activity through its interaction with sigma receptor . last, er stress induces atf cleavage and its er-to-golgi translocation and finally an atf transcriptional program, whose main target genes consist of er chaperones (i.e., grp /bip or grp ) and erad components (shoulders et al., ) . thus, activation of the atf branch enhances the er protein-folding capacity and homeostatic balance (adachi et al., ) . overexpression of the sars-cov s protein leads to the activation of both grp and grp promoters (siu et al., ) , suggesting atf activation. notably, the small amounts of bip found on the cell surface have been proposed to help cov entry and have been suggested as a target to impede sars-cov- infection (ha et al., ) . atf cleavage and nuclear translocation of the released cytosolic fragment were observed following sars-cov orf ab transfection. in addition, orf ab has also been shown to localize to the er and to interact with the luminal domain of atf (sung et al., ). these observations are consistent with the fact that many proteins that interact with cov- orf are encoded by atf target genes. moreover, the cytosolic atf fragment has been shown to be phosphorylated and activated by p mapk (luo and lee, ) , one of the most active kinases upon sars-cov- infection , supporting a role for atf in cov- infection. collectively, these studies indicate the importance of evaluating the precise impact of cov- proteins on the activation of er stress receptors and induction of the upr. interestingly, cov- m and nsp proteins were found to bind molecules involved in the regulation of er morphology. among them, receptor expression-enhancing proteins are implicated in formation and restructuring of the er network, as well as intracellular transport of receptors to the plasma membrane. yif a recycles between the er and the golgi with a major ergic localization, maintaining homeostasis of the early secretory pathway . rab a modulates er morphology by controlling er stress responses (mateus et al., ) . thus, these cov- interactions may be crucial to controlling er morphology and homeostasis, limiting induction of er stress and apoptosis. other links between er structure and cov- infection are the orf c-scap interaction and nsp /gorasp or golga binding (table ). in particular, scap regulates the er-to-golgi translocation and activation of sterol regulatory elementbinding protein (srebp). srebp target genes are involved in lipid biosynthesis and metabolic homeostasis, two key processes during virus infection . orf c-mediated scap binding may be used to promote lipid synthesis via srebp activation for generating the envelopes needed for cov- replication. thus, available scap-targeting drugs and pre-srebp inhibitors that have shown some inhibitory effects on middle east respiratory syndrome-cov replication, together with treatment using the cholesterol metabolite -hydroxycholesterol, could be interesting to test . nsp binds gorasp and golga and other proteins regulating golgi structure and vesicular transport (table ) . especially during er stress, the gorasp -golga complex mediates the unconventional trafficking of select glycoproteins to the cell membrane. together, these results suggest that cov- may control er-golgi homeostasis maintenance by acting on diverse er-golgi-related structures and pathways. targeting er homeostasis taken together, the above observations suggest that cov- reshapes the entire secretory pathway to its own advantage. therefore, repositioning available compounds to target the secretory pathway and upr components could represent a strategy against cov- . inhibitors for er stress receptors are available and employed in numerous pathologies, including cancer and metabolic diseases. perk/eif α inhibitors are gsk , salubrinal/guanabenz, and integrated stress response inhibitor. the use of these drugs may lead to exaggerated protein load in the er lumen and increased er stress induction by preventing eif α phosphorylation and translation inhibition (matsuoka and komoike, ; hetz et al., ) . ire rnase inhibition with kinase-inhibiting rnase attenuators (kiras; kira and kira ), toyocamycin, stf- , µ c, mkc series, and b-i could be used to dampen xbp mrna splicing and ridd activity (cubillos-ruiz and glimcher, ) . although no drugs directly targeting atf are available, indirect inhibition was obtained using nelfinavir. this compound targets human immunodeficiency virus proteases and also inhibits the proteases responsible for atf cleavage (guan et al., ; sicari et al., ) . to slow down viral spread, it could be ideal to target the protein quality control machinery, which in turn would dampen viral assembly and maturation. for instance, compounds that target glycosylation enzymes, such as kinefusine and iminosugar derivative -deoxynojirimycin, which, respectively, inhibit mannosidase and α-glucosidase, are used in the clinic to dampen the morphogenesis of enveloped viruses. in addition, these drugs alter ace receptor glycosylation and transport to the cell surface, thus contributing to prevention of virus entry (winchester, ) . exacerbating er stress to induce apoptosis could be obtained by targeting er enzymes or chaperones. several protein disulfide isomerase inhibitors are available, including pacma , which specifically targets the cysteine residue present in the active site of protein disulfide isomerase proteins and blocking their capability to bind cargoes (kaplan et al., ) . finally, downstream stations of the secretory pathway could be targeted using golgicide a (ga), an inhibitor of er-golgi transport vesicles. it was already shown that ga is able to inhibit protein secretion, shiga toxin trafficking in the endosomal compartment, and replication of coxsackievirus (alborzinia et al., ) . in this perspective, ga could be used to block virus spreading and maturation, acting on golgi proteins that interact with nsp . eeyarestatin i targets erad by binding p , thus eliciting er stress. interestingly, this drug interferes with the trafficking of a shiga-like toxin (wang et al., ) . moreover, er/golgi/sec pathway-related compounds have been proposed by gordon et al. ( ) . among the u.s. food and drug administration-approved drugs, they suggested ps (er protein processing, sec inhibitor); ihvr- (er protein processing); fk- (fkbp binder); zotatifin (eif e /h, eif a inhibitor); rapamycin and rapalogs (larp , fkbp , fkbp / , mammalian target of rapamycin inhibitor); diverse sigmar / modulators, such as chloroquine, pb , and haloperidol e- , pd- , and rs-ppcc; as well as diverse sigmar / direct modulators, such as the antagonist pd- and the agonist rs-ppcc (gordon et al., ) . however, owing to the huge numbers of substrates of some of the above targets, these protocols might lack the specificity needed to guarantee a reasonable therapeutic window. to evaluate whether the select interaction of cov- with components of the secretory pathway could represent an actionable strategy, we sought to identify therapeutic targets through signature-driven pharmacogenomic analysis using three distinct ontological vocabularies (mgi mammalian phenotype, reactome, and gene ontology). to this end, the whole cov- human host interactome (gordon et al., ) was imported into bioinfominer (bioinfominer.com; lhomond et al., ) . this allowed mapping the viral interactome to a consensus semantic tree graph that provides a systemic functional overview. this was used to interrogate the l connectivity map repository, statistically selecting nontrivial network perturbagens, namely compounds that, when administered in cellular models, elicit perturbation in gene subsets of the input signature (table ) . this approach differs in the sense that it exploits the whole vector of the derived signature, which in our case integrates core regulatory proteins linking broadly perturbed modules of the host cellular physiology, in order to prioritize compounds that affect a critical subset of them, ordered according to the significance of the enrichment score, measuring their overlap with the proposed targeted gene sets of the l repository. furthermore, because bioinfominer enables delineation of the systemic tree snapshot of the mode of host infection, we exploited this feature to provide a detailed comparison of viral interactome functional profiles. this network-aided phylogenetic analysis measured the enrichment of the vertices of this graph, estimated the functional similarities of the semantic tree graphs of the viral interactomes, and applied agglomerative clustering for the pathogens under analysis. thus, cov- is robustly found to functionally share the highest similarities with enteroviruses (rhinovirus c and coxsackievirus a ; fig. ). the ontological terms obtained from the whole cov- interactome with the three bioinfominer vocabularies (mgi mammalian phenotype, reactome, and gene ontology) revealed many proteins related to the secretory pathway among the perturbed genes (last columns in table ). starting from the idea that cov- exploits host-derived secretory pathway components for assembly, budding, and spreading, we repeated the same bioinformatic analysis using the secretory pathway component list (table ) . we observed enrichment for five host-derived virus-interacting proteins (golgb , pde dip, tor a, hmox , and hyou ) involved in different processes and related to quality control and er-golgi homeostasis maintenance. specifically, golgb interacts with cov- nsp , and it is key for golgi organization and long intercisternal communication. interestingly, it is also an antigen in chronic rheumatoid arthritis. on the basis of our analysis, golgb could be a target of diverse classes of compounds, such as catechins, ganciclovir, and cetirizine-dihydrochloride. catechins, already proposed as potential anti-influenza virus agents, inhibit neuraminidase and specifically interact with ha (müller and downard, ) . moreover, because catechins also alter the viral membrane, they the table highlights compounds, ordered according to their statistical scoring, which perturb gene subsets of the input signature (last column), when administered in the indicated cell line. the input gene signature has been derived from the bioinfominer analysis, using the mgimp ontology, for the cov- secretome ( protein interactions). may also interfere with particle maturation. cetirizines present anti-inflammatory effects and decrease expression of leukocyte intercellular adhesion molecule membrane receptors in nasal epithelial cells (ciprandi et al., ) . this receptor is a major target for respiratory viruses and reasonably also for sars-cov- . finally, ganciclovir is an anti-herpesviridae family drug (cytomegalovirus, epstein-barr virus, or herpes simplex virus); it blocks dna replication, and it has already been tested in patients with cov- infection in combination with two other antiviral drugs to minimize the potential for septic shock and inflammation (yan et al., ) . a second possible therapeutic target is pde dip, which anchors components of the campdependent pathway involved in cell movement and migration to the golgi stacks (robinson et al., ; andruska et al., (andruska et al., ). nsp interaction with pde dip may help viral particle movement during the exit step. among pde dip-targeting drugs, estrone has been employed against influenza virus infection and other respiratory diseases (robinson et al., ) . a third target that came up in our analysis is tor a, a member of the aaa+ atpase superfamily. because its depletion impairs herpes simplex virus replication and induces improper envelope formation (maric et al., ) , tor a might facilitate envelope formation of sars-cov- . hmox and hyou are er residents triggered by oxygen deprivation that interact with orf and orf a, respectively. their presence in the top hits supports the involvement of hypoxia and er stress in cov- infection, possibly reflecting an antiapoptotic role. hmox and hyou are targets of radicicol, an hsp inhibitor, which blocks vesicular stomatitis virus replication (born et al., ) . the sars-cov- genome encodes for nonstructural and accessory proteins, each with different specific host-protein binding patterns. although none of them is essential for replication in other covs, some of them help with virus assembly and virulence, recruiting elements of the host cell's secretory pathway compartments. indeed, these represent a considerable part of the sars-cov- host-protein interactome, whose pathophysiological significance in the context of sars-cov- infection requires further functional studies. our bioinformatic analyses unveiled that some of those proteins, such as tor a, golgb , or hmox , could represent attractive targets for diverse classes of compounds (table ) . however, we still do not know how these proteins impact cov- biology. in-depth studies on their structure and localization during cov- infection are necessary to better characterize the related phenotypes. synergistic effects may be obtained when canonical treatments (protease and/or rna polymerase inhibitors) are accompanied by drugs targeting secretory pathway components, the rationale being that viral propagation depends on the efficiency of secretory organelles. given the strong interconnections with apoptosis and innate immunity, the upr elicited by cov- may modulate host antiviral responses from different points of view. reducing er stress and enhancing protein folding, it may promote chaperone production and massive production of suitably glycosylated s proteins, helping cov replication. the expansion of the er (likely facilitated by scap-srebp) may provide a source of membranes for dmv production and budding. erad may be important to guarantee stoichiometric virus assembly, though its role during infection remains to be understood. accumulation in apoptotic bodies might shield viruses from immune recognition (hay and kannourakis, ) . cov- highlights once more the jekylland-hyde dualism of the upr. on the one hand, the upr attenuates global translation and activates innate immunity. on the other hand, exaggerated immune response activation is associated with tissue damage and immunopathogenesis, typical in figure . network-aided phylogenetic analysis of viral pathogen infection models. the graphs depict functional comparisons of virus-host protein interactomes, using bioinfominer with the indicated vocabularies (gene ontology, mgi mammalian phenotype, and reactome). for each graph, the comparison estimates the degree of their semantic similarities via agglomerative clustering to construct the phylogenetic tree. the similarity of two viruses was calculated by averaging the values derived from three different semantic similarity measures (resnik, ; aggregate ic [song et al., ] ; and xgrasm [mazandu et al., ] ) in conjunction with the average best matches approach (mazandu et al., ) . cov infection (dandekar and perlman, ) . for all these reasons, studies on the interconnections between covs, inflammation, and upr ramifications are needed. to conclude, identification of new secretory 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and m the -nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame dynamic retention of ero α and ero β in the endoplasmic reticulum by interactions with pdi and erp coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication semantic similarity in a taxonomy: an information-based measure and its application to problems of ambiguity in natural language β-estradiol protects females against influenza by recruiting neutrophils and increasing virus-specific cd t cell responses in the lungs modulation of the sigma- receptor-ire pathway is beneficial in preclinical models of inflammation and sepsis progressive quality control of secretory proteins in the early secretory compartment by erp anthrax toxin requires zdhhc -mediated palmitoylation of its surface-processing host enzymes stressindependent activation of xbp s and/or atf reveals three functionally diverse er proteostasis environments mutant p improves cancer cells' resistance to endoplasmic reticulum stress by sustaining activation of the upr regulator atf comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus hku advanced tutorial: input uncertainty quantification the intracellular sites of early replication and budding of sars-coronavirus multi-level proteomics reveals host-perturbation strategies of sars-cov- and sars-cov epidemiology, genetic recombination, and pathogenesis of coronaviruses protein quality control in the secretory pathway the ab protein of sars-cov is a luminal er membrane-associated protein and induces the activation of atf two novel protein o-glucosyltransferases that modify sites distinct from poglut and affect notch trafficking and signaling a ph-regulated quality control cycle for surveillance of secretory protein assembly the coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mrna concentrations receptor recognition by the novel coronavirus from wuhan: an analysis based on decade--long structural studies of sars coronavirus the erad inhibitor eeyarestatin i is a bifunctional compound with a membrane-binding domain and a p / vcp inhibitory group structural basis of ph-dependent client binding by erp , a key regulator of protein secretion at the er-golgi interface zinc regulates erp -dependent protein quality control in the early secretory pathway sitespecific glycan analysis of the sars-cov- spike iminosugars, from botanical curiosities to licensed drugs a new coronavirus associated with human respiratory disease in china the first days of novel coronavirus (sars-cov- ) outbreak: recent advances, prevention, and treatment pofut promotes endometrial decidualization by enhancing the o-fucosylation of notch yipf and yif a recycle between the er and the golgi apparatus and are involved in the maintenance of the golgi structure srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target a highly conserved cryptic epitope in the receptor binding domains of sars-cov- and sars-cov virus-induced er stress and the unfolded protein response strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit we thank dr. van der goot and dr. saraste for their critical comments on fig. .e. chevet was funded by the institut national de la santé et de la recherche médicale, agence nationale de la recherche submitted: june revised: july accepted: july key: cord- -vlk cebh authors: kolter, thomas title: ganglioside biochemistry date: - - journal: isrn biochem doi: . / / sha: doc_id: cord_uid: vlk cebh gangliosides are sialic acid-containing glycosphingolipids. they occur especially on the cellular surfaces of neuronal cells, where they form a complex pattern, but are also found in many other cell types. the paper provides a general overview on their structures, occurrence, and metabolism. key functional, biochemical, and pathobiochemical aspects are summarized. together with glycoproteins and glycosaminoglycans, glycosphingolipids (gsls) contribute to the glycocalyx that covers eukaryotic cell surfaces. gangliosides are sialic acidcontaining glycosphingolipids and provide a significant part of cell surface glycans on neuronal cells. gsls are lipids that contain a sphingoid base and one or more sugar residues [ ] . sialic acids ( figure ) are nine-carbon sugars biosynthetically formed from n-acetylmannosamine and phosphoenolpyruvate [ , ] . with a mean pk a value of around . , they are more acidic than the majority of carboxylic acids and negatively charged at most physiological ph values. the name "ganglioside" was coined by the german biochemist klenk ( klenk ( - and assigned to a group of acidic gsls that he isolated from ganglion cells [ , ] and from the brains of patients who suffered from the so-called amaurotic idiocy [ , ] . sialic acid was first isolated from submaxillary mucin in [ ] . its structure was elucidated in the nineteen fifties by different groups and it was found to be identical to that of the n-acetylneuraminic acid isolated by klenk and faillard. the first structure of a ganglioside was elucidated in by kuhn and wiegandt [ ] . in , svennerholm suggested a nomenclature of brain gangliosides [ , ] . the biochemical defects underlying the diseases formerly known as amaurotic idiocy, gm gangliosidosis [ ] , tay-sachs- [ ] , and sandhoff disease [ ] were identified by sandhoff and others in the s. in their structures, gangliosides combine a glycan and a lipid portion and contribute to both, the cellular lipidome and the glycome/sialome [ ] . a great variety of carbohydrate sequences are found within the gsls [ ] , including the gangliosides [ ] . although carbohydrate residues of different structure, linkage, and anomeric configuration occur in gsls, only a limited number of the so-called series with characteristic carbohydrate sequences are found within evolutionary related organisms (table ) . within the gangliosides, sialic acids can be attached only to a few of the gsl series, in adult mammals especially to the ganglio series. among the sialic acids, n-acetylneuraminic acid is the most frequently found member in humans, but also nglycolylneuraminic acid is abundant in many other species (figure ) . a total of more than different sialic acids have been described [ , ] . they can be o-acetylated in positions , , or [ ] , but also n-deacetylated, omethylated, sulfated, or modified by lactonization [ ] (see figure ). the nomenclature of gsls specifies the glycan part of these lipids. two ganglioside nomenclature systems are currently in use to assign names to the corresponding structures. most researches prefer the short-hand nomenclature according to svennerholm, which was initially based on the migration order of ganglio-series gangliosides in chromatography [ ] . later on, it has been extended to other root structures. the more comprehensive iupac system [ ] is less frequently applied. according to svennerholm, a core structure of neutral sugars define the name of a respective series, in which the pyranose forms of dgalactose (gal), d-n-acetyl-glucosamine (glcnac), or d-n-acetylgalactosamine (galnac) are attached in defined order and linkage to lactosylceramide (galβ , glcβ cer) or β-galactosylceramide (galβ cer). the names contain information about the series ("g" = ganglio, "l" = lacto), the number of sialic acids ("a" = , "m" = , "d" = , "t" = , "q" = , "p" = , "h" = , "s" = ), and, indirectly, on the number of uncharged carbohydrates: initially it has been assumed that this number cannot exceed , so that the name "ganglioside gm " indicates that this ganglioside contains ( − " " = ) neutral sugars of the ganglio series. this series is defined by the sequence galβ - galnacβ - galβ - glccer. sialic acids can be attached once, twice, or severalfold to different positions within the core structures. most often, they are found in α , -linkage to the "inner" or "outer" galactosyl residue, and in α , -linkage to other sialic acids. ganglioside gm bears one sialic acid moiety connected to the -oh-group of the galactosyl residue in position ii of the gangliotetraose moiety (see also figure ). the corresponding iupac-iubmb short-hand name is ii neu acgg cer. structures of ganglio-series gangliosides can also be derived from the scheme of ganglioside biosynthesis (see below; figure ). in general, ganglio-series gsls of the -series bear no sialic acids on the galactose in position ii, of the a-series bear one, of the b-series bear two, and of the c-series bear three sialic acid residues. however, gm b and gd c have a "b" and "c" in their names, although both are -series gangliosides (see the scheme of ganglioside biosynthesis, figure ). gm is a gala-series ganglioside, although the "g" suggests ganglio series. figure shows the structure of ganglioside gq b, one of the most abundant gangliosides in adult human brain (g = ganglio series, q = sialic acids, − = neutral carbohydrate residues, and bseries = sialic acids attached to the "inner" galactose). ganglioside core structures can be additionally modified; they can be elongated, such as in gd agalnac [ ] ( figure ). this ganglioside occurs, for example, on spinal neurons [ , ] and can give rise to autoantibodies as a cause of variant forms of the guillain-barré syndrome [ , ] and other neuropathies [ ] . a modified gm derivative that contains taurine in amide linkage to the sialic acid carboxyl group has been identified in the brain of patients with tay-sachs disease [ ] . hybrid-type gsls and gangliosides with postglycosylation modifications add further complexity to this substance class [ ] . as an example, lacto-ganglio hybrid-type gangliosides have been identified in bovine brain [ ] . most gangliosides found in adult mammals belong to the ganglio, gala, lacto, and neolacto series. ganglioside gm (figure ), a member of the gala series, has the structure neuacα , galβ cer and is often found with an α-hydroxyfatty acid within the ceramide moiety. during development, also gangliosides with other core structures are transiently formed, such as the stage-specific embryonic antigen ssea- , a ganglioside of the globo series [ ] (figure ). in adults, globo-series gangliosides occur on human erythrocytes [ ] , in human kidney [ ] , and on various stem cells [ ] . for example, ssea- , but not ssea- or globo-h (figure ), is expressed in cord blood-derived mesenchymal stem cells [ ] . with the exception of echinoderms (marine organisms of typically pentaradial symmetry), gangliosides are usually absent from invertebrates. arthropods, for example, form acidic gsls with a manβ , glcβ , cer core, which contain glucuronic acid instead of sialic acids. for gangliosides of echinoderms [ ] [ ] [ ] [ ] [ ] , there is no systematic short-hand nomenclature. they show structural features uncommon to mammalian gangliosides, such as sialic acid residues within the oligosaccharide moieties (e.g., lg- , figure ), α , linked sialic acids (e.g., llg- , figure ), sialic acid methylation or sulfation, or a glycosyl inositolphosphoceramide core, for example, [ , ] . in cultured neurons, echinodermal gangliosides show neuritogenic and growth-inhibitory activities. in this regard, they are more potent than other figure : structure of gq b, one of the most abundant gangliosides in adult human brain, which is involved in long term potentiation, synaptic plasticity, and improvement of cognitive function [ ] . gangliosides [ , ] and potentiate the neuritogenic effect of nerve growth factor. heterogeneity is not only found within the glycan part, but also within the ceramide moiety. this can consist of different sphingoid bases [ ] , sphinganine, sphingosine, and phytosphingosine of different chain lengths ( figure ), which can be further modified by o-acetylation [ ] . in higher animals, c -and c -sphingosine are the most abundant sphingoid bases of gangliosides. the fatty acids found in the ceramide part of gangliosides are mostly saturated. α-hydroxylated fatty acids [ ] are not frequently found in brain gangliosides, but are, for example, abundant in gangliosides from intestine, liver, or kidney, and in gm . to specify the lipoform of a ganglioside, designations such as (d : / : )gm are used for a ii neu aclaccer with a sphingosine (d = dihydroxy, = one double bond; see also figure ) of carbons and a stearoyl residue ( : ) within the ceramide portion. the functional consequences of the heterogeneities in the lipid component are largely unknown, but the lipid part can mask the receptor function of ganglioside glycans via interaction with membrane cholesterol [ , ] . as another example, the ceramide portion of gm dictates retrograde transport of cholera toxin bound to gm , and only gm with unsaturated acyl chains is sorted from the plasma membrane to the trans-golgi network and the er [ ] . ganglioside profiling with respect to glycan and ceramide structures is more and more in the focus of ganglioside analysis. gangliosides are especially abundant in the brain, where their occurrence in the grey matter is about -fold higher than in white matter. in adult human brain regions, the β galt-v β galnact figure : formation of ssea- from globotriaosylceramide (gb cer). ssea, stage-specific embryonic antigen; t, transferase; fut, fucosyltransferase (modified from [ ] figure : examples for gangliosides from echinoderms: lg- from the starfish astropecten latespinosus [ ] and llg- from the starfish linckia laevigata [ ] [ ] [ ] . d-ribo-phytosphingosine values range from to μg lipid-bound sialic acid/mg protein [ ] . in the brain, ganglioside expression correlates with neurogenesis, synaptogenesis, synaptic transmission, and cell proliferation [ , ] . in cultured murine hippocampal neurons, axonogenesis, but not dendritogenesis, is accompanied by an increase in the formation of complex gangliosides and by a shift from the a-to b-series [ ] . in extraneural tissues, the ganglioside content is one-to twoorders of magnitude lower than in the brain; relatively high concentrations of ganglio-series gangliosides are found in bone marrow, erythrocytes, intestine, liver, spleen, and testis, gm in kidney, and ssea- in embryonic stem cells. cellular gangliosides form in part complex, cell-type-and tissue-specific glycan patterns [ ] . these are not stable with time, but change with physiological and pathophysiological processes such as cell growth, differentiation, viral transformation, ontogenesis, oncogenesis, embryogenesis [ , ] , lactation, or tumor progression [ ] . gangliosides of the ganglio-series are especially found in the nervous system, where they contribute to - % of the lipid content [ ] . during brain development, the ganglioside pattern changes from the prevalence of the simple gangliosides gm and gd to more complex ones such as gd a and gt b [ ] (for structures, see figures and ) . ganglioside content and composition of the brain change also during aging: for example, the amount of lipid-bound sialic acid decreased from μg/g wet weight in a -year-old healthy proband to μg/g wet weight in a -year-old individual. despite this, the concentrations of gq b, gt b, and gd b increase with age at the expense of gm and gd a [ ] (for structures, see figures and ) . changes in ganglioside composition with age also occur in liver [ ] . there are only indications on the functional consequences of such changes [ ] . gangliosides are also found in serum. there, especially gm , gd , gd a, gm , gt b, sialylneolactotetraosylceramide ( figure ), gd b, and gq b are present, where about % of them are transported by serum lipoproteins, predominantly by ldl ( %), followed by hdl ( %) and vldl ( %) [ ] . after the discovery of extracellular microvesicles (formerly called microparticles) [ ] , which were not distinguished from lipoproteins in earlier experiments, it might turn out that these assignments have to be revised. experiments in rats have shown that after injection of [ c]sialic acid-labeled gangliosides gm and [ h]sphingosine-containing labeled ganglioside gm , the gm and gm probes had serum half-lives of . and . h, respectively. after three hours, % of the gm and % of the gm probes were taken up by the liver, and a smaller extent in the central nervous system, kidneys, and lung [ ] . subcellularly, the majority of gangliosides resides in the plasma membrane [ ] . however, gangliosides also occur in organellar membranes such as in mitochondria, where gd regulates apoptosis [ ] , and in the nucleus, where they are involved in ca + balance [ , ] . the glycans found in gangliosides are sometimes modified by the acylation of sialic acid residues in different positions [ ] . o-acetylated sialic acids in gangliosides occur especially in growing cells and tissues and are regarded as oncofetal markers present on different tumors [ ] . they also serve as receptors for influenza c viruses or coronaviruses [ ] . another modified sialic acid is n-glycolylneuraminic acid (neu gc) [ ] . with the exception of certain tumors and in fetuses, it is found only in trace amounts in human tissues [ ] . as a component of glycoconjugates, neu gc is known as the hanganutziu-deicher antigen [ ] . it is abundant in many species of the deuterostome lineage, including simians, mice, rat, beef, pork, or lamb, but is nearly absent from birds and reptiles [ ] . neu gc on glycoconjugates contributes to xenoantigenicity in pig-human xenotransplantation [ ] , and in cats, neu gc distinguishes the blood groups a and b: [neu gc] gd is found in feline blood group a erythrocytes, [neu ac] gd on blood group b, and feline blood group ab erythrocyte membranes contain [neugc] gd , [neu ac,neu gc]gd , and [neuac] gd [ ] . humans cannot synthesize neu gc due to an irreversible inactivation of the cmah gene on chromosome p . encoding cytidine monophosphate-n-acetylneuraminic acid hydroxylase [ ] . this enzyme converts cmpneu ac to cmpneu gc and its function is thought to be lost during a "sialoquake" in human evolution [ , ] . determination of neu gc and neu ac-containing gangliosides is either achieved by classical chromatographic techniques combined with antibody staining [ ] , or, with higher sensitivity, by combination of chromatography with esi-ms [ ] . a potential application is the immunochemical detection of [neu gc]gm as biomarker of nonsmall-cell lung cancer [ ] . also ganglioside lactones ( figure ) have been detected in various tissues, for example, gd lactone in mouse brain [ ] and gd b lactone in human brain [ ] . ganglioside lactones are more immunogenic than gangliosides [ ] and occur on tumor cells such as melanoma as tumor-associated antigens. in vitro, lactonization of gangliosides can be followed by a strong negative cotton effect at nm in cd spectroscopy [ ] . temporal and spatial differences are also observed for the ganglioside lipid part. in undifferentiated neuronal cell cultures, gangliosides with c sphingosine are present only in trace amounts, but their content increases with the onset of cell differentiation [ ] . in rat brain, the fraction of gangliosides containing c sphingosine increases with age [ ] in cerebellum [ ] or forebrain [ ] . spatial differences regarding the sphingoid base chain length have also been detected in mice: while gangliosides containing c species were widely distributed throughout the frontal brain, c species are selectively localized along the entorhinalhippocampus projections [ ] . fatty acid and sphingoid base composition is also different between human motor and sensory nerves [ ] . nutrition. since gangliosides are components of most vertebrate cell types, they are ingested with the nutrition, for example, with egg yolk (gm , gm , and gd ), meat, or in milk [ ] . milk contains gangliosides, especially gd and gm , in the membrane fraction of the fat globule. dietary gangliosides modify the intestinal microflora and prevent infections during early infancy [ ] . in infants, more than % of dietary gangliosides survive the passage through the stomach, in part with acid-catalyzed lactonization, and are absorbed in the intestine [ ] . ingestion of dietary gangliosides leads to an increase of gangliosides in serum. in human nutrition, sialic acid derived from gangliosides and other glycoconjugates is an essential nutrient for the rapidly growing brain in infants [ ] . the pathophysiological consequences of nutritional neu gc uptake are unknown. in the past, ganglioside structures and levels were obtained by comprehensive chemical analysis, while nowadays this is attempted within lipidomics using mass spectrometry as the key technology [ ] . in general, gangliosides are isolated from tissues and body fluids by chloroform-methanol extraction [ , ] . extraction efficiency can increase when small amounts of water are present in the extraction solvent [ ] , for example, using the solvent system chloroform : methanol : water ( : : ) [ ] . when extraction is followed by a partition step such as that developed by folch et al. [ ] , gangliosides-in contrast to the majority of other lipid classes-partition into the upper, aqueous phase. from there, they can be isolated by a solid phase extraction and separated from neutral gsls by anion exchange chromatography [ ] , such as with deae (=diethylaminoethyl) sephadex [ ] . o-acetylation of sialic acids and also ganglioside lactonization [ ] are modifications that are lost under alkaline conditions. these are often applied to remove glycerophospholipids that contain fatty acids in ester linkage [ ] . if information on these modifications is desired, gangliosides from tissues can be determined without alkaline treatment, for example, after chloroform/methanol extraction in a ratio of : and a subsequent partition step [ ] . separation of gangliosides according to their glycan composition is achieved by thin layer chromatography (tlc) [ ] and by hplc and other techniques that can be coupled to mass spectrometry [ ] . this facilitates their identification by mass spectrometry and is required for their characterization by staining with suitable antibodies [ , ] , lectins, or other binding proteins [ ] . although not required for mass-spectrometric profiling, separated ganglioside classes can also be further separated according to their ceramide structure by reversed phase chromatography [ ] [ ] [ ] . quantification can be achieved by staining and densitometry, or-if suitable standard substances are availableby mass spectrometry. since the biosynthetic machinery generates heterogeneities within both, the lipid and the glycan part, comprehensive ganglioside analysis is a highly demanding task within lipidomics [ ] . in addition to glycoforms that are also well known from glycoprotein analysis, "lipoforms" [ ] become increasingly important to understand ganglioside metabolism and function. various protocols for ganglioside determination by mass spectrometry have been developed [ ] . they are largely based on electrospray mass spectrometry as ionization technique; but also maldi plays a role. the available methods range from preanalytics to bioinformatic data handling and include imaging methods using maldi and secondary ion mass spectrometry (sims) to determine the spatial distribution of the analytes [ ] . in addition to their constitution, little is known about the conformation of gangliosides in their native, membrane-bound surroundings. x-ray data are not available for gangliosides, although isolated glycans have been investigated by various means. for a simulation of gm conformations in a bilayer, compare [ ] , which, for example, shows that the glucose moiety of gm is buried within phosphatidylcholine head groups. the diversity of cell surface glycans, including that of gangliosides, is generated within the golgi apparatus [ ] , and the heterogeneities within the ceramide part result from the biosynthesis of ceramide at the endoplasmic reticulum (er). de novo synthesis of gangliosides can be distinguished from salvage processes [ , ] , in which sialic acids, sugars, fatty acids, and sphingoid bases are recycled. the latter process can predominate by far in differentiated cells. ganglioside biosynthesis starts with the formation of ceramide ( figure ) at the cytoplasmic leaflet of the er membrane [ ] [ ] [ ] . the first step, the condensation of l-serine and a coenzyme a-activated fatty acid is catalyzed by the pyridoxal phosphate-dependent serine palmitoyltransferase (spt) [ ] . the incorporation of l-serine into gsls can be used to monitor their de novo biosynthesis using l-serine radiolabelled in the position isrn biochemistry (the carbon in position is lost as carbon dioxide). in the brain, the external supply of l-serine by astrocytes is essential for neuronal lipid biosynthesis and brain development [ ] . in agreement with this observation, genetically engineered rodents with deficient phosphoglycerate dehydrogenase required for l-serine formation from d-glucose show drastically reduced ganglioside levels, defects in brain morphogenesis, and drastically reduced lifespan [ , ] . the next step in sphingolipid biosynthesis is the nadph-dependent reduction of -ketosphinganine to sphinganine by -ketosphinganine reductase, followed by acylation of sphinganine to dihydroceramides of different chain lengths [ ] . during salvage, also other sphingoid bases are acylated by n-acyltransferases of the lass family. lass encodes ceramide synthase , which is expressed in the brain and involved in the formation of the membrane anchor of gangliosides. in mice, spontaneous recessive mutations in the lass gene are associated with cerebellar ataxia and purkinje cell degeneration [ ] . although the ceramide part of brain gangliosides contains mostly nonhydroxylated fatty acids, apparently all members of the lass family are also able to transfer the corresponding -hydroxy-fatty acids [ ] . dihydroceramides are dehydrogenated to ceramide by the dihydroceramide desaturase des [ ] , or hydroxylated to phytoceramides by des . ceramide is the common precursor of gsls and sphingomyelin and is transported to the golgi apparatus at least in part in a protein-dependent manner by the transport protein cert [ ] [ ] [ ] . formation. gsl synthesis continues by the stepwise transfer of nucleotide-activated monosaccharide units first on ceramide and then on gsls with growing glycan chains. glycosidation is coupled to exocytosis through the golgi apparatus to the plasma membrane [ ] at the rate of bulk vesicle flow [ ] . the complex ganglioside and gsl glycoforms on eukaryotic cell surfaces are generated by only a few enzymes that act within a combinatorial biosynthetic pathway [ , ] . the first glycosyltransferases involved in ganglioside biosynthesis have been characterized in the laboratories of roseman and basu [ ] . according to the number of sialic acids connected to the "inner" galactosyl residue, ganglioseries gangliosides are classified into members of the -, a-, b-, and c-series ( figure ). b-series gangliosides contain the neu acα , neu ac sequence, which is commonly not found in glycoproteins. higher members of these different subseries can be formed by the action of the same glycosyltransferases, which show less specificity than those acting early in the pathway [ ] [ ] [ ] [ ] [ ] . the glycosyltransferases and sialyltransferases [ , ] of the ganglioside biosynthetic pathway are expressed in a cell-type-and developmental-dependent fashion. ganglioside pattern changes during the development of the brain [ ] , and after differentiation, differences in glycolipid composition have even been found between different neuronal cell types [ ] . in addition, ganglioside patterns vary between different cell types and change with the differentiation of the cell. as an example, β , -n-acetylglucosaminyl-transferase expression, which leads to the formation of glycolipids of the lacto and neolacto series ( figure ), is high during murine embryonic development and decreases after birth to undetectable levels in most cell types [ , ] . in adult animals, expression is high in spleen [ ] , and in cerebellum, it is restricted to purkinje cells [ ] . gsls including gangliosides are formed biosynthetically at intracellular membranes from which they are transported to the plasma membrane by exocytotic membrane flow [ ] . while many human diseases are known that are due to defects in gsl and sphingolipid degradation, the only known human disease caused by a defect glycosyltransferase of ganglioside biosynthesis is the human autosomal recessive infantile-onset symptomatic epilepsy syndrome, which is caused by a nonsense mutation in the gene encoding gm synthase [ ] . a principal difference between ganglioside biosynthesis in the golgi apparatus and degradation in the endolysosomal compartment is that during gsl formation, membranebound glycosyltransferases interact with their membranebound glycolipid substrates by diffusion within the twodimensional plane of the lipid bilayer. therefore, reaction rates can become independent of the reaction volume and obey two-dimensional enzyme kinetics. this means that kinetic constants can be normalized on lipid surface area instead of reaction volume, for example, in terms of the amount of membrane protein [ ] . as a consequence, glycosyltransferases that lack their transmembrane domain lose most of their activity towards membrane bound substrates [ ] . during degradation in endosomes and lysosomes, the glycosidases are soluble enzymes, and the substrates are membrane-bound. this explains in part the requirement for endosomal and lysosomal lipid-transfer proteins for the degradation of gsls with short glycan chains, which is not the case in biosynthesis. in addition to ganglioside biosynthesis in the golgi apparatus, there are also indications ganglioside formation by plasma membrane-associated glycosyltransferases [ ] . in the monoglycosylceramides glucosylceramide (glccer) and galactosylceramide (galcer), which are also called cerebrosides, the hexosyl residues are present in βanomeric configuration. galcers with α-configuration occur only in lower organisms [ ] and are highly immunogenic for mammals [ ] . most gangliosides are biosynthetically derived from glccer; only ganglioside gm is derived from galcer. ganglioside gm has been discovered as a minor component of human brain gangliosides [ ] , where it is localized within myelin [ ] . it also occurs, for example, on erythrocytes, kidney, and in the intestine and is abundant in some fish species. however, the most frequently found members of the gala series are galcer and sulfatide (galcer- -sulfate) in oligodendrocytes, schwann cells, kidney, testis, and intestine. they are present in high concentrations in the multilamellar layers of the myelin where they are required for glial adhesion [ ] , apparently via interaction between the carbohydrate head groups of sulfatide and galcer on different myelin layers [ ] . myelin lipids contain the highest fraction of -hydroxy-fatty acids, which are formed by fatty acid hydroxylase- [ ] . their presence in gala-series gsls contributes to carbohydrate-carbohydrate interactions between the gsls [ ] . in contrast to galcer synthase, glccer synthase appears to be dispensable for oligodendrocytes [ ] . while ceramide galactosylation catalyzed by udp-glucose:ceramide galactosyltransferase (galt ) [ ] occurs at the er membrane, the later steps of gala-series gsl biosynthesis, formation of sulfatide [ ] , digalactosylceramide [ ] , and ganglioside gm take place in the lumen of the golgi apparatus. in contrast to most glycosyltransferases in ganglioside biosynthesis, which are type ii transmembrane proteins, ceramide galactosyltransferase is a type i transmembrane protein with the catalytic domain on the luminal side of the er [ ] . according to data obtained in zebrafish and mice, gm can be formed by st gal v, which can also make gm . therefore, gm and gm formation appear to depend on the availability of their precursors, galcer and laccer [ ] . little is known about the function of gm . it can interact with the myelin basic protein, shows immunosuppressive properties, and can prevent experimental allergic encephalomyelitis in guinea pigs [ ] . glucosylceramide. the first step in the biosynthesis of most gangliosides is the transfer of a glucose residue from udp glucose to ceramide catalyzed by udp-glucose:ceramide glucosyltransferase [ , ] . although glccer and galcer synthases catalyze similar reactions, their cdnas share no sequence homology. ceramide glucosyltransferase is a type iii transmembrane protein. it forms noncovalent dimers or oligomers [ ] with their cterminal catalytic domains in the cytosol [ ] . since the formation of glccer occurs on the cytoplasmic face [ ] and that of laccer on the luminal site of the golgi membrane [ ] , glucosylceramide has to be translocated across a membrane. this is mediated by a flippase of unknown identity: the abc-transporters, abc-b and -c , translocate short chain glccer analogs through the golgi membrane [ , ] . transversal translocation can be carried out after transport of glccer by the cytoplasmic lipid-transfer protein fapp (four-phosphate adaptor protein ) either to the er [ ] , where it might be translocated by an uncharacterized flippase [ ] , or at the trans-golgi [ ] . a part of the glccer pool can reach the cytosolic leaflet of the plasma membrane where it can be degraded by the β-glucosidase gba [ ] . candidate cytosolic glccertransporters are the glycolipid transfer protein gltp and fapp . the biosynthesis of higher gangliosides occurs on the luminal face of the golgi apparatus [ ] , so that their glycan chains are orientated extracytoplasmic. laccer is formed by galactosyltransferase i, which transfers a galactose residue from udp galactose to glucosylceramide [ ] . further carbohydrate residues are transferred in a stepwise manner to the growing glycan chains. laccer and its sialylated derivatives, the hematosides gm , gd , and gt ( figure ) serve as precursors for complex gangliosides of the -, a-, b-, and c-series. these different series ( figure ) are characterized by the presence of no ( -series), one (a-series), two (b-series) or three sialic acids residues linked to the position of the "inner" galactosyl residue. in adult mammalian brain, gangliosides from the -and c-series are found only in trace amounts, and gm b and gd α are transiently expressed during chick brain biogenesis [ ] . -series gangliosides (gm b, gd c, and gd α) are found in genetically engineered mice deficient in st gal v (gm synthase), where they are present in amounts that correspond to the total ganglioside content of normal animals [ ] . these mice are not able to form gm and higher gangliosides of the a-c-series. they display altered glucose homeostasis with an accelerated insulin receptor signalling pathway, a key finding that demonstrates the inhibition of the insulin receptor by gm or a higher ganglioside derived from it in vivo [ ] . c-series gangliosides (figures and ) are formed during mammalian brain development where they are thought to be involved in growth, differentiation, and migration of neuronal cells. they are abundant in fish brain, and in adult rats; they occur in liver, kidney, and pancreas [ ] and in tumors such as glioma. the transferases that catalyze the first steps in ganglioside biosynthesis show high specificity towards their glycolipid substrates. the relative amounts of laccer, gm , gd , and gt seem to determine the amount of -, a-, b-, and c-series gangliosides. the glycosyltransferases that act late in this pathway represent a kind of assembly line and transfer the respective carbohydrates to glycosyl acceptors that differ only in the number of sialic acid residues bound to the "inner" galactose residue. the complex "α-"gangliosides with sialic acid moieties in α , -glycosidic linkage to n-acetylgalactosamine residues is specific for cholinergic neurons [ ] and has been added later to the biosynthetic scheme [ ] . in mice, the sialyltransferases that form gangliosides gd a and gt b have been identified as st gal ii and st gal iii [ ] . a significant advance towards understanding the function of the complex ganglioside pattern found in eukaryotic cells is the development of mice with defects in distinct biosynthetic steps [ ] . a mouse melanoma cell line deficient in glccer and glccer-derived gsls was viable and showed only minor changes in cellular morphology and growth rate. from these observations it was concluded that gsls including gangliosides are not essential for animal survival [ ] . later, it was reported that mice with targeted disruption of the ceramide glucosyltransferase gene displayed no cellular differentiation beyond the primitive germ layers and died around day . of embryonic development [ ] . mice deficient in b galnt i (gm synthase) are not able to form gm , gd , and higher gangliosides derived from them. although these animals show only subtle impairment of brain function [ ] , they exhibit multiple defects, such as axonal degeneration, defects in myelination [ ] and motor function [ ] , or an impaired response of t cells to interleukin [ ] , only to mention a few. later studies showed that cd -and cd -positive t cells require different ganglioside subsets for activation [ ] . the mutant male mice are sterile and also show morphological and functional defects in the testis [ ] . further examination of galnac-transferase deficient mice revealed that gm -deficiency is accompanied by parkinsonlike symptoms, which could be rescued by l-dopa or the membrane permeable gm -analog liga (see figure ) [ ] . this is in agreement with a series of reports that gm can alleviate symptoms in models of parkinson disease, for example, [ ] . in parkinson disease, anionic lipids and especially gm inhibit aggregation of α-synuclein to cytotoxic fibrils [ ] . mice deficient in st sia i (gd -synthase) do not form gd and b-series gangliosides. they have a normal life span and are without detectable developmental defects [ ] . when these mice were crossbred with mice carrying a disrupted b galnt i gene, the resulting double mutant mice express only ganglioside gm as their major ganglioside. these "gm -only-mice" are extremely susceptible to sound stimuli, develop lethal seizures, and display a sudden death phenotype [ ] . double knockout mouse deficient in b galnt i and st gal v (gm -synthase) are not able to form any ganglioside of the ganglio-series. these animals are severely diseased and show elevated levels of laccer, laccersulfate, and traces of other gangliosides that are present also in normal brain [ ] . sphingolipid biosynthesis is a highly regulated process and also coordinated with sterol and glycerolipid biosynthesis. sphingolipids are major regulators of lipid metabolism and activate sterol-regulatory element binding proteins (srebps) [ ] . the sphingomyelin synthaserelated synthase, the ceramide transporter cert, and proteins of the orosomucoid-(orm)-familie seem to play key roles in sphingolipid homeostasis [ ] . ganglioside pattern are characteristic for a cell type in a certain differentiation state, and, for example, mice deficient in gm -synthase that cannot form the typical brain gangliosides show a ganglioside content similar to that of normal animals [ ] . how exactly the relative amounts of gangliosides are controlled is not clear [ ] , but the transcriptional regulation of transferase genes seems to be a key point [ ] . the picture gets more complicated by the fact that different transferase isoforms with different properties can be present: three murine gm -synthase isoforms that arise from two transcripts have been characterized. one is resident in the er membrane, the two others in the golgi, but with different half-life [ ] . in addition, the kinetic parameters of the transferases, their topological organization within the golgi apparatus, or spatial neighborhood to other transferases will influence the resulting ganglioside pattern. an attempt has been made to calculate glycolipid pattern on the bases of the kinetic constants of the transferases, that were estimated from the steady state concentrations of the glycolipid substrates in intact cells [ ] . contradictory results have been reported on the subcellular localization of the glycosyltransferases involved in the biosynthesis of ganglioseries gangliosides [ ] . an additional feature of ganglioside biosynthesis and its regulation [ ] is the formation of functional complexes, as predicted by roseman [ ] . in these complexes [ ] , the glycosyltransferases do not only form functional platforms, but can also show altered activity and suborganellar localization [ ] . one of these complexes characterized in certain cho cells [ ] comprises b galnt i and b galt iv (figure ), so that it can accept gm and release gm . this might explain why the brain contains large amounts of gm and gd a, but little gm . also galt i, st gal v, and st sia i can form such a complex [ ] . . . general. the constitutive degradation of gangliosides takes place in endosomes and lysosomes. in addition, also the plasma membrane-associated sialidase neu [ , ] can degrade gangliosides and is, for example, highly expressed on melanoma cells [ ] . even the nuclear envelope contains sialidases, with neu in the inner and neu in the outer nuclear membrane [ ] . lysosomal ganglioside degradation takes place after the endocytosis of parts of the plasma membrane at intraendosomal and intralysosomal membranes and related lipid aggregates. this requires the presence of suitable glycosidases [ ] , of an appropriate ph, in some cases also of lipid-transfer proteins, and of an appropriate composition of the ganglioside-containing membranes [ ] . as proposed in , two different membrane pools are present in endosomes and lysosomes [ ] (figure ). they differ in lipid-and protein composition and function. while the luminal membrane pool that is derived from the plasma membrane or by autophagy is degraded, the perimeter membrane ( figure ) is protected from degradation by various means [ ] . this ensures the integrity of the compartment, which can be abolished during apoptosis [ ] . a marker lipid that is exclusively found in luminal membranes [ ] is bis(monoacylglycero)phosphate (bmp; figure ) , chemically incorrect also named as lysobisphosphatidic acid (lbpa). bmp plays a key role for membrane degradation [ ] and is formed from phosphatidylglycerol [ ] . due to its sn , sn configuration, it is only slowly degraded by lipases and persists on inner membranes, in which it can amount up to % of total phospholipids [ ] . with a predicted pk a value of about , bmp is negatively charged even at lysosomal ph. in vitro studies show that negatively charged lipids are required for binding of lysosomal proteins to membranes. although other negatively charged lipids such as dolichol phosphate or phosphatidylinositol can be present on luminal membranes, bmp appears to be the key factor that distinguishes this membrane pool from the perimeter membrane. on the other hand, the perimeter membrane of endosomes and lysosomes shows an entirely different lipid and protein composition. it is protected by a glycocalyx formed by highly n-glycosylated integral membrane proteins [ , ] , and ganglioside gm present in this membrane is resistant to degradation [ ] . ganglioside degradation starts with the action of glycosidases that cleave off monosaccharide units from the non-reducing end of the ganglioside glycan chains. this happens in a sequential manner, which explains the different human diseases that are associated with defects in this pathway. the glycosidases are soluble enzymes in the lumen of endosomes and lysosomes. it turned out that their activity is not sufficient towards gsl substrates with cleavage sites in proximity to the intralysosomal membrane surface. although also other factors play a role, this can be attributed to steric hindrance by adjacent membrane components that impede the access of the soluble enzyme. for example, in wild-type and gm -activator deficient fibroblasts, radiolabelled gd agalnac (figure ), which has the same terminal trisaccharide as gm , is degraded in the absence of the gm -activator protein, while the degradation of gm itself is strictly dependent on the presence of the activator [ ] . as glycosidase substrates, gsls with four carbohydrate residues or less require the additional presence of small lipid binding glycoproteins, either the gm activator protein or one of the four saposins a-d. these act in part as lipid-transfer proteins that extract the membrane-bound substrates and present them to the hydrolases. they have different specificities and mechanisms of action [ ] . in the case of gangliosides, at least the gm -activator protein and saposin-b participate in the degradation of gm , gm , and gm ( figure ). in vitro, in addition to enzymes and activator proteins, also an appropriate membrane-lipid composition of the ganglioside-containing membrane is required for degradation [ ] . saposin-a [ ] and saposin-b [ ] extract membrane lipids much better from membranes that are rich in bmp and poor in cholesterol. bmp also increases the ability of the gm activator to solubilize lipids [ ] and stimulates the hydrolysis of membrane-bound gm by gm β-galactosidase [ ] and of ganglioside gm by β-hexosaminidase a [ ] . bmp also stimulates hydrolysis of the kidney sulfatide with ganglio-series gsl-core sm (gangliotriaosylceramide-ii sulfate) by β-hexosaminidases a and s in the presence of the gm activator [ , ] . cholesterol, which is known to stabilize lipid bilayers, has to be transported from intraendosomal membranes to the npc protein resident in the endosomal perimeter membrane by the soluble lipid-transfer protein npc . in vitro, this transfer is greatly stimulated by bmp and strongly inhibited by sphingomyelin [ ] . saposins are small, water-soluble lysosomal lipid-binding and -transfer proteins of about - kda molecular weight. they are derived from a common precursor protein, prosaposin, by proteolytic processing. saposins belong to a family of proteins with conserved three-dimensional fold [ ] and occur as homo-and heterodimers and -oligomers. the first saposin has been characterized in as the so-called sulfatide activator since it enables the degradation of sulfatide by arylsulfatase a [ ] . today this protein is known as saposin-b or sap-b. saposin-b has many functions: it is a lipid-binding protein with broad specificity [ ] and forms water-soluble lipidprotein complexes [ ] . with respect to gangliosides, it is able to stimulate the degradation of ganglioside gm by gm -β-galactosidase [ ] . studies in cultured human skin fibroblast derived from saposin-b and prosaposin-deficient patients show that it is also required for the degradation of gm [ , ] . it is important to note that glycosylation of saposin-b is essential for some of its functions and that human patients without this postranslational modification die, although the unglycosylated variant protein is present in lysosomes [ ] . mechanistically, saposin-b dimers seem to act similar to the gm activator: x-ray data indicate that they can adopt two conformations, an open one and a closed one [ ] . according to a model view, the open conformation interacts directly with the membrane and extracts the lipid ligand. this is accompanied by a change to the closed conformation in which the ligand is exposed to the degrading enzyme in a water-soluble activator-lipid complex. human patients with an inherited deficiency of saposin-b develop an atypical form of metachromatic leukodystrophy with the accumulation of sulfatides, digalactosylceramide, and globotriaosylceramide [ ] (see figure for structures). saposin-b knockout mice show enhanced levels of sulfatides especially in brain and kidney [ ] . activator. the gm activator is a small glycoprotein of . kda in its deglycosylated form and is required for the degradation of ganglioside gm by βhexosaminidase a in vivo [ ] . inherited deficiency of the gm -activator protein leads to the ab variant of gm gangliosidoses [ ] . based on the x-ray structure [ , ] and data from photoaffinity labeling [ ] , in some respects the gm -activator acts in a way similar to saposin-b. a more detailed picture of the binding mode was derived from binding studies using a spin-labelled gm activator to phosphatidylcholine bilayers [ ] . the protein can extract a variety of lipids, which has been exploited for assay development [ ] . however, its major function is to form a water-soluble gm -protein complex that is the native michaelis-menten substrate of β-hexosaminidase a [ ] . negatively charged lipids such as bmp, dolichol phosphate, or phosphatidylinositol increase the extraction efficiency towards gm [ ] , gm [ ] , and other lipids [ ] from liposomal membranes. binding characteristics of the gm activator are altered by the presence of a his tag [ ] . in langmuir experiments, the gm -activator protein is able to penetrate into a phospholipid monolayer, but only when the lateral pressure is below a critical value, which depend on the lipid composition and is in the range from to mn/m [ ] . in addition to its function as a ganglioside-transfer protein, the gm activator binds also other lipids like phosphatidylcholine [ ] and platelet activating factor (paf) and inhibits its action [ , ] . it is not clear whether the gm activator displays inherent hydrolytic activity towards lipid substrates such as platelet activating factor [ ] or phosphatidylcholine [ ] . apparently unrelated to its gm transfer property is the function of the gm activator as adipokine [ ] . gm activator orthologs might serve different functions in other organisms, for example as a pheromone-binding protein in drosophila [ ] , or an inhibitor of paf-induced chemotaxis in nematodes [ ] . the saposins and the gm -activator play major roles in the transfer of lipid antigens to membrane-resident cd proteins [ , ] . degradation. gm -β-galactosidase is a protein of kda, which is derived from an -kda precursor [ , ] . an alternatively spliced, enzymatically inactive β-galactosidase form of kda is an elastin/laminin-binding protein [ ] . gm -β-galactosidase is part of a lysosomal multienzyme complex, together with the so-called protective protein (carboxypeptidase a), sialidase, and n-acetylaminogalactose- -sulfate sulfatase [ ] . gm -β-galactosidase catalyzes the hydrolytic cleavage of several β-galactosides. the hydrolysis of ganglioside gm to gm requires the presence of either the gm -activator protein, or saposin-b [ ] , or, in vitro, of an appropriate detergent. degradation. gm is degraded by the cleavage of the n-acetylgalactosaminyl residue by β-hexosaminidases. in mice, the substrate specificity of the murine lysosomal sialidase allows for a significant cleavage also of the sialic moiety in gm (to yield ga ) [ ] . cleavage of the galnac residue requires the presence of the gm -activator protein in vivo, or of an appropriate detergent in vitro. three gene products participate in gm hydrolysis, the β-hexosaminidase αand β-chains, and the gm -activator protein. β-hexosaminidases are dimers that result from the combination of their αand β-subunits and differ in properties such as stability and substrate specificity. β-hexosaminidase a with subunit composition α,β cleaves terminal β-glycosidically linked n-acetyl-glucosamine and n-acetylgalactosamine residues from negatively charged and uncharged glycoconjugates by a retaining doubledisplacement mechanism. the enzyme has two active sites, one on the α-chain and the other on the β-chain [ ] . β-hexosaminidase b (ββ) [ , ] predominantly cleaves uncharged substrates such as ga and oligosaccharides with terminal n-acetyl-hexosamine residues (see also figure ). β-hexosaminidase s (αα) is thermolabile and of secondary significance for gm degradation, but it contributes to the degradation of glycosaminoglycans and sulfated glycolipids [ ] . defects in enzymes and other proteins required for lysosomal degradation of complex lipids and of oligomeric or polymeric biomolecules lead to inherited diseases, the lysosomal storage diseases [ ] . they can be classified according to the stored substances, as sphingolipidoses [ ] , mucopolysaccharidoses, mucolipidoses, glycoprotein-, and glycogen-storage diseases [ , ] . ganglioside degradation is impaired in the gangliosidoses and secondarily also in other sphingolipid storage diseases [ ] . the principles [ ] governing pathogenesis [ , ] and therapy of sphingolipidoses [ ] are also valid for the ganglioside storage diseases. key factors are the residual activity of the degrading system, which determines the course of the disease [ , ] , and the cell-type-specific expression of storage material. due to the cell-type-specific expression of gangliosides, the central nervous system is especially affected in the gangliosidoses. in sphingolipidoses in general, the storage lipids coprecipitate other hydrophobic substances present in the endolysosomal compartment, lipids and proteins, as secondary storage products [ ] . in niemann-pick disease, type c, which is a primary defect of endosomal cholesterol transport, a secondary accumulation of sphingomyelin (therefore the name niemann-pick) and of gangliosides is observed that is also of therapeutic relevance [ , ] ; for a remarkable treatment of niemann pick c fibroblasts with a histone deacetylase inhibitor, compare [ ] . secondary storage of gangliosides gm and gm occurs also in hurler disease [ ] (mucopolysaccharidosis type i; α-liduronidase deficiency). lipid storage produces a kind of traffic jam [ , ] , which interferes with lipid transport and lysosomal function. primary and secondary storages substances can impair nutrient delivery via the endolysosomal system: as demonstrated in mouse models of gm gangliosidoses and in a variant form of the gm gangliosidoses, sandhoff disease, iron homeostasis is impaired in the animals, and supplementation of the animals with iron ions increased their life expectancy by nearly % [ ] . since also autophagy can be impaired in lysosomal storage diseases [ ] , both pathways may lead to a shortage of nutrients. ganglioside degradation is impaired in the gangliosidoses. in another disease, galactosialidosis, the primary defect of carboxypeptidase a (protective protein), leads to a secondary loss of β-galactosidase and sialidase neu accompanied by gm storage [ ] . gangliosidoses are caused by defects in the genes encoding glycosidases or lipidtransfer proteins that are required for lysosomal ganglioside degradation. the theoretical basis for the therapeutic approaches towards gangliosidoses is the "threshold theory" [ ] , which predicts that the ratio of substrate influx into the lysosomes and the degradation capacity determine the course of the diseases. both parameters can be addressed by different therapeutic approaches [ ] . . . gm gangliosidosis. gm gangliosidosis is caused by an inherited deficiency of gm -β-galactosidase (acid βgalactosidase; glb ; ec . . . ) [ ] . after the description of the first patients [ ] it became also known as landing diseases [ ] . it is a rare disease with an autosomal recessive mode of inheritance and characterized by the accumulation of gm and ga (figure ) in neuronal cells [ ] . according to the substrate specificity of the variant enzyme in the patients, an inherited defect of the β-galactosidase can also lead to another disease, morquio disease, type b. three clinical forms of gm gangliosidosis can be distinguished, infantile (type ) gm gangliosidosis with the developmental arrest and progressive deterioration of the nervous system in early infancy and a life expectancy of about years, late infantile/juvenile form (type ), and an adult/chronic form (type ). dysmorphic changes characteristic for morquio disease type b are less prominent or completely absent in these clinical forms. in addition to gm , other enzyme substrates accumulate, such as ga (figure ) [ ] , oligosaccharides from glycoproteins, and intermediates of keratin sulfate degradation [ ] . these substances are stored in different organs, according to their major site of biosynthesis. lysosomal gm accumulation in neurons leads to the degeneration of the nervous system. like in other storage diseases, an inflammatory response [ ] , neurorestorative properties of excess ganglioside gm [ ] in the plasma membrane, and an unfolded protein response [ ] contribute to pathogenesis. such as in other sphingolipidoses [ ] , severity and progression of the disease correlate with the residual enzymatic activity in cells and body fluids. morquio type b disease clinically resembles a mild phenotype of morquio a disease, where keratan sulfate accumulates due to n-acetyl-galactosamine- -sulfatase deficiency. like gm gangliosidosis, morquio type b is due to the inherited defect of gm -β-galactosidase. it is characterized by the predominant storage of keratan sulfate and oligosaccharides with terminal β-galactosyl residues. patients show generalized skeletal dysplasia without involvement of the nervous system and without hepatosplenomegaly; for a clinical description, compare [ ] . differences between gm gangliosidosis and morquio b disease can be attributed to a lower affinity and activity of β-galactosidase variants towards substrates with gal-β , -glcnac motifs in morquio patients compared to the gal-β , -galnac motive present in ganglioside gm [ ] . there is no causal therapy available for gm -gangliosidosis; however, progress is made towards the development of pharmacological chaperones also for this lysosomal disease [ ] [ ] [ ] . the gm -gangliosidoses are caused by defects in degradation of ganglioside gm [ ] . the three variant forms of the gm -gangliosidoses are named according to the hexosaminidase isoenzyme that remains intact. the b-variant, in its infantile course better known as tay-sachs disease, is caused by the deficiency of hexosaminidases a and s, but with normal hexosaminidase b. the variant, or sandhoff disease, is caused by the deficiency of the β-chain and the resulting deficient activity of β-hexosaminidases a and b (therefore, none of the major enzymes is intact), however with the remaining activity of β-hexosaminidase s. the ab-variant-β-hexosaminidases a and b (and s) intact-results from mutations in the gm -activator gene; so that tissue samples from the patients are able to degrade gm in detergent-containing enzyme assays. clinically, the b variant of gm gangliosidoses can be subclassified into infantile, juvenile, chronic, and adult onset forms. the infantile form, tay-sachs disease, has a higher prevalence among ashkenazi jews with a heterozygote frequency of : . affected children are normal at birth and show first symptoms, such as mild motor weakness, a cherry red spot in the central retina, and increased startle reaction between and months of life. progressive deterioration with weakness, hypotonia, or poor head control leads to a vegetative state and death often between the second and fourth year of life. juvenile and adult course is observed in patients with a higher residual activity of the variant hexosaminidase a [ ] . symptoms are very heterogeneous; for a clinical description, compare [ ] . the b variant of gm gangliosidoses [ , ] was very difficult to elucidate: synthetic uncharged substrates used for diagnosis such as mufglcnac (figure ; for kinetic parameters see [ ] ) were cleaved, suggesting the presence of β-hexosaminidase, and also the gm activator was present. as it turned out, the b variant differs enzymatically from the b variant by an altered substrate specificity of the variant β-hexosaminidase a. while uncharged substrates are cleaved, no activity is detected towards gm and towards sulfated, negatively charged [ ] synthetic fluorogenic substrates. in the b variant, the function of the α-chain active site is defective, but subunit association, enzyme processing, and the activity of the β-chain are not impaired. homozygous patients with the b mutation show the course of the juvenile disease; compound heterozygotes with a b and a null allele show a late infantile course. disease. the variant of gm gangliosidosis was the first gangliosidosis for which the underlying enzymatic defect was identified [ ] . due to the deficiency of two enzyme activities, β-hexosaminidases a and b, storage of negatively charged glycolipids characteristic for tay-sachs disease and, in addition, of uncharged substrates such as ga in the brain and globoside in visceral organs (figure ) is observed. in infantile sandhoff disease, patients show clinical and pathological manifestations of tay-sachs disease (infantile b variant) and in addition also organomegaly and slight bone deformations. for further symptoms and the description of juvenile and adult forms, compare [ ] . the ab variant is due to the deficiency of the gm -activator protein [ ] , with intact β-hexosaminidases a and b (and s), therefore the name. the disease is characterized by accumulation of gm and ga (for structures, see figure ). the clinical picture [ ] resembles that of tay-sachs disease with a delayed appearance of symptoms; an animal model is available [ ] . although lysosomal gm as the major storage compound in gm gangliosidoses is neither toxic nor immunogenic, its accumulation induces inflammatory responses as demonstrated for glycoconjugates in the murine model of sandhoff disease [ ] . huge axon hillock enlargements, the so-called meganeurites, have been observed in neurons of patients with different lysosomal storage diseases, which might be attributed to the storage substance gm and contribute to synaptic dysfunction [ ] . as in other sphingolipidoses [ ] , the corresponding (more toxic) lysolipid, in this case lysogm (figure ), is elevated [ , ] and contributes to the pathogenesis. lysogm has been suggested as a biomarker for tay-sachs and sandhoff disease [ ] ; for occurrence and role of lysogsls in acquired diseases, compare [ ] . despite naturally occurring animal models of gm gangliosidoses in dogs, cats, and pig, murine models are used for therapy studies. since the mouse model of tay-sachs disease is largely asymptomatic, the mouse model of sandhoff disease is used for most studies [ ] . despite some success in the experimental treatment of juvenile and adult patients as well as in the animal models, there is no causal therapy available for the severe forms of the gm gangliosidoses. the limitations of the substrate reduction approach, which reduces the gm influx into the lysosomal compartment, have been evaluated by a genetic experiment: sandhoff-disease mice were crossbred with mice defective in gm synthase. the lifespan of these animals was much longer than that of sandhoff-disease mice, but instead of gm storage they developed a oligosaccharide storage, neurological disease [ ] . therapeutic approaches such as bone-marrow transplantation [ ] , enzyme-replacement therapy with recombinant highly phosphomannosylated β-hexosaminidase a [ ] , or transplantation of neural stem cells [ ] have been investigated in the animal model of the variant, substrate-reduction therapy with n-butyl deoxynojirimycin [ , ] , and with pyrimethamine as pharmacological chaperone [ , ] in adult patients and gene therapy in endothelial cells [ ] . treatment of the accompanying inflammation is beneficial [ ] . aspects. in addition to inherited diseases, ganglioside levels can also be altered in several acquired diseases [ ] . for example, gangliosides play roles in neurological diseases such as alzheimer's [ ] , parkinson, or huntington's disease [ ] . in cancer, ganglioside expression can also be altered in tumor cells with an impact on signalling and tumor-host interactions [ ] . [neu gc]gm [ ] , gd , gd , gm , and fucosylgm are regarded as tumor-associated antigens [ ] and are targets for the immunotherapy of cancer [ ] . also several neuropathies including variant forms of guillain-barré and miller-fisher syndrome are caused by serum antibodies against gangliosides [ ] . there are only a few therapeutic roles for gangliosides, especially since they can induce neuropathies. in the past, gangliosides isolated from bovine brain have been investigated and also applied to human patients to improve neural repair and for the treatment of stroke [ , ] . also the direct application of ganglioside gm into the brain of patients with alzheimer disease has been evaluated [ ] . as indirect roles, the inhibition of ganglioside biosynthesis for the treatment of insulin resistance [ ] , the interference with microbial binding to gangliosides [ ] , or the reduction of neurotoxicity with liga [ ] has to be mentioned. a plethora of functions has been attributed to gangliosides [ ] , for example, for gm [ ] , the most abundant ganglioside in most mammalian cell types, but not in neurons, or for gm [ ] . in general, gangliosides mediate their function via interaction with soluble or membranebound binding molecules outside the cell ("trans" interaction), or by influencing properties of proteins within the same membrane ("cis" interaction) [ , [ ] [ ] [ ] [ ] . "trans" interactions occur between the glycan part of gangliosides on the one side with lectins on the other side. also gangliosides contribute to the chemical high-density sugar code of cell surfaces [ ] . for example, gm can be recognized by galectin- [ ] and sialic acids in α , figure : structure of liga , a semitruncated and halogenated gm analog that can pass the blood-brain barrier. are recognized by the sialic acid binding immunoglobulin lectins siglec- , α , -sialosides by siglec- , and α , sialosides by siglec- [ ] . also carbohydrate-carbohydrate interactions can play a role [ , ] . although interactions between individual carbohydrate residues [ ] are weak, clustering of gangliosides offer the possibility for multivalent interactions, if they are not buried under glycoprotein glycans. apparently, gm on mouse melanoma b cells can mediate cell adhesion to mouse lymphoma l cells by binding to ga (gangliotriaosylceramide, galnacβ , galβ , glcβ , cer; see figure or figure ) [ ] . within the nervous system, gangliosides act in a "trans" manner with the myelin-associated glycoprotein mag. mag recognizes neuacα - galβ - galnactermini on axonal gangliosides, an interaction that is essential for axon-myelin stability and axon regeneration [ ] . "cis" interactions can happen via a direct interaction, or, indirectly, via the properties of the membrane or of putative-membrane domains [ ] . this way gangliosides influence the activities of receptor-tyrosine kinases in the plasma membrane, such as the receptors of epidermalgrowth factor, nerve growth factor, and insulin and therefore cell signaling [ ] . for example, gm enhances trka neurotrophin receptor-activation in a "cis"-manner [ ] ; for a brief overview of proteins affected by certain gangliosides, compare, for example, [ ] . since the characterization of lipid microdomains in living cells is difficult, some conclusions can be drawn from in vitro experiments. for example, gm inhibits the autophosphorylation of purified egfr reconstituted into the proteoliposomes of defined lipid compositions, but not the egf binding [ ] . there are indications that gangliosides may not act only in an autonomous manner, but might also support the formation of distinct membrane phases, although it is a matter of debate to which extent this operates in vivo. it is believed that gangliosides are not homogeneously distributed on the cell surface, but segregate into membrane domains together with gpi-anchored proteins, sphingomyelin and cholesterol. such rafts have been supposed to be the physiological surroundings of many membrane proteins, although no rigid proof for their existence has been provided. ganglioside plays a largely unexplored role for membrane structure [ ] . due to their large hydrated head groups, they stabilize membrane areas with positive curvature [ ] . a multitude of reports propose a segregation of gangliosides and other gsls with cholesterol and gpi-anchored proteins into the lipid platforms known as "rafts" in the membranes of living cells [ ] [ ] [ ] [ ] . since most of the applied methods (detergent extraction, antibody, and toxin staining) constitute a bias towards the formation of such domains [ ] , the existence, size, and lifetime of rafts are a matter of debate. from thermodynamic considerations, it is clear that procedures such as detergent extraction can (or have to) produce artificial results and are not suitable for raft characterization [ ] [ ] [ ] [ ] . although it has been demonstrated that the treatment of cellular membranes with detergents causes the redistribution of gangliosides and gpi-anchored proteins [ , ] , these techniques are still applied and not always critically examined. experiments in living cells using sted microscopy [ ] and other visualization techniques [ ] point to an upper limit of lifetime and size of such domains in the range of ms and nm. in addition, sialic acids and oligosialic acids present on gangliosides can modulate membrane surface charge density, the ph at the membrane surface, and membrane potentials [ ] . in planar lipid bilayers, ganglioside gd a can increase the excitability of voltage-dependent sodium channels [ ] . . . infection. "cis" and "trans" interactions of gangliosides play multiple roles in the immune system [ ] and in infectious diseases [ , ] , where gangliosides act as cellular receptors and coreceptors for viruses, bacteria, and microbial toxins. the most prominent example is gm as the receptor for cholera toxin [ ] ; other examples are the toxin of clostridium botulinum and the saba adhesin of helicobacter pylori [ ] that bind to cell surface gangliosides of the host [ ] . binding of sialylated cell surface glycoconjugates to siglecs [ ] on white blood cells is used within innate and adaptive immune responses to distinguish between self and nonself and to dampen autoimmune responses [ ] . many pathogens use sialic acids on cell surface glycoconjugates for cellular entry, for example, periodontal pathogens [ ] . recent examples include ganglioside gt b, which seems to be the host cell receptor for the merkel cell polyomavirus [ ] . this virus has been identified as the cause of merkel cell carcinoma, an aggressive type of skin cancer. also sialidase-insensitive rotaviruses recognize sialic acid, for example, on ganglioside gm , which is not substrate of all sialidases due to its branched structure [ ] and the glycan present in ganglioside gd a serves as host receptor for the adenoviruses that cause epidemic keratoconjunctivitis [ ] . gangliosides are secondary gene products. their function can be analyzed by knockout experiments, where in cells, tissues, or organisms their formation or degradation is interrupted by genomic, posttranscriptional, or chemical strategies. especially valuable were genetically engineered mice with defects in ganglioside biosynthesis [ ] , which revealed, for example, a role of gangliosides in calcium homeostasis [ ] , neural repair [ ] , or neurological diseases [ ] . also investigations in human patients [ ] , genetically engineered mammals [ ] , and other organisms [ ] allowed insight into various aspects of ganglioside metabolism and transport. also mutant and silenced cells have been applied for functional studies. in vitro systems such as liposomes or planar monolayers allow investigations that are to difficult to be carried out in cells. for example, when gm is incorporated into liposomes, a phase separation into gm -rich and gm -poor phases occurs above a certain gm content [ ] . this would fit to reports on gm -enriched microdomains in living cells. in experimental approaches, ganglioside biosynthesis can be modulated by inhibitors [ ] . also the enhancement of ganglioside biosynthesis can be used, for example, chemically, or by the introduction of glycosyltransferase encoding cdna in cultured cells [ , ] . an enantiomer of the glucosylceramide synthase inhibitor d-threo-pdmp (pdmp = -phenyl- -decanoylamino- -morpholino- -propanol), lthreo-pdmp, acts as an enhancer of ganglioside biosynthesis by upregulating glycosyltransferases. this was accompanied by increased neurite outgrowth [ ] . additional possibilities are the generation of mutant cells, for example for gm synthase [ ] or by posttranscriptional silencing like rna interference. while even complex systems like cultured cells can survive without gsls, they are required for the development of multicellular organisms [ ] . gangliosides. structurally homogenous gangliosides and ganglioside probes that are modified by isotopes, fluorescence, chemical reporter groups, photoaffinity, or affinity ligands are valuable tools for the analysis of ganglioside function, metabolism, and transport. these tools are available by total or partial chemical synthesis, or by biosynthetic incorporation of suitable-for example, photolabile-n-acylmannosamine precursors into gangliosides [ ] using the methodology for biosynthetic sialic acid modification developed by kayser et al. [ ] . for enzymological, transport, and crosslinking studies, tritium and c are incorporated into different positions of gangliosides, but also radioiodination is possible in the presence of aryl residues [ , ] . ganglioside total synthesis is a time-consuming and demanding task and usually performed only in specialized laboratories for examples, see [ , ] . it relies predominantly on the sequential glycosidation of a -o-protected azidosphingosine [ ] with suitably protected and activated glycosyl donors. this includes the trichloroacetimidates [ ] as well as methods for α-selective sialylation reactions [ ] . also chemoenzymatic procedures have been developed, where different glycosidation steps are catalyzed by glycosyltransferases [ ] or glycosidases [ ] , or where oligosaccharyl fluorides are coupled to native or fluorescent ceramide anchors using an engineered endoglycoceramidase (glycosynthase) [ , ] . ganglioside oligosaccharides, such as those of gm , gm , gm , gd , and gt , can be produced by genetically engineered bacterial strains. for the biotechnological production of ganglioside head groups, lactose can be internalized in e. coli as a precursor to be used as acceptor for glycosyltransferases [ ] [ ] [ ] [ ] [ ] [ ] . an application of the ganglioside biosynthetic machinery is the preparative production of neoglycolipids with ganglioside head groups [ ] figure : example of a gm analog [ ] spin labelled with a , -dimethyl-oxazolidine- -oxyl-(doxyl-) residue and a fluorescent gm analog used for sted microscopy [ ] . using a lung squamous-cell carcinoma line (rerf-lc-a) and -azidododecyl β-lactoside as a suitable primer [ ] . gsls isolated from natural sources can be used for the preparation of chemically modified derivatives [ ] like labelled gsls [ , ] , or those of enhanced metabolic stability [ ] . the chemical release of the ganglioside glycan chain can be achieved by osmium tetroxide/periodate treatment of protected gangliosides [ , ] , or by ozonolysis of native gangliosides [ ] . initially, both methods give rise to ganglioside aldehydes, which are not isolated but subsequently fragmented by alkaline treatment, or, if desired, are isolated for further applications [ ] . the glycan part can also be released enzymatically by ceramide glycanase [ , ] . lysogangliosides that lack the acyl moiety at the sphingosine nitrogen can be prepared by chemical procedures [ , ] , or by enzymatic treatment of gangliosides with sphingolipid n-deacylase [ ] [ ] [ ] . lysogangliosides (see figure ) can be used for the introduction of fluorescence, spin, or radiolabels or other modifications into the lipid backbone of gangliosides. a semitruncated, dihalogenated gm analog that is able to pass the blood-brain barrier is liga [ ] (figure ). there are also several approaches to the synthesis of photoactivatable gsl derivatives [ ] . it has to be noted that when gangliosides are added to the medium of cultured cells, they are largely present as oligomers in the form of micelles or vesicles, as monomers bound to proteins, and as free monomers. in aqueous surroundings, gangliosides form aggregates of different size and shape [ , ] . in most cases these are micellar structures of - kda [ ] , in the case of the gangliosides with small head groups, gm and gm also vesicles [ , , ] . the critical micellar concentrations of gsls are in the range of − - − m [ ] and depend on temperature, ph, and, in part, on the method of determination. typical values are . × − m for gm to . × − m for gt b [ ] . uptake of exogenously added gangliosides by cells in culture [ ] can proceed in different ways [ , ] ( figure ) . with the aid of radiolabelled [ ] and spinlabelled gangliosides [ ] (figure ), three modes of adherence have been distinguished: - % of the exogenous ganglioside consist in loosely associated micelles and also monomers, which can be removed by delipidated serum proteins. a second fraction is attached to cellular proteins in a trypsin-labile fashion, and, finally, a trypsin-stable fraction is presumably inserted into the plasma membrane of the cell. only the last fraction is in the topologically correct, native orientation. when bound to proteins, the offrate of gangliosides with native alkyl chain lengths can be very low. this is not the case for synthetic, semitruncated derivatives of higher solubility, which are frequently used, but show different intracellular transport behaviour compared to native gangliosides [ ] . fluorescently labelled glycosphingolipids [ ] have been applied, but also their properties can differ significantly from the ones of native glycosphingolipids. gangliosides can also be transferred from cultured donor to acceptor cells that are separated by a membrane [ ] . by this process known as "shedding" [ ] , tumor cells can release up to . % of their gangliosides per hour [ ] . fluorescent ganglioside probes that bear the fluorophore in at the membrane-water interphase ( figure ) behave physicochemically more like native gangliosides. such compounds have been used as probes for sted microscopy [ ] or to quantify the transfer capacity of gm activator in a liposomal fret assay system [ ] . despite the fast development of analytical and biophysical tools, the analytical determination of ganglioside pattern, their spatial resolution, and their correlation with function is still a challenge. especially a convincing characterization of ganglioside-containing membrane domains in living cells and of their roles at the cellular level would constitute a considerable advance in the field. n-acetyl-neuraminic acid glc: glucose gal: galactose galnac: n-acetyl-galactosamine. the author has no direct financial relation with any commercial identity mentioned in the paper that might lead to a conflict of interests. a comprehensive classification system for lipids primer on genes encoding enzymes in sialic acid metabolism in mammals sialic acids Über die ganglioside, eine neue 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models of gm and gm gangliosidosis enhanced susceptibility to kainate-induced seizures, neuronal apoptosis, and death in mice lacking gangliotetraose gangliosides: protection with liga , a membrane-permeant analog of gm gm -ganglioside-mediated activation of the unfolded protein isrn biochemistry response causes neuronal death in a neurodegenerative gangliosidosis imbalanced substrate specificity of mutant β-galactosidase in patients with morquio b disease chemical chaperone therapy for gm -gangliosidosis the potential action of galactose as a "chemical chaperone": increase of beta galactosidase activity in fibroblasts from an adult gm -gangliosidosis patient evaluation of nnonyl-deoxygalactonojirimycin as a pharmacological chaperone for human gm gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy the gm gangliosidoses a review and predictive models of ganglioside uptake by biological membranes incorporation of ganglioside analogues into fibroblast cell membranes. a spin-label study direct observation of the nanoscale dynamics of membrane lipids in a living cell variant of gm -gangliosidosis with hexosaminidase a having a severely changed substrate specificity biochemical and molecular aspects of late-onset gm -gangliosidosis: b variant as a prototype diagnosis and carrier detection of tay-sachs disease: direct determination of hexosaminidase a using -methylumbelliferyl derivatives of β-n-acetylglucosamine- -sulfate and β-nacetylgalactosamine- -sulfate tay-sachs disease: one-step assay of β-n-acetylhexosaminidase in serum with a sulphated chromogenic substrate mouse model of gm activator deficiency manifests cerebellar pathology and motor impairment distortion of neuronal geometry and formation of aberrant synapses in neuronal storage disease occurrence of lysoganglioside lyso-g(m ) (ii -neu ac-gangliotriaosylsphingosine) in g(m ) gangliodosis brain accumulation of lysosphingolipids in tissues from patients with gm and gm gangliosidoses lyso-gm ganglioside: a possible biomarker of tay-sachs disease and sandhoff disease a view on sphingolipids and disease a genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder bone marrow transplantation prolongs life span and ameliorates neurologic manifestations in sandhoff disease mice highly phosphomannosylated enzyme replacement therapy for gm gangliosidosis neural stem cell transplantation benefits a monogenic neurometabolic disorder during the symptomatic phase of disease substrate reduction therapy with miglustat in chronic gm gangliosidosis type sandhoff: results of a -year follow-up substrate reduction therapy an open-label phase i/ii clinical trial of pyrimethamine for the treatment of patients affected with chronic gm gangliosidosis (tay-sachs or sandhoff variants) crystal structure of β-hexosaminidase b in complex with pyrimethamine, a potential pharmacological chaperone induced secretion of β-hexosaminidase by human brain endothelial cells: a novel approach in sandhoff disease? nsaids increase survival in the sandhoff disease mouse: synergy with n-butyldeoxynojirimycin pathological significance of ganglioside clusters in alzheimer's disease functional roles of gangliosides in neurodevelopment: an overview of recent advances aberrant glycosphingolipid expression and membrane organization in tumor cells: consequences on tumor-host interactions ngcgm ganglioside: a privileged target for cancer vaccines cancer vaccines and carbohydrate epitopes immunology in the clinic review series, focus on cancer: glycolipids as targets for tumour immunotherapy a common mechanism and a new categorization for anti-ganglioside antibody-mediated neuropathies the therapeutic role of gangliosides in neurological disorders gangliosides, ngf, brain aging and disease: a mini-review with personal reflections alzheimer disease-effect of continuous intracerebroventricular treatment with gm ganglioside and a systematic activation programme inhibition of ganglioside biosynthesis as a novel therapeutic approach in insulin resistance receptor mimicry as novel therapeutic treatment for biothreat agents semisynthetic sphingoglycolipid liga is neuroprotective against human immunodeficiency virus-gp -mediated apoptosis gangliosides as modulators of cell function ganglioside gm and its biological functions in search of a solution to the sphinx-like riddle of gm functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains glycosynaptic microdomains controlling tumor cell phenotype through alteration of cell growth, adhesion, and motility gangliosides in cell recognition and membrane protein regulation glycolipid-mediated cell-cell recognition in inflammation and nerve regeneration beyond glycoproteins as galectin counterreceptors: tumor-effector t cell growth control via ganglioside gm galectin- is a major receptor for ganglioside gm , a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture carbohydrate-to-carbohydrate interaction, through glycosynapse, as a basis of cell recognition and membrane organization carbohydrate-carbohydrate interaction in basic cell biology carbohydrate-carbohydrate interactions in cell recognition specific interaction between gangliotriaosylceramide (gg ) and sialosyllactosylceramide (g(m ) as a basis for specific cellular recognition between lymphoma and melanoma cells brain gangliosides in axon-myelin stability and axon regeneration gangliosides and the multiscale modulation of membrane structure receptor modifications in glycobiology neural functions of glycolipids lipid rafts: keys to neurodegeneration regulation of human egf receptor by lipids gangliosides as components of lipid membrane domains membrane organization and lipid rafts revitalizing membrane rafts: new tools and insights lipid rafts as a membraneorganizing principle lipids and cholesterol as regulators of traffic in the endomembrane system principles of microdomain formation in biological membranes-are there lipid liquid ordered domains in living cellular membranes? pip signaling in lipid domains: a critical reevaluation rafts defined: a report on the keystone symposium on lipid rafts and cell function detergentresistant membranes should not be identified with membrane rafts triton promotes domain formation in lipid raft mixtures membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation imaging of mobile long-lived nanoplatforms in the live cell plasma membrane membrane oligo-and polysialic acids ganglioside gd a increases the excitability of voltage-dependent sodium channels multifarious roles of sialic acids in immunity sphingolipids in infectious diseases viruses and sialic acids: rules of engagement cholera toxin-a foe & a friend helicobacter pylori and complex gangliosides sialic acids in human health and disease siglecs and their roles in the immune system siglecs as sensors of self in innate and adaptive immune responses sialic acid, periodontal pathogens and tannerella forsythia: stick around and enjoy the feast! ganglioside gt b is a putative host cell receptor for the merkel cell polyomavirus sialic acid dependence in rotavirus host cell invasion the gd a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis knockout mice and glycolipids cerebellar neurons lacking complex gangliosides degenerate in the presence of depolarizing levels of potassium regulatory mechanisms of nervous systems with glycosphingolipids functions of sphingolipid metabolism in mammals -lessons from genetic defects sphingolipids and membrane biology as determined from genetic models phase behavior in multilamellar vesicles of dppc containing ganglioside gm with a c : sphingoid base and a : acyl chain (gm ( , )) observed by x-ray diffraction glycosphingolipids-nature, function, and pharmacological modulation enhancement of malignant properties of human osteosarcoma cells with disialyl gangliosides gd /gd gm /gd /ga synthase expression results in the reduced cancer phenotypes with modulation of composition and raft-localization of gangliosides in a melanoma cell line chapter neurotrophic and neuroprotective actions of an enhancer of ganglioside biosynthesis mutant ng - cells (ng-cr ) deficient in gm synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium-induced apoptosis: they are rescued with liga- biomolecule function: no reliable prediction from cell culture metabolism of diazirine-modified n-acetylmannosamine analogues to photocross-linking sialosides biosynthesis of a nonphysiological sialic acid in different rat organs, using n-propanoyl-d-hexosamines as precursors tubulin anchoring to glycolipid-enriched, detergent-resistant domains of the neuronal plasma membrane hplc-based procedure for the preparation of carbenegenerating photoreactive gm and gm ganglioside derivatives radioiodinated to high specific radioactivity with chloramine t as an oxidant azidosphingosine glycosylation in glycosphingolipid synthesis glycosyl trichloroacetimidates selective α-sialylation enzymatic approaches to o-glycoside introduction: glycosyltransferases enzymatic glycosylation by transferases glycosphingolipid synthesis employing a combination of recombinant glycosyltransferases and an endoglycoceramidase glycosynthase biosynthesis of conjugatable saccharidic moieties of gm and gm gangliosides by engineered e. coli the genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, campylobacter jejuni genetic engineering of escherichia coli for the economical production of sialylated oligosaccharides highly efficient biosynthesis of the oligosaccharide moiety of the gd ganglioside by using metabolically engineered escherichia coli a new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria large-scale in vivo synthesis of the carbohydrate moieties of gangliosides gm and gm by metabolically engineered escherichia coli large scale biosynthesis of ganglioside analogues by rerf-lc-ai cells cultured in hyperflask use of sphingolipid analogs: benefits and risks a simple and novel method for tritium labeling of gangliosides and other sphingolipids preparation of radiolabeled gangliosides uptake and metabolism of exogenous glycosphingolipids by cultured cells structure and function of glycosphingolipids and sphingolipids: recollections and future trends release of carbohydrates from sphingoglycolipid by osmium-catalyzed periodate oxidation followed by treatment with mild alkali carbohydrate components of extraneuronal gangliosides from bovine and human spleen, and bovine kidney structural and functional glycosphingolipidomics by glycoblotting with an aminooxy-functionalized gold nanoparticle a unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide preparation of homogenous oligosaccharide chains from glycosphingolipids synthesis of lysogangliosides lysogangliosides: synthesis and use in preparing labeled gangliosides facile method for the preparation of lyso-gm and lyso-gm design of a covalently bonded glycosphingolipid microarray anti-ganglioside antibodies bind with enhanced affinity to gangliosides containing very long chain fatty acids sphingolipid photoaffinity labels thermodynamicgeometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine aggregative properties of gangliosides in solution lipid membrane domains in glycobiology micellar properties of glycosphingolipids in aqueous media shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins characterization of the cellular binding of exogenous gangliosides chemical synthesis of fluorescent glycero-and sphingolipids synthesis, shedding, and intercellular transfer of human medulloblastoma gangliosides: abrogation by a new inhibitor of glucosylceramide synthase shedding and immunoregulatory activity of yac- lymphoma cell gangliosides shedding of human neuroblastoma gangliosides synthesis of novel nbd-gm and nbd-gm for the transfer activity of gm -activator protein by a fret-based assay system support from professor dr. k. sandhoff, the dfg, and the european community ( th framework program "lipidomicnet", proposal no. ) is gratefully acknowledged. key: cord- -ldn pj authors: inoue, satoshi; tanaka, kazunori; tanaka, hiromitsu; ohtomo, kohei; kanda, toshio; imamura, morikazu; quan, guo‐xing; kojima, katsura; yamashita, tetsuro; nakajima, tasuku; taira, hideharu; tamura, toshiki; mizuno, shigeki title: assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of l‐chain for protection of α , ‐mannose residues in n‐linked oligosaccharide chains of fibrohexamerin/p date: - - journal: eur j biochem doi: . /j. - . . .x sha: doc_id: cord_uid: ldn pj silk fibroin of bombyx mori is secreted from the posterior silk gland (psg) as a . ‐mda elementary unit, consisting of six sets of a disulfide‐linked heavy chain (h‐chain)–light chain (l‐chain) heterodimer and one molecule of fibrohexamerin (fhx)/p . fhx/p , a glycoprotein, associates noncovalently with the h–l heterodimers. the elementary unit was found and purified from the endoplasmic reticulum (er) extract of psg cells. a substantial amount of fhx/p unassembled into the elementary unit was also present in er. in normal‐level fibroin‐producing breeds (j‐ and c ), the elementary unit contained fhx/p of either kda (major) or kda (minor). the ‐kda fhx/p was produced from the ‐kda form by digestion with the bacterial α , ‐mannosidase in vitro. the elementary unit in the er extract contained only the ‐kda fhx/p , whereas both ‐ and ‐kda forms of fhx/p were present in the er plus golgi mixed extracts. in naked‐pupa mutants [nd( ), nd‐s and nd‐s (d)], extremely small amounts of fibroin were produced and they consisted of one molecule of ‐kda fhx/p and six molecules of h‐chain but no l‐chain. when the nd‐s (d) mutant was subjected to transgenesis with the normal l‐chain gene, the (h‐l)( )fhx( )‐type elementary unit containing the ‐kda fhx/p , was produced. these results suggest that fhx/p in the elementary unit is largely protected from digestion with golgi α , ‐mannosidases when l‐chains are present in the unit. models suggesting a role of l‐chain for the protection of α , ‐mannose residues of fhx/p are presented. silk fibroin of the silkworm, bombyx mori, is synthesized in the posterior silk gland (psg) cells, secreted into the lumen of psg, associated with sericin in the lumen of the middle silk gland (msg) and spun out from the anterior silk gland as silk fibers to form a cocoon. the silk fibroin is secreted in a form of a . -mda protein complex designated as the elementary unit of fibroin [ ] , which consists of six sets of heavy chain (h-chain; -kda fibrous protein)-light chain (l-chain; kda) disulfide-linked heterodimer and one molecule of a glycoprotein, fibrohexamerin (fhx)/p [ , ] . a single disulfide bond is formed between cys of l-chain and cys-c ( th residue from the c-terminus) of h-chain [ ] . fhx/p contains three n-linked oligosaccharide chains at asn , asn , asn [ ] and exists either in a -kda (major) or -kda (minor) molecular form [ , ] , which has been suggested to have different compositions of oligosaccharide chains [ ] . fhx/p associates with (h-l) mainly by hydrophobic interactions [ ] and is centrally important in the maintenance of the elementary unit, because treatment of the elementary unit with % triton x- , n-glycosidase f or endo-h caused partial displacement or deglycosylation of fhx/p and simultaneously remarkable disintegration of the elementary unit [ ] . on the other hand, treatment of the elementary unit with . mm dithiothreitol dissociated l-chains completely from h-chains without affecting the gross integrity of the once-formed elementary unit [ ] . in the three fibroinsecretion-deficient naked-pupa mutant silkworms; nd( ), nd-s and nd-s d , the level of secretion of fibroin is less than % of that in the normal level fibroin-producing breeds [ , ] and the extremely small amounts of fibroin produced by these three mutant silkworms lack l-chains and show the composition of h fhx [ ] . the disulfide linkage between h-and l-chains is not formed in nd-s and nd-s d mutants because of the lack of cys- in the mutant l-chain [ ] . it is of interest to note that the fhx/p in the secreted fibroin of the three naked-pupa mutants is only of the -kda molecular form [ ] . in the present study, we aimed at answering the following three specific questions: (a) where is the site of assembly of the elementary unit in psg cells?; (b) what is the difference between -kda and -kda fhx/p ?; (c) does the l-chain play a role in the formation of the -kda fhx/p ? fertilized eggs of b. mori j- and c , which are normal-level producers of fibroin, and three fibroinsecretion-deficient naked-pupa mutants [nd ( ) , nd-s and nd-s d ] were supplied by the national institute of agrobiological sciences, tsukuba, japan and their larvae were reared in the authors' laboratories (tohoku university and national institute of agrobiological sciences). the nd( ) mutation is linked to the fibroin h-chain gene, fibh [ ] . nd-s and nd-s d are mutations of the fibroin l-chain gene, fibl [ , ] . fifty pairs of psgs were excised from larvae of b. mori j- at the fifth day of the fifth instar and layers of giant psg cells were separated as described [ ] . the psg cell layers were washed in tmk [ mm tris/hcl (ph . ), mm mgcl , mm kcl], immersed in ml of tmk containing . m sucrose and mm phenylmethylsulfonyl fluoride for min at °c, and were forced to pass successively through nylon meshes of -mm and . -mm pore sizes. cells passed through the two nylon meshes were gently disintegrated by passing through a -gauge needle six times to yield psg cell lysate. the lysate was centrifuged at g for min at °c to sediment nuclei. the supernatant ( ml per tube) was layered on . - . m linear sucrose gradient in tmk ( . ml per tube), centrifuged at g in a sw ti rotor (beckman) for h at °c, and . -ml fractions were collected. fractions containing er were monitored by the nadph-cytochrome c reductase assay [ ] and by western blotting with the rabbit anti-(drosophila calnexin-peptide) ig raised against a synthetic peptide, caqteesntkrkrqarke [ ] . for the former assay, a -ll portion of each fraction was mixed with ll of the assay mixture [ . mm cytochrome c (sigma), . mm nadph, mm sodium azide, mm tris/hcl (ph . )], incubated at °c for min and a was measured. fractions containing golgi complex were monitored by the assay for golgi a-mannosidase ii [ ] [ ] [ ] as follows. a -ll portion of each fraction was mixed with ll of . mm -metylumbelliferyl-a-d-mannopyranoside (nacalai tesque inc., kyoto, japan), . · nacl/p i , . % triton x- , incubated at °c for min, and then ll of . m glycine, . m na co was added to stop the reaction. the intensity of fluorescence was measured by fluorescence spectrometer f- (hitachi) at the exciting wavelength of nm and the emission wavelength of nm. fractions containing er only or er plus golgi were pooled, sonicated briefly and used as the er extract or the er plus golgi mixed extracts. the er extract (about ml) was concentrated to about ml using molcut ii (millipore). the elementary unit was isolated from the concentrated er extract by two successive gel filtration column chromatographies as described [ ] . fibroin samples from psg lumen and cocoons were prepared as described [ ] . fhx/p was purified from the fibroin secreted into the lumen of psg of j- or partially purified (before the step of reverse-phase hplc) from following sources; cocoons of nd( ), nd-s, nd-s d , c or l · transgenic line, and protein samples from er extract or er plus golgi mixed extracts of c or nd-s d as described [ ] . western blotting of fibroin samples were carried out as described previously [ ] with the following antibodies: the rabbit polyclonal anti-h-chain or the anti-l-chain ig [ ] , the mouse polyclonal anti-fhx/p ig [ ] , the rabbit anti-l peptide ig specific to the normal l-chain [ ] , the mouse polyclonal anti-(nd-s d -peptide) ig specific to the c-terminal sequence of the nd-s d mutant l-chain [ ] , or the rabbit anti-(drosophila calnexin-peptide) ig as described above [ ] . the -and -kda bands of fhx/p , detected by western blotting with the anti-fhx/p polyclonal antibody, were subjected to a densitometric analysis using gs- imaging densitometer (bio-rad). purified fhx/p was digested with endo-h (new england biolabs) under the conditions as described previously [ ] . purified fhx/p from j- ( ng) or partially purified fhx/p from nd( ), nd-s or nd-s d (about ng each) was dissolved in ll of nacl/p i ( mm nacl, . mm kcl, . mm na hpo , . mm kh po ), mixed with ll of lgaell ) solution of the purified bacillus sp. a , -mannosidase (ec . . . ) [ ] , and incubated at °c for h. fibroin samples from cocoons and purified or partially purified fhx/p were subjected to sds/page, transferred to a nitrocellulose membrane, and subjected to the reaction with biotinylated cona (honen). the reaction was detected with alkaline phosphatase-conjugated streptavidin (gibco-brl) as described [ ] . quantification of h-chain, l-chain and fhx/p in the elementary unit, psg cellular fractions or in cocoon proteins was carried out by elisa with specific antibodies as described [ ] . construction of a recombinant transforming plasmid and production of transgenic silkworms a ¢-flanking region () to + ) of the l-chain gene containing a putative promoter was amplified by pcr from the cosmid clone pkyfl - [ ] . the cdna sequences encoding l-chain and the ¢-flanking region plus a part of poly(a) stretch (+ to + ) were amplified by pcr from the cdna clone pfl [ ] . these fragments were inserted into pbac( xp -dsred ) transforming vector [ ] to construct pbac( xp -dsred + l-chain promoter/normal l-chain cdna) for simultaneous expression of dsred and the normal l-chain. the recombinant plasmid was purified using qiagen midi kits (qiagen). fertilized eggs of the nd-s d mutant silkworm within h after oviposition were treated with . m hcl for h at °c to break embryonic diapause. two to three nanoliters of a mixture of the helper plasmid pha pig [ ] and the above recombinant plasmid ( . lgaell ) dna, each) in . mm phosphate buffer (ph . ), mm kcl was injected into each of the eggs at h after the hcl treatment. transgenic embryos at the first (g ) or second (g ) generation or moths derived from them were selected by examining the expression of dsred in the stemmata and nervous tissues [ ] under leica mzfl iii fluorescence microscope. ecori-digested genomic dnas from moths were subjected to southern blot hybridization with the l-chain ¢-flanking region (from + to + ) from pfl [ ] as a probe for the normal l-chain gene. total rna from psg or msg was prepared as described [ , ] and subjected to northern blot hybridization with following probes, each labeled with alkphos direct labeling system (amersham pharmacia biotech): a part of the l-chain cdna sequence (+ to + ) which is common to normal and nd-s d l-chains amplified by pcr from pfl [ ] and the b. mori elongation factor a- isoform cdna sequence (from + to + ) amplified by pcr from pbmef- a- [ ] . it has been speculated that the elementary unit of fibroin is assembled in er and transported to golgi complex [ ] by analogy with other oligomeric secretory proteins [ ] . in order to examine this notion, er was isolated from the psg cell extract of a normal breed (j- ) by sucrose density gradient centrifugation under nondenaturing conditions (fig. a) . distributions of er markers (the reaction with anticalnexin-peptide antibody and the nadph-cytochrome c reductase activity) and a golgi marker (golgi a-mannosidase ii activity) across the gradient indicated that fractions containing sufficiently pure er, in terms of its separation from golgi, were obtained (fractions - ) but fractions showing activity of the golgi-marker enzyme (fractions - ) still contained substantial er materials (fig. a) . recentrifugation of fractions - through the similar sucrose gradient could not yield a sufficiently pure golgi fraction (data not shown). the h-and l-chains, and fhx/p were all present in the er fraction (fractions - ) as detected by specific antibodies. the peak er fractions (fractions - in fig. a ) were pooled and subjected to brief sonication to yield an er extract. the er extract was then separated on a gel filtration column of tsk-gel hw -f, and each fraction was examined by western blotting with the anti-h-chain, the anti-l-chain, or the anti-fhx/p ig (fig. c ). protein complexes containing h-and l-chains, and fhx/p were eluted between fractions and , forming two peaks. because the elution profile of the first peak (fractions and , bracketed) was identical, in comparison with the elution profile of b-connectin ( . mda), to that of the elementary unit isolated from psg cells [ ] , these fractions were pooled, concentrated, and subjected to re-chromatography on a tsk-gel hw -s gel filtration column (fig. d ). the protein complex was eluted as a single peak at the position for approximately . -mda and consisted of h-chain, l-chain, and fhx/p as shown by western blotting (fig. d ). when fractions - (bracketed) were pooled and analyzed by the quantitative elisa, the molar ratio of h-and l-chains, and fhx/p was shown to be close to (h-chain) : (l-chain) : (fhx/p ) ( table ) . because all these properties are identical to those of the elementary unit isolated from the total psg cell extract (table and [ ] ), we interpreted these results to indicate that the elementary unit of fibroin is assembled in er. when the total psg cell extract was subjected to the quantitative elisa, the molar ratio of h-chain : l-chain : fhx/p was . : . : (table ). these results suggested that excess fhx/p which was not assembled into the elementary unit was present in psg cells. a similar molar ratio was obtained when the total er extract was subjected to the quantitative elisa ( table ) , suggesting that a substantial fraction of fhx/p molecules which was not assembled into the elementary unit was retained in er. this notion was supported by the fact that the lower molecular-mass fraction of the er extract (fractions - in fig. c ) showed the molar ratio of h-chain : l-chain : fhx/p which was similar to that of the total er extract ( table ) . the -and -kda molecular forms of fhx/p are suggested to be caused by the presence or absence of a , -mannose residues in the n-linked oligosaccharide chains it has been shown that fhx/p in the secreted fibroin of the normal breed j- is either -kda (major) or -kda (minor), whereas only the -kda form is present in the table . determination of molar ratios of h-chain, l-chain and fhx/p in different psg cellular fractions of b. mori j- (a normal breed). protein samples in low mw fraction from er extract were prepared from fractions ( - ) of gel filtration chromatography as shown in fig. (fig. , lane ). the presence of n-linked oligosaccharide chains in the -kda fhx/p molecules from the three nakedpupa mutants was shown previously by their positive reactions to biotinylated con a and their susceptibility to the endo-h digestion [ ] . it was noted that fhx/p of the normal breed j- , as detected with the anti-fhx/p antibody, was present as a single band ( kda) in sucrose-gradient fractions containing er (fig. a, fractions - ) , whereas it was present as two bands ( and kda) in fractions containing both er and golgi complex (fig. a, fractions - ) . in order to confirm these observations, fhx/p was partially purified from cocoon shells, er extract, or er plus golgi mixed extracts of the normal breed c and subjected to western blotting with the anti-fhx/p antibody or to the lectin blotting with biotinylated cona before or after digestion with the bacterial a , -mannosidase or endo-h (fig. a) . fhx/p was detected as a -kda single band in the er extract (fig. a , lane ) but as two bands ( and kda) in the er plus golgi mixed extracts (fig. a, lane ) or in cocoons (fig. a, lane ) . the -kda fhx/p was converted to the -kda single band by digestion with the bacterial a , -mannosidase (fig. a, lanes - ) , and the -and -kda fhx/p were converted to the -kda single band by digestion with endo-h (fig. a , left panel, lanes - ). the reaction of fhx/p with biotinylated cona became undetectable after digestion with endo-h (fig. a, right panel, lanes - ) . these results suggest that the -kda component is the er form of fhx/p and the -kda component represents fhx/p whose n-linked oligosaccharide chains lost their terminal a , -mannose residues by digestion with a , -manosidases in golgi complex, and further imply that fhx/p in the elementary unit of the normal-level fibroin-producing breeds is largely resistant to the action of a , -mannosidases in golgi complex and secreted as the er-type -kda form. the psg cell extract of nd-s d mutant was subjected to the sucrose density gradient centrifugation to separate er and er plus golgi mixed fractions as for the normal breed j- (fig. b) , and each fraction was sonicated briefly to yield an extract. when electrophoretic mobilities of partially purified fhx/p molecules from cocoon shells, the er extract and the er plus golgi mixed extracts from nd-s d mutant were compared, it was confirmed that only the -kda fhx/p was present in the extremely small amount of fibroin secreted into cocoons (fig. b, lane ) , whereas only the -kda form was present in the er extract and both -(major) and -kda (minor) forms were present in the er plus golgi mixed extracts (fig. b, lanes , ) as for the normal breed c (fig. a) . it is thus conceivable that in the nd-s d mutant silkworm, fhx/p in the l-chain-free h fhx -type elementary unit (table ) is processed efficiently by the action of golgi a , -mannosidases to yield only the -kda molecule in the secreted fibroin. in order to examine a role of l-chain in the protection of a , -mannose residues of fhx/p in the elementary unit, the nd-s d mutant silkworm was subjected to transgenesis with the normal l-chain promoter/cdna sequence together with a marker gene of dsred (fig. a) , and two transgenic lines l · and l - were selected. the l · line expressed dsred strongly and the l-chain sequence was suggested to be integrated into two major genomic loci by southern blot hybridization (fig. b, lanes and ) with a probe specific to the normal l-chain gene (fig. a) . northern blot hybridization indicated that both normal l-chain mrna and nd-s d mutant l-chain mrna were produced at high levels in a psg-specific manner in the l · line (fig. c, lanes and ) . another transgenic line l - expressed extremely low-levels of dsred and the normal l-chain mrna as judged by reverse transcription (rt)-pcr (data not shown). in consistency with these results, the mean weight of cocoons produced (n ¼ each) was mg for l · and . mg for l - as compared with mg for c and . mg (consisting mostly of sericin) for nd-s d . proteins from cocoon shells of c ( . lg; fig. , lanes and ), nd-s d ( lg; fig. , lanes and ) and two transgenic lines [l · ( lg ; fig. , lanes and ) and l - ( lg; fig. , lanes and ) ] were subjected to western blotting with the anti-h-chain (fig. a) , the anti-l-chain (fig. b) , or the anti-fhx/p ig (fig. c) , and to the lectin blotting using biotinylated cona (fig. d ) before (fig. , lanes - ) or after (fig. , lanes - ) the reductive cleavage of disulfide bonds. in these experiments, a large excess of cocoon protein samples was used from nd-s d and the transgenic line l - in order to obtain similar signal intensity. in all the cocoon protein samples, h-chain (fig. a ) and fhx/p (fig. c ) were detected. the normal l-chain was detected clearly after cleavage of disulfide bonds (fig. b ) in the normal (c ; lane ) and the two transgenic lines (lanes and ) but the mutant l-chain was undetectable in the cocoon protein sample from the nd-s d mutant (fig. b, lanes and ) . the anti-l-chain polyclonal antibody [ ] used in these experiments could detect the nd-s d mutant l-chain as well in the psg cell extract (fig. b) . it was suggested from these results that the relatively large amount of normal l-chain expressed in the l · transgenic line contributed to the restoration of the high-level secretion of fibroin. in the two transgenic lines (l · and l - ), fhx/p was detected as two bands of and kda (fig. c , lanes , and , ) like in the normal breed c (fig. c, lanes and ) . the slight decrease in electrophoretic mobility of fhx/p after cleavage of disulfide bonds was interpreted as the loss of the compact structure of fhx/p due to the cleavage of intramolecular disulfide bonds [ ] . in the transgenic lines, fhx/p molecules of and kda were both reactive to biotinylated cona indicating that n-linked oligosaccharide chains existed in both types of molecules (fig. d) . it was demonstrated by the quantitative elisa that the molar ratios of h-chain, l-chain, and fhx/p were close to : : for the fibroin secreted into cocoons of both transgenic lines ( table ). it was demonstrated in this study that the elementary unit of fibroin, having the same molar ratio of h-and l-chains, and fhx/p as of the elementary unit present in the psg extract, could be isolated from the er extract of psg cells of a normal-level fibroin-producing silkworm table . determination of molar ratios of h-chain, l-chain, and fhx/p in the fibroin secreted into cocoons of a normal breed (c ), nd-s d mutant, or transgenic lines of b. mori. a values were obtained by elisa. protein samples (all in ll) assayed were ng for h-chain, ng for lchain and ng for fhx/p from cocoons of c and the transgenic line l · , or lg for h-chain, lg for l-chain and lg for fhx/p from naked-pupa cocoons of nd-s d and cocoons of the transgenic line l - . n ¼ ; ±, sd; nd, not detected. breed j- . it has been shown that the assembly of oligomeric protein complexes occurs generally in er [ ] but some protein complexes, such as connexin of gap junctions [ ] and m protein of coronavirus [ ] , have been shown to be assembled in golgi complex. in the case of silk fibroin, a vast amount of newly synthesized, high molecular-mass ( kda) fibrous h-chain molecules in er must be transported efficiently, without being denatured, to golgi complex for secretion from psg cells. the assembly of the elementary unit in er is most likely a crucial event to meet the er quality control [ ] and to ensure the efficient intracellular transport and secretion of h-chains. there seem to be two key events in the assembly of the (h-l) fhx -type elementary unit: (a) the formation of disulfide-linked h-l heterodimers and (b) the formation of the three-component protein complex in which the noncovalent association of the glycoprotein fhx/p is centrally important. the clue to the first event has been obtained from studies [ ] on the naked-pupa mutants, nd-s and nd-s d . in these mutant silkworms, chimeric l-chains are formed because of the exon-shuffling mutation of the l-chain gene. these chimeric l-chains lack cys which normally forms a disulfide bond with cys-c (the th residue from the c-terminus) of the h-chain. the free sulfhydryl group of cys-c on h-chains seem to be harmful for the transport of the elementary unit from er to golgi, because (a) the level of fibroin secretion is less than and ) , nd-s d (lanes and ) or l · (lanes and ) with the -bp l-chain cdna probe (encoded by exons i, ii, and iii) common to c and nd-s d l-chain mrnas or with the cdna probe for the b. mori elongation factor a- isoform as an internal control. % of the normal level, (b) er is unusually enlarged in psg cells, (c) the development of psg is significantly retarded and (d) a thin, naked-pupa-type cocoons are formed in these mutants [ ] . the present results that (a) the normal l-chain is expressed (b) the normal elementary unit is assembled and (c) the nd-s d phenotypes with respect to the retardation of psg development and the formation of thin cocoons were largely converted to the normal phenotypes in the l · transgenic line expressing a significant level of the normal l-chain strongly support the above notion. with respect to the second event in the assembly of the elementary unit, it is of interest to note that the extremely small amount of fibroin secreted into thin cocoons of the nd-s d mutant formed the h fhx -type complex ( table ). these results suggest that, although very inefficient in the absence of l-chain, one molecule of fhx/p could associate noncovalently with six molecules of h-chains. we demonstrated previously that n-linked oligosaccharide chains of fhx/p were important in maintaining the integrity of the elementary unit [ ] . however, considering the facts that oligosaccharide chains play important roles in the assembly of vesicular stomatitis viral glycoprotein [ , ] , influenza virus hemagglutinin [ , ] , and igm [ ] in er, a further role of n-linked oligosacharide chains of fhx/p in the process of assembly of the elementary unit in er may be speculated. with regard to this notion, the present finding that a relatively large amount of fhx/p molecules, i.e. about one third of molecular numbers of h-chain and l-chain (table ) , exists in er without being assembled into the elementary unit is interesting. this observation seems to be consistent with the previous observation that concentrations of fhx/p mrna and h-chain mrna are roughly equimolar in psg cells during the last intermolt stage [ ] . the deduced sequence of fhx/p contains a typical signal peptide sequence at the n-terminal region [ ] and the sequence is cleaved at the n-terminal side of gly in the secreted fhx/p [ ] . however, fhx/p does not contain the er retention signal sequence [ ] in its c-terminal region. thus, it could be speculated that the retention of fhx/p molecules which were not assembled into the elementary unit in er might be attained by interaction with other er-resident proteins but the real reason remains to be elucidated. we would like to speculate that fhx/p molecules having n-linked oligosaccharide chains interact noncovalently with nascent h-chains, perhaps during their translation and translocation into er, as a sort of molecular chaperone to prevent denaturation of h-chains. subsequently l-chains form a disulfide bond with cys-c of h-chains, then assembly of the (h-l) fhx complex takes place. the successfully assembled elementary units are allowed by the er quality control system and transported efficiently to golgi complex. it has been shown that most of the glycoproteins expressed in lepidopteran insect cells have either high-mannose type (glcnac man ) ) or truncated tri-mannosyl type [glcnac man (± fuc)] n-linked oligosaccharide chains, most likely due to the high activity of b-n-acetylglucosaminidase in golgi, which prevents further processing to the complex-or hybrid-type chains [ ] [ ] [ ] . as suggested from the deduced primary structure of fhx/p [ , ] , the presence of three n-linked oligosaccharide chains per a molecule of fhx/p was demonstrated [ ] . furthermore, conversion of the -kda form of the purified or partially purified fhx/p to the -kda form was achieved by digestion with the bacterial a , -mannosidase (this study). we can calculate molecular masses of fhx/p containing different numbers of mannose residues as follows: . kda for a molecule containing three chains of glcnac man and . kda for a molecule containing three chains of glcnac man , which are close to the -and -kda forms of fhx/p , respectively. we think it most probable that the -kda form of fhx/ p is produced in the golgi complex by the action of golgi a , -mannosidases, because the present results showed that only the -kda form was present in the er extract and the -kda form was detected in the golgi plus er mixed extracts from psg cells. in the giant polyploid cells of psg, er is fully developed [ ] and we could not succeed in isolating a pure golgi fraction which is not contaminated with er. we also tried to compare digestibility of the terminal a , -mannose residues of n-linked oligosaccharide chains of fhx/p in the isolated intact elementary unit or in the l-chain-free elementary unit produced by the treatment with dithiothreitol as described [ ] with the bacterial a , -mannosidase in vitro. however, this enzyme could not cleave the a , -mannose residues of fhx/p in either protein complex (data not shown), although the same enzyme could digest a , -mannose residues efficiently in purified or partially purified fhx/p . we suspect that the dynamic configuration of the isolated elementary unit is different from that during the intracellular transport or the size of the bacterial a , -mannosidase dimer ( kda · ¼ kda) employed [ ] is too large to access the oligosaccharide chains of fhx/p which have been suggested to be located internally of the elementary unit [ ] . a possible mechanism of the protection of a , -mannose residues of fhx/p in the elementary unit in the presence of l-chain two possibilities exist with respect to the role of l-chain in the protection of terminal a , -mannose residues of fhx/p in the elementary unit from the action of golgi a , -mannosidases: a direct model (fig. a ) and an indirect model (fig. b) . in the direct model, the l-chain has dual functions: formation of the disulfide-linked h-l heterodimer which is essential for the efficient transport of the h-chain-containing protein complex from er to golgi and protection of the terminal a , -mannose residues of fhx/p in the elementary unit. we suggested previously that the six sets of h-l dimers surrounded the centrally located fhx/p molecule in the elementary unit based on the observation that biotinylated cona was accessible to the n-linked oligosaccharide chains of fhx/p only after the treatment of the elementary unit with m urea or % triton x- [ ] . the l-chains in the majority of elementary units may interact noncovalently with the oligosaccharide chains of fhx/p or act as structural barriers and cause protection of the terminal mannose residues from digestion with a , -mannosidases in golgi complex. in the latter model, the function of the l-chain is to accelerate transport of the assembled elementary unit from er to golgi. a vast flow of elementary units containing h-l dimers and fhx/p into golgi may result in conditions where molecular numbers of golgi a , mannosidases become substantially insufficient and thus the majority of elementary units are allowed to leave golgi for secretion without losing the a , -mannose residues of fhx/p . it is of interest to note that -and -kda forms of fhx/ p are present in either transgenic line; l · producing a high level or l - producing an extremely low level of normal l-chain. it implies that numbers of the normal elementary unit are assembled in proportion to the available numbers of the normal l-chain, and the n-linked oligosaccharide chains of fhx/p in those elementary units are protected to the similar extent from the action of golgi a , mannosidases. these results seem to favor the direct model. on the other hand, the presence of -kda fhx/p as a minor component in elementary units produced in the normal breeds seems to favor the indirect model. perhaps the above two models may not be mutually exclusive. although further experimental evidences are required to judge which mechanism is more likely, present results suggest that not only fhx/p but also l-chain play respective roles in the formation of the integral structure of the elementary unit of fibroin. silk fibroin of bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of h-chain, l-chain, and p , with a : : molar ratio complete nucleotide sequence of the gene encoding the bombyx mori silk protein p and predicted amino acid sequence of the protein amino acid sequence and putative conformational characteristics of the kd silk protein of bombyx mori determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by bombyx mori hydrophobic interaction of p , containing asn-linked oligosaccharide chains, with the h-l complex of silk fibroin produced by bombyx mori reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm nd ( ) further evidence for importance of the subunit combination of silk fibroin in its efficient secretion from the posterior silk gland cells production of a chimeric fibroin light-chain polypeptide in a fibroin secretion-deficient naked pupa mutant of the silkworm bombyx mori linkage analysis of the fibroin gene in the silkworm, bombyx mori genetic analysis of the nd-s mutation in the silkworm, bombyx mori expression of the human udp-galactose transporter in the golgi membranes of murine had- cells that lack the endogenous transporter n-linked oligosaccharide chains of sendai virus fusion protein determine the interaction with endoplasmic reticulum molecular chaperones a new a-d-mannosidase occurring in golgi membranes lysosomal a-d-mannosidase of rat liver isolation of subcellular organelles immunological identification of the major disulfide-linked light component of silk fibroin an a , -mannosidase from a bacillus sp.: purification, characterization, and mode of action strucure of the bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain primary structure of the silk fibroin light chain determined by cdna sequencing and peptide analysis transgenic silkworms produce recombinant human type iii procollagen in cocoons germline transformation of the silkworm bombyx mori l. using a piggybac transposon-derived vector ) xp -egfp marker facilitates screening for transgenic silkworm bombyx mori l. from the embryonic stage onwards nucleotide sequence of the cdna encoding silk gland elongation factor a protein oligomerization in the endoplasmic reticulum multisubunit assembly of an integral membrane channel protein, gap junction con-nexin , occurs after exit from the er oligomeization of a trans-golgi/trans-golgi network retained protein occurs in the golgi complex and may be part of its retention quality control in the secretory pathway a single n-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface assembly of influenza hemagglutinin trimers and its role in intracellular transport folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin biogenesis and function of igm: the role of the conserved l-chain tailpiece glycans developmental variations of a nonfibroin mrna of bombyx mori silkgland, encoding for a low-molecular-weight silk protein structural organization of the p gene of bombyx mori and comparative analysis of its ¢ flanking dna with that of the fibroin gene a c-terminal signal prevents secretion of luminal er proteins insect cells contain an unusual, membrane-bound b-n-acetylglucosaminidase probably involved in the processing of protein n-glycans n-acetyl-b-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expression in insect cells from a baculovirus vector sialylation of n-glycans on the recombinant proteins expressed by a baculovirus-insect cell system under b-n-acetylglucosaminidase inhibition the structure and ultrastructure of the silk gland we thank kazuko seo and hiroko yamazaki, national institute of agrobiological sciences, for technical assistance, and dr masahiro tomita and dr katsutoshi yoshizato, hiroshima tissue regeneration project, for providing pbac( xp -dsred ) vector. this work was supported in part by the ministry of agriculture, forestry and fisheries, japan and by the program for the promotion of basic research activities for innovative bioscience, japan. key: cord- -b xvycyk authors: nan title: envelope glycoprotein interactions in coronavirus assembly date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: b xvycyk coronaviruses are assembled by budding into smooth membranes of the intermediate er-to-golgi compartment. we have studied the association of the viral membrane glycoproteins m and s in the formation of the virion envelope. using coimmunoprecipitation analysis we demonstrated that the m and s proteins of mouse hepatitis virus (mhv) interact specifically forming heteromultimeric complexes in infected cells. these could be detected only when the detergents used for their solubilization from cells or virions were carefully chosen: a combination of nonionic (np- ) and ionic (deoxycholic acid) detergents proved to be optimal. pulse-chase experiments revealed that newly made m and s proteins engaged in complex formation with different kinetics. whereas the m protein appeared in complexes immediately after its synthesis, newly synthesized s protein did so only after a lag phase of > min. newly made m was incorporated into virus particles faster than s, which suggests that it associates with preexisting s molecules. using the vaccinia virus t -driven coexpression of m and s we also demonstrate formation of m/s complexes in the absence of other coronaviral proteins. pulse-chase labelings and coimmunoprecipitation analyses revealed that m and s associate in pre-golgi membranes because the unglycosylated form of m appeared in m/s complexes rapidly. since no association of m and s was detected when protein export from the er was blocked by brefeldin a, stable complexes most likely arise in the er-to-golgi intermediate compartment. sucrose velocity gradient analysis showed the m/s complexes to be heterogeneous and of higher order, suggesting that they are maintained by homo- and heterotypic interactions. m/s complexes colocalized with alpha-mannosidase ii, a resident golgi protein. they acquired golgi-specific oligosaccharide modifications but were not detected at the cell surface. thus, the s protein, which on itself was transported to the plasma membrane, was retained in the golgi complex by its association with the m protein. because coronaviruses bud at pre-golgi membranes, this result implies that the envelope glycoprotein complexes do not determine the site of budding. yet, the self-association of the mhv envelope glycoproteins into higher order complexes is indicative of its role in the sorting of the viral membrane proteins and in driving the formation of the viral lipoprotein coat in virus assembly. m and s associate in pre-golgi membranes because the unglycosylated form of m appeared in m/s complexes rapidly. since no association of m and s was detected when protein export from the er was blocked by brefeldin a, stable complexes most likely arise in the er-to-golgi intermediate compartment. sucrose velocity gradient analysis showed the m/s complexes to be heterogeneous and of higher order, suggesting that they are maintained by homo-and heterotypic interactions. m/s complexes colocalized with a-mannosidase ii, a resident golgi protein. they acquired golgi-specific oligosaccharide modifications but were not detected at the cell surface. thus, the s protein, which on itself was transported to the plasma membrane, was retained in the golgi complex by its association with the m protein. because coronaviruses bud at pre-golgi membranes, this result implies that the envelope glycoprotein complexes do not determine the site of budding. yet, the self-association of the mhv envelope glycoproteins into higher order complexes is indicative of its role in the sorting of the viral membrane proteins and in driving the formation of the viral lipoprotein coat in virus assembly. udding through cellular membranes is the final step in the assembly of enveloped viruses. it results in the envelopment of the viral nucleocapsid (nc) by a membrane modified by the viral envelope proteins. complex protein-protein interactions between the envelope proteins control the timing, location, and specificity of budding. the envelopment of the nc is believed to be driven by its interactions with the envelope proteins (simons and garoff, ; dubois-dalcq et al., ; simons (stephens and compans, ) , while others bud into intracellular compartments (pettersson, ; griffiths and rottier, ) . the location of virus budding is thought to be dictated by the envelope proteins because they usually accumulate at the site of budding (dubois-dalcq et al., ; stephens and compans, ; pettersson, ; hobman, ) . when the semliki forest virus spike proteins were arrested in the golgi complex by monensin, budding took place there rather than at the plasma membrane (griffiths et al., ) . however, the accumulation of the vesicular stomatitis virus (vsv) g protein in the trans-golgi network by incubation at °c prevented assembly (griffiths et al., ) . thus, although a local accumulation of envelope proteins may be instrumental in budding, additional yet unidentified factors may be involved in localizing this process. we studied the assembly of the mouse hepatitis coronavirus (mhv), strain a- . mhv is a large, enveloped, positive-strand rna virus with a simple protein composition (spaan et al., ; holmes, ) . the viral genome is packaged by the nucleocapsid (n) protein into an nc with helical symmetry, and this in turn is enveloped by a lipid bilayer containing the membrane (m) protein ( - kd) and the spike (s) protein ( kd). a small, nonglycosylated membrane protein (e) of ~ kd was recently identified as a minor third constituent of the viral envelope (yu et al., ) . coronaviruses are assembled at smooth membranes of the intermediate compartment (ic) (tooze et al., ; klumperman et al., ; krijnse-locker et al., ) . because coronaviruses lack a matrix protein, envelopment probably involves direct interactions between the nc and one or more of the envelope proteins. the m protein is a likely candidate because of its abundancy, and because it was found to associate with the nc in vitro (sturman et al., ) . moreover, the s protein is probably dispensable for budding since spikeless virions were produced when infected cells were treated with tunicamycin (holmes et al., ; rottier et al., ) . the role of the e protein is presently unknown. we have shown previously that neither of the envelope glycoproteins accumulates at the site of budding when expressed independently: the m protein alone localizes to the golgi complex (rottier and rose, ; krijnse locker et al., a; klumperman et al., ) , whereas the s protein is transported to the plasma membrane (vennema, h., and p. j. m. rottier, unpublished data) . the different fates of the independently expressed glycoproteins suggested to us that m and s, which are synthesized from separate mrnas in infected cells, are involved in intermolecular interactions during virus assembly. the present study was therefore aimed at detecting and characterizing such interactions in mhv-infected cells and in cells expressing m and s from their cloned genes. using proper solubilization conditions, m/s complexes were identified by immunoprecipitation and sedimentation analysis. we investigated whether the association of m and s is the factor determining the site of budding, as it might preclude their transport beyond the budding compartment. in addition, we asked whether lateral interactions between m and s lead to the formation of large envelope glycoprotein assemblies indicative of their role in driving the formation of the viral envelope. sac(-) cells were maintained in dulbecco's minimal essential medium containing % fcs, penicillin, and streptomycin (dmem- % fcs). bhk- cells were maintained in dmem- % fcs. ost - cells (elroy-stein and moss, ) , a kind gift of b. moss (national institutes of health, bethesda, md) were maintained in dmem- % fcs supplemented with ~g/ml g- (geneticin; gibco laboratories, grand island, ny). mhv-a was propagated in sac(-) cells as described previously (spaan et al., ) . the recombinant vaccinia virus vtf - expressing the bacteriophage t rna polymerase (fuerst et al., ) was obtained from b. moss. the production of the rabbit polyclonal antiserum to mhv-a , the rabbit antipeptide serum to the m protein, and the rabbit polyclonal antiserum to vesicular stomatitis virus have been described previously (krijnse locker et al., b; rottier et al., , vennema et al., b . the mabs j . and j . against s and m, respectively, were kindly provided by j. fleming (university of southern california, los angeles, ca). the polyclonal rabbit serum against a-mannosidase ii (man ii) (moremen et al., ) was a kind gift of k. moremen (university of georgia, athens, ga). mhv infection. subconfluent monolayers of sac(-) or ost - cells in or -mm dishes were washed with pbs containing ~g of deae-dextran per ml and % fcs (pbs-deae-i% fcs) and inoculated with mhv-a for min at a multiplicity of infection of - in pbs-deae- % fcs at °c. for expression of cloned genes, subconfluent monolayers of ost - cells in -mm dishes were washed with dmem and inoculated with vtf - at a multiplicity of infection of ~ in dmem for rain at °c. ceils were then washed with dmem and transfected with plasmid dna. the following vectors were used: ptum-m (opstelten et al., ) , ptum-s (the mhv s gene cloned as a bamhi fragment [vennema et al., b] into ptug ), and ptuv-g, which contain a cdna copy of the mhv-m, the mhv-s, and the vsv-g protein, respectively, under the control of the t promotor. routinely, ixl dmem containing - txg plasmid dna was mixed with ixl lipofectin reagent (gibco brl, life technologies, inc., gaithersburg, md) and added to the cells. after a -rain incubation at room temperature, ~ dmem was added and the cells were incubated further at °c. coexpression of the mhv m and s proteins in double-transfected ost- cells was monitored by immunofluorescence. approximately % of the cells expressed detectable levels of the viral glycoproteins at h after infection; > % of these cells expressed both m and s. labeling. the incubation temperature of vtft- -infected bhk and ost - cells was shifted to °c at h after inoculation. the incubation temperature of mhv-infected ost - cells was shifted to °c min before labeling. at . or . h after inoculation, the ceils were starved for min in mem without methionine (gibco laboratories). when indicated, brefeldin a (bfa) (boehringer mannheim biochemicals, indianapolis, in) was added to a concentration of ~g/ml. cells were pulse labeled with - p.ci s-in vitro labeling mix (amersham corp., arlington heights, il) for the times indicated, then washed once with dmem- % fcs supplemented with mm hepes, mm l-methionine, and mm l-cysteine (chase medium) and chased for various times in chase medium. the cells were lysed on ice in mm tris (ph . ), . mm edta, . % np- , . % na-deoxycholate (detergent solution) containing mm pmsf. the lysates were spun for min at , g at °c to remove nuclei and cell debris. in the experiment of fig. a cells were also lysed using % np- or % triton x- in mm tris (ph . ), . mm edta or % triton x- in mnt ( mm mes, mm tris [ph . ], mm naci, . mm edta, mm egta). samples of cell lysates were mixed with % (wt/wt) sucrose in detergent solution to a final concentration of % (wt/wt) before loading on gradients consisting of - % (wt/wt) sucrose in detergent solution. the gradients were centrifuged for min at , rpm in an sw . rotor (beckman instruments, inc., fullerton, ca) at °c and fractions of ~ p. were collected from the bottom of the tubes. aliquots of each fraction were subjected to immunoprecipitation with specific antibodies and the precipitates analyzed by sds-page. the sedimentation of the marker molecules catalase ( . s ,~) and thyroglobulin ( . s ,~) was performed in a parallel gradient. aliquots of the fractions were analyzed in an sds-page, and the proteins visualized by staining with coomassie brilliant blue. viral proteins were immunoprecipitated with the polyclonal mhv-a antiserum ( }~l), the mab j . ct m ( }xl), or with the mab j . ct s ( i~l). antibodies were added to aliquots of cell lysates diluted with detergent solution to a final vol of p~l. after overnight incubation at °c, immune complexes were collected using - }xl of a % (wt/vol) suspension of formaldehyde-fixed and heat-inactivated staphylococcus aureus cells (gibco brl, life technologies, inc.). after a -min incubation at °c the cells were washed three times with detergent solution and finally suspended in }~l . mm tris-hcl (ph . ), mm dtf, % sds, % glycerol (sample buffer). the samples were heated for min at °c before loading on or % sds-pag. endoglycosidase h (boehringer mannheim biochemicals) treatments were carried out as described by machamer er al. ( ) . quantification of the radioactivity in the protein bands in the dried gels was carried out using a phosphoimager and imagequant (version . ; molecular dynamics, inc., sunnyvale, ca) according to the manufacturer's instructions. transfected cells were labeled with s-in vitro labeling mix from - . h after infection and chased for h in chase medium. the culture media cleared by centrifugation for min at , rpm, °c were diluted with a / volume of a times concentrated stock detergent solution and subjected to immunoprecipitation using mabs against m and s. plates were put on ice and the cells washed with pbs/ % fcs and incubated for h in p, pbs/ % fcs containing the mab j . etm ( p, ), and/or mab j . as ( }~l), and/or the polyclonal anti-vsv serum ( p~l). thereafter, cells were extensively washed with pbs/ % fcs and lysed with detergent solution containing mm pmsf. the lysates were spun for min at , g at °c, and }~l of a % (wt/vol) suspension of formalin-fixed s. aureus cells was added to the supernatants to collect the immune complexes. after a -min incubation at °c, the cells were pelleted by centrifugation, washed three times with detergent solution, and finally suspended in sample buffer. the primary supernatants were subjected to a second round of immunoprecipitation using the same antibodies. ost - or bhk- cells grown on -mm, gelatin-coated coverslips, were infected and transfected as described above. h after infection cells were fixed with % paraformaldehyde for - min and washed three times with pbs containing mm glycine (pbs-glycine). cells were permeabilized with pbs/ % triton x- for rain followed by three washes with pbs-glycine. they were then treated for min with a mixture of two an-tisera diluted in pbs-glycine- % fcs: mab j . ~s ( : ); m peptide antiserum ( : ); polyclonal man ii rabbit antiserum ( : ). antibodies were washed away, and the cells were stained for min with affinitypurified rhodamine-conjugated goat anti-rabbit ig and fluorescein-conjugated goat anti-mouse ig (protos immunoresearch, san fransisco, ca) that were diluted : and : in pbs-glycine- % fcs, respectively. all incubations were done at room temperature. finally, the coverslips were washed extensively and mounted in fluorosave tm (calbiochem corp., la jolla, ca). fluorescence was viewed with a microscope (bhs-f; olympus corp., precision instruments division, success, ny). complex formation between the m and s protein of coronaviruses has never been demonstrated. we reasoned that this might have been due to a disruption of the interactions during analytical procedures since maintenance of the integrity of the complexes might require specific conditions. by analyzing the effects of different solubilization conditions we were able to demonstrate the existence of m/s complexes and found that the choice of the detergents is crucial for the preservation of the interaction between m and s. this is illustrated by the experiment shown in fig. irrespective of the detergent used, the anti-mhv serum precipitated the viral structural proteins n, m, and the s precursor (gp ). the mabets precipitated the s protein, as expected, but in one case also the m protein, when a combination of the nonionic detergent np- and the ionic detergent deoxycholic acid was used. the amount of coprecipitated m was similar to that obtained with the anti-mhv serum; virtually no n protein nor any cellular proteins was observed in the precipitate. we conclude that in infected cells a large fraction of m is physically complexed with s, and that the stability of these complexes is dependent on the detergents used for solubilization. to determine whether m and s associate in the absence of other coronaviral proteins, we used vaccinia virus ttdriven coexpression of their respective genes in ostt- cells (elroy-stein and moss, ) . the results with the anti-mhv serum show that m and s were efficiently labeled in cells coexpressing the proteins (fig. b) . when using the s-specific antibodies m was also precipitated, in addition to s, indicating that the proteins do associate independent of other coronaviral proteins. to exclude the possibility that their association had occurred after cell lysis, we performed the immunoprecipitations with a mixture of lysates from cultures in which m and s had been expressed separately. in this case m was not coprecipitated. we never observed specific coprecipitation of other labeled proteins: some bands appearing after longer exposures were also detected in analyses of cells expressing m or s alone. note that the rabbit anti-mhv serum precipitates some non-mhv proteins from expressing cells, not from mhv-infected cells. the identity of these proteins is unclear, but they are likely to be derived from vaccinia virus. the almost quantitative coprecipitation of m from mhv-infected cells indicates that all forms of the protein were associated with s. the incubation temperature ( °c) did not affect complex formation since the same pattern was observed at °c ( fig. a) . in contrast, predominantly glycosylated forms of m were associated with s in transfected cells. to determine the kinetics of protein association in infected cells we carried out a pulse-chase experiment. mhv-infected cells were labeled for min and chased for various time periods as indicated in fig. . the material precipitated with the anti-mhv serum represents the total pool of labeled viral structural proteins present in infected cells after the various chase periods. their amount decreased during the chase period due to assembly into virions and subsequent release from the cells. the changes in the mobilities of the glycoproteins show that they were processed; the unglycosylated form of m was converted into various slower-migrating species as a result of posttranslational o-glycosylation. the addition of the first sugar, n-acetyl-galactosamine, takes place in the ic (tooze et al., ; krijnse-locker et al., ) while the addition of galactose and sialic acid occur after the protein has reached the golgi complex (krijnse locker et al., a) . the s protein is synthesized as a core glycosylated precursor s/gp which is slowly converted into s/gpl (not clearly resolved in this gel) by maturation of its oligosaccharides. a fraction of this species is cleaved into subunits s and $ , both with a molecular mass of ~ kd (s/gp ). m/s complexes were analyzed by immunoprecipitation with the monospecific antibodies against m and s. using the mabe~s, coprecipitation of m was again observed after the pulse. the amount of coprecipitated m protein rapidly increased and reached its maximum after -- min of chase. apparently, newly synthesized m associated with s very quickly as evidenced also by the presence of the unglycosylated form of m in the m/s complexes. these observations indicate that the proteins associate in a pre-golgi compartment. the m protein present in m/s compl.exes was converted from its unglycosylated form into the various glycosylated species, which shows that its processing occurs after its association with s. when the m a b a m was used instead, coprecipitation of labeled s was not observed immediately, but only after - min of chase. later, the amount of coprecipitated s, including its cleaved form s/gp , gradually increased. these observations indicate that s associated with m before its processing to the mature forms s/gpl and s/gp . m and s thus engage in complex formation at different rates: m rapidly associates with s, while s does so only after a considerable lag time. this implies that newly synthesized m molecules associate with s molecules already present. if these interpretations were correct, the m and s proteins would be incorporated into virus particles with different kinetics. we therefore performed a pulse-chase labeling of infected cells and monitored the appearance of labeled proteins in extracellular virus. because mhv is assembled intracellularly, such an analysis provides an indirect measurement of the kinetics of incorporation of newly synthesized proteins into virus particles. virus was purified by pelleting and analyzed by sds-page (fig. a) . the results of the quantifications of the radioactivities in the bands representing m and s (s/gp + s/gp ) are shown in fig. b. virions containing labeled s protein appeared in the medium after a -min lag period. thereafter, roughly equal amounts of labeled s were found to be released during each chase interval for at least min. in contrast, labeled m protein started to appear in the culture medium already during the - min chase period, increased rapidly during the -- min chase period, and declined thereafter. this implies that newly synthesized m was incorporated into virions faster than s. in addition, the s molecules were assembled into virions in a more protracted fashion than the m molecules. to investigate whether the association of m and s is the factor determining the site of virus budding we analyzed the intracellular localization of m/s complexes by indirect double immunofluorescence. for this purpose, we coexpressed m and s in bhk- cells because these cells are appropriate for immunolocalization. both proteins localized to a distinct perinuclear region (fig. , a and b) . in contrast, when expressed by itself, s had a faint reticular appearance (fig. d) while it was also observed at the surface of nonpermeabilized cells (not shown). the intracellular localization of the m protein appeared not to be affected by s because its distribution was similar to that in cells which expressed m only (fig. c) . the suggestion that m and s coaccumulated in the golgi complex was confirmed by visualizing this compartment using a serum against man ii, a resident golgi protein (moremen et al., ) . the localization of man ii clearly overlapped with that of the s protein (fig. , e and f). the conclusion that m had retained s in the golgi region is consistent with the observation that m/s complexes predominantly contained mature forms of m (fig. b) . to analyze the transport of m/s complexes biochemically we labeled double-transfected cells for min and chased them for different periods. equal fractions of the cell lysates were subjected to immunoprecipitation with the anti-mhv serum (fig. a) , the mabols (fig. a) , or the mabetm (fig. b) . the precipitates obtained using the mab~m were split to perform an endoglycosidase h sensitivity assay. the time courses with which newly synthesized m and s engage in heterocomplexes in the absence of other viral components appeared to be similar to those observed in mhv-infected cells (fig. ) . after the pulse, a small frac-tion of m was coprecipitated by the mabots (fig. a) ; its amount increased during the chase and reached a maximum between and min of chase. in contrast, coprecipitation of labeled s appeared only after min of chase and reached its highest level ~ min later (fig. b) . the m protein already associated with s while still in its unglycosylated form (fig. a) indicating that the complexes are formed in a pre-golgi compartment. consistently, the s precursor gpl , coprecipitated by the m-specific antibodies ( fig. b) , appeared to be completely endoglycosidase h-sensitive, which means that it had not yet passed the medial-golgi. the finding that m/s complexes eventually consisted of golgi-modified forms both of m and of s confirms their transport to the golgi complex. surprisingly, the pulse-chase assay did not reveal any transient accumulation of the unglycosylated precursor form m nor of m . apparently, the accumulation of m/s complexes in the ic, which supposedly occurs in mhv-infected cells, does not occur when the proteins are coexpressed. instead, m/s complexes are transported efficiently beyond the budding site to the golgi complex in the absence of other coronaviral proteins. although the golgi-modified forms of m and s were all found in m/s complexes, a fraction of m and s remained immature and incompetent to associate even after longer chase periods. as far as the s protein is concerned, this is similar to what we found in mhv-infected cells (fig. ) . in that case, however, the m protein associated with s very rapidly and almost quantitatively. this suggests that a fraction of m is not properly processed in this vaccinia virusbased expression system, a phenomenon also found with other viral proteins (marquardt and helenius, ; vennema, h., g.-j. godeke, and p. j. m. rottier, unpublished data). however, the less efficient transport of m seems not to be directly caused by the vaccinia virus infection, since the m protein expressed by a recombinant vaccinia virus was transported almost quantitatively to the golgi complex (krijnse locker et al., a) . the data obtained so far suggest that the coexpressed m and s proteins associate during their transport to the golgi complex while they form complexes already in the er of mhv-infected cells (fig. b) . to establish whether m and s associate in the er or beyond this compartment we analyzed the formation of m/s complexes in the presence -schwartz et al., -schwartz et al., , klausner et al., ) . in the experiment of fig. , bfa was added to mhv-infected cells and to cells coexpressing m and s at min before the labeling, and it was kept present during further incubations. the activity of the drug was evident from its effect on the maturation of s. in the presence of bfa its conversion into s/gp and its subsequent cleavage were completely inhibited, consistent with the block of transport to a late golgi compartment. the maturation of the m protein was not affected by bfa. this indicates that the enzymes catalyzing the formation of m and m relocated into the er upon the addition of bfa. this result is somewhat different from findings in another cell type (krijnse locker et al., a) where n o m species was synthesized when bfa was present. in the coexpression system bfa prevented the formation of stable m/s complexes as judged by the absence of m in the immunoprecipitate of s. this indicates that transport of the coexpressed m and s proteins from the er is required for their complexation. in contrast, in mhvinfected cells the association of m and s was not affected by bfa since equal amounts of m were coprecipitated from bfa-treated and -untreated samples. thus, the failure of m and s to interact in coexpressing cells was not artificially induced by bfa. the effects of bfa appeared to be reversible; after its washout a significant fraction of m was coprecipitated. in addition, the appearance of s/gp and s/gp , though weak, indicates that transport from the er to the golgi complex was also restored to some extent. the immunofluorescence data indicated that s is retained intracellularly by its interaction with m. apparently, the signal that mediates the retention of m in the golgi complex (rottier and rose, ; krijnse locker et al., a , , is also functional in complexes of m with s. in contrast to m, the s protein is normally transported to the cell surface (vennema, h., and p. j. m. rottier, unpublished data; see below) . to test whether the efficacy of golgi retention of the m protein is affected by its association with s we performed cell surface immunoprecipitations. cells expressing s or both m and s proteins were labeled for min and chased for h to allow the proteins to reach their final destination. the culture media were screened for the presence of viral proteins and the mabs as and am, which recognize the ectodomain of m or s, were used for surface immunoprecipitations. the intracellular pool of viral proteins was collected in a second round of immunoprecipitation with the same antibodies. the m and s proteins were virtually absent from the medium and the plasma membrane of double-transfected cells, although most of them had reached the golgi complex as evidenced by their maturation state inside the cells (fig. ) . in contrast, we specifically detected s/gp and s/gp at the surface of cells expressing s alone; the precursor s/gp was only detected intracellularly confirming the reliability of the assay. some s protein also appeared at the plasma membrane when expressed with m; a tiny fraction apparently escapes from interacting with m and is transported to the cell surface. accordingly, cells coexpressing m and s still fused, albeit more slowly and less extensively than when s was expressed alone (not shown). relatively more s/gp accumulated in the presence of m, whereas most of it was cleaved in its absence. moreover, s/gp was detected predominantly inside the cells coexpressing m and s while it appeared more prominently at the cell surface when expressed on its own. in the figure . intracellular transport of m/s complexes, vtft- -infected ost - cells were cotransfected with ptum-m and ptum-s dna. cells were pulse labeled at h after infection for min and immediately lysed or chased for the times indicated. equal fractions of the cell lysates were used for immunoprecipitation using polyclonal anti-mhv serum (a), the mabas (a), or the mabam (b). precipitates obtained using mabam were split; one-half was mock treated and the other half was treated with endoglycosidase h (b). viral proteins were analyzed in two sds- % pags. latter situation a significant amount of s/gp was found in the culture medium presumably representing the s subunit dissociated from the membrane-anchored $ subunit. the results indicate that s, when caught in m/s complexes, was arrested in the golgi complex. despite its interaction with s, the m protein does not leak to the plasma membrane. to check whether the accumulation of m in the golgi complex does prevent protein transport through this compartment nonspecifically we expressed m together with the vsv-g protein. the result shows that the g protein appeared quantitatively at the plasma membrane. we conclude that golgi retention of the s protein is mediated by its specific interaction with the m protein and that trans- figure . effects of bfa on the formation of m/s complexes. vtf - -infected ost - cells were cotransfected with ptum-m and ptum-s dna (m/s). parallel cultures were infected with mhv. at h after infection cells were pulse labeled for min followed by a -min chase. when indicated bfa was added to the cells at . h after infection and it was kept present during labeling and chase (+). in one case, bfa was washed out and cells were further chased for another min in the absence of bfa (+/-). equal portions of the lysates were used for immunoprecipitation using polyclonal anti-mhv serum or mabas. viral proteins were analyzed in an sds- % page. port of other membrane proteins to the cell surface is unimpeded by the presence of m. to investigate the complexity of the m/s complexes we have analyzed their sedimentation in sucrose velocity gradients. cells coexpressing m and s were radiolabeled for min and lysed either immediately or after and min of chase. the cleared lysates were analyzed in - % sucrose gradients. after fractionation, aliquots of gradient fractions were subjected to immunoprecipitation with the mabas or with the mabam. newly synthesized s protein was almost exclusively detected at the top of the gradient, while some m protein was coprecipitated from samples of the lower part of the gradient (fig. a) . the virtual absence of labeled s in the latter samples indicates that newly synthesized m was associated with preexisting, unlabeled s. during the chase the m protein in the complexes matured and gradually more labeled s protein sedimented into the gradient (fig. , b and c). the m/s complexes sedimented heterogeneously and were found predominantly between fraction and the bottom of the gradient. in addition, some m coprecipitated with s from the two top fractions; this material might represent partially disrupted complexes. using the m-specific antibodies we found that the complexes formed after a min chase (fig. d) contained s precursor gpl , as well as s/gpl . in contrast, when the s protein was expressed alone, its mature forms s/gpl and s/gp remained at the top of the gradient indicating that the protein does not nonspecifically associate into large aggregates (fig. e) . we have used the sedimentation behavior of catalase and thyroglobulin in parallel gradients as markers to get an estimate of the size of m/s complexes. these proteins peaked in fractions and , respectively (data not shown). this indicates that most complexes migrated as structures with sedimentation values higher than s. both unglycosylated and glycosylated forms of m were found in heterocomplexes formed during the pulse. the figure . intracellular accumulation of m/s complexes, vqtt- -infected ost - cells were (co)transfected with ptum-s, ptum-m, and/or ptuv-g dna. cells were pulse labeled for min and chased for min. the media (m) were cleared and subjected to immunoprecipitation (ripa) using the mabets and mabotm. the cell-surface (s) immunoprecipitations were carried out as follows: antibodies against the expressed proteins were added to the cells to allow binding at the plasma membrane. after a -h incubation on ice, the cells were washed extensively and then lysed. the immune complexes were precipitated with s. aureus cells after which the remaining intracellular (i) pool of viral proteins was collected in a second round of immunoprecipitation with the same antibodies. viral proteins were analyzed in an sds- % page (a). fig. b represents a longer exposure of a part of fig. a. presence of the unglycosylated form again indicates (see fig. ) that the protein is taken up in large heterocomplexes before its arrival in the golgi complex. the m and s proteins of mhv have different destinations in cells when expressed independently, yet they coassemble into virions during infection. here we describe the molecular basis for this peculiarity by showing that the two proteins exhibit an intrinsic affinity for each other. the association of m and s occurs in an early compartment since newly synthesized, unmodified m molecules are immediately taken up into complexes with s, indicating that they are formed before or during budding. in contrast, newly made s associates slowly and less efficiently, and m and s are therefore assembled into virions at different rates. when coexpressed in cells, m and s form large heteromultimeric complexes that are transported beyond the site of budding and accumulate in the golgi complex. under conditions of analysis, the stability of the envelope glycoprotein complexes is critically dependent on the detergents used for their solubilization. m-s interactions are preserved in a combination of np- and sodium deoxycholate. several observations indicated that m/s complexes do not form nonspecifically after solubilization. first, other proteins were not significantly coprecipitated with m and s. second, newly synthesized s started to associate with m only after a lag time of about min. third, m/s complexes were not formed upon mixing lysates containing m and s that had been separately expressed. moreover, flotation analysis ruled out the possibility that m and s were coisolated as part of detergent-insoluble membranes (data not shown). collectively, these data provide evidence for the existence and specificity of m-s interactions. newly synthesized m and s molecules enter into heterocomplexes with different kinetics, probably due to their different maturation rates. folding of s occurs slowly and involves the formation of intramolecular disulfide bonds (opstelten et al., ) , the addition and processing of n-linked sugars, and the assembly into homo-oligomers (vennema et al., a) . in contrast, the m protein acquires its final conformation in the er rapidly, without being glycosylated, without the formation of disulfide bonds (opstelten et al., ) , and even without the need for atp . thus folding of s is probably rate limiting in its association with m. newly made m interacts with preexisting folded s, as evidenced by the presence of unlabeled s in complexes after short labelings. only completely oxidized s molecules associate with m, and s is unable to interact when its folding has been inhibited by in vivo reduction with dtt (opstelten et al., ; our unpublished results) . a similar situation has been observed for bunyaviruses: heterodimerization of uukuniemi virus glycoproteins occurs between newly made g and presynthesized g due to the slow maturation of the latter (persson and pettersson, ) . mhv buds in the rer late in infection (tooze et al., ; . several observations indicate that m and s associate in this compartment. first, m/s complexes are rapidly formed, much of m was associated with s after a min pulse. second, the presence of unglycosylated m in the complexes indicates its association before addition of the first sugar (n-acetyl-galactosamine). third, treatment of infected cells with bfa, a drug that prevents exit of proteins from the er, did not inhibit the formation of m/s complexes. although these data suggest that the interaction between m and s precedes budding, we cannot exclude that association occurs synchronously with assembly. the association of coexpressed m and s also occurs in an early compartment since newly synthesized, unmodified m molecules appear in m/s complexes rapidly. however, the failure to detect the interactions in the presence of bfa suggests that stable complexes are formed only after the proteins have left the er, i.e., in the ic. this suggests that other factors are involved in the early formation and/or stabilization of m/s complexes in infected cells. one such factor might be the nc. its binding to the envelope glycoprotein complexes might promote and stabilize the inter-actions between m and s and create nucleation sites for conglomeration of the complexes. alternatively, the difference might be just the result of a concentration effect since multimerization reactions are dependent on the con- centrations of the reacting molecules (braakman et al., ) . thus, under our coexpression conditions, stable association of m and s might not have occurred in the e r because the proteins did not accumulate to sufficient lev-els. membrane glycoproteins are indeed concentrated during their export from the er (copeland et al., ; balch et al., ) , and this might induce the formation of stable m/s complexes. in the course of an mhv infection, the synthesis of the envelope proteins reaches much higher levels than during expression. thus, the association of m and s in infected cells might take place in the ic initially and in the er at later stages of infection, coinciding with the temporal pattern of virus budding. the m/s complexes do not determine the site of budding, because their transit through the ic was neither blocked nor delayed. the envelope proteins of other viruses generally accumulate at the site of assembly (pettersson, ; griffiths and rottier, ; hobman, ) while this is clearly not the case for mhv. because the m and s protein are not retained in the ic individually, we had anticipated that their association might endow the complexes with new retention information. their accumulation in the golgi complex, however, indicates that (an)other viral factor(s) determines the site of budding. a likely candidate is the nc that might bind and recruit the m/s complexes early in the exocytic pathway. another candidate is the small envelope protein (e) which was recently identified as a minor virion component (yu et al., ) . due to its association with m, the s protein accumulated in the golgi complex, the intrinsic residence of the m protein. because the cell-surface expression of the vsv-g protein was not affected by m, we conclude that m specifically retains the s protein. similarly, the g protein of punta toro virus is transported to the cell surface when expressed by itself, but accumulates in the golgi complex after heterodimerization with g that contains the signal for golgi retention (chen et al., ; matsuoka et al., ) . cosorting by association is a well-known principle of protein targeting and retention in eukaryotic cells. recently, nilsson et al. ( nilsson et al. ( , proposed kin recognition as a mechanism for sorting and retention of golgi enzymes. accordingly, sorting of the enzymes is based on specific interactions between kin-oligomers, while retention is achieved by their involvement in complexes too bulky to enter transport vescicles. the self-association of m and s has similar features: the proteins interact specifically, segregate from other proteins, and are subsequently targeted to and retained in the golgi complex. however, the association of m and s into large complexes in pre-golgi membranes apparently does not inhibit their further transport to the golgi complex. the interaction between m and s probably functions to incorporate the s protein into virions. s is dispensable for particle assembly since spikeless virions are released from infected cells treated with tunicamycin (holmes et al., ; rottier et al., ) . moreover, recent data from our laboratory show that the assembly of coronavirus-like particles also occurs independently of the s protein (vennema, h., g.-j. godeke, and p. j. m. rottier, unpublished data) which implies that envelope formation does not depend on m-s interactions. we therefore assume that the higher-order m/s complexes are maintained primarily, if not only, by m-m interactions. this is supported by our recent findings that m associates into large complexes when expressed by itself (krijnse locker et al., ) . extensive envelope protein interactions have important functions in coronavirus assembly. first, they are responsible for host protein exclusion. second, local membrane domains harboring these large envelope protein assemblies serve as the sites for budding. third, the clustering of envelope proteins may alter the fluidity of the lipid bilayer, for instance to facilitate the induction of curvature (dubois-dalcq et al., ; simons and fuller, ) . this factor could promote budding. finally, the envelope protein clusters might serve as a template for the condensation of the nc. the nc of coronaviruses are long, loosely coiled strands in the cytoplasm of infected cells that condensate at the membranes of the budding compartment; other viruses having a helical nc, e.g., vsv, encode a matrix protein that controls the coiling of the nc (newcomb et al., ) . the emerging picture of coronavirus budding shows extensive local rafts (patches) of laterally interacting m molecules in pre-golgi membranes. only few copies of the e protein and variable numbers of s molecules are encountered in these structures. ncs bind to the cytoplasmically exposed domains of the m proteins, surround themselves with the modified membrane, and pinch off into the lumen. the rafts are continuously being replenished by the arrival and incorporation of newly synthesized viral envelope proteins. further work should show whether this model is correct and establish the interactions that compound it. vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic 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complex oligomerization of a trans-golgi/trans-golgi network retained protein occurs in the golgi complex and may be part of its retention rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er microtubule-dependent retrograde transport of proteins into the er in the presence of brefeldin a suggests an er recycling pathway nucleocapsid-glycoprotein interactions required for assembly of alphaviruses the e glycoprotein of an avian coronavirus is targeted to the cis golgi complex misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum a signal for golgi retention in the bunyavirus g glycoprotein novel purification of the catalytic domain of golgi ct-mannosidase ii in vitro reassembly of vesicular stomafitis virus skeletons kin recognition. a model for the retention of golgi enzymes kin recognition between medial golgi enzymes in hela cells disulfide bonds in folding and transport of mouse hepatitis coronavirus glycoproteins formation and intracellular transport of a heterodimeric viral spike protein complex protein localization and virus assembly at intracellular membranes coronavirus e glycoprotein expressed from cloned cdna localizes in the golgi region viral protein synthesis in mouse hepatitis virus strain a -infected cells: effects of tunicamycin signal recognition particledependent insertion of coronavirus el, an intracellular membrane glycoprotein the budding of enveloped viruses: a paradigm for membrane sorting? the budding mechanisms of enveloped animal viruses isolation and identification of virus-specific mrnas in cells infected with mouse hepatitis virus (mhv-a ) coronaviruses: structure and genome expression assembly of animal viruses at cellular membranes isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid spike protein-nucleocapsid interactions drive the budding of alphaviruses replication of coronavirus mhv-a in sac-cells: determination of the first site of budding of progeny virions site of addition of n-acetyl-galactosamine to the e glycoprotein of mouse hepatitis virus-a biosynthesis and function of the coronavirus spike protein intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly mouse hepatitis virus gene b protein is a new virion envelope protein we thank h. vennema and g.-j. godeke for providing the plasmid dnas used in this work.received for publication february and in revised form june . key: cord- -u lb w authors: donaldson, julie g.; jackson, catherine l. title: arf family g proteins and their regulators: roles in membrane transport, development and disease date: - - journal: nature reviews molecular cell biology doi: . /nrm sha: doc_id: cord_uid: u lb w members of the adp-ribosylation factor (arf) family of guanine-nucleotide-binding (g) proteins, including the arf-like (arl) proteins and sar , regulate membrane traffic and organelle structure by recruiting cargo-sorting coat proteins, modulating membrane lipid composition, and interacting with regulators of other g proteins. new roles of arf and arl proteins are emerging, including novel functions at the golgi complex and in cilia formation. their function is under tight spatial control, which is mediated by guanine nucleotide exchange factors (gefs) and gtpase-activating proteins (gaps) that catalyse gtp exchange and hydrolysis, respectively. important advances are being gained in our understanding of the functional networks that are formed not only by the gefs and gaps themselves but also by the inactive forms of the arf proteins. guanine nucleotide exchange factors (gefs) . proteins that promote the release of gdp from guanin e-nucleotide-binding (g) proteins, which allows gtp to bind. these proteins often stabilize the nucleotide-free form and then are released upon gtp binding. gtpase-activating proteins (gaps) . proteins that promote gtp hydrolysis on gtp-bound guanine-nucleotide-binding (g) proteins. for adp-ribosylation factor (arf) proteins, gaps are critical, as arfs have negligible intrinsic gtpase activity. the catalytic regions of gaps often include an arg-finger motif that inserts into the gtp-binding pocket to stimulate hydrolysis of gtp. signalling is more complex and that gefs and gaps can initiate their own physiological responses. we see evidence of arf proteins acting in pairs or in series in the endoplasmic reticulum (er)-golgi system and at the plasma membrane. in this review, we emphasize how arf proteins function as a network in which the arf regulators participate. these regulators also integrate arf activities with other g protein signalling networks, as well as initiating their own distinct signalling pathways. we highlight new arf and arl activities, discuss a lipid modification, occurring co-or post-translationally, in which a myristoyl moiety is attached to a gly residue at the second position from the amino terminus, after cleavage of the n-terminal met residue. gdp dissociation inhibitor (gdi) . a protein that binds specifically to the gdp-bound form of a gtp-binding protein, preventing guanine nucleotide exchange. how gefs and gaps can act as scaffolds, both as effectors and in initiating signalling responses, and describe how they participate in development and disease. the reader is referred to two excellent prior reviews: one deals comprehensively with arf and arf function and the other more broadly with arf, arl, gef and gap proteins . arfs localize to membranes throughout the cell, including the plasma membrane and the membranes of the secretory, endosomal and lysosomal pathways. a distinguishing feature of arf family g proteins is the presence of an n-terminal amphipathic helix that is critical for membrane binding (fig. a,b ). in addition, all arf proteins are modified by myristoylation at the n terminus and this co-translational modification is required for membrane recruitment and biological activity. the myristoyl group and associated n-terminal amphipathic helix are inserted into the membrane upon gtp binding . thus, in addition to changes in the effector-binding regions upon exchange of gdp for gtp, arf proteins undergo a second change in conformation that brings them into very close contact with the membrane (fig. ) . this distinguishes them from other small g proteins of the ras superfamily, including the ras, rho and rab families, which have a long carboxy-terminal linker to which their lipid membrane anchor is attached . arf effectors are thus constrained to a position close to the membrane surface, in contrast to those of rab and rho, which can be located some distance from the membrane . some arl proteins are myristoylated but most seem to lack this modification. in arl b, loss of hydrophobic residues in the amphipathic helix abolishes lysosomal localization . interestingly, arfrp (arl in saccharomyces cerevisiae), arl a and arl b are acetylated rather than myristoylated at their n terminus . in sar , the n-terminal amphipathic helix binds directly to membrane s and induces membrane curvature . unlike for rab and rho g proteins, no gdp dissociation inhibitor (gdi) proteins have been identified for arfs or arls. arf and arf appear to be released from membranes on gtp hydrolysis in cells. arf , however, remains bound to membranes in its gdp-bound conformation, and there is evidence that arf and arf remain bound to er-golgi intermediate compartment (ergic) membranes in their gdp-bound form , . this raises the possibility that arf proteins that are bound to membranes in their gdp-bound form might interact with membrane-localized partners and mediate signalling. evidence for this idea is emerging for arf (see below), and suggests that distinct signalling pathways might be coordinated through the nucleotide state of these constitutively membrane-bound arf family proteins. sar and some of the arls, such as arl , arl and arl , are cytosolic when gdp-bound, similarly to arf (refs , ) , and it remains to be determined whether this is true for other arl proteins. in humans, there are arf gefs, which are divided into six subfamilies, as well as the sar gef sec (table ) . no specific arl gefs have yet been identified, although the arf gef syt (suppressor of ypt ) in yeast apparently also has activity towards arl (ref. ). the identified mammalian arf gaps fall into nine major subgroups based on their domain structure (table ) . two arl gaps have been identified (see below). gefs and gaps are recruited to very specific sites within cells to not only catalyse gtp exchange and hydrolysis, respectively, but also to assemble protein complexes at these sites independently of their catalytic activity (fig. c) . in this way, versatile signalling networks can be assembled that can respond dynamically to extracellula r and intracellular signals. following activation on membranes, gtp-bound arfs recruit coat proteins, lipid-modifying enzymes, tethers and other effector molecules that influence membrane trafficking and organelle structure , (table ) . for example, arf recruits the cytosolic coatomer complex i (copi) to golgi membranes, allowing sorting of cargo proteins into copi-coated vesicles . arf proteins at the trans-golgi network (tgn) also recruit heterotetrameric clathrin adaptor protein (ap ), ap and ap , as well as the three monomeric golgi-localized, γ-ear-containing, adp-ribosylation factor-binding figure | the domain structure and regulation of arf and arls. a | a schematic of representative adp-ribosylation factor (arf), sar and arf-like (arl) proteins, indicating the conserved amino-terminal amphipathic helix and the protein-specific lipid modifications at the n terminus. these include myristoylation (myr) and acetylation (acet), both of which ensure tight membrane association. the effector regions of the guaninenucleotide-binding (g) protein, switch (sw ) and sw , and the interswitch region between them, are depicted. these regions change conformation upon exchange of gdp for gtp and are involved in interactions with effectors. b | arf•gdp reversibly associates with the membrane surface, and the myristoylated n-terminal helix ensures tight membrane association of arf•gtp. the switch and interswitch regions are also shown, and these undergo a conformational change upon gtp binding to enter the hydrophobic pocket that the n-terminal amphipathic helix occupies in the gdp-bound form. c | arf family g proteins undergo a cycle of gtp binding and hydrolysis, mediated by guanine nucleotide exchange factors (gefs) and gtpase-activating proteins (gaps), respectively. the gtp-bound form is thought to carry out g protein functions through interaction with 'classical effectors', including vesicle coat proteins and enzymes that can modify membrane lipid composition; however, increased attention has focused on networks of effectors that are targeted by proteins that interact with gefs and gaps themselves and unique effectors that associate specifically with the gdp-bound form of arf proteins. a slender extension on the cell surface. a non-motile, primary cilium is present on nearly all epithelial cells in the body and serves as a sensory organ that is important for regulating cell differentiation and division. proteins (ggas), gga , gga and gga . these various coat proteins specifically bind cargo proteins and incorporate them into newly forming vesicles for sorting and transport to their correct destinations. arfs can also recruit and activate enzymes that alter membrane lipid composition. phospholipase d (pld), which hydrolyses phosphatidylcholine to generate phosphatidic acid, is activated by all arf proteins and also by arl (ref. ). all arf proteins can both recruit and activate ptdins- -phosphate -kinase (ptdins p k), an enzyme that phosphorylates ptdins p at the -position to generate ptdins- , -bisphosphate (ptdins( , ) p ) . for example, arf at the cell periphery directly affects the activity of ptdins p k at the plasma membrane, and thus regulates ptdins( , )p levels there . at the golgi, arf recruits and stimulates the activity of ptdins -kinase (ptdins k), forming ptdins p, which is an important membrane lipid for golgi function . arf also binds to ptdins p-specific pleckstrin homology (ph) domains contained in a family of oxysterolbinding proteins that are believed to function in lipid homeo stasis at the golgi . new functions for golgi-associated arfs. the five arf proteins in humans, arf , arf , arf , arf , and arf , are ubiquitously expressed. studies to date have focused mainly on arf at the golgi and arf at the plasma membrane, but arf , arf and arf are also present on golgi membranes (fig. a) . surprisingly, depletion experiments using rna interference (rnai) show that no single arf, including arf , is required for golgi function; instead, arfs function in pairs at particular steps in golgi transport . for example, arf and arf act redundantly during transport in the early secretory pathway . consistent with this observation, arf localizes to the ergic and cis-golgi and, together with arf at the cis-golgi, it organizes traffickin g between these compartments . arf and arf are identical except for seven amino acid differences in their n-terminal and c-terminal regions, and previously they were thought to function and localize identically. however, a golgitargeting sequence contained within the α helix of arf and arf targets a chimaera of arf and arf to the early golgi . furthermore, arf localizes specifically to the tgn (fig. a) , and this localization depends on four arf -specific amino acids contained in the n-terminal amphipathic helix, which are conserved among arf homologues . arf , but not arf , becomes cytosolic at o c, the temperature at which exit from the tgn is blocked . thus, arf might have an additional crucial role during exit from the golgi. several important functions for class ii arfs at the tgn have now been defined (fig. a) . in an elegant series of studies, arf was found to specifically recognize the vxpx cytosolic targeting motif in retinal rhodopsin to facilitate its transport into the rod outer segment, which is a specialized cilium (fig. b) . this ciliary targeting complex includes, in addition to arf , rab , fip (a shared arf and rab effector) and asap (arf gap containing sh , ankyrin repeat and ph domains ) . exactly how this complex facilitates the packaging of rhodopsin into post-golgi carriers has yet to be determined but, interestingly, rhodopsin itself initiates complex formation by recruiting arf . the rhodopsin-binding site on arf is in the α helix , the same region that in arf binds the snare protein membrin (also known as gosr ) to mediate targeting to the early golgi ; thus, this region might generally allow arf protein binding to membrane receptors. arf and arf can also directly bind to caps (calcium-dependent activator protein for secretion), which regulates exocytosis of dense core vesicles from nerve terminals . it is the gdp-bound form of the arf that binds to the ph domains of caps proteins, and knockdown of caps, arf or arf causes retention of chromagrannin (a marker for dense core vesicles) in the golgi, suggesting that arf and arf , together with caps, regulate the release of dense core vesicles from the golgi (fig. a) . how these roles of arf and arf at the tgn in specialized cells can be reconciled plants have numerous adp-ribosylation factors (arfs) that are homologous to human arf (ref. ) and were originally thought to lack class iii arf -like proteins. however, in arabidopsis thaliana, arfb (also called arfb a) localizes to the plasma membrane and lacks the golgi-targeting motif (mxxe) that is found in other arf homologues in plants and in mammals . nevertheless, only the gbf and big subfamilies of arf guanine nucleotide exchange factors (gefs) seem to be present in plants, and these function in both endocytic and golgi trafficking pathways . a. thaliana gnom (also known as emb ) is a homologue of mammalian gbf but acts at endosomes and the plasma membrane during the polar transport of the plant hormone auxin during development , . another gbf-like protein in a. thaliana, gnom-like (gnl ), functions at the golgi similarly to mammalian gbf , but is also involved in endosomal trafficking . big (also known as ben and atmin ) was identified in a screen for a. thaliana mutants defective for internalization of the pin auxin transporter from the plasma membrane. this arf gef is most closely related to big and big in mammalian cells, localizes predominantly to the trans-golgi network (tgn) and early endosomes, and is involved in early endosomal trafficking . interestingly, big is targeted for degradation by a plant bacterial pathogen, pseudomonas syringae, to protect the latter from host defence systems at the cell wall . a. thaliana arf gaps include four members of a family of mammalian acap homologues that are known as vascular network (van )-like after the first member to be characterized . van (also known as scarface and agd ) regulates formation of plant vascular networks , . in addition to its roles on endosomes, van cooperates with gnom during clathrin-mediated endocytosis of the pin auxin transporter . another arf gap in a. thaliana, arf gap domain (agd ; also known as nevershed), is a homologue of yeast arf gap effector (age ) and mammalian smap family arf gaps that localizes to the tgn . agd is required for floral organ cell separation and regulates membrane trafficking though tgn-early endosomal compartments to trigger organ abscission . interestingly, the protozoan parasite trypanosoma brucei expresses a single arf protein that has characteristics of both arf and arf . t. brucei arf is a basic protein with a calculated isoelectric point (pi) value of . , which is similar to that of human arf , but t. brucei arf contains the golgi-targeting motif mxxe that is found in human arf and arf (ref. ). depletion of t. brucei arf by small interfering rna causes a major decrease in endocytosis and the formation of intracellular flagella, but the golgi remains intact . trypanosomes also express an arf-like (arl ) homologue, which is involved in microtubule biogenesis and cytokinesis , and an arl homologue, which is important for golgi structure and exocytosis of glycosyl phosphatidylinositol (gpi)-anchored proteins . arf and arl proteins in trypanosomes are myristoylated, a modification that is required for their activity. trypanosomes cause african sleeping sickness, a disease with no successful therapy. a selective inhibitor of trypanosomal n-myristoyl transferase has been shown to be effective in blocking trypanosome viability in a mouse model of this disease . with findings of arf localization to, and arf and arf functioning at, the early golgi in other cells , , is not known. recent discoveries show that arf regulates lipid transfer proteins within the golgi and promotes the formation of lipid droplets at the ergic (fig. a) . at the golgi, arf recruits the lipid transfer proteins ceramide transfer (cert) and fapp (ref. ) through interaction with their ph domains, which can also bind ptdins p. cert mediates the non-vesicular transport of ceramide from the er to the golgi and fapp mediates the transfer of glucosylceramide from the cytosolic side of the early golgi to the trans-golgi . exactly how the directionality of this transfer occurs, and the role that arf has, is not yet clear. the finding that arf associates with gbf and copi during lipid droplet formation was un expected. these proteins were identified in an rnai screen of lipid droplet formation in d. melanogaster and also appeared in proteomic analyses of lipid droplets along with other trafficking proteins, which led to the idea that lipid droplets interface with multiple membrane trafficking pathways . in particular, the delivery of two proteins, adipose triglyceride lipase (atgl) and adipose differentiation-related protein (adrp; also known as adipophilin), to the surface of lipid droplets requires arf , gbf and copi, and possibly the copii machinery, in mammalian cells ; similar results were obtained in d. melanogaster s cells . another arf family membe r, arfrp , is highly expressed in adipocytes, and mice that lack arfrp in adipose tissue show severe defects in lipid storage and enhanced lipolysis . finally, in some cell types arf at the plasma membrane affects endocytosis of proteins anchored to the membrane by a glyco syl ptdins (gpi) linkage . this may also require the arf gef gbf (ref. ) and could be related to the other lipid-regulating functions of arf . a great deal of work on arf function has been summarized in a pre vious review , so here we focus on more recent advances. in mammals, arf is not required for early embryonic development, but arf -knockout mice die at and arf localize to the early cis-golgi and arf specifically localizes to the trans-golgi network (tgn). in addition to the recruitment of coat proteins (coatomer complex i (copi), gga (golgi-localized, γ-ear-containing, adp-ribosylation factor-binding protein) and adaptor protein (ap )) to the golgi, arf binds to ceramide transfer (cert) and fapp to mediate the transport of ceramide and glucosylceramide lipids from the cis-golgi to the trans-golgi. at the er-golgi intermediate compartment (ergic), arf and its guanine nucleotide exchange factor (gef) gbf act with copii to regulate the formation of lipid droplets and for the replication of several viruses. caps (calcium-dependent activator protein for secretion), which is involved in regulated secretion, is recruited to the tgn by arf and arf . at the er, sar , activated by sec , recruits copii to allow vesicle transport to the golgi. b | in retinal cells, arf binds specifically to rhodopsin in the tgn membrane and, together with fip , asap (arf gap containing sh , ankyrin repeat and ph domains) and rab , it facilitates the transport of rhodopsin in transport vesicles from the inner segment to the outer segment, which is a specialized cilium. arf-like (arl ) has been found to be localized to the connecting cilium, and retinitis pigmentosa (rp ; also known as xrp ), an arl gap, localizes to the tgn, the basal body and the membrane adjacent to the connecting cilium. c | in primary cilia, arl recruits the bbsome coat complex that facilitates the transport of membrane proteins into the cilium. arl is localized to the cilium and has been implicated in intraflagellar transport. adrp, adipose differentiation-related protein (also known as adipophilin); atgl, adipose triglyceride lipase; ptdins k, phosphatidylinositol -kinase. new end take-off a switch in cellular growth of fission yeast, from monopolar extension to bipolar extension. mid-gestatio n or shortly after birth and exhibit impaired liver development . this phenotype suggests that the critical physiological roles of arf take place after birth and is consistent with reported effects of arf on cell adhesion, cell migration, wound healing and metastasis. arf is present at the plasma membrane and influences both the cortical actin cytoskeleton and endosomal membrane recycling (fig. ) . at the plasma membrane, arf changes the membrane lipid composition through activation of ptdins p k and pld, resulting in the generation of ptdins( , )p and phosphatidic acid. these lipids are important for sorting proteins within the membrane, for the formation of clathrin-coated pits during endocytosis, and for the recruitment and activation of rho family g proteins, such as rac, to alter actin poly merization. there is some evidence that arf can interact with ap (ref. ) and clathrin during g proteincoupled receptor (gpcr) cell signalling . a recent study has found that arf enters cells in clathrin-coated vesicles to facilitate the rapid recycling of the transferrin receptor back to the plasma membrane through interaction with the microtubule motor adaptor protein jnkinteracting protein (jip ) after clathrin uncoating . in some cells, arf associates with endosomal membranes derived from clathrin-independent forms of endo cytosis and mediates recycling of this membrane back to the plasma membrane . recycling endosomes return membrane proteins that are important for cell adhesion and migration back to the plasma membrane , . arf regulation of such endosomal membrane trafficking is required for the polarized delivery of cdc , rac and the par complex to the leading edge of migratin g cells , which can alter adhesion to the extracellular matrix through focal adhesions and actin-based protrusions. hence, regardless of the mode of endocytosis, arf is important for membrane recycling. the crucial functions of arf in membrane lipid modification, establishment of cell polarity and promotion of endocytic recycling are conserved in yeast and d. melanogaster . arf , the yeast arf homologue, contributes to ptdins( , )p levels at the plasma membrane and also affects polarization events, such as bud site selection in s. cerevisiae and new end take-off growth in schizosaccharomyces pombe . arfb, the arf homologue in the filamentous fungi aspergillus nidulans, localizes to both the plasma membrane and endomembranes, and regulates endocytosis and polarity establishment during hyphal growth . in d. melanogaster, figure | the localization and function of arf and arl proteins in endosomal-lysosomal trafficking. at the plasma membrane, adp-ribosylation factor (arf ) activates phosphatidylinositol- -phosphate -kinase (ptdins p k) to generate ptdins- , -bisphosphate (ptdins( , )p ) and, together with arf-like (arl ), recruits cytohesin (also known as arno) guanine nucleotide exchange factors (gefs) that can lead to further activation of arf or arf . cytohesins associate with the ipcef (interactor protein for cytohesin exchange factors)-dock complex, which activates rac, but another rac gef, kalirin, can be recruited to membranes by arf •gdp. arf at the plasma membrane can regulate the membrane lipid composition, alterations in cortical actin to drive protrusions (for example, during cell migration), and endocytosis of ligand-activated guanine-nucleotide-binding (g) protein-coupled receptors (gpcr) via clathrin-dependent endocytosis. arf and the microtubule motor adaptor protein jnk-interacting protein (jip ) promote rapid recycling of endosomal membrane back to the cell surface, and arf , together with the exocyst complex, also affects slow recycling from sorting endosomes. arf has been implicated in clathrin-independent endocytosis of glycosyl ptdins (gpi)-anchored proteins in some cells. arf and the arf gefs cytohesin and brag have been implicated in both assembly and disassembly of adherens junctions. two arf gtpase-activating proteins (gaps), asap (arf gap containing sh , ankyrin repeat and ph domains ) and git , localize to focal adhesions that mediate adhesion to the extracellular matrix (ecm), and git interacts with pix, a gef for cdc . arl is required for fusion of multivesicular late endosomes with lysosomes and is involved in transport along microtubules. hgf, hepatocyte growth factor. cellular adhesions that connect epithelial cells to form a polarized epithelium. made up of homotypic cadherin interactions and associated intracellular proteins. (roundabout homologue ). acts as a receptor for slit protein and regulates vascular integrity. an adhesive, ring-like, actin-rich structure that is formed on the ventral surface of cells. a complex of proteins that facilitates membrane traffic into the cilium. mutant forms of several bbs components have been identified as causative agents for various ciliopathies. deletion of the arf homologue blocks the rapid endocytic recycling required for cytokinesis in spermatocytes, resulting in male sterility, but no other phenotypes were reported . interestingly, in mammalian cells arf interacts with jip to control a motor switch mechanism regulating endosomal trafficking in cytokinesis . the crystal structure of arf in complex with jip shows that residues adjacent to the switch regions are structural determinants for the specific binding of jip to arf (ref. ). arf has been implicated in both the assembly and disassembly of adherens junctions in polarized epithelial cells (fig. ) . during adherens junction formation, par recruits a scaffolding protein, frmd a, that binds to cytohesin gefs, which leads to activation of arf (ref. ). treatment of fully polarized epithelial cells with hepatocyte growth factor leads to activation of arf , most likely through the arf gef brag (ref. ), and activation of rac, which causes disassembly of adherens junctions by stimulating endocytosis of epithelial cadherin (e-cadherin) . hence, depending on the signalling complex assembled, either formation or dis assembly of adherens junctions can be achieved through activation of arf . there is also some evidence that the arf gef efa affects tight-junction assembly . arf activation has also been reported at the onset of tubulogenesis (a developmental progression from polarized epithelia to tubular structures), and perturbation of the arf gtp-gdp cycle inhibits tubule formation . importance of turning off arf . arf proteins carry out their actions through a regulated cycle of gtp binding and hydrolysis. this allows arfs to engage and disengage with their effectors with spatial and temporal specificity, and in some cases may allow arf•gdp to bind other classes of effector. arf •gdp binds several tbc (tre -bub -cdc ) domain-containing proteins, which often have rab gap activity . arf •gdp binds both tbc domain family member (tbc d ; a protein mutated in familial infantile myoclonic epilepsy ) and the tre oncogene . tre binding to arf increases its activation ; although tre does not itself have gef activity towards arf , it may facilitate interaction of arf with another gef. arf •gdp also binds to the kalirin family of rho gefs, through their spectrin-like repeat domain , and recruits kalirin to the membrane, where it subsequently activates rac and rhog to regulate actin dynamics (fig. ) . hence, arf •gdp and arf •gtp both interact with regulatory proteins of other small g proteins, allowing alternative signalling pathways to be activated depending on which nucleotide is bound (fig. c) . this raises the intriguing possibility that other gdp-bound arf or arl proteins might also bind unique effector proteins. turning off arf is important for its biological function. in some cells, expression of the constitutively active mutant of arf , q l, leads to the accumu lation of early endosomes containing plasma membrane proteins that enter cells independently of clathrin; failure to in activate arf blocks further trafficking of this membrane towards recycling or to other destinations . immediately upon platelet activation, arf •gtp levels fall, and this inactivation precedes, and is required for, the subsequent activation of rac . arf is important for the disassembly of adherens junctions and, more recently, active arf was shown to disrupt the formation of epithelial cysts . the slit -robo signalling pathway is important for maintaining barrier function in the vascular network, and robo interacts with paxillin to recruit arf gap proteins, such as git , to inactivate arf (ref. ); this arf inactivation suppresses protrusive activity of the endothelial cells and neovascularization. git and arf inactivation are also important for maintaining the podosome, an actin-rich sealing zone in osteoclasts . finally, non-canonical ubiquitylation of arf , catalysed by fbx (an f -box and sec domaincontaining protein) seems to be another, unusual, way to turn off arf (ref. ). fbx is diminished or lacking in several cance r cell lines, which is consistent with roles for arf in cance r cell metastasis . similarly to arf , arl and arl arose early in evolution and share common effectors in plants, yeast and mammals. arl recruits grip-domain golgins to the tgn . it also mediates tgnlocalization of arf-interacting proteins (arfaptins), which contain bin-amphiphysin-rvs (bar) domains that induce the formation of tubules and vesicles at the tgn . whereas arl functions in vesicle trafficking similarly to arfs, arl has a highly conserved function in regulating microtubule-based processes . arl is closely related to arl , but is found only in cells with cilia, where it regulates microtubule-based processes at the cilial basal body , (fig. b) . elmod has been reported to be a gap for arl , but also has activity against arf and arf , which is surprising given that it has no homology to arf gaps ; the physiological relevance of this activity remains to be determined. retinitis pigmentosa (rp ; also known as xrp ) acts as a gap for arl during intraflagellar transport and ciliogenesis. arl , arl and arl affect intraflagellar transport and ciliogenesis (fig. b,c) . cilia are vital for cell signalling and differentiation, and their impaired formation is responsible for many genetic disorders . bardet-biedl syndrome is a complex genetic disease that can be caused by mutation in any one of genes associated with ciliogenesis. transport of membrane proteins into the cilium is driven by a complex of proteins, called the bbsome. bbsome subunits have 'coat-like' attributes and simila r structural folds to those found in copi and adaptor protein complexes, suggesting that the bbsome can sort specific cargo for transport (fig. c) . arl is a bbs subunit (bbs ) and is required in its gtp-bound form to recruit the bbsome onto the plasma membrane to drive cargo sorting into cilia . structural and biochemical analyses have shown that one of the mutations in arl that causes bardet-biedl syndrome, t r, leads to a non-functional arl that cannot bind gtp . this supports the idea that arl recruits the bbsome complex to membranes for formation of bbsome-coated vesicles. arl is mutated in patients with joubert syndrome, which is a rare, complex cerebral disorder that is characterized by developmental delays and cognitive dis ability. it is also involved in intraflagellar transport (fig. c) and, in c. elegans, arl- associates with the doublet segment of the cilium and its loss results in shortened cilia , . retinitis pigmentosa is a retinal degeneration disease, and mutations in the rp gene are responsible for a large fraction of the most severe x-linked form. rp was identified as a gap for arl , and mutations associated with retinitis pigmentosa compromise arl gap activity . arl localizes to the photoreceptor segment connecting to the cilium (fig. b) , and arl -/mice have abnormal kidney and photoreceptor development, indicating the importance of this protein in primary cilia . rp localizes to the basal body and centriole at the base of the photoreceptor cilium, but also to the adjacent golgi and apical plasma membrane . furthermore, rp promotes vesicle trafficking from the golgi to the base of the cilium in mammalian cells , presumably acting together with arf , asap and fip . intriguingly, d. melanogaster arl (also called dead end) regulates actin polymerization and vesicular trafficking to the plasma membrane, which are important for tracheal morphogenesis . hence, arl appears to link microtubule-based processe s and vesicular trafficking during development. arl might also coordinate microtubule and vesicular trafficking. arl localizes to late endosomes and lysosomes (fig. ) in both humans and worms, and mediates transport of endocytic proteins between these two compartments . arl also facilitates the axonal transport of presynaptic cargo proteins in vesicles, preventing their premature aggregation . exactly how these two functions of arl are related is not clear but they might both involve transport along microtubules . a great deal of progress has been made in identifying arf gefs, and an unexpectedly broad range of roles has been revealed for these regulators, including both the coordination of membrane trafficking with lipid homeostasis and signalling at the plasma membrane (table ) . because gefs ensure the precise temporal and spatial activation of arfs, their own localization mechanisms are crucial for understanding their cellular roles. these mechanisms are turning out to be quite complex, even for the simplest of the arf gefs, the members of the cytohesin (also known as arno) family. membrane trafficking is crucial to numerous developmental and physiological processes, and the specific functions of different arf gefs in these pathways and their links to disease are now being revealed. there is particular interest in understanding how arf gefs are recruited to membranes to regulate arf activation. big and big localize to the tgn and endosomes, where they have both distinct and overlapping functions , . by contrast, gbf localizes predominantly to the cis-golgi (fig. a) , where it controls transport of membrane proteins through the secretory pathway . the activity of phosphodiesterase a is important for recruitment of big and big to the trans-golgi . however, rab (ref. ) and ptdins p generated by ptdins kiiiα are involved in recruitment of gbf to membranes. other close connections between golgi arfs and ptdins p have emerged recently. in yeast there is an interesting synergy observed between the arf gef gea and ptdins p produced by pik (the yeast homologue of ptdins kiiiβ). both are simultaneously required to activate the aminophospholipid translocase (flippase) drs at the tgn during formation of ap -clathrin vesicles . ptdins ks are essential for viral replication, and notably produce the ptdins p-enriched membrane environment that recruits the enteroviral rna polymerases . gbf is required for the replication of numerous viruses, including enteroviruses, hepatitis c virus and corona viruses [ ] [ ] [ ] [ ] . in enteroviral systems, gbf and ptdins kiiiβ are recruited coordinately to membranes by the viral a protein to promote formation of functional viral replication complexes near er exit sites (fig. a) . yel is an efa -like gef for the arf orthologue arf in yeast, and localizes to the plasma membrane of the emerging bud . similarly to its mammalian orthologues, the ph domain of yel is required for membrane targeting but, interestingly, multiple regions of the protein are important for precise spatial localization of this gef . brag , an arf gef, also has a ph domain that is critical for membrane targeting and in breast cancer cells is specifically recruited to the egf receptor upon egf stimulation, through direct interaction of its ph domain with the egf receptor . this interaction requires phosphorylation on specific tyr residues and thus the recruitment of brag couples receptor activation to arf activation . brag is overexpressed in many breast cancer cell lines and depletion of brag by small interfering rna blocks cell invasion in vitro and in animal tumour models . these observations add to others that have implicated arf and its activation in a number of models of cancer cell invasion and metastasis , . autoinhibition of cytohesin gefs. at the cell periphery, the cytohesin gefs function in plasma membraneendosomal membrane trafficking pathways, and in signal transduction pathways that are important for cell proliferation, immune response and growth control , . members of this gef family can catalyse exchange on both arf and arf in vitro and in cells, although in vitro they are more efficient gefs for arf (ref. ). recent insights have been gained into how cytohesin activation is spatially regulated, and how its autoinhibition is relieved (fig. ) . in addition to phosphoinositide binding at the membrane, the ph domains of cytohesin family members interact with the gtp-bound forms of arf (ref. ) and arl (refs , ) , leading to cytohesin recruitment and further activation of arf or arf at the membrane. a crystal structure of the sec domain in tandem with the ph domain of cytohesin (also known as grp ) revealed that it adopts an autoinhibited conformation. the c-terminal helix that phagocytosis a cellular endocytic process for engulfing large particles, such as bacteria, and bringing them inside the cell. follows the ph domain and the linker between the sec and ph domains block the catalytic site . interaction of the ph domain with arf •gtp and phosphoinositides (either ptdins( , )p or ptdins- , , -trisphosphate (ptdins( , , )p )), as well as the interaction of the polybasic c terminus of cytohesin with acidic phospholipids, all contribute to relieving this autoinhibition (fig. ) . reconstitution of the cytohesin-exchange assay on liposomes, in the presence of both activating arf •gtp and substrate arf , revealed that mutations in the ph domain of cytohesin that abolished interaction with arf •gtp were completely inactive . together, these studies demonstrate how precise spatial regulation of cytohesin activation is achieved. a specific phosphoinositide (ptdins( , )p and/or ptdins( , , )p ), additional acidic phospholipids and an active arf localized in the plasma membrane must all coincide to relieve autoinhibition, thus restricting the membrane domain at which these gefs can become active. we do not know whether arf , arf or both are the primary substrates for the cytohesins. however, arf •gtp is more efficient in relieving autoinhibition of cytohesins than arf •gtp, both in vitro and in cells , . the activation of cytohesins by a gtp-bound arf family member raises the question of whether they can engage in a positive feedback loop, whereby the substrate itself can stimulate further exchange. indeed, such a loop has been demonstrated in vitro for arf (ref. ). there is also evidence that cytohesins might mediate a cascade of activation from arf to arf . cells expressing constitutively active arf q l have increased levels of arf •gtp . arf affects several processes at the plasma membrane, including recruitment of proteins to focal adhesions and during phagocytosis. in the forming phagocytic cup, arf •gtp is recruited earlier than arf •gtp, at a stage that requires rapid insertion of new membrane . hence, the arf -cytohesin-arf cascade might ensure a high level of activated arf protein here. arf is less abundant than arf in cells, and as both arf and arf can recruit effectors such as ptdins p k and pld, processes requiring acute activation of such effectors may rely on the more abundant arf to provide an adequate supply. in support of this idea, both arf and arf , through cytohesins, contribute to activation of ptdins p k and pld in the insulin signalling pathway . in addition to arf -cytohesin-arf or possible arl -cytohesin-arf cascades, there is a conserved arl cascade, in which yeast arl •gtp recruits arl to tgn membranes . in this case, it is not known whether an arl gef is involved. hence, arf family cascades could be common and could explain the golgi arfs that act in pairs. use of the specific cytohesin inhibitor secinh has revealed roles for this family of gefs in the insulin and erbb receptor tyr kinase signalling pathways [ ] [ ] [ ] . cytohesins are positive activators of insulin signalling in both d. melanogaster and mammalian cells, and they are important for cell growth and for insulin sensitivity in human liver cells , . they regulate insulin signalling by binding cnk , a scaffolding molecule that is important for ras, phosphoinositide -kinase (pi k) and akt signalling . cnk recruits cytohesins in an insulin-dependent manner to the plasma membrane, where they generate a ptdins( , )p enriched micro domain that is essential for pi k-akt activation. other scaffolding proteins interact with the coiled-coil domain of cytohesin; these proteins include golgi reassembly-stacking protein (grasp) and ipcef (interactor protein for cytohesin exchange factor s), which mediate the interaction of dock with cytohesin . interestingly, assembly of this scaffolding complex promotes rac activation and cell migration, indicating that these scaffolds assemble a signalling complex that determines a specific downstream output upon arf activation . cytohesins also affect integrin signalling in the immune system, and cytohesin can activate β integrins in dendritic cells , possibly through a scaffoldin g role of cytohesins. levels of arf and the efa and cytohesin family gefs markedly increase in the mammalian brain after birth, suggesting important roles in postnatal nervous system development . experiments in isolated hippocampal neurons indicate that arf , efa and the cytohesins might affect neurite and dendritic spine development , . in humans, mutations in the arf gef big are linked to autosomal recessive periventricular heterotopia (arph), a disease in which the cerebral cortex is severely underdeveloped owing to failure of neurons in the lateral ventricular proliferative zone to migrate to the cortex . this impaired migration arises from a figure | the recruitment of an arf gef to the membrane is coupled to relief of autoinhibition. an active gtp-bound adp-ribosylation factor (arf) family member (either arf-like (arl ) or arf ), phosphoinositides (phosphatidylinositol- , -bisphosphate (ptdins( , )p ) or ptdins- , , -trisphosphate (ptdins( , , )p )), and additional acidic phospholipids such as phosphatidylserine, are all required for membrane recruitment of the cytohesin (also known as arno) guanine nucleotide exchange factor (gef), to convert it from its cytosolic inactive form to its fully active membrane-bound form. before recruitment, the sec catalytic gef domain, the pleckstrin homology (ph) domain and the carboxy-terminal α-helix of cytohesin are in an autoinhibited conformation (left), with the c-terminal α-helix (charged residues within this are shown as '+') and linker situated between the catalytic sec domain and the ph domain, which blocks the arf-binding site. upon binding of the ph domain to the gtp-bound gef at the membrane, the catalytic site is released from autoinhibition (right). this can in turn drive further activation of arf proteins, such as arf , at the membrane, and may form the basis of an arf protein activation cascade. long-term depression (ltd) . a reduction in the efficacy or strength of neuronal synapses that is linked to learning and memory formation. defect in vesicular trafficking that alters the adhesive properties of these neurons . disease alleles include an early frameshift mutation that deletes most of the big protein . members of the brag (or iqsec) family of arf gefs are extremely abundant in neuronal postsynapti c densities, and can serve as gefs for arf (ref. ). brag (also known as iqsec ) and brag are vital for neuronal development. brag is mutated in x-linked nonsyndromic intellectual disability (also referred to as mental retardation). three point mutations isolated from patients map to the sec domain and result in proteins that cannot activate arf normally . brag has been linked to alterations in synaptic content during long-term depression (ltd). signalling through ampa (α-amino- -hydroxy- -methyl- -isoxazole propionic acid)-type glutamate receptors facilitates ltd, and downregulation of activated ampa receptors is normally regulated by ampa receptor-mediated recruitment of brag , which in turn activates arf and endocytosis . thus, brag gefs and arf are vital for neuronal developmen t and learning. cytohesin gefs may affect signalling through epidermal growth factor (egf) or erbb receptor tyr kinase receptors independently of their gef activity. egf receptors (egfrs) undergo ligand-induced dimerization and subsequent transphosphorylation, mediated by conformational changes in their cytoplasmic portion. cytohesins bind directly to these cytoplasmic domains and promote conformational changes that increase phosphorylation . furthermore, treatment of an egf receptor-dependent lung cancer cell line with the cytohesin inhibitor secinh reduced proliferation . surprisingly, this function of the cytohesins does not require their gef activity. similarly, in c. elegans, the gef efa- regulates microtubule dynamics at the cell cortex independently of its substrate arf (ref. ). furthermore, essential functions of gbf in poliovirus replication are independent of arf activation . the extent to which other arf gefs may have broader roles beyond arf activation warrants further investigation. there are also suggestions that some multidomain arf gap proteins have functions that are independent of their gap activity. all arf gaps contain the conserved zinc-finger arf gap catalytic domain in addition to other domains responsible for membrane recruitment, regulation of gap activity and other scaffolding functions (table ) . arfgap , the first arf gap to be cloned , is golgilocalized and, together with arfgap and arfgap , mediates most arf-bound gtp hydrolysis at the golgi. the complex, multidomain structure of the other arf gap families has stimulated much research. here, we highlight a few examples of how these multidomain arf gaps, by recognizing the gtp-bound form of their substrate arf, act as downstream effectors in addition to signal terminators. information about other arf gaps can be found in an excellent review article . the asap proteins are the prototypical multidomain gaps that interact with many signalling molecules, including src and focal adhesion kinase (table ) . asap resides in focal adhesions but, in response to src activation, it facilitates formation of podosomes , which are discrete actin-based structures that are formed at the cell substratum to degrade matrix. the crystal structure of arf in complex with the catalytic domain of asap revealed that a catalytic arg-finger of asap is responsible for gtp hydrolysis , similarly to many other gaps, a finding that is consistent with an earlier structure of the gap domain of asap (ref. ). there is also some evidence that calcium might bind to the complex and regulate gap activity , although this needs to be confirmed with full-length asap and arf and in cells. the asaps all have n-terminal bar domains that can induce membrane curvature and tubule formation in transfected cells and in cell-free systems. the bar domain in asap negatively regulates its gap activity towards arf (ref. ) , and binding of the rab effector fip to the bar domain of asap stimulates its gap activity . as mentioned earlier, asap also promotes ciliary targeting together with arf and fip (ref. ) (fig. b) . asap is upregulated in breast, pancreatic and colorectal cancer . cbl-interacting protein (cin ; also known as sh kbp ) binds to asap , recruiting the e ubiquitin ligase cbl, to trigger the mono ubiquitylation of asap ; this modification is important for invasion of breast cancer cells but the role for ubiquitylation of asap in cell invasion is not known. one caveat to observations made when asap is expressed in cells is that a study designed to systematically look at arf gap function and arf specificity failed to detect an effect of asap expression on either arf •gtp or arf •gtp levels in cells . this raises the possibility that the gap activity of asap might not always be critical for some of asap 's specific functions. the arf gap git , originally identified as a gpcr kinase-interacting protein, can coordinate signalling by acting as a scaffold. git and its substrate arf affect ligand-stimulated endocytosis of several gpcrs through either clathrin-dependent or clathrin-independent endocytic pathways . among the proteins interacting with git are the cdc and rac gef pix, focal adhesion kinase and paxillin. git , similarly to asap , is sometimes observed in focal adhesions and its influence on the activation of cdc and rac suggests that arf inactivation and rac activation are coordinated (fig. ) . d. melanogaster git is required for muscle morphogenesis and the git -knockout mouse is defective in fear learning and dendritic spine formation . rac interacts with git , disrupting git binding to paxillin; this in turn stimulates git gap activity, presumably towards arf (ref. ), and inhibits cell spreading and neuritogenesis. in endothelial cells, robo interacts with paxillin, which recruits git to inactivate arf , and this leads to vascular stability, blocking cellular protrusions and neovascular leak . thus, these examples provide insights into how modular arf gaps promote spatially and temporally restricted assembly of signalling complexes, and allow a precise physiological output in response to a signal. intracellular pathogens can use a fascinating gapblocking mechanism to rewire the host cell's signalling network for their own purposes. enterohaemorrhagic escherichia coli produce the espg protein, which binds to gtp-bound arf and arf , blocking their access to gaps and disrupting the function of both early golgi and recycling endosomes . moreover, espg simultaneously binds to p -activated kinase (pak), an effector of a distinct g protein family member, cdc , and promotes pak localization at golgi membranes rather than at the plasma membrane. this raises the possibility that espg assembles its own signalling complex on intra cellular membranes to subvert membrane trafficking and polarit y processes in host cells. conclusions & perspectives arf activity is regulated in a spatiotemporal manner by the gefs and gaps, underlining the importance of precise localization of these regulators. in the case of cytohesins, such specificity can be achieved through a coincidence-detection mechanism, requiring both an activating arf or arl protein and a specific lipid composition. this example also reveals the existence of arf family activation cascades and how relief of autoinhibition can be coupled to precise spatial cues. it will be interesting to see how widespread these mechanisms are among arf family members. arf cascades, similarly to those demonstrated for rab g proteins, could transform one membrane domain into another during highly dynamic membrane trafficking. these transformations involve coordinated changes in the lipid and protein composition of each membrane domain, a specialty of many arf family members, which recruit both lipidmodifying enzymes and protein effectors such as coats and tethers. the signature feature of arf family proteins, their n-terminal membrane-binding amphipathic helix, ensures that they are closely associated with the lipid bilayer in their gtp-bound form. future studies on how arf family proteins function will therefore require in vitro reconstitution on model membranes. there appears to be a particularly important link between arf function and ptdins p, a lipid that has a central role in the function of the golgi, which parallels the coordination of membrane trafficking and ptdins( , ) p signalling by arf at the plasma membrane. the gaps and gefs for the arf family proteins are multidomain proteins that can assemble signalling complexes and so place the arfs and arls into larger networks. these networks include cytoskeleton regulators, and it appears that some arl proteins (arl , for example) have evolved exclusively to regulate the cytoskeleton. the role of arf in networks linking membrane trafficking to the actin cytoskeleton also involves interaction of arf with gefs and gaps of the rac and rho small g proteins, actin cytoskeleton regulators. another emerging concept is that some arf family members remain membrane-bound in their gdp-bound form so that they can interact with signalling complexes and promote alternative signalling pathways. ultimately, these arf family signalling networks will need to be studied through systems level analysis. so far, no gefs and only two gaps that are specific for an arl have been identified. several arl proteins affect ciliogenesis and, in some cases, ciliopathies; other arls function in neurons and have been associated with neurodegenerative disorders. hence, increased understanding of arls and their regulators should inform both fundamental questions in cell biology and disease mechanisms. finally, the use of model organisms to complement studies in mammalian cells has already provided valuable insights into the physiological roles of arf family proteins. this approach holds great promise for uncovering the unknown functions of most arls, as well as defining the full range of activities of all arf and arl proteins. arf proteins: roles in membrane traffic and beyond the small g proteins of the arf family and their regulators n-terminal hydrophobic residues of the g-protein adp-ribosylation factor- insert into membrane phospholipids upon gdp to gtp exchange toward a structural understanding of arf family:effector specificity an n-terminally acetylated arf-like gtpase is localised to lysosomes and affects their motility sar p n-terminal helix initiates membrane curvature and completes the fission of a copii vesicle differential membrane association properties and regulation of class i and class ii arfs characterization of class i and ii adpribosylation factors (arfs) in live cells: gdp-bound class ii arfs associate with the er-golgi intermediate compartment independently of gbf an arf-like small g protein, arl- , promotes the axonal transport of presynaptic cargoes by suppressing vesicle aggregation syt p promotes activation of arl p at the late golgi to recruit imh p the copi system: molecular mechanisms and function coat proteins: shaping membrane transport phospholipid-and gtp-dependent activation of cholera toxin and phospholipase d by human adp-ribosylation factor-like protein (harl ) protein-lipid interactions in membrane trafficking at the golgi complex isoform-selective effects of the depletion of adpribosylation factors - on membrane traffic adp ribosylation factors and and group via phospholipase a regulate morphology and intraorganellar traffic in the endoplasmic reticulum-golgi intermediate compartment targeting of arf- to the early golgi by membrin, an er-golgi snare arf is activated uniquely at the trans-golgi network by brefeldin a-inhibited guanine nucleotide exchange factors rhodopsin c terminus, the site of mutations causing retinal disease, regulates trafficking by binding to adp-ribosylation factor (arf ) ciliary targeting motif vxpx directs assembly of a trafficking module through arf shows that the ciliary targeting motif in rhodopsin, vxpx, binds to arf and regulates its association with the tgn, where a ciliary targeting complex selects and packages cargo for delivery to the cilium interaction of calcium-dependent activator protein for secretion (caps ) with the class ii adp-ribosylation factor small gtpases is required for dense-core vesicle trafficking in the trans-golgi network glycosphingolipid synthesis requires fapp transfer of glucosylceramide functional genomic screen reveals genes involved in lipid-droplet formation and utilization dynamic activity of lipid droplets: protein phosphorylation and gtp-mediated protein translocation demonstrates a novel pathway -requiring arf , gbf and copi, as well as copii -by which the lipid droplet-associated proteins atgl and adrp are delivered copi complex is a regulator of lipid homeostasis the arf-like gtpase arfrp is essential for lipid droplet growth and is involved in the regulation of lipolysis arf is directly involved in dynamin-independent endocytosis analysis of endocytic pathways in drosophila cells reveals a conserved role for gbf in internalization via geecs crucial role of the small gtpase arf in hepatic cord formation during liver development the small g-protein arf gtp recruits the ap- adaptor complex to membranes arf regulates angiotensin ii type receptor endocytosis by controlling the recruitment of ap- and clathrin decoupling of activation and effector binding underlies arf priming of fast endocytic recycling pathways and mechanisms of endocytic recycling arf and microtubules in adhesion-dependent trafficking of lipid rafts cdc localization and cell polarity depend on membrane traffic yeast arf p modulates plasma membrane ptdins( , )p levels to facilitate endocytosis role for arf p in development of polarity, but not endocytosis, in saccharomyces cerevisiae adp-ribosylation factor arf p may function as a molecular switch of new end take off in fission yeast aspergillus nidulans arfb plays a role in endocytosis and polarized growth spermatocyte cytokinesis requires rapid membrane addition mediated by arf on central spindle recycling endosomes arf interacts with jip to control a motor switch mechanism regulating endosome traffic in cytokinesis the crystal structure of arf in complex with its effector jip , which acts as a microtubule motor adaptor frmd a regulates epithelial polarity by connecting arf activation with the par complex gep /brag : activator of adpribosylation factor for regulation of cell adhesion and actin cytoskeleton via e-cadherin and α-catenin efa , exchange factor for arf , regulates the actin cytoskeleton and associated tight junction in response to e-cadherin engagement arf -dependent activation of erk and rac modulates epithelial tubule development analysis of gtpase-activating proteins: rab and rab are key rabs required to maintain a functional golgi complex in human cells tbc d , an arf -interacting protein, is mutated in familial infantile myoclonic epilepsy the tbc (tre- /bub /cdc ) domain protein tre regulates plasma membraneendosomal trafficking through activation of arf arf recruits the rac gef kalirin to the plasma membrane facilitating rac activation discovery of new cargo proteins that enter cells through clathrin-independent endocytosis arf plays an early role in platelet activation by collagen and convulxin unregulated arf activation in epithelial cysts generates hyperactive signaling endosomes and disrupts morphogenesis slit -robo signalling promotes vascular stability by blocking arf activity src-dependent repression of arf is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts fbx makes arf refractory to function via ubiquitination the egfr-gep -arf -amap signaling pathway specific to breast cancer invasion and metastasis arfaptins are localized to the trans-golgi by interaction with arl , but not arfs arl and arl regulate different microtubule-dependent processes elmod is an arl gtpase-activating protein that also acts on arfs trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier? shows that arl , the bbs subunit, recruits the coat-like bbsome onto the plasma membrane to sort cargo for transport into cilia bardet-biedl syndrome-associated small gtpase arl (bbs ) functions at or near the ciliary gate and modulates wnt signaling the small gtpases arl- and arl- coordinate intraflagellar transport and ciliogenesis joubert syndrome arl b functions at ciliary membranes and stabilizes protein transport in caenorhabditis elegans the retinitis pigmentosa gene product is a gtpaseactivating protein for arf-like adp-ribosylation factor-like is involved in kidney and photoreceptor development the retinitis pigmentosa protein rp links pericentriolar vesicle transport between the golgi and the primary cilium the drosophila dead end arf-like gtpase controls vesicle trafficking during tracheal fusion cell morphogenesis the arf-like gtpase arl mediates delivery of endocytosed macromolecules to lysosomes in caenorhabditis elegans redundant roles of big and big , guanine-nucleotide exchange factors for adpribosylation factors in membrane traffic between the trans-golgi network and endosomes specific functions of big and big in endomembrane organization localization of large adp-ribosylation factor-guanine nucleotide exchange factors to different golgi compartments: evidence for distinct functions in protein traffic dissecting the role of the arf guanine nucleotide exchange factor gbf in golgi biogenesis and protein trafficking interaction of phosphodiesterase a with brefeldin a-inhibited guanine nucleotideexchange proteins big and big and effect on arf activity rab b interacts with gbf and modulates both arf dynamics and copi association the phosphatidylinositol -kinase pi kiiiα is required for the recruitment of gbf to golgi membranes regulation of a golgi flippase by phosphoinositides and an arfgef shows that specific enteroviral proteins promote recruitment of ptdins kiiiβ to viral replication membranes to produce ptdins p, which the enteroviral rna polymerase binds to directly and requires for its membrane recruitment a critical role of a cellular membrane traffic protein in poliovirus rna replication mouse hepatitis coronavirus rna replication depends on gbf -mediated arf activation gbf , a guanine nucleotide exchange factor for arf, is crucial for coxsackievirus b rna replication identification of gbf as a cellular factor required for hepatitis c virus rna replication identification of a guanine nucleotide exchange factor for arf , the yeast orthologue of mammalian arf shows that brag is recruited by its ph domain to the activated egfr, coupling egf stimulation to arf activation. depletion of brag , which is overexpressed in many breast cancers, inhibits cell invasion and metastasis in animal models regulation of arf activation: the sec family of guanine nucleotide exchange factors guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction active arf recruits arno/cytohesin gefs to the pm by binding their ph domains the arl family of small g proteins can recruit the cytohesin arf exchange factors to the plasma membrane arl d recruits cytohesin- /arno to modulate actin remodeling structural basis and mechanism of autoregulation in -phosphoinositide-dependent grp family arf gtpase exchange factors presents the crystal structure of the cytohesin catalytic sec domain and the ph domain in tandem, revealing an autoinhibited conformation that can be relieved by binding of arf and phosphoinositides to the ph domain kinetic studies of the arf activator arno on model membranes in the presence of arf effectors suggest control by a positive feedback loop a phosphatidylinositol- -kinase-dependent signal transition regulates arf and arf during fcγ receptor-mediated phagocytosis the cnk scaffold binds cytohesins and promotes insulin pathway signaling shows that cytohesin arf gefs facilitate conformational changes in the cytoplasmic portion of egf or erbb receptor tyr kinase receptors that promote ligand-induced signalling, and that inhibition of these gefs reduces proliferation of lung cancer cells the cytohesin steppke is essential for insulin signalling in drosophila inhibition of cytohesins by secinh leads to hepatic insulin resistance grasp and ipcef promote arf-to-rac signaling and cell migration by coordinating the association of arno/cytohesin with dock cytohesin- controls the activation of rhoa and modulates integrin-dependent adhesion and migration of dendritic cells distinct spatiotemporal expression of efa d, a guanine nucleotide exchange factor for arf , among the efa family in mouse brain regulation of dendritic development by the arf exchange factor arno arf and efa a regulate the development and maintenance of dendritic spines mutations in arfgef implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia mutations in the guanine nucleotide exchange factor gene iqsec cause nonsyndromic intellectual disability ampa receptor signaling through brag and arf critical for long-term synaptic depression ltd, mediated by down-regulation of neuronal receptors by clathrin-mediated endocytosis, is shown in this study to require activation of arf by brag caenorhabditis elegans efa- limits microtubule growth at the cell cortex poliovirus replication requires the n-terminus but not the catalytic sec domain of arfgef gbf the arf gtpase-activating protein: zinc finger motif and golgi complex localization arf gaps and their interacting proteins src-dependent phosphorylation of asap regulates podosomes the structure of an arf-arfgap complex reveals a ca + regulatory mechanism crystal structure of the arf-gap domain and ankyrin repeats of pyk -associated protein β autoinhibition of arf gtpase-activating protein activity by the bar domain in asap arf gtpase-activating protein asap interacts with rab effector fip and regulates pericentrosomal localization of transferrin receptor-positive recycling endosome cin , a cbl-interacting protein, is a component of amap -mediated breast cancer invasion machinery substrate specificities and activities of azap family arf gaps in vivo regulation of receptor trafficking by grks and arrestins the drosophila homologue of arf-gap git , dgit, is required for proper muscle morphogenesis and guidance during embryogenesis impaired fear response in mice lacking git impaired spine formation and learning in gpcr kinase interacting protein- (git ) knockout mice rac inhibits adhesion and differentiation of neuronal cells by modifying git downstream signaling the assembly of a gtpase-kinase signalling complex by a bacterial catalytic scaffold analysis of the small gtpase gene superfamily of arabidopsis correct targeting of plant arf gtpases relies on distinct protein domains role of the gnom gene in arabidopsis apical-basal patterning -from mutant phenotype to cellular mechanism of protein action adp-ribosylation factor machinery mediates endocytosis in plant cells an arf-gef acting at the golgi and in selective endocytosis in polarized plant cells fluorescence imaging-based screen identifies arf gef component of early endosomal trafficking a bacterial virulence protein suppresses host innate immunity to cause plant disease van arf-gap-mediated vesicle transport is involved in leaf vascular network formation scarface encodes an arf-gap that is required for normal auxin efflux and vein patterning in arabidopsis agd is a gtpase-activating protein at the trans-golgi network in a screen for mutations affecting floral organ shedding in a. thaliana, this study identified mutations in an arf gap protein named nevershed, which localizes to the tgn and endosomes and is required for trafficking of cargo molecules involved in cell separation trypanosoma brucei arf plays a central role in endocytosis and golgi-lysosome trafficking the small gtpase arl is required for cytokinesis in trypanosoma brucei functional analysis of tbarl , an n-myristoylated golgi protein essential for viability in bloodstream trypanosomes n-myristoyltransferase inhibitors as new leads to treat sleeping sickness consensus nomenclature for the human arfgap domain-containing proteins we apologize to authors whose work we could not cite owing to space limitations. we thank c. eyster, l. maldonado-baez, j. ménétrey and c. le clainche for critical reading of the manuscript. work in our laboratories is supported by the division of intramural research in the national heart, lung, and blood institute, us national institutes of health (j.g.d.) and grants from the agence nationale de la recherche and the centre national de la recherche scientifique, france (c.l.j.). the authors declare no competing financial interests. the authors would like to note that catherine l. jackson's address was incomplete as it appeared in the original version of this article. this has been corrected in the online version. key: cord- -q jx n l authors: nan title: localization and targeting of the saccharomyces cerevisiae kre p/mnt p alpha , -mannosyltransferase to a medial-golgi compartment date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: q jx n l the yeast kre p/mnt p alpha , -mannosyltransferase is a type ii membrane protein with a short cytoplasmic amino terminus, a membrane- spanning region, and a large catalytic luminal domain containing one n- glycosylation site. anti-kre p/mnt p antibodies identify a -kd integral membrane protein that is progressively n-glycosylated in an mnn -dependent manner. kre p/mnt p is localized in a golgi compartment that overlaps with that containing the medial-golgi mannosyltransferase mnn p, and distinct from that including the late golgi protein kex p. to determine which regions of kre p/mnt p are required for golgi localization, kre p/mnt p mutant proteins were assembled by substitution of kre p domains with equivalent sequences from the vacuolar proteins dpap b and pho p. chimeric proteins were tested for correct topology, in vitro and in vivo activity, and were localized intracellularly by indirect immunofluorescence. the results demonstrate that the nh -terminal cytoplasmic domain is necessary for correct kre p golgi localization whereas, the membrane-spanning and stem domains are dispensable. however, in a test of targeting sufficiency, the presence of the entire kre p cytoplasmic tail, plus the transmembrane domain and a -amino acid residue luminal stem region was required to localize a pho p reporter protein to the yeast golgi. cesses have been studied from genetic and biochemical perspectives (kukuruzinska et al., ; pryer et al., ) resulting in a functionally defined golgi apparatus that has been subdivided into several subcompartments on the basis of asparagine-linked (n-linked) oligosaccharide modifications (franzusoff and schekman, ; graham and emr, ; wilsbach and payne, ; gaynor et al., ) . in yeast, secretory pathway glycoproteins can acquire two types of n-linked oligosaccharides, either a simple core carbohydrate or one extended by an outer-chain glycan structure. the n-linked core oligosaccharide elaborated in the er is mainly constituted of mansglcnac and may undergo golgi maturation resulting in man . _ glcnac . in other cases, glycoproteins traversing the golgi apparatus have their core oligosaccharide extended by outer chains (see fig. ; ballou, ; herscovics and orlean, ) . the initial step in outer-chain synthesis takes place and defines an early golgi compartment in which a backbone of ed, - inked mannose residues is attached to the core oligosaccharide. this golgi compartment has been likened to the mammalian cis-golgi network (wilsbach and payne, ) . outer chain elaboration is then brought to completion: the al, - inked mannose structure is enlarged in a sequential fashion by al, -and al, -mannosyltransferases in a subsequent distinct com-partment defining the yeast medial-golgi compartment. mature glycosylated proteins are finally transported to a late golgi compartment where proteolytic processing of secreted protein precursors occurs. some evidence suggests that this golgi subcompartment is also involved in vacuolar protein sorting, making this late golgi compartment comparable to the mammalian tgn (wilsbach and payne, ) . in s. cerevisiae, the various golgi compartments have not been morphologically well characterized. the yeast golgi complex is not structurally similar to the perinuclear stacked cisternal subcompartments characteristic of the golgi apparatus of mammalian cells. immunoelectron microscopy revealed that the yeast golgi is composed of as many as concave, disklike membranous cisternae scattered in the cytoplasm and most often not organized in parallel stacks (preuss et al., ) . in indirect immunofluorescence using antibodies to different yeast golgi proteins, the isolated cisternae are visualized as dispersed punctate spots (franzusoff et al., ; redding et al., ; antebi and fink, ; cooper and bussey, ; roberts et al., ) . the establishment and maintenance of the polarized organization of the golgi apparatus relies on the existence of complex sorting and transport mechanisms permitting specific golgi proteins to be delivered to their precise cisternal destinations. resident proteins of the secretory pathway of eukaryotic cells require particular targeting signals that specify their final location (pelham and munro, ; gleeson et al., ; low and hong, ) . mammalian membrane glycoproteins not possessing positive sorting sequences are transported to the cell surface through a bulk flow of proteins and lipids (pelham and munro, ) . in yeast, secretory pathway membrane glycoproteins lacking targeting sequences are brought by default to the vacuole (cooper and bussey, ; roberts et al., ; wilcox et al., ; nothwehr et al., ; gaynor et al., ; hill and stevens, ) . proteins associated with glycan modifications constitute a major class of resident golgi proteins. enzymes belonging to different mammalian glycosyltransferase families share a similar type ii structural organization, but lack amino acid sequence homology or discernible targeting motifs even if situated in the same golgi compartment (shaper and shaper, ; kleene and berger, ) . it has been established that the membrane-spanning domain of animal glycosyltransferases plays a central role in golgi localization (munro, ; nilsson et al., ; swift and machamer, ; aoki et al., ; burke et al., ; colley et al., ; tang et al., ; teasdale et al., ; wong et al., ; gleeson et al., ; low and hong, i ) . to define more precisely the s. cerevisiae golgi complex and to better understand how posttranslational modifications occur, we have studied the glycosyltransferase, kre p/mntlp. kre /mnt was isolated as a gene implicated in cell-wall assembly conferring k killer toxin resistance when mutated (hill et al., ) and found to encode an cd, -mannosyltransferase (h/iusler and . kre /mnt encodes a -amino acid residue predicted type ii membrane protein containing a putative transmembrane domain near its nh terminus and one potential luminal n-glycosylation site. kre p/mntlp specifically adds a third mannose during the linear elongation of o-linked carbohydrate chains that may contain up to five mannose residues (h~usler et al., ) and is also apparently involved in asparagine-linked glycosylation (hill et al., ; lussier et al., b) . here we report further characterization of the kre / mntl-encoded mannosyltransferase. we demonstrate that kre p/mntlp possesses a type ii orientation, is progressively n-glycosylated in an mnnl-dependent fashion, and is localized in a golgi subcompartment that overlaps with the mnnlp medial-golgi compartment. in addition, we show that the short nh -terminal cytoplasmic tail domain of kre p/mntlp is required for correct golgi localization, whereas the membrane-spanning and stem domains are dispensable. however, localization of a reporter protein to the yeast golgi requires a region of kre p/mntlp encompassing the cytoplasmic tail, the transmembrane domain (tmd), and a segment of the luminal stem domain. all yeast constructions used strains sey (mata, (boone et al., ) . strains were transformed using the lithium acetate procedure using sheared, denatured carrier dna (gietz et al., ) . transformants were selected on synthetic minimal medium with auxotrophic supplements. levels of sensitivity to k killer toxin were evaluated in sey by a seeded plate assay using a modified medium consisting of . % ynb, . % required amino acids, . % bacto agar, . % methylene blue, % glucose, and buffered to ph . with halvorson minimal medium (lussier et al., (lussier et al., , a brown et al., ) . (underlined) . finally, kpkp was obtained by replacing the kre p tmd in kkkp with that of pho p using the same oligonucleotide that was used to obtain kpkk all chimeric protein constructions were verified by dna sequencing using the dideoxy chain termination procedure (sanger et al., ) with the sequenase enzyme (united states biochemical corp., cleveland, oh), a-ass-datp and specific dna primers. kre p antibodies were raised in rabbits against a bsa-coupled synthetic peptide corresponding to the last amino acid residues of the protein (nh -kpknwkkfre-cooh; obtained from the sheldon biotechnology centre, mcgill university, montr al, qu bec, canada). initially, rabbits were injected with ~g of conjugated peptide in freund's complete adjuvant, followed by three subsequent injections with equivalent amounts of peptide in freund's incomplete adjuvant at - -wk intervals. the conjugated kre p peptide was coupled to cyanogen bromide-activated sepharose cl- b (pharmacia lkb biotechnology, inc., montr al, canada) and used in a column to affinity purify the antiserum as described by raymond et al ( ) . yeast total-ceu protein extracts were prepared from cultures growing exponentially in yeast nitrogen base selective medium by cell lysis with glass beads in the presence of protease inhibitors. membrane fractions were prepared as described (nakayama et al., ) by centrifuging cell lysates at , g for min and by centrifuging the resulting supernatant at , g for h. the high speed pellet contained the insoluble membrane fraction. yeast proteins were separated by sds-page, and immunoblots were carried out mainly as described (lussier et al., a) . briefly, blots were treated in tbst buffer ( mm tris, ph . , mm nac , . % tween , % nonfat dried milk powder) and subsequently incubated with affinity-purified anti-kre p antibodies in the same buffer. after antibody binding, membranes were washed in tbst and a second antibody directed against rabbit immunoglobulins and conjugated with alkaline phosphatase, was then added. the blots were again washed and proteins detected using an enhanced chemiluminescence procedure (amersham canada, oakviue, ontario). analysis of [ s]methionine-labeled proteins was as described (cooper and bussey, ) . briefly, cells were grown to an od of . - . in selective medium (ynb) and then labeled with trans s ( ~ci; icn biochemicals inc., montr al, canada) for min at °c. yeast cells treated with tunicamycin ( ~g m - ) were preincubated before radiolabeling for min at °c. tunicamycin is a hydrophobic analogue of udp-n-acetylglucosamine that blocks the addition of n-acetylglucosamine to dolichol phosphate, the first step in n-linked oligosaccharide formation. chase conditions were achieved by addition of l-methionine and l-cysteine to a final concentration of i mm. txl, -mannosyltransferase activity assays were performed essentially as described (lewis and ballou, ; hhusler and robbins, ) . s. cerevisiae cells ($ background) were grown in selective medium to an od of . - . and lysed with glass beads in the presence of protease inhibitors. high speed pellet fractions including golgi and vacuolar membranes were prepared by centrifuging cell lysates at , g for min and by centrifuging the resulting supernatant at , g for h at °c. incubation mixtures contained mm hepes, ph . , mm mnci , . % triton x- , . mm gdp-[lgc]mannose, and mm c~-d-methylmanno-pyranoside as an acceptor (sigma chemical co.) and p, of membrane fraction in a total vol of tzl. reaction mixtures were incubated for -- min at °c and then passed through a resin (ag -x ; bio-rad laboratories, hercules, ca) column to remove unreacted gdp-mannose. neutral products were eluted with ml of water and radioactivity was measured. control assays were conducted in which the saccharide acceptor was omitted and counts obtained in these assays were subtracted from values obtained in assays made with the sugar acceptor. enzymatic activities (see fig. ) are expressed as percentages of specific activity (mmol/h/mg of membrane protein) for each chimeric protein. yeast cells were grown in selective medium to an od of . - . . and immediately fixed by the addition to cultures of potassium-phosphate (ph . ) to . m, and formaldehyde to . %. after gentle agitation for rain, cells were pelleted and resuspended in a fixation buffer containing . m potassium-phosphate (ph . ) and . % formaldehyde. cells were further fixed for another rain. formaldehyde-treated ceils were washed with . m potassium-phosphate (ph . ) buffer, resuspended in a solution of ioo mm tris-hc , ph , mm dtr, mm edta, and . m sorbitol and incubated for min at °c with gentle agitation. after washing of the fixed cells, cell walls were removed by treatment with zymolyase t (icn biochemicals) at a final concentration of ixg/ml in . m potassium phosphate (ph . ) . m sorbitol for - min at °c. fixed spheroplasted cells were washed in . m potassium-phosphate (ph . ) . m sorbitol and resuspended in the same buffer. cells were subsequently adsorbed to poly-l-lysine coated microscope slides, permitted to stand for min and washed with pbs. slides were then immersed in - °c methanol for - min, and then for s in - °c acetone, depending on yeast strains. treated slides were air-dried and used immediately or stored at °c until needed. fixed mounted cells were incubated with primary antibodies diluted in pbs containing . mg/ml bsa for h at °c or overnight at °c. anti-kre p ab was used at dilutions of : - : . for dual-labeling experiments involving kre p and medialor late golgi markers, the influenza hemagglutinin virus epitope (sequence ypy-dvpdya) was inserted by oligonucleotide-directed mutagenesis directly at the cooh-terminal domain of mnnlp and in the region corresponding to the kexlp luminal domain. epitope-tagged mnnlp and kexlp were detected with the ca monoclonal antibody (kolodziej and young, ) . the latter was used at dilutions ranging from : -- : , . mab dll which recognizes the -kd subunit of the yeast vacuolar membrane h+-atpase (kane et al., ) was used at dilutions of : - : as a vacuolar marker for colocalization studies with kre p chimeric proteins. fluorescence signals were obtained by subsequent incubation of treated cells with rhodamine x sulfonyl chloride (texas red)-conjugated goat anti-rabbit igg ( : - : ) and fftc-conjugated goat anti-mouse igg ( : - : ) which were used as secondary antibodies. nuclei and mitochondria were visualized by staining with ', -diamidino- -phenyl-indole (dapi). cells were examined with an epifluorescence microscope (axiophot; carl zeiss, inc., thornwood, ny), and photographed with film (t-max ; eastman kodak co., rochester, ny). the extent of colocalization of kre p with mnnlp or kexlp was scored by quantitating in a given dual localization experiment cells that contained clearly defined signals representing - individual fluorescent punctate spots for each antigen. compiled data revealed that mnnlp gave rise to approximately the same number of punctate spots per cell as kre p and % of the punctiform fluorescent spots from texas red and fitc overlapped. kexlp gave rise to ~ % fewer punctate spots per cell compared with kre p-associated signals, and % of the punctiform fluorescent spots emanating from both proteins did not overlap. finally, the intracellular localization of chimeric protein kkp was quantitatively scored by examining individual ceils containing clearly defined signals. punctiform fluorescence different from nucleus, er, or vacuole was defined as golgi localization. vacuolar localization was determined by colocalization with the -kd vacuolar membrane h+-atpase subunit. the product encoded by the kre gene is a predicted type ii membrane protein of amino acid residues with a short cytoplasmic nh terminus, a hydrophobic transmembrane region, and a large luminal enzymatic domain containing one potential n-glycosylation site ( fig. a) . to identify and subsequently analyze kre p, a specific rabbit antiserum was raised, affinity purified, and used to detect antigen-antibody complexes by western blotting of total-cell protein extracts of yeast strains harboring a kre disruption or containing a kre multicopy plasmid to facilitate immunological detection. the affinity-purified ab detected kre p as a - kd protein in the kre overexpressing strain that was absent from the kre ::trp strain ( fig. b) , and not detected by preimmune antiserum (data not shown). the in vivo-produced kre p is ~ . kd larger than the . -kd molecular mass predicted from the d n a sequence. the possible integral membrane protein nature of kre p was examined by using reagents extracting cytoplasmic, vesicle-enclosed, and peripheral membrane proteins but leaving intact tightly associated membrane proteins. ceils expressing kre at high levels were lysed with glass beads and treated with sodium carbonate or urea. after high-speed centrifugation of the treated cell lysates, the distribution of kre p in the pellet and supernatant fractions was assessed by western immunoblotting. as can be seen in fig. c, kre p was found only in membrane pellet fractions. the difference between the expected ( . kd) and observed molecular mass of kre p ( fig. b) is likely to be at kre p is oriented as a type ii membrane-anchored protein, a topology characteristic of all isolated glycosyltransferases (shaper and shaper, ; kleene and berger, ; gleeson et al., ) . kre p consists of a short amino-terminal cytoplasmic domain, a hydrophobic transmembrane domain, and a large carboxy-terminal luminal catalytic domain. the catalytic domain is linked to the transmembrane domain by a polypeptidic "stem" region. the stem region is generally thought to be devoid of secondary structure. (b) immunological detection of kre p. yeast total-protein extracts from kre ::trp in sey and from the same strain expressing kre from the multicopy plasmid yep (hill et al., ) , were immunoblotted with affinity-purified anti-kre p polyclonal antibodies (see materials and methods). the molecular mass standards are shown in kilodaltons. (c) kre p is membrane associated. sey cells overexpressing kre were lysed, and cell debris was removed by centrifugation. the resulting crude homogenate was aliquoted, and fractions were rendered . m with na co or . m with urea. after min, each fraction was centrifuged at , g for h and after sds-page, kre p was detected by immunoblotting with the anti-kre p ab in pellet (p) and supernatant (s) fractions. least partly due to n-glycosylation, since kre p is expected to act in the secretory pathway and the protein possesses a single site for n-glycosyl attachment in its predicted luminal domain (asna -gln-thr) (see fig. ). to test for kre p n-glycosylation, yeast cells were [ s]methionine labeled in the presence or absence of tunicamycin, an inhibitor of n-glycosylation. immunoprecipitation and s d s -p a g e analysis of labeled cell lysates revealed that kre p was n-glycosylated (see fig. , lanes and ), with the position of the sole n-glycosylation attachment site at a s n being consistent with a type ii topology for kre p. the molecular mass of kre p in the presence of tunicamycin is about kd, still . kd larger than its pre-dicted molecular mass. the membrane nature of kre p or other posttranslational modifications could explain this discrepancy. to establish possible golgi modifications that kre p might acquire, and to attempt to assess to which golgi subcompartments it had been exposed, the size of the kre plinked oligosaccharide chains was examined in specific n-linked glycosylation mutants. the mnn strains carry mutations at various loci resulting in glycosylation defects ( fig. ; ballou, ; ballou et al., ) . the n-linked carbohydrates from an mnn strain lack the outer chain. the mnnl strain produces glycoproteins with n-linked chains lacking terminal c~l, -mannosyl residues (fig. ) . wild-type, mnnl, and mnn mutant yeast strains carrying kre were [ s]methionine labeled for rain and chased for min, and the extent of glycosylation of immunoprecipitated kre p was examined (fig. ) . the size of kre p produced in a wild-type strain after a -min radiolabeling is ~ kd (major band, lane ). after an additional -min chase, an apparent increase of molecular mass from to ~ - kd was seen (fig. , lanes and ). this apparent - kd increase in mass suggests that kre p may undergo a post-er modification not involving extensive outer chain elaboration. the molecular mass of kre p produced in an mnn strain after a -min pulse (fig. , lanes and ) and a min chase (lanes and ) was identical to the mass of kre p from a wild-type cell, indicating that kre p does not receive an outer chain oligosaccharide. we tested whether the time-dependent additional carbohydrate modification required the mnn encoded golgi al, -mannosyltransferase, an enzyme that acts on both core and outer chains ( fig. ; ballou et al., ; graham and emr, ; graham et al., ; yip et al., ) . after a short pulse, kre p synthesized in a mnnl strain is approximately of wild-type size (fig. , lane ) . after a -min chase, however, the mnnl-produced kre p was ~ - kd smaller than the wild-type protein (lanes - ) indicating that kre p is normally exposed to a golgi compartment where the mnnlp cxl, -mannosyltransferase adds at least three mannose residues to the kre p n-glycosyl core. mnnlp therefore contributes to most of the observed kre p post-er modifications (fig. ) . glcnac carbohydrate structure. in other cases, core structures may be extended by an outer chain of variable size (up to mannose residues) that is composed of a backbone of al, -mannosyl residues to which are attached branched al, -and cd, -mannosyl side chains. recent evidence suggests that the initiating ochlp al, -mannosyltransferase (nakayama et al., ) defines a very early golgi compartment that appears to be distinct from a subsequent early golgi compartment responsible for cxl, -mannosyl side chains elongation (gaynor et al., ) . the ~ , -and al, mannosyl side chain modifications are predicted to occur in the medial-golgi. the possible s. cerevisiae n-linked oligosaccharide structures are shown (adapted from ballou, and ballou et al., ) . arrows depict [ , , al, , cd, , and cd, linkages between mannoses of the core and outer chain. x = on average. the mannose units not present in the mnn mutation are indicated. the mnnlp-terminal al, -mannosyltransferase is responsible for the addition of all al, -mannosyl residues in a medial-golgi compartment (ballou, ; graham et al., ; yip et al., ) . [] represents mannose residues that are thought to be added to the kre p core oligosaccharide in a mnnl-dependent manner (see fig. ). fully glycosylated proteins are then transported to a late golgi compartment which includes the proteolytic enzymes (kex p, kexlp, and dpap a) responsible for maturation of secreted protein precursors. glycoproteins that are not retained in the golgi complex can be subsequently directed to the vacuole through an endosomal compartment or to the cell surface where they can be (a) incorporated into the plasma membrane, (b) secreted and retained in the periplasmic space/cell wall, or (c) secreted extracellularly. both the function of kre p as a txl, -mannosyltransferase and its slow receipt of mnnl-dependent modifications imply localization to a golgi compartment. kre p subcellular localization was examined by indirect immunofluorescence and showed a punctate pattern of fluorescent signals indicative of a golgi association (fig. c) . between and structures per cell can be seen, depending on individual cells and the plane of focus. punctiform fluorescence was never seen with the anti-kre p antibody in kre :: trp cells (fig. a) . the kre p signal distribution did not overlap with nuclei or mitochondria viewed by d n a staining with dapi ( fig. d) . to attempt to define in which golgi compartment kre p is localized, its intracellular localization was compared with that of a known medial-golgi protein, the mannosyltransferase mnnlp (ballou, ; graham et al., ; yip et al., ) , by double-label immunofluorescence (fig. ) . quantitative evaluation of punctate spot distribution (described in materials and methods) revealed that % of the kre p corresponding texas red-labeled structures coincided with the fitc-labeled mnnlp fluorescence spots. the location of kre p was also compared with that of a late golgi protein, the carboxypeptidase kexlp (see figs. and ; dmochowska et al., ; bussey, , ) . a quantitative scoring of punctiform fluorescence distribution indicated that % of the kre p corresponding structures did not coincide with the kexlp fluorescence patches. to examine the basis of localization of a yeast golgi glycosyltransferase, an analysis of kre p noncatalytic domains was made. the roles of the cytoplasmic nh terminus, tmd, and luminal stem region of kre p in golgi targeting were tested by constructing chimeric proteins in which kre p-specific segments were substituted with the corresponding domains of the dap or ph gene products (fig. ) . dap encodes the vacuolar dipeptidyl aminopeptidase b (roberts et al., ) , dpap b, and is a type ii integral membrane glycoprotein that lacks apparent vacuolar targeting information. no individual domain of dpap b was shown to be required for its transport to the vacuole besides a nonspecific hydrophobic tmd (roberts et al., ) . ph encodes a vacuolar alkaline phosphatase. it is also a type ii membrane protein (kaneko et al., ; klionsky and emr, ; nothwehr et al., ) that is thought to be transported to the vacuole by default (nothwehr et al., ) . substitutions designed with vacuolar proteins were used to avoid potential problems with cryptic golgi-targeting sequences. indirect immunofluorescence detection of chimeric proteins was undertaken to determine their intracellular location(s). initially, the role of the membrane-spanning domain of kre p was assessed with fusion protein kdkk (fig. ) , in which the kre p tmd was removed and substituted with that of dpap b. kdkk was found, like wild-type kre p, to be localized to small punctate structures of the yeast golgi (fig. ) . to exclude the possibility that the dpap b tmd is able to function as a kre p tmd in the context of chimeric protein kdkk by sharing some unknown common features not apparent at the amino acid level, a chimeric protein consisting of kre p with the pho p membrane-spanning domain was made. as was the case with kdkk, kpkk was also found to be localized to the golgi hill et al., ) and the influenza hemagglutinin virus epitope-tagged mnn on multicopy plasmid yep (ura ; hill et al., ) or epitope-tagged kex gene (see materials and methods) under an adh gene promoter on centromeric-based plasmid prs (ura ; sikorski and hieter, ) were fixed, spheroplasted, attached to polylysine-coated glass slides, and then incubated with affinity-purified anti-kre p and ca mab (kolodziej and young, ) . fluorescence signals were obtained by subsequent incubation of treated cells with texas red-conjugated goat anti-rabbit igg and fitc-conjugated goat antimouse igg which were used as secondary antibodies. arrows indicate colocalization of punctiform fluorescence. apparatus (fig. ) indicating that the kre p tmd is dispensable for golgi retention. the function of the cytoplasmic domain of kre p was examined with chimeric protein d k k k which possesses the cytoplasmic nh terminus of kre p replaced by that of d p a p b. d k k k could not be visualized in sey (data not shown) suggesting possible degradation of this protein in the yeast vacuole as found with other mistargeted golgi proteins (cooper and bussey, ; roberts et al., ; wilcox et al., ; nothwehr et al., ) . to figure . schematic representation of chimeric proteins used in the analysis of kre p golgi targeting. the kre protein is composed of amino acid residues. the length of the different kre p domains are as follows: nh terminus, residues; tmd, residues; stem, ~ residues and the catalytic domain is ~ amino acid residues long. dpap b is an -amino acid residue type ii vacuolar membrane protein with cytoplasmic nh -terminal and membrane-spanning domains of and amino acid residues, respectively, and a luminal domain consisting of amino acid residues. the vacuolar alkaline phosphatase pho p is a -amino acid residue type ii membrane protein. the length of the different pho p domains are as follows: nhz terminus, residues; tmd, residues; and the luminal domain is amino acid residues long. the different chimeric constructions were made by replacing or fusing specific kre p domains with those of vacuolar dpap b and pho p. chimeric proteins are represented (not to scale) as an assembly of different domains which are distinguished by a letter specifying their origin. k, d, and p, respectively, denote kre p, dpap b, and pho p. mr represents the two remaining amino acid residues in the cytoplasmic nh terminus of kre p after truncation. putative n-glycosylation sites are indicated (y). for details on constructions, see materials and methods. test this possibility, d k k k was expressed in strain $ which contains mutations in the major vacuolar hydrolases and was found to be exclusively mislocalized to the yeast vacuole as shown by colocalization with a vacuolar membrane marker (fig. ) . in contrast, when wild-type kre p was expressed from a p~-based plasmid in strain $ , all positive cells showed a punctate pattern of fluorescence. however, % of these stained cells also showed fluorescence associated with the vacuole (data not shown). these vacuolar stained cells appeared to be expressing high levels of kre p as they displayed high fluorescence levels. the kre p cytoplasmic tail nh terminus was deleted to further explore its role in targeting. chimeric construct mr/kkk lacks the kre p cytoplasmic tail, with the exception of the initiating methionine codon and the arg residue lying directly adjacent to the tmd. mr/kkk was figure . golgi localization of chimeric proteins kdkk, kpkk, and kd-k by indirect immunofluorescence. yeast ceils (sey ) carrying a kre disruption (kre ::trp ) and containing the different chimeric constructions on yep were fixed, spheroplasted, attached to polylysine-coated glass slides, and incubated with anti-kre p antibodies followed by a subsequent incubation with a texas red-conjugated secondary ab and dapi. found to be mislocalized to the vacuole (fig. ) , again demonstrating that the cytoplasmic nh -terminal domain of kre p is required for correct golgi localization. some er retention of this mr/kkk protein was seen in ~ % of positive cells, indicative of an increased transit time through this organelle. the role of the luminal amino acid residues flanking the kre p tmd was examined with fusion protein kd-k. it comprises the kre p nh terminus, the membrane-spanning domain of dpap b, the kre p stem region lacking the first amino acid residues following the tmd, and the catalytic domain of kre p. kd-k was found to be localized in the golgi complex (fig. ) suggesting that neither the deleted stem region nor the tmd is necessary for golgi retention in the context of a kre p catalytic domain. in parallel with the localization studies, all chimeric proteins were assessed functionally. they were verified to be membrane associated and properly glycosylated (data not shown), showing that they possess a type ii membrane orientation (see fig. ). in addition, their in vitro and in vivo enzymatic activities were assayed since low levels of mannosyltransferase activity would imply abnormal secondary structure of a particular chimera and possible mislocaliza-tion in the secretory pathway. the in vitro cd, -mannosyltransferase activity was assayed in cell-membrane fractions. the specific activity of each chimeric protein is displayed in fig. , where kre expressed in a kre a strain was arbitrarily determined to be %, a value just slightly lower than that of wild-type mannosyltransferase activity from a genomic copy of kre . kdkk, kpkk, dkkk, as well as mr/kkk appear to be fully active. kd-k displays intermediate levels of mannosyltransferase activity suggesting that the stem region from position to is necessary for folding or proper catalytic activity. the capacity of chimeric proteins to function in vivo in golgi-localized mannosylation was assessed by using a k killer toxin sensitivity assay (fig. ). k killer yeast strains secrete a small pore-forming toxin that requires a cell-wall receptor for function (bussey, ) . kre null mutations lead to shorter mannose chains on cell-wall mannoproreins disrupting the toxin receptor and leading to resistance (hill et al., ; h/~usler et al., ) . yeast cells containing different chimeric proteins were assayed for killer toxin sensitivity and comparisons were made between a sensitive wild-type strain, a resistant kre null strain, and the null strain containing plasmids bearing wild-type kre or different chimeric constructions. as can be seen in fig. , the kre null mutant is toxin resistant, showing no killing zone, but the same strain containing the kre gene, or expressing kdkk or kpkk displays a large clear killing zone of ,-~ mm similar to wild-type cells consistent with correct golgi localization. chimeric protein kd-k has also been shown to be localized to the golgi complex but has a reduced zone size ( mm) likely a consequence of its reduced mannosyltransferase activity. the reduced killing zone ( mm) of cells expressing dkkk which has wild-type enzymatic activity in vitro indicates that it is not correctly retained in the kre p golgi subcompartment. mr/kkk has also been shown to be localized to the vacuole, but displays an almost wild-type zone size ( . mm). its catalytic domain appears to have a normal conformation as it possesses wild-type mannosyltransferase activity, but it is partially retained in the er (see above). thus, mr/kkk while slowly passing through the secretory pathway en route to the vacuole, is likely able to correctly mannosylate cell-wall proteins. finally, these results indicate that for kre p the default compartment for mislocalization is the vacuole, a conclusion previously reached for late golgi membrane proteins (cooper and bussey, ; roberts et al., ; wilcox et al., ; nothwehr et al., ) and some er membrane proteins (gaynor et al., ; hill and stevens, ) . to determine which regions of kre p are sufficient to target a reporter protein to the golgi complex, chimeric proteins kkp and kkkp were constructed (see fig. and materials and methods for details). kkp consists of the kre p cytoplasmic and membrane-spanning domains fused to the pho p luminal domain. kkkp contains the kre p nh terminus, tmd, and a segment encompassing the first amino acid residues of the stem domain fused to and containing the different chimeric constructions on yep were fixed, spheroplasted, attached to polylysine-coated glass slides, and incubated with anti-kre p antibodies which was followed by a subsequent incubation with a fitc-coupled anti-rabbit secondary ab and dapi. colocalization of vacuolar localized chimeric proteins with the yeast vacuolar membrane h÷-atpase was achieved by coincubation of fixed cells with mab dll (kane et al., ) with subsequent incubation of treated cells with texas red-conjugated anti-mouse secondary antibodies. the pho p luminal domain. both chimeric proteins were first verified to be membrane associated and properly glycosylated (data not shown), indicative of a correct type ii membrane orientation (fig. ) . chimeric protein kkkp was found, like wild-type kre p, to be typically localized to punctiform golgi structures (fig. ) . however, when the partial stem domain was removed, the majority of the resulting chimeric protein (kkp) was found to be localized in the vacuole. the intracellular localization of chimeric protein kkp was quantitatively scored (see materials and methods); % of positive cells displayed a vacuolar signal and % of the cells had vacuolar and punctate fluorescent signals (typical results are shown in fig. ) , whereas % showed exclusively punctiform fluorescence. thus, the kre protein region encompassing the nh terminus, tmd, and partial stem domain is sufficient for full golgi retention of a vacuolar protein. these results were extended biochemically by examining the processing kinetics of both chimeric proteins. the phosp alkaline phosphatase is activated in the vacuole (klionsky and emr, ; nothwehr et al., ) where the catalytic luminal domain is processed by the proteinase a (ammerer et al., ; woolford et al., ) . kkkp was found not to be cleaved, whereas in contrast, the majority of kkp was processed in accord with its mostly vacuolar location (data not shown). lastly, to assess if the kre p tmd is required for proper golgi retention in the context of an heterologous luminal domain, we constructed fusion protein kpkp in which the kre p tmd of kkkp was removed and substituted with that of phosp (see fig. ). golgi retention of this fusion protein would indicate that the kre p nh terminus cytoplasmic and stem domains are sufficient to achieve correct golgi intracellular localization. immunofluorescence studies of kpkp revealed that this chimeric protein was retained in the er (data not shown), and thus uninformative. kre p is part of the yeast glycosylation machinery and was anticipated to be localized in a medial-golgi compartment where n-and o-modified proteins receive al, - inked mannose residues (fig. ) . evidence presented here is consistent with this expectation. we have demonstrated that in situ detection of kre p by indirect immunofluorescence reveals intracellular punctiform staining typical of the yeast golgi, in agreement with chapman and munro ( ) . kre p is an al, -mannosyltransferase that receives an mnnlp-dependent ctl, modification, the next sequential step in the orderly glycosylation of yeast proteins. thus, kre p must reach a golgi compartment harboring the mnnlp cd, -mannosyltransferase (fig. ) . these ed, and al, - inked activities could be colocalized, or, if they are situated in two distinct and consecutive cisternae, kre p would have to reach the mnnlp compartment and then be retrieved to its own preceding organelle. kre p and mnnlp have been shown to colocalize to a considerable extent, placing kre p in or close to the mnnlp medial-golgi compartment. consistent with this assignment, previous biochemical studies provide evidence that kre p is localized in a compartment distinct from the late golgi. immunoisolated late golgi organelles containing the endoproteinases kexlp, kex p, and dpap a were shown to be devoid of kre p (bryant and boyd, ) . our immunocytochemical colocalization results also indicate that kre p is not in the same compartment as kexlp. the % of kre p and kexlp punctiform fluorescence signals that do colocalize could be due to two or more stacked cisternae that are seen in ~ % of all cisternae in a given yeast cell (preuss et al., ) . however, the possibility remains that proteins from the medial-golgi (kre p) and from the late kre ,~ % figure . activity of chimeric proteins. enzymatic activity was measured in vitro by ed, -mannosyltransferase assays of membrane preparations, and in vivo mannosylation of cell-wall proteins was assessed by verifying k killer toxin sensitivity in a wild-type strain and a kre null mutant containing kre and chimeric constructs expressed from the centromeric-based plasmid prs (sikorski and hieter, ) . enzymatic activity is expressed as percentages of specific activity (mmot/h/mg) for each chimeric protein. a kre null strain expressing kre from prs arbitrarily represents % specific activity. concentrated k killer toxin was spotted on a lawn of ~, /ml cells from a fresh culture of each strain (see materials and methods). golgi (kexlp) are not always in mutually exclusive cisternae and their intracellular distribution may partially overlap. this can be the case in mammals where a pair of trans-golgi and medial-golgi glycosyltransferases showed substantial overlap (nilsson et al., ) . the kre p/mnnlp compartment remains to be precisely defined but some evidence suggests it could be identical to, or overlap with, compartments containing the golgi type ii membrane n-glycoprotein guanosine diphosphatase, gdalp (abeijon et al., (abeijon et al., , . subcellular fractionation experiments indicated that gdalp and kre p activities cosediment (abeijon et al., ) , whereas gdalp and the late golgi marker, kex p, are in distinct compartments (bowser and novick, ) . mnnlp also cosediments with gdalp and was found in fractions different from those containing kex p . these results are in accord with our immunofluorescence data indicating that kre p and mnnlp are in the same golgi compartment. the apparent double role of kre p in o-and n-linked glycosylation hill et al., ; lussier et al., b) is most simply explained if both types of modification are carried out in the same golgi compartments. the observations that mnnlp also acts on o-and n-linked chains (ballou, ; herscovics and orlean, ) , and that the o-glycosyltransferase kre p is itself n-glycosylated, are consistent with a common set of compartments for these modifications. however, more complex scenarios could be envisaged with specific subcompartments for o-and n-glycosylation. kre p is predicted to act in a functionally and genetically defined compartment where various cd, -mannosyl residues are added to oligosaccharide chains. this compartment(s) is also likely to include other ~l, -mannosyltransferases responsible for n-linked outer chain elaboration (fig. ) . a family of putative kre p-like mannosyltransferases has been identified (hill et al., ; bussereau et al., ; lussier et al., ; mallet et al., ) , and some members have been shown bioehemically to be golgi mannosyltransferases (lussier et al., b) . immunolocalization results from the different chimeric proteins demonstrate that the kre p nh -terminal cytoplasmic domain was required for localization to the golgi complex. chimeric proteins lacking the -amino acid residue cytoplasmic domain are fully active as a mannosyltransferase in vitro but fail to be properly retained and are mislocalized to the vacuole. a trivial explanation for the localization of d k k k to the vacuole is that the cytoplasmic domain of dpap b contains a cryptic vacuolar targeting signal that overrides the golgi-targeting sequence of kre p. arguing against this view is the demonstration that the nh -terminal domain of dpap b is not necessary for vacuolar targeting (roberts et al., ; nothwehr et al., ) . to ensure that mislocalization of dkkk was not caused by some previously unrecognized vacuolar targeting signal of the nh -terminal domain of dpap b, chimeric protein mr/kkk was constructed in which the kre p cytoplasmic tail was deleted. as with dkkk, mr/ kkk was mislocalized to the vacuole, showing that the nh -terminal domain of kre p is necessary for proper golgi targeting. chimeric proteins k d k k and kpkk could not be distinguished from wild-type kre p, permitting the conclusion that the kre p tmd in the context of a native protein is not necessary for correct golgi localization. chimeric construct kd-k which comprises the kre p cytoplasmic tail, the dpap b membrane-spanning domain, a kre p partial stem region, and the kre protein mannosyltransferase domain was correctly localized to the golgi complex. this indicates that the first amino acid residues of the stem region of kre p are dispensable for golgi targeting. the results obtained with this fusion protein again show that the kre p tmd is not required for golgi retention. the fact that in the kd-k construct, the kre p tmd and part of the stem region are not present, argues that in the context of a protein containing a kre p luminal catalytic domain the cytoplasmic tail of kre p is sufficient to correctly target such a chimera to the golgi complex, un- figure i . localization of chimeric proteins kkkp and kkp. yeast cells carrying a ph disruption (pho ::trp ) in a sey background and containing the different chimeric constructions on yep were fixed, spheroplasted, attached to polylysine-coated glass slides, and incubated with anti-pho p antibodies followed by a subsequent incubation with a fitccoupled anti-rabbit secondary ab and dapi. colocalization of vacuolar localized chimeric proteins with the yeast vacuolar membrane h+-atpase was achieved by coincubation of fixed cells along with mab dll (kane et al., ) followed by incubation of treated cells with texas red-conjugated anti-mouse secondary antibodies. arrows point to golgi punctiform structures. less some remaining luminal sequence also plays some role in retention. to analyze further the targeting role of the three nonenzymatic domains of kre p and to remove the possible additional complexity of a targeting mechanism involving the mannosyltransferase portion of kre p, combinations of noncatalytic domains of kre p were tested to assess what region of kre p was sufficient to target a pho p reporter protein to the golgi complex. as opposed to the results obtained with fusion protein kd-k, all three noncatalytic domains of the kre protein were found to be required for full golgi retention of the pho p luminal region (kkkp). these results are in agreement with those of chapman and munro ( ) who found that a fusion protein containing the nhz terminus, the membrane-spanning domain, and a partial stem region of kre p linked to a reporter protein was retained in the golgi complex. the mostly vacuolar localization of kkp demonstrated that the first part of the kre p stem region which is not required for retention in the context of a kre p catalytic domain (kd-k), is necessary, in combination with the kre p cytoplasmic tail and tmd for the targeting of the pho p catalytic portion to the golgi complex (kkkp). taken together, the intracellular localizations of chimeric proteins kd-k, kkp, and kkkp suggest that the kre p luminal domain does play a role in golgi localization. data obtained with chimeric proteins kdkk, kpkk, kd-k, and kkp clearly show that the kre p tmd is dispensable for golgi retention and not sufficient to retain a reporter protein in the yeast golgi complex. our results are, however, at variance with conclusions reached in a recent study of chapman and munro ( ) and we address this below. in a first set of experiments, chapman and munro ( ) tested the role of the kre p tmd with a chimeric protein consisting of a full-length fusion of kre p/mntlp linked to the suc -encoded invertase by a small region containing a kex p-dependent cleavage site (chimeric protein mmmi). the mislocalization of this protein was expected to result in production of soluble invertase when passing through the kex p-containing late golgi compartment. this fusion protein was inferred to be in the golgi complex as no invertase was secreted from the cell. when the tmd of kre p was replaced with that of pho p (mpmi) in this protein, it appeared to be mislocalized to some extent, as some invertase was secreted from the cell. this result was interpreted to indicate a role for the kre p tmd in golgi retention. however, the intracellular localization of this chimeric protein was not visualized by immunofluorescence. we replaced the kre p tmd in the native protein with two different nonrelated tmd's, and showed that both proteins (kdkk and kpkk) were golgi localized by using indirect immunofluorescence microscopy. in addition, we tested that the proteins had the right conformation by measuring their glycosylation in vivo, and their mannosyltransferase activity in vitro and in vivo. these proteins (kdkk and kpkk) behaved phenotypically as wild-type kre p in our tests with no indication that the kre p tmd in the context of a native protein is necessary for proper retention. a possible explanation of the chapman and munro ( ) results is that the kre p tmd is somehow necessary for golgi retention in the context of those large heterologous proteins perhaps because some retention property of the luminal domain of kre p has been disrupted in the invertase fusion. unfortunately, the er retention of chimeric protein kpkp did not permit resolution of this question. second, chapman and munro ( ) reported that a pho chimeric protein containing a kre p tmd (chimeric protein pkp) slowed vacuolar processing of the luminal portion of pho p. they inferred from their results that pkp was at least partially localized in the golgi complex and was retained by the kre p tmd. again, the pkp chimeric protein was not localized intracellularly. this is clearly different from our results with pho p chimeras, which show that the kre p cytoplasmic nhz-terminal and transmembrane domains (kkp) are not sufficient for proper golgi retention and the presence of the kre p cytoplasmic tail, tmd, and stem region is required for full retention of a pho p reporter protein (kkkp) in the yeast golgi. to resolve this apparent inconsistency, we obtained the pkp construct and, on examination of its cellular location, found that it was exclusively localized to the vacuole displaying no punctiform fluorescence (data not shown). this finding is entirely consistent with our localization results with kkp, and offers no support for the chapman and munro conclusion that the kre p tmd will at least partially retain a heterologous protein in the yeast golgi. the membrane-spanning domain plays a crucial role in the localization of resident animal golgi glycosyltransferases. in contrast to kre p, alterations of the tmd of a certain number of mammalian glycosyltransferases results in some mislocalization of the modified protein (munro, ; aoki et al., ; teasdale et al., ; burke et al., ; gleeson et al., ) . the hydrophobic tmd of several mammalian glycosyltransferases appears to be both necessary and sufficient to confer complete golgi retention (nilsson et al., ; aoki et al., ; shaper and shaper, ; teasdale et al., ; gleeson et al., ; low and hong, ) whereas in other cases, some additional flanking sequences are required to achieve effective golgi targeting (munro, ; burke et al., ; shaper and shaper, ; dahdal et al., ; burke et al., ; gleeson et al., ; low and hong, ) . the exact nature of these nhz-terminal and stem sequences remains the be precisely determined but do not appear to involve the whole of each domain but mainly the short stretches of hydrophilic residues directly flanking the lipid-embedded tmd (munro, ; burke et al., ; shaper and shaper, ; dahdal et al., ; gleeson et al., ; low and hong, ) . it has been postulated that the targeting of glycosyltransferases carrying a tmd sorting signal could be due to interactions between the membrane-spanning domain and compartment specific membrane lipids (pelham and munro, ) . there is no evidence that kre p is retained by such a mechanism, since in the series of constructs we have devised, the kre p tmd is not implicated in retention. in this lipid interaction model, the length of the membranespanning domain is important for the proper sorting of animal glycosyltransferases (munro, ; pelham and munro, ) . in the case of kdkk the tmd of dpap b is three amino acid residues shorter than the kre p tmd, and in the case of kpkk the tmd of pho p is seven amino acid residues longer than the kre p tmd, yet both are retained in the golgi complex. none of the studied animal glycosylation enzymes appear to need an intact cytoplasmic domain to achieve golgi retention (munro, ; aoki et al., ; colley et al., ; shaper and shaper, ; teasdale et al., ; dahdal et al., ; burke et al., ; gleeson et al., ; low and hong, ) . kre p, thus, constitutes the first demonstration of an eukaryotic glycosyltransferase requiring a short cytoplasmic amino-terminal domain for correct intracellular localization. the -amino acid residue-targeting domain of kre p seemingly parallels that of the late yeast golgi enzymes kexlp, kex p and dpap a (cooper and bussey, ; wilcox et al., ; nothwehr et al., ) . it was shown for dpap a that a -amino acid region within the cytoplasmic domain was both required and sufficient for proper retention (nothwehr et al., ) , and for kex p, a -amino acid region was found to be essential (wilcox et al., ) . the accurate golgi targeting of kex p and dpap a is clathrin dependent and aromatic residues are thought necessary for this retention process (wilcox et al., ; nothwehr et al., ; wilsbach and payne, ) . while unrelated at a sequence level with kex p and dpap a, the amino-terminal domain of kre p contains a single aromatic phenylalanine residue (malfls-krllr) which was mutated to an alanine, an alteration showing no effect on targeting and, therefore, providing no evidence for clathrin-based retention of kre p (lussier, m., a.-m. sdicu, t. ketela, and h. bussey, unpublished results) . the mnnlp al, -mannosyltransferase is the only other yeast glycosyltransferase where targeting has been examined. mnnlp targeting appears different from that of kre p since a mutant mnnlp lacking its nh -terminal cytoplasmic tail is properly localized to the golgi complex . correct retention of mnnlp is clathrin dependent but, contrary to kex p and dpap a, does not seem to be mediated by a direct interaction through its cytoplasmic tail. the reason for the kre p requirement for its cytoplasmic tail remains unclear. it could be clathrin dependent but not involving an aromatic residue, or through some other process. the gdalp type ii golgi membrane protein (abeijon et al., ; abeijon el al., ) has been shown to be properly localized in a strain lacking clathrin heavy chains (seeger and payne, ; wilsbach and payne, ) . the mnnlp, kre p, and gdalp enzymes appear to be in close proximity or even in the same golgi compartment (this work; abeijon et al., ; graham et al., ) . this raises the possibility that specific golgi membrane proteins showing a similar compartmental distribution may be retained by more than one mechanism. our results indicate that although only the cytoplasmic nh terminus has been shown to be required for golgi retention, no single domain is able to specify correct golgi localization. a chimeric protein including a kre p cytoplasmic tail and enzymatic domain (kd-k) was properly targeted. in contrast, a luminal reporter protein could only be properly retained in the golgi complex by the kre p cytoplasmic tail, plus the membrane-spanning and a amino acid residue stem domain (kkkp). the entire kre p cytoplasmic tail and membrane-spanning domain were not sufficient to retain pho p in the golgi complex (kkp). therefore, it appears that a combination of kre p topological domains is needed to achieve proper golgi localization. the presence or requirement of more than one specific targeting signal in a given secretory pathway membraneassociated protein has been hinted at in a few cases recently. the tmd might not be the sole targeting motif present in particular animal glycosyltransferases as some cytoplasmic and luminal sequences appear to be involved in retention (munro, ; burke et al., ; shaper and shaper, ; dahdal et al., ; burke et al., ; gleeson et al., ; low and hong, ) . the trans-golgi network protein tgn appears to achieve proper golgi localization using two nonoverlapping targeting domains: a tyrosine-based cytoplasmic retrieval signal and a retention signal found in its membrane spanning domain (bos et al., ; humphrey et al., ; wong and hong, ; ponnambalam et al., ) . for the cgn sed p syntaxin, the intracellular localization is only partially specified by its tmd and an additional localization signal involving its cytoplasmic domain appears to be involved (banfield et al., ) . targeting studies on the p type ii membrane protein indicate that the cytoplasmic, membrane-spanning and luminal domains are all necessary for proper er-golgi intermediate compartment localization (hauri and schweizer, ; schweizer et al., schweizer et al., , . finally, the wbpl type i membrane protein of the yeast er possesses a cytoplasmic kkxx golgi-to-er retrieval motif. when this targeting motif is inactivated, wbpl is still er retained indicating that another targeting signal is present in the protein (gaynor et al., ) . the fact that kre p requires more than one topological segment to achieve proper golgi localization, emphasizes that there may be distinct, but not necessarily mutually exclusive, mechanisms of retention. golgi proteins that possess a cytoplasmic targeting signal have been proposed to be sorted via a receptor-mediated mechanism (wilsbach and payne, ; gleeson et al., ; low and hong, ) . overexpression of the late golgi yeast proteins kexlp, kex p, and dpap a leads to some vacuolar mislocalization, indicating saturation of the capacity of a receptor-based sorting process (cooper and bussey, ; wilcox et al., ; nothwehr et al., ; wilsbach and payne, ) . kre p overexpression also results in some mistargeting to the vacuole, and could be similarly explained. when wild-type kre p is expressed from a xbased multicopy vector in a background with mutations in the major vacuolar hydrolases, all positive cells display a punctiform pattern of fluorescence with % of the stained cells also showing vacuolar fluorescence. this small percentage of doubly stained cells are likely expressing high levels of kre p as determined by the intensity of the fluorescence. in contrast, overexpression of animal glycosyltransferases does not bring about saturation of the retention mechanism (munro, ; aoki et al., ; burke et al., ; colley et al., ; teasdale et al., ; gleeson et al., ) . interpreting our data in the simplest way, the kre p segment encompassing the nh terminus along with the tmd and stem region could interact directly with a medial-golgi localized receptor spanning both sides of the golgi lipid bilayer. for kre p to be retained in the golgi, an interaction between the putative receptor and the nh -terminal domain is essential. part of the presumed kre p receptor complex could be part of the golgi extracisternal space matrix which in mammals was recently shown to bind medial-golgi enzymes presumably through their cytoplasmic tails . interestingly, examination of the cytoplasmic nhz-terminal domains of the six members of the kre mannosyltransferase family (lussier et al., ; mallet et al., ) reveals that the kre p (malflskrllr) sequence resembles only that of ktr p (mrflskrilk; mallet et al., ) where the sequence flskr(i/l)l(k/r) is conserved in both enzymes. this may imply a common recep-tor for the two proteins. interaction of the presumed receptor protein with a chimeric construct lacking the kre p stem domain but including a reporter luminal domain (kkp) may be partial, and insufficient to retain the fusion protein in the golgi complex. to achieve full golgi retention of a reporter luminal protein, interaction of the postulated receptor with the three nonenzymatic domains would be required. in the case of kd-k, only partial interaction would occur with the putative receptor and the observed golgi retention may now reveal an additional mechanism involving a segment of the kre p luminal region possibly by oligomerization/kin recognition that has been implicated in the retention of certain mammalian golgi'membrane proteins. it has been proposed that protein oligomers are assembled through their tmd and/or luminal domains in a particular golgi cisternae and because of their highorder structure are consequently excluded from entering forward-moving secretory vesicles (weisz et al., ; gleeson et al., ; low and hong, ; nilsson et al., ; schweizer et al., ; yamaguchi and fukuda, ) . applying such a model to a functionally active kre protein involves self-association or formation of heterooligomers between kre p and other medial-golgi protein(s) either through the catalytic domain or possibly with some contiguous stem sequences retained in construct overall, our results suggest that proper targeting of kre p in the yeast medial-golgi may involve two different mechanisms. the saturability of the kre p retention system implies a receptor-based retention mechanism that could involve protein-protein interactions over a tail/tmd/ partial stem region. in addition, a second mechanism that may involve oligomerization would operate in some way through the kre p luminal domain. in conclusion, our observations on kre p targeting may point to differences in golgi localization mechanisms between yeast and animal glycosyltransferases or could indicate that multiple retention or retrieval mechanisms are used to varying extents in both systems. topography of glycosylation in yeast: characterization of gdp-mannose transport and lumenal guanosine diphosphatase activities in golgi-like vesicles guanosine diphosphatase is required for protein and sphingolipid glycosylation in the golgi lumen of saccharomyces cerevisiae pep gene of saccharomyces cerevisiae encodes proteinase a, a vacuolar enzyme required for processing of vacuolar precursors the yeast ca +-atpase homologue, pmr is required for normal golgi function and localizes in a novel golgi-like distribution golgi retention of a trans-golgi membrane protein. galactosyltransferase requires cysteine and histidine residues within the membrane-anchoring domain isolation, characterization, and properties of saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects revision of the oligosaccharide structures of yeast carboxypeptidase y localization of sed , a putative vesicle targeting molecule, to the eis-golgi network involves both its transmembrane and cytoplasmic domains yeast kre genes provide evidence for a pathway of cell wall [ -glucan assembly tgn is maintained in the trans-golgi network by a tyrosine-containing motif in the cytoplasmic domain secl protein, an essential component of the exocytic apparatus, is associated with the plasma membrane and with a soluble . s particle the k killer toxin: molecular and genetic applications to secretion and cell surface assembly. in molecular genetics of yeast: a practical approach immunoisolation of kex p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single golgi compartment the transmembrane and flanking sequences of , -n-acetylglucosaminyltransferase i specify medial-golgi localization medial-golgi retention of n-acetylglucosaminyltransferase i a . kb segment on the right arm of chromosome ii from saccharomyces cerevisiae including part of the dur gene, contains five putative new genes k killer toxin, a pore-forming protein from yeast the functioning of the yeast golgi apparatus requires an er protein encoded by anp , a member of a new family of genes affecting the secretory pathway the signal anchor and stem regions of the [ -galactoside ~x , -sialyltransferase may each act to localize the enzyme to the golgi apparatus characterization of the yeast kex gene product: a carboxypeptidase involved in processing of secreted precursor proteins yeast kexlp is a golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane specific sequences in the signal anchor of the -galactoside a- , -sialyltransferase are not essential for golgi retention yeast kex gene encodes a putative protease with a carboxypeptidase b-like function involved in killer toxin and alpha factor precursor processing functional compartments of the yeast golgi apparatus are defined by the see mutation localization of components involved in protein transport and processing through the yeast golgi apparatus signal-mediated retrieval of a membrane protein from the golgi to the er in yeast studies on the transformation of intact yeast cells by the liac/ss-dna/peg procedure targeting of proteins to the golgi apparatus compartmental organization of golgi-specific protein modification and vacuolar protein sorting events defined in yeast sec (nsf) mutant clathrin-dependent localization of a- , mannosyltransferase to the golgi complex of s. cerevisiae the er-golgi intermediate compartment glycosylation in saccharomyces cerevisiae: cloning and characterization of an a-l, -mannosyltransferase structural gene yeast glycoprotein biosynthesis: mnt encodes an c~-l, -mannosyltransferase involved in o-glycosylation glycoprotein biosynthesis in yeast yeast/e. coli shuttle vectors with multiple unique restriction sites yeast kre defines a new gene family encoding probable secretory proteins, and is required for the correct n-glycosylation of proteins vma p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar h+-atpase complex localization of tgn to the trans-golgi network: involvement of a cytoplasmic tyrosine--containiug sequence assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar h+-atpase structural characteristics of the ph gene encoding repressible alkaline phosphatase in saccharomyces cerevisiae the molecular and cell biology of glycosyltransferases membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. embo (eur mot epitope tagging and protein surveillance protein glycosylation in yeast rapid and efficient sitespecific mutagenesis without phenotypic selection separation and characterization of two al, -mannosyltransferase activities from saccharomyces cerevisiae protein trafficking in and out of the golgi apparatus ktr : a new member of the kre mannosyltransferase gene family protein o-glycosylation in yeast. the pmt gene specifies a second protein o-mannosyltransferase that functions in addition to the pmtl-encoded activity functional characterization of the kre /mnt mannosyltransferase gene family nucleotide sequence analysis of an . kb fragment of yeast chromosome i including bem , a new gene of the wd- repeat family and a new member of the kre /mnt family sequences within and adjacent to the transmembrane segment of a- , -sialyltransferase specify golgi retention ochi encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides. embo (eur mot the membrane spanning domain of - , -galactosyltransferase specifies trans-golgi localization overlapping distribution of two glycosyltransferases in the golgi apparatus of hela cells kin recognition between medial golgi enzymes in hela cells membrane protein retention in the yeast golgi apparatus: dipeptidyl aminopeptidase a is retained by a cytoplasmic signal containing aromatic residues sorting of membrane proteins in the secretory pathway the tgn glycoprotein contains two nonoverlapping signals that mediate localization to the trans-golgi network characterization of the saccharomyces golgi complex through the cell cycle by immunoelectron microscopy vesicle-mediated protein sorting molecular analysis of the yeast vps gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle immunolocalization of kex protease identifies a putative late golgi compartment in the yeast saccharomyces cerevisiae structure, biosynthesis, and localization of dipeptidyl aminopeptidase b, an integral membrane glycoprotein of the yeast vacuole membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment targeting, disruption, replacement, and allele rescue: integrative dna transformation in yeast molecular cloning: a laboratory manual dna sequencing with chainterminating inhibitors characterization of a novel kda membrane protein. implications for the organization of the er-to-golgi pathway retention of p in an er-golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers selective and immediate effects of clathrin heavy chain mutations on golgi membrane protein retention in saccharomyces cerevisiae enzymes associated with glycosylation a system of shuttle vectors and yeast host strains designed for efficient manipulation of dna in saccharomyces cerevisiae isolation of a matrix that binds medial-golgi enzymes a golgi retention signal in a membrane-spanning domain of coronavirus e protein the transmembrane domain of n-glucosaminyltransferase i contains a golgi retention signal the signal for golgi retention of bovine , -galactosyltransferase is the transmembrane domain oligomerization of a membrane protein correlates with its retention in the golgi complex mutation of a tyrosine localization signal in the cytosolic tail of kex p protease disrupts golgi retention and results in default transport to the vacuole dynamic retention of tgn membrane proteins in saccharomyces cerevisiae the -residue transmembrane domain of -galactoside ct , -sialyltransferase is sufficient for golgi retention the sxyqrl sequence in the cytoplasmic domain of tgn plays a major role in the trans-golgi network localization the pep gene encodes an aspartyl protease implicated in the posttranslational regulation of saccharomyces cerevisiae vacuolar hydrolases golgi retention mechanism of - , -galactosyltransferase cloning and analysis of the saccharomyces cerevisine mnn and mnn genes required for complex glycosylation of secreted proteins we thank dr. annette herscovics for critically reading the manuscript, the members of the bussey laboratory for helpful comments and suggestions, antony cooper and kathryn hill for all their help and advice, tom stevens for kindly providing strains, plasmids, and antibodies, anne camirand for advice with the mannosyltransferase assays, rowan chapman for discussion and plasmids, v. mackay and c. yip for the mnn clone, carole smith and guy i'heureux for photographic work, and diane oki for assistance with manuscript preparation.supported by an operating grant from the natural sciences and engineering research council of canada. m. lussier was the recipient of a postdoctoral fellowship from the medical research council of canada.received for publication may and in revised form july . key: cord- - d l f authors: li, jie; ahat, erpan; wang, yanzhuang title: golgi structure and function in health, stress, and diseases date: - - journal: the golgi apparatus and centriole doi: . / - - - - _ sha: doc_id: cord_uid: d l f the golgi apparatus is a central intracellular membrane-bound organelle with key functions in trafficking, processing, and sorting of newly synthesized membrane and secretory proteins and lipids. to best perform these functions, golgi membranes form a unique stacked structure. the golgi structure is dynamic but tightly regulated; it undergoes rapid disassembly and reassembly during the cell cycle of mammalian cells and is disrupted under certain stress and pathological conditions. in the past decade, significant amount of effort has been made to reveal the molecular mechanisms that regulate the golgi membrane architecture and function. here we review the major discoveries in the mechanisms of golgi structure formation, regulation, and alteration in relation to its functions in physiological and pathological conditions to further our understanding of golgi structure and function in health and diseases. receive proteins and lipids from the endoplasmic reticulum (er) by the cis cisternae and export them from the trans cisternae and the trans-golgi network (tgn) to other intracellular membranes such as the endosomes, lysosomes, plasma membrane, and outside of the cell (tang and wang ; wang and seemann ) . while traversing the golgi stack, cargo molecules are modified and processed. the sub-compartments of the golgi stacks house a set of glycosidases and glycosyltransferases responsible for the synthesis of glycoproteins and glycolipids. in the tgn, many secretory proteins are proteolytically cleaved by lumenal proteinases zhang and wang ; huttner et al. ) . many efforts have been made to understand the mechanism of golgi structure formation. the formation of the golgi ribbon depends on golgi matrix proteins and an intact microtubule organization. cytosolic dynein moves golgi membranes along centrosome-derived microtubules toward the (-) end of the microtubules (matteis et al. ; rabouille and kondylis ; wei and seemann ) . subsequently, golgi-oriented microtubules maintain golgi stacks in the proximity and facilitate tubular connections between them (zhu and kaverina ) . while dynein and microtubules are required for the concentration of golgi stacks in the pericentrosomal region and golgi ribbon formation, they are not essential for the generation and maintenance of the stacked structure, as depolymerization of microtubules disrupts the golgi ribbon but not the stacks (thyberg and moskalewski ) . from the s, morphological studies have shown connections in the space between cisternae that might be involved in the adhesion of cisternae into stacks (franke et al. ; mollenhauer ; cluett and brown ) , which were later identified as golgi matrix proteins. these include golgi stacking proteins and membrane tethers, as discussed below. in , the concept of "golgi matrix" proteins was first introduced (slusarewicz et al. ) . since then, several golgi matrix proteins have been identified to be responsible for maintaining the unique architecture and function of the golgi apparatus. major components of the golgi matrix are summarized in table . . key proteins involved in golgi structure formation are discussed below. among all golgi matrix proteins, the golgi reassembly stacking proteins grasp and grasp (grasps, also called gorasp and gorasp , respectively) are best characterized for their roles in golgi structure formation, including stacking , bekier et al. , shin et al. , barr et al. ) , ribbon-linking (puthenveedu et al. ; tang et al. ; feinstein and linstedt ) , cargo transportation (kuo et al. ; table . -kctd (dementieva et al. ) • golgi stacking xiang and wang ; bekier et al. ) • golgi ribbon formation (feinstein and linstedt ) • cell cycle control (duran et al. ) • transport of specific cargo (kuo et al. ; d'angelo et al. ) • p cargo receptor retention • autophagy (zhang et al. ; zhang and wang a, b) • unconventional secretion (dupont et al. ; rabouille and linstedt ; vinke et al. ; gee et al. ; piao et al. ) • (barr et al. ; xiang and wang ; bekier et al. ; shin et al. ) • golgi ribbon formation (puthenveedu et al. ; tang et al. ) • cell cycle progression (preisinger et al. ; sutterlin et al. ; yoshimura et al. ; duran et al. ; tang et al. b) • mitotic spindle formation (sutterlin et al. ) (continued) • unconventional secretion (gee et al. ) • apoptosis (lane et al. ) golgins cis p rab (weide et al. ) -p (nakamura et al. ) -grasp -syntaxin -akap (rivero et al. ) -zfpl (chiu et al. ) -tuba (kodani et al. ) • golgi ribbon formation (puthenveedu et al. ) • copii vesicle tethering (moyer et al. ; alvarez et al. ) • non-centrosomal microtubule organization (rivero et al. ) • centrosome regulation (kodani and sutterlin ; kodani et al. ) • spindle formation (kodani and sutterlin ) • cell migration (preisinger et al. ) • apoptosis (walker et al. ) • purkinje neuron development (liu et al. ) p cis p rab (allan et al. ) -gm (nakamura et al. ) -giantin (sonnichsen et al. ) syntaxin , gos- • post-mitotic golgi reassembly brandon et al. ; dirac-svejstrup et al. ) • snare assembly • membrane tethering nakamura et al. ; alvarez et al. ; seemann et al. ; allan et al. ) • apoptosis (chiu et al. ) • nuclear import (mukherjee and shields ) giantin/ golgb cis/medial tmd rab / (rosing et al. ) -p (sonnichsen et al. ) -gm (sonnichsen et al. ) -gcp /acbd ) • golgi stack organization (rosing et al. ; koreishi et al. ) • ribbon organization (koreishi et al. ) • membrane tethering (sonnichsen et al. ; linstedt et al. ; lowe et al. ; misumi et al. ; alvarez et al. ; munro ) • er-to-golgi trafficking • apoptosis (lowe et al. ) • cilia formation (bergen et al. ) golgin- / blzf medial/ trans p rab (short et al. ) -grasp (short et al. , zhao et al. ) • golgi stacking (short et al. , zhao et al. • membrane tethering (short et al. ) golgin- / golga b tmd a • uncharacterized golgin- / golga cis tmd rab (satoh et al. ) -casp (osterrieder et al. ) -cog complex (sohda et al. ) • golgi stacking and reassembly (satoh et al. ) • golgi ribbon formation (diao et al. ) • er-golgi tethering and protein transport (osterrieder et al. ) • membrane tethering (malsam et al. ) (continued) (shin et al. (shin et al. , • tgn-to-pm trafficking of e-cadherin (lock et al. ) • endosome-to-tgn trafficking (lu and hong ; lu et al. ) • retrograde transport from recycling endosomes to the tgn (jing et al. ) • poxvirus morphogenesis (alzhanova and hruby ) golgin- / golga / gcp cis p -gcp /acbd (sbodio et al. ; sbodio and machamer ) -gcp (ohta et al. ) -β ar (gilbert et al. ) -romk, pist (bundis et al. ) • post-golgi trafficking (bundis et al. ; gilbert et al. ) • apoptosis (mancini et al. , maag et al. , sbodio et al. golgin- / golga / p trans grip arl / (wu et al. ; sinka et al. ) rab / / / (sinka et al. ) -tbc d (shin et al. (shin et al. , • tgn-to-pm traffic (lu and hong ) • phagophore formation (sohda et al. ) gcp acylation -golgin- /golga / gcp (ohta et al. ) • golgi-to-pm trafficking (ohta et al. ) gcc trans grip arl / (sinka et al. ), rab / / (sinka et al. ) • tgn organization and endosome-to-tgn trafficking (luke et al. ; lieu et al. ) • golgi ribbon formation (gosavi et al. ) • actin organization (makhoul et al. -clasp (efimov et al. ) • golgi ribbon formation • golgi integrity (lin et al. ) • membrane tethering reddy et al. ; ganley et al. ) • mpr recycling (reddy et al. ) • attachment of non-centrosomal microtubules (efimov et al. ) acbd -poliovirus a protein (greninger et al. ) -numb (zhou et al. ) • golgi integrity and er-to-golgi trafficking • nuclear translocation of caspasegenerated golgin- fragments (residues - ) machamer , sbodio et al. ) • asymmetric cell division (zhou et al. ) • aichi virus and poliovirus replication (greninger et al. ) bicaudal-d/ bicd p -dynactin, p -dynamitin (hoogenraad et al. ) • recruitment of the dynein-dynactin complex (hoogenraad et al. ) (continued) • copi-independent golgi-to-er transport (matanis et al. ) • endosome-to-golgi transport (wanschers et al. ) • autosomal-dominant spinal muscular atrophy (neveling et al. , martinez-carrera and wirth ) casp/coy cis tmd -golgin- (osterrieder et al. ) -cog , sed , sly , gos , sft /gs (anderson et al. • er-golgi tethering and protein transport (osterrieder et al. ) • retrograde-directed copi vesicles (anderson et al. ) cg-nap (takahashi et al. ) • centrosome amplification (nishimura et al. ) • microtubule nucleation coh / vps b -thyroid receptor (chen et al. ) • golgi ribbon formation (rios et al. ) • membrane tethering (drin et al. ) • er-to-golgi trafficking (gillingham et al. ) • γ-tubulin recruitment (rios et al. ) • sorting to primary cilia (follit et al. ) • interacts with thyroid hormone receptor beta (chen et al. • cell cycle progression (cao et al. ; fu et al. ; parisi et al. ) • protein aggregation (ghosh et al. ; ristic et al. ; yi and yuan ; kobayashi et al. ; song et al. ; nishikori et al. ) • autophagy (papadopoulos et al. ) • dna repair (van den boom et al. ; fujita et al. ; davis et al. ; meerang et al. ; partridge et al. ) • er-stress-induced gene transcription (marza et al. ) • antiviral signaling (hauler et al. , hao et al. acbd barr et al. ) , unconventional secretion (dupont et al. ; rabouille and linstedt ; vinke et al. ; gee et al. ; piao et al. ) , cell cycle regulation (preisinger et al. ; sutterlin et al. ; yoshimura et al. ; duran et al. ; tang et al. b) , apoptosis (lane et al. ) , and autophagy (zhang et al. ; zhang and wang a, b) , although the mechanisms are less well understood. both grasps share a similar structure: a conserved n-terminal grasp domain consisting of two pdz domains (pdz and pdz ) and an intrinsically disordered c-terminal serine/proline-rich (spr) domain with multiple phosphorylation sites (zhang and wang b) ( fig. . ). both grasp and grasp are peripheral membrane proteins that are attached to the golgi membranes via an n-terminal myristic acid modification and the interaction with their membrane-bound partner proteins (gm and golgin- , respectively) and therefore are concentrated at the interface between the cisternae where stacking occurs (short et al. ; barr et al. ) . grasp is concentrated on the cis-golgi cisternae, whereas grasp localizes to the medial/trans-golgi cisternae (barr et al. ; ). both play complementary roles in golgi stack formation . mechanistically, grasp proteins form homodimers via the n-terminal pdz domains, and dimers from adjacent golgi cisternae further oligomerize in trans and function as the "glue" that tethers the cisternae into a stack (wang et al. , ). an in vitro study using modified grasp domain peptides indicated that insertion of the myristic acid moiety is required for the oriented association to golgi membranes, which ensures the protein-protein interaction in trans. furthermore, the conformational change caused by myristoylation affects the tendency of grasp domain for self-interaction (heinrich et al. ). depletion of either grasp or grasp reduces the number of cisternae per golgi stack, whereas depletion of both grasps leads to disassembly of the entire golgi stack (sutterlin et al. , bekier et al. ). the grasps are tightly modulated by a phosphorylation and dephosphorylation cycle during cell division, resulting in mitotic disassembly and post-mitotic reassembly of the golgi (feinstein and linstedt ; cervigni et al. ; lin et al. ; wang et al. ; preisinger et al. ; tang et al. ; xiang and wang ; tang et al. ; truschel et al. ) . recent studies have identified novel grasp-binding proteins involved in golgi biogenesis and morphology modulation. recent research of the crystal structure of grasp bound to the golgin- c-terminal peptide revealed that golgin- promotes the oligomerization of grasp by forming a new interaction between two neighboring pdz molecules to play an important role in golgi stacking (zhao et al. ) . meanwhile, using an optimized in vitro system, mammalian enabled homologue (mena) and dnaj homolog subfamily a member (dja ) were identified as grasp binding partners with potential functions on golgi structure maintenance li et al. ) . mena is an actin elongation factor recruited to the golgi membranes by grasp to facilitate actin polymerization and grasp oligomerization and thus functions with actin as bridging proteins of grasp in golgi ribbon linking. dja is a co-chaperone of heat shock cognate kda protein (hsc ), but the activity of dja in golgi structure formation is independent of its co-chaperone activity or hsc , rather, through dja -grasp interaction to promote grasp oligomerization. thus, dja facilitates golgi structure formation through an unconventional hsc -independent pathway. these studies further confirmed grasp as a multifaceted protein in golgi structure formation and indicated that an array of grasp binding proteins could play important roles in golgi morphology maintenance ( fig. . ). in addition, grasp was reported to be involved in glucose starvation-induced autophagy (zhang et al. fig. . structure, modification, and binding sites on grasp (a) and grasp (b). rat grasp and grasp sequences are used for illustration. both grasps share a similar structure: a conserved n-terminal grasp domain consisting of two pdz domains (pdz and pdz ) and a c-terminal serine/proline-rich (spr) domain with multiple phosphorylation sites (indicated by asterisks) that are involved in grasp modulation during the cell cycle. both grasp and grasp are peripheral membrane proteins attached to the golgi membranes via n-terminal myristoylation and the interaction with their membrane-bound partner proteins (gm and golgin- , respectively). grasp -binding proteins mena and dja have been identified to enhance golgi ribbon linking and stacking, respectively. grasp is regulated by o-glcnacylation depending on the glucose level and interacts with lc and lamp to facilitate glucose starvation-induced autophagy interacts with lc on autophagosomes and lamp on lysosomes to facilitate autophagosome maturation, which will be discussed in later sections. golgins are a family of golgi-associated coiled-coil proteins that are necessary for vesicle tethering at the golgi and maintenance of golgi integrity (muschalik and munro ; witkos and lowe ; gillingham and munro ) . most golgins are peripheral membrane proteins anchored on golgi membranes via their c-terminus and are associated with small gtpases of rab, arf, and arl families (table . ) (munro ; sinka et al. ). these interactions mediate both membrane attachment and selective localization of a specific golgin to a specific sub-compartment of the golgi (witkos and lowe ) . golgins lack significant sequence homology between the family members and localize to different regions of the golgi to play distinct roles in tethering events, membrane traffic, and golgi organization. the coiled-coil regions provide the golgins with an extended structure required for the tethering function, while the interactions with rab gtpases control these molecules in their open (extended) or closed (folded) confirmation (cheung et al. ) . in addition, golgins often contain specific sequence and structural features at the n-and c-terminal ends, which allow them to recognize vesicles and golgi cisternal membranes based on the curvature and lipid composition of the membranes (drin et al. ; drin et al. ; magdeleine et al. ) . detailed information about golgins and their functions are summarized in table . . gm was the first identified golgi matrix protein and is predominantly found in the central region of the cis-golgi (nakamura et al. ) , where it forms a stable complex with grasp ( barr et al. ). depletion of gm results in the disruption of the golgi ribbon and causes protein glycosylation defects (puthenveedu et al. ) . there are two possible ways gm contributes to golgi ribbon formation. first, gm targets grasp to the rim of the cisternae where grasp promotes lateral linking of cisternae via oligomerization (puthenveedu et al. ) . mena and actin cytoskeleton may facilitate grasp in this action . second, gm recruits a-kinase anchoring protein (akap ) onto the cis-golgi and allows golgi-associated nucleation of microtubules, which arranges golgi stacks in close proximity to form a ribbon (rivero et al. ). similarly, other golgins may also work with the microtubule cytoskeleton in a similar way to facilitate golgi structure organization. for example, gmap- , another cis-golgi-localized golgin, recruits the γ-tubulin-containing complexes to the golgi membranes and promotes the formation of tubulin oligomers on golgi membranes. depletion of gmap- results in extensive golgi fragmentation, suggesting a role in golgi ribbon formation (rios et al. ). in addition to grasp , gm also interacts with p and giantin to form the gm -p -giantin tethering complex (sonnichsen et al. ). the rab effector p , when recruited to coat protein complex (cop) ii vesicles during the budding from the er, interacts with the soluble atpases n-ethylmaleimide-sensitive factor (nsf) attachment protein receptors (snares), a specialized set of copii v-snares, to form a cis-snare complex that promotes vesicle targeting to the golgi apparatus (allan et al. ) . meanwhile, p also binds giantin on copi vesicles and works with gm on golgi membranes to provide a bridging role for vesicle tethering (sonnichsen et al. ). thus, gm and p are two major tethering factors in er-to-golgi trafficking (munro ) . other than the well-studied grasp -gm and gm -p -giantin complexes, the grip domain containing golgins are another group of proteins associated with the golgi structure. most grip domain-containing golgins localize to the tgn via their grip domains and are involved in golgi organization (luke et al. ) . gcc is reported to localize independently of arl on tgn and plays an essential role in golgi structure formation. depletion of gcc results in fragmentation of both cis-and trans-golgi that are dispersed throughout the cytoplasm ). on the other hand, another tgn golgin gcc is reported to play a role in tgn organization and ribbon-linking. overexpression of gcc causes a loss of the compact golgi ribbon and dispersal of mini-stacks throughout the cytoplasm, while knockdown of gcc results in a longer ribbon structure (gosavi et al. ) . a recent report suggests that gcc -induces golgi ribbon dispersal via actin and non-muscle myosin iia. in addition, a novel gcc -binding partner, the long isoform of intersectin- (itsn- ), a guanine nucleotide exchange factor for cdc , is identified to be involved in this process (makhoul et al. ). besides the golgi matrix proteins and their cofactors described above, other proteins including snares, kinases, methyltransferases, and gtpases have also been reported to be related to golgi structural organization and function. a few examples are discussed below. vesicle-associated membrane protein (vamp ), a v-snare protein located on the tgn, was first shown to play a role in retrograde trafficking from early endosomes to the tgn (steegmaier et al. ) . it was later reported that depletion of vamp led to fragmentation of the golgi ribbon, although golgi membranes remained in the juxtanuclear area. em studies revealed shortened golgi stacks with a normal arrangement. depletion of the cognate snares of vamp , syntaxin , syntaxin , and vti a also disrupted the golgi ribbon. these findings suggest that the maintenance of the golgi ribbon structure requires normal retrograde trafficking, which is likely mediated by the formation of vamp -containing snare complexes (shitara et al. ) . serine/threonine-protein kinase h (pskh ) was primarily characterized with multiple intracellular localizations, including brefeldin a-sensitive golgi compartment, centrosomes, nucleus, and cytoplasm (brede et al. ) . pskh targeting to golgi depends on dual n-terminal acylation, myristoylation on glycine , and palmitoylation on cysteine . expression of palmitoylation site mutant pskh results in the disassembly of the golgi apparatus to a diffused cytoplasmic pattern without interrupting the microtubule cytoskeleton (brede et al. ) . the substrates of this kinase on the golgi are so far unidentified. protein arginine methyltransferase (prmt ) localizes to the golgi apparatus and forms complexes with several components, including gm , which was later identified as a substrate of prmt . n-terminal methylation of gm does not affect its golgi localization but is critical for golgi ribbon formation. depletion of prmt and expression of methylation-defective gm mutants result in fragmentation and dispersal of the golgi ribbon (zhou et al. ) . in addition to the proteins mentioned above, rab small gtpases are another group of key regulators of mammalian golgi organization. many rab proteins, including but not limited to rab , rab and rab (aizawa and fukuda ) , rab and rab (dejgaard et al. ) , rab / (goud et al. ; martinez et al. ; liu et al. ), and rab (kelly et al. ) , have been shown to play a role in golgi structure organization and reviewed previously in detail (goud et al. ) . considering that rab proteins switch between inactive gdp-bound and active gtp-bound forms, it has been proposed that golgi organization-related rab proteins are divided into two categories. with class rabs, the golgi ribbon is disrupted by rab inactivation but appears normal with overexpression, whereas with class rabs, rab inactivation has little effect on golgi ribbon organization, while overexpression leads to the redistribution of golgi enzymes to the er (liu and storrie ) . these results indicate that rabs control the golgi structure through modulating membrane tethering and trafficking. the golgi undergoes a series of sophisticated cell cycle-dependent disassembly and reassembly processes, including the deformation and reformation of golgi ribbon, stacks, and cisternae. at the onset of mitosis, the golgi ribbon unlinks into ministacks, which further undergo unstacking and vesiculation. these processes ensure the equal distribution of golgi compartments into the two daughter cells (wang ; wei and seemann ) . in telophase, the golgi vesicles fuse into cisternae and form stacks. the new stacks then accumulate in the perinuclear region and further link into a ribbon. the molecular factors that control these processes include golgi matrix proteins, kinases and phosphatases, ubiquitin ligases and deubiquitinating enzymes, vesicle budding and fusion factors, and actin and microtubule cytoskeleton. an in vitro system has been developed to replicate the golgi disassembly and reassembly process through sequential treatments of purified golgi membranes with mitotic (mc) and interphase (ic) cytosol, or with purified proteins (tang et al. , a . this system provides a powerful tool for testing key proteins in golgi structure formation, which makes it possible to identify the minimal machinery and key components that control mitotic golgi disassembly and post-mitotic reassembly . the first step of golgi disassembly at the onset of mitosis is golgi ribbon unlinking. at this step, golgi stacks in the ribbon are disconnected and dispersed. this step involves disconnecting the tubules between the stacks by the membrane fission protein ctbp/bars, which is crucial for g /m transition (hidalgo carcedo et al. ; colanzi et al. ). further, grasps undergo mitotic phosphorylation which are also required for ribbon-unlinking. the extracellular-signal-regulated kinase (erk) directly phosphorylates grasp and blocks its activity in both golgi ribbon formation and trans-oligomerization (feinstein and linstedt ) , while grasp is phosphorylated by c-jun n-terminal kinase (jnk) on serine (s ), which causes the separation of the golgi stacks (cervigni et al. ) . sequential phosphorylation of grasp on multiple sites by cyclin-dependent kinase (cdk ) and polo-like kinase (plk ) results in its conformational changes and subsequent de-oligomerization (lin et al. ; preisinger et al. ; wang et al. ; tang et al. ; vielemeyer et al. ). on the other hand, grasp is phosphorylated by erk and partially by cdk . in addition to the unique phosphorylation sites in the spr domains of grasps, s within the grasp domain of grasp is modified by plk , which causes conformational change and impaired self-association (sengupta and linstedt ). an in vivo membrane-tethering activity assay using a construct of full-length grasp fused to the c-terminal mitochondrial anchoring sequence shows that the tethering activity is diminished by introducing a phosphomimic s d mutant. this result suggests that s might be a plk target site on both grasps, although direct evidence remains to be provided (truschel et al. ). grasp is dephosphorylated by pp a in late mitosis and the trans-oligomer reformation is therefore rehabilitated to promote cisternae stacking . the unstacked cisternae further disassemble into vesicles, which depends on copi vesicle formation and blockage of vesicle docking and membrane fusion. as mentioned above, the gm -p -giantin complex promotes copii vesicle docking to the golgi. cdk phosphorylation of gm on s during mitosis inhibits p -interaction and therefore blocks vesicle docking ). inhibition of cdk causes golgi vesiculation failure, suggesting an essential role of cdk activity in mitotic golgi vesiculation. however, expression of the gm s a non-phosphorylatable mutant in gm -depleted cells causes no apparent defects in golgi vesiculation and mitotic progression, indicating the existence of gm s phosphorylation-independent pathways that ensure golgi vesiculation and mitotic progression in mammalian cells (sundaramoorthy et al. ) . recently, the recruitment and activation of aurora kinase family member aurora a at the centrosomes in m phase was reported to depend on golgi ribbon unlinking in g phase (barretta et al. ) . aurora a functions in centrosome maturation, mitotic entry, and bipolar spindle formation during mitosis (nikonova et al. ; carmena et al. ; kimura et al. ) . this finding indicates a potential link between aurora a activity and cell cycle-associated golgi structure modulation. indeed, it was later confirmed that knockdown or inhibition of aurora a induces golgi dispersal without affecting the gm protein level in interphase. further investigation revealed that interference of aurora a causes golgi dispersal only after mitosis via the dissociation of the golgi and centrosome (kimura et al. ) . these studies revealed a novel relationship between g phase golgi unlinking, m phase aurora a activation, and interphase golgi structure formation. two aaa atpases, nsf and p /vcp, are involved in membrane fusion during post-mitotic golgi reassembly, and their activities are regulated by phosphorylation during mitosis . for nsf-catalyzed fusion, the p -gm tethering complex is disrupted by gm phosphorylation during mitosis , while post-mitotic phosphorylation of p by a casein kinase ii (ckii)like enzyme is required for cisterna reassembly (dirac-svejstrup et al. ) . contemporarily, homotypic fusion of golgi membranes mediated by p is also blocked upon phosphorylation of p and p . p uses these two distinct cofactors for its membrane fusion function: p is essential for the regrowth of golgi cisternae from mitotic golgi fragments (kondo et al. ), while p is required for the maintenance of the golgi structure in interphase as well as for its reassembly in late mitosis (uchiyama et al. ). both pathways are regulated by cdk -mediated phosphorylation. phosphorylation of p on s abolishes its binding to golgi membranes, resulting in mitotic inhibition of the p /p pathway (uchiyama et al. ) . phosphorylation on s and threonine (t ) disables p from binding to golgi membranes and consequently blocks p /p -mediated golgi membrane fusion at late mitosis (kaneko et al. ). in addition to phosphorylation, p /p -mediated golgi membrane fusion is also regulated by ubiquitination (tang and wang ) . tang et al. discovered that the homologous to the e -ap carboxyl terminus (hect) domain containing ubiquitin ligase hace is targeted to the golgi membrane through the interaction with rab and participates in post-mitotic golgi biogenesis ). depletion of hace or expression of an inactive mutant impairs post-mitotic golgi membrane fusion. the identification of hace as a golgi-localized ubiquitin ligase provides evidence that ubiquitin has a critical role in golgi biogenesis during the cell cycle . later, the golgi t-snare syntaxin was identified as a ubiquitination substrate ). syntaxin is monoubiquitinated by hace in early mitosis and deubiquitinated by the de-ubiquitinase vcip in late mitosis (wang et al. ). the monoubiquitination of syntaxin at lysine (k ) in the snare domain impairs the interaction between syntaxin and the cognate v-snare bet but increases its binding to the p adaptor p through the uba domain of p , which is required for post-mitotic golgi membrane fusion (meyer et al. ) . expression of the syntaxin k r mutant in cells impairs post-mitotic golgi reassembly. therefore, monoubiquitinated syntaxin recruits p /p to the mitotic golgi fragments and promotes post-mitotic golgi reassembly upon ubiquitin removal by vcip . vcip was originally identified as a p interacting protein (kano et al. ) . it was later shown to be a de-ubiquitinating (dub) enzyme involved in p /p -mediated membrane fusion (wang et al. ) . vcip dub activity, as well as its interaction with p and association with golgi membranes, is regulated by phosphorylation (zhang and wang a; zhang et al. ) . in mitosis, vcip is phosphorylated at s by cdk and thus is inactivated, allowing syntaxin to be ubiquitinated by hace ; in telophase, vcip is dephosphorylated and reactivated, removing ubiquitin from syntaxin to allow p -mediated membrane fusion wang ) . these studies revealed a novel mechanism that monoubiquitination regulates golgi membrane dynamics during the mammalian cell cycle. as stated above, the golgi apparatus in mammalian cells forms a unique stacked structure under normal growth conditions, which undergoes a regulated disassembly and reassembly process during the cell cycle. however, the golgi structure and function could be impaired under stress conditions, such as dna damage, energy and nutrient deprivation, and pro-apoptotic conditions. this could be attributed to perturbation of microtubule organization or phosphorylation, degradation, or cleavage of golgi structural proteins. additionally, many signaling molecules have been identified to be associated with the golgi. thus, it has been proposed that the golgi could sense and transduce stress signals and therefore serves as a hub in the cellular signaling network (farhan and rabouille ; mayinger ; makhoul et al. ). apoptosis, also known as programmed cell death, is a cell suicide mechanism carried out by organelle-directed regulators such as the bcl- proteins and ultimately executed by the caspase family proteases (nicholson and thornberry ) . organellar response to apoptotic initiation includes death receptor endocytosis, mitochondrial and lysosomal permeabilization, er calcium release, and golgi fragmentation. the golgi is one of the first organelles to be affected during apoptosis (mukherjee et al. ; aslan and thomas ) . during apoptosis, several golgi matrix proteins related to golgi structure maintenance are cleaved by caspases, leading to golgi fragmentation (hicks and machamer ) . apoptotic golgi fragmentation is one of the most extensively studied golgi stress responses. reported caspases-cleaved golgi proteins include grasp , golgin- , gm , p , syntaxin , and giantin (lane et al. ; mancini et al. ; walker et al. ; chiu et al. ; lowe et al. ; machamer ) , as summarized in table . and discussed below. golgin- is a golgin that plays a role in vesicle tethering and trafficking . it is cleaved by caspase- , caspase- , and caspase- during apoptosis. under pro-apoptotic conditions stimulated by staurosporine, the golgi senses and transduces apoptotic signals using a local caspase, caspase- . caspase- is special in a way that it has both the property of initiator caspases and the substrate specificity of executioner caspases (mancini et al. ) . although it is unclear how caspase- is activated by pro-apoptotic signals, in vitro and in vivo caspase cleavage assays showed that caspase- cleavage of golgin- at aspartate (d ) happens prior to golgin- cleavage by caspase- and at d and d (mancini et al. ) . expression of the d a cleavage-defective mutant of golgin- delays golgi disintegration under staurosporine treatment (machamer ; hicks and machamer ) . golgi fragmentation (mancini et al. ; mukherjee et al. ; nozawa et al. ; hicks and machamer ; maag et al. ) grasp anisomycin; sts caspase- d , d , d golgi fragmentation (lane et al. ; cheng et al. (lowe et al. ; nozawa et al. ) ca carminomycin i, ch an anti-fas monoclonal antibody, d aspartic acid, sts staurosporine subsequently, it was shown that an n-terminal amino acid fragment of golgin- contains both a golgi localization signal and a nuclear localization signal (hicks and machamer ) . expression of a non-cleavable golgin- mutant inhibits er stress or ligation of death receptor-induced apoptosis (maag et al. ) . latterly, yeast two hybrid screening revealed that gcp preferentially binds to one of the caspase cleavage products of golgin- , aa - , to inhibit its nuclear localization (sbodio et al. ) . overexpression of gcp sensitizes cells to staurosporineinduced apoptosis, while nuclear localization of a golgin- apoptotic cleavage fragment (aa - ) protects cells from apoptosis. however, another report indicates that golgin- depletion does not affect the golgi morphology nor constitutive secretion (williams et al. ) . therefore, the mechanism of how golgin- transduces apoptotic signals and regulates the apoptotic response needs to be further studied. grasp is cleaved in apoptosis induced by oxygen-and glucose-deprivation (ogd) as in ischemia-induced cerebral vascular endothelial injury (yin et al. ) and in staurosporine-or fas ligand-induced apoptosis (lane et al. , cheng et al. . in apoptosis, grasp is cleaved by caspase- on d , d , and d . expression of a cleavage-resistant form of grasp delays golgi fragmentation in apoptosis and protects cells from fas/cd -mediated apoptosis, whereas expression of an n-terminal caspase-cleaved fragment dramatically sensitizes cells to fas/cd -mediated apoptosis (lane et al. , cheng et al. . further results revealed that the c-terminal fragments of grasp produced by caspase cleavage promotes fas/cd -mediated apoptosis via being targeted to mitochondria by binding to bcl-x l (cheng et al. ). however, the mechanism of how the c-terminal cleavage fragment of grasp regulates apoptosis at the mitochondria and the role of bcl-x l in this process are still unknown. the golgi fragmentation phenotype induced by apoptotic grasp cleavage is similar to that of grasp phosphorylation in mitosis (warren ) . there is evidence that several kinases involved in mitotic grasp phosphorylation such as cdk and erk are activated during apoptosis and regulate apoptosis by phosphorylating caspase and bcl- family proteins (terrano et al. ; yamaguchi et al. ; lu et al. ) . whether these kinases directly regulate apoptotic golgi fragmentation by phosphorylating grasp or other golgi proteins remains unclear (ji et al. ). the er-to-golgi membrane tether, p , is cleaved by caspase- and caspase- during apoptosis. expression of a caspase-resistant form of p delays golgi fragmentation in apoptosis. exogenous expression of a p c-terminal apoptotic fragment leads to apoptosis and golgi fragmentation (chiu et al. ) . the extreme c-terminal fragment, generated by caspase cleavage during apoptosis, translocates into the nucleus and further activates the apoptosis machinery. interestingly, translocation of the p c-terminal fragment happens prior to major golgi structural changes, indicating it as an early event (mukherjee and shields ) . the p c-terminus is sumoylated, which regulates its nuclear translocation and amplification of apoptosis signals in a p -dependent manner (how and shields ; mukherjee and shields ) . in a high-throughput screen, carminomycin i (ca) was discovered to inhibit cell proliferation of von hippel-lindau (vhl) defective clear cell renal cell carcinoma (vhlÀ/À ccrcc) (woldemichael et al. ) . ca activates caspase- and caspase- to cleave p , which inhibits ccrcc proliferation (woldemichael et al. ). several other golgi structural proteins are also involved in apoptosis or cleaved by caspases. the level of gm is reduced during fas-mediated apoptosis but not in staurosporine-induced apoptosis (walker et al. ). however, it is not clear whether the reduction is due to gm cleavage or degradation. syntaxin and giantin are also cleaved by caspase- during apoptosis, which inhibits er-to-golgi transport (lowe et al. ). golgin- and golgin- are cleaved during necrosis but not apoptosis (nozawa et al. ) . cleavage of golgin- and golgin- during necrosis is also caspase-dependent, since pretreatment of the cells with pan-caspase inhibitor, zvad-fmk, abolished the cleavage of these two proteins (nozawa et al. ) . some of the golgi proteins are also reported to regulate apoptosis. the golgi snare gs is involved in cisplatin-induced apoptosis in a p -dependent manner (sun et al. ) . overexpression of gs sensitizes hek cells to the apoptosisinducer cisplatin via the accumulation of p and bax and the stimulation of p pro-apoptotic phosphorylation at s . it was also shown that gs forms a complex with the p ubiquitin e ligase murine double minute (mdm ) to inhibit its function and consequential p ubiquitination and degradation (sun et al. ) . therefore, gs promotes cisplatin-induced apoptosis by stabilizing and regulating pro-apoptotic phosphorylation of p . a well-studied mediator of intracellular vesicle fusion, nsf attachment protein α (αsnap), has been reported to have pro-survival functions (naydenov et al. ). depletion of αsnap triggers apoptosis in epithelial cells by reducing the antiapoptotic protein bcl- . depletion of αsnap in p null or bax null cells still results in apoptosis, indicating that the anti-apoptotic function is independent of p and bax. interestingly, αsnap depletion induces apoptosis independent of the cleavage of golgi proteins such as grasp , golgin- , and p but rather by dysregulation of er-golgi vesicle cycling and possibly through er stress (naydenov et al. ) . some other golgi proteins, including human golgi antiapoptotic protein (h-gaap) (gubser et al. ; saraiva et al. ) and golgi integral membrane protein (golim ) (bai et al. ) , are also reported to have anti-apoptotic functions. golim is overexpressed in some head and neck cancers, and depletion of golim reduces cell proliferation and cell viability by inducing apoptosis (bai et al. ) . although microtubule and actin filaments play important roles in golgi orientation and structure, golgi fragmentation in apoptosis occurs prior to cytoskeleton disorganization (mukherjee et al. ; yadav and linstedt ) . furthermore, the level of actin and tubulin did not change during apoptosis, while golgi structural proteins are cleaved as discussed above. therefore, golgi fragmentation in early apoptosis is independent of microtubule and actin filament disorganization. the golgi phosphoprotein (golph ) is a peripheral membrane protein that regulates vesicle budding and tgn-to-plasma membrane trafficking (dippold et al. ). golph is localized to the tgn by binding to phosphatidylinositol -phosphate (pi p). depletion of pi p leads to golph dissociation from the tgn. golph also binds to the actin-based motor protein myo a to link golgi membranes with the actin cytoskeleton. this bridging effect creates a tension required for vesicle budding, trafficking, and maintenance of the golgi ribbon. depletion of golph or myo a leads to the loss of the tensile force, resulting in the shrinkage of the golgi ribbon and a reduction of vesicles formed at the tgn (dippold et al. ; bishe et al. ; ng et al. ) . golph is an oncogene known to be overexpressed in some solid tumors, including lung cancer and breast cancer (scott et al. ; zeng et al. ) . it is also reported that golph increases cell proliferation and cell size by regulating cell proliferation through the interaction with the retromer complex and activation of the mammalian target of rapamycin mtor (scott et al. ). golph induces cell proliferation in breast cancer cells by inhibiting the tumor suppressor transcription factor foxo through activating akt (zeng et al. ). these findings demonstrate that a trans-golgi protein can serve as an oncogene (scott et al. ; buschman et al. ; kuna and field ) . interestingly, dna damage causes golgi dispersal in a golph -dependent manner. dna damage activates the dna-pk kinase to phosphorylate golph on t /t , which aberrantly increases the tensile force for the golgi to fragment. golgi fragmentation in this scenario increases cell survival with an unknown mechanism (farber-katz et al. ). depletion of golph or myo a increases cancer cells' sensitivity to dna damage inducing agents, suggesting that golph phosphorylation-induced golgi fragmentation may serve as a protective mechanism (farber-katz et al. ) . considering that golph is overexpressed in many solid tumors, it is reasonable to speculate that this may be a mechanism of how cancer cells escape dna damage-induced apoptosis (farber-katz et al. ; buschman et al. ; li et al. b) . in addition to its high expression level in some cancer cells, golph overexpression is also reported in mouse n a cells under oxygen-glucose deprivation and reoxygenation (ogd/r), a model mimicking severe oxidative injury (li et al. a) . in this ogd/r model, golph is overexpressed and forms puncta in the cytosol, which induces the formation of reactive oxygen species (ros) and lipidation of lc . opposed to its anti-apoptotic role in cancer cells, depletion of golph in ogd/r desensitizes the cells to apoptosis (li et al. a ). most recently, the golgi stacking protein grasp was reported to regulate autophagy upon energy deprivation (zhang et al. ; zhang and wang a, b) . under normal growth condition, grasp is o-glcnacylated and localizes in the medial-and trans-golgi for stacking. however, under glucose starvation, a pool of de-o-glcnacylated grasp translocates to the interface between autophagosomes and lysosomes to facilitate autophagosome-lysosome fusion. after a short-term energy deprivation, the golgi structure is only mildly affected, possibly due to sufficient grasp molecules remaining in the golgi to maintain its structure. among over a dozen golgi proteins tested, only grasp , but not grasp , gm , or golgin- , is o-glcnacylated under growth conditions and targets to autophagosomes upon energy deprivation, indicating that grasp serves as an energy sensor on the golgi to regulate both intracellular trafficking and autophagy. significantly, the same scenario may be seen in autophagy induced by amino acid starvation and inhibition of mtor (zhang et al. ) . in addition to golgi fragmentation, gcc overexpression also induces autophagy via reducing the activity of mtor. a considerable pool of mtor is localized and activated on the golgi, which is dependent on the ribbon structure for recruitment but independent of lysosomal mtor activation (gosavi et al. ) . these findings indicate the golgi ribbon as an important location for the functional regulation of mtor activity. additionally, autophagosomes may directly form on golgi membranes (guo et al. ) or obtain membranes from the golgi . the only recognized transmembrane atg protein, atg , localizes at the trans-golgi network and late endosomes and is essential for autophagosome formation (yamamoto et al. ) , although the detailed mechanism awaits further investigation (orsi et al. ) . recently, the endoplasmic reticulum-golgi intermediate compartment (ergic) is proposed to serve as a key membrane source for autophagosome formation . under normal condition, copii vesicles are generated from the er-exit sites (eres) for er-golgi membrane trafficking, while upon starvation, the copii assembly activator prolactin regulatory element-binding protein (preb)/sec relocates to the ergic and triggers ergic-copii vesicle formation as membrane templates for lc lipidation. golgi structure defects and dysfunctions have been observed in many diseases, including pathogen infection, neurodegenerative diseases, and cancer (aridor and hannan ) . generally, the mechanisms of golgi fragmentation include imbalanced membrane flux, altered microtubule dynamics, and posttranslational modifications or proteolytic cleavage of golgi structural proteins (wei and seemann ) . in many cases, the correlation between golgi defect and disease progression is unclear. a few interesting cases reported recently are discussed below. ad is an age-related neurodegenerative disease of the central nerve system characterized by progressive loss of cognition and memory. golgi fragmentation occurs in neurons of patients with ad since the earliest stages of disease development . some cases of early-onset ad are related to mutations in the amyloid precursor protein (app) or presenilin and (psn and ). the amyloid-beta (aβ) peptide is a proteolytic product of app, which is considered to be the major inducer of ad (selkoe and hardy ) . aβ accumulation is likely the direct cause of golgi fragmentation, as aβ-treatment causes reversible golgi fragmentation in cultured neurons (joshi et al. ) . present research supports that activation of cyclin-dependent kinase (cdk ) by aβ accumulation via the [ca + ]-calpain-p pathway may be the major trigger of golgi fragmentation in ad joshi et al. ; joshi and wang ; evin ; ayala and colanzi ) . cdk may function in two ways. first, cdk phosphorylates gm at s and inhibits its interaction with the golgi tethering protein p (sun et al. ) . second, cdk phosphorylates grasp at t /t , which inhibits grasp function in golgi stack formation and ribbon linking (joshi et al. ) . furthermore, inhibiting cdk or expressing non-phosphorylatable grasp mutants both rescued the golgi structure and reduced aβ secretion by elevating the α-cleavage of app (joshi and wang ; joshi et al. ) , indicating that grasp phosphorylation may be the main reason for ad-induced golgi fragmentation. these studies not only provide molecular mechanisms for golgi fragmentation but also suggest golgi as a potential drug target for ad treatment (joshi et al. ; joshi and wang ; ayala and colanzi ) . als is a fatal neurodegenerative disorder specifically targeted to motor neurons. fragmented golgi has been observed in numerous models of superoxide dismutase (sod ), tdp- , fus, and optineurin-associated als (fujita et al. ; soo et al. ; van dis et al. ; wallis et al. ) . sod inhibits er-to-golgi transport and causes golgi fragmentation (atkin et al. ) by reducing the β-cop protein level, accumulating the er-golgi v-snares gs and gs , and destabilizing microtubules by the upregulation of stathmins and (bellouze et al. ; atkin et al. ) . fus and tdp- impair the incorporation of secretory cargo into copii vesicles (soo et al. ) . furthermore, expression of als-related optineurin mutants impairs myosin vi-mediated protein trafficking from golgi to plasma membrane, which also induces golgi fragmentation . conclusively, impairment of distinct protein trafficking pathways by different als-linked proteins are specific triggers for golgi fragmentation in als. pd is pathologically characterized by the loss of dopamine-containing neurons and by the formation of intracellular protein aggregates known as lewy bodies in which α-synuclein has been recognized as a major constituent (forno ; wakabayashi et al. ) . golgi fragmentation can be detected in early-stage pd brains (fujita et al. ) and is strongly correlated to the presence of prefibrillar α-synuclein (gosavi et al. ) . since then, emerging studies have provided important insights into the mechanisms of how α-synuclein causes pathological golgi fragmentation and neuronal degeneration. the primary effect of α-synuclein aggregation is the inhibition of er-to-golgi transport (lashuel and hirling ) , which can be rescued by the overexpression of rab and rab and depletion of rab and syntaxin (rendon et al. ; coune et al. ) . in a most recent report, mutations in the leucine-rich repeat kinase (lrrk ), a major genetic cause of autosomaldominantly inherited pd, markedly enhance rab l phosphorylation on s , resulting in tgn fragmentation (fujimoto et al. ). golgi disorganization may be related to cancer progression and metastasis in the following aspects: aberrant glycosylation, abnormal expression of ras gtpase, dysregulation of kinases, and hyperactivation of myosin motor proteins (petrosyan ) . perturbation of the golgi morphology in cancers results in an increase of sialylation which is associated with a metastatic cell phenotype (schultz et al. ). overexpression of sialylated antigens is significantly correlated with tumor progression and therapy resistance due to an anti-apoptotic effect (lee et al. ; park et al. ; petrosyan et al. ) . over-activation of rabs, which coordinate with golgins in protein transportation and golgi structure maintenance, has been reported in different types of cancers (goldenring ) . furthermore, golgi disorganizationrelated kinases, including src, erk , and p -activated protein kinase (pak ), are found elevated in tumor cells (chia et al. ; ching et al. ; weller et al. ). the ubiquitin ligase hace , which regulates p -mediated golgi membrane fusion as discussed above, is reported as a tumor suppressor downregulated in multiple tumors including wilms' tumor (anglesio et al. ) , resulting in golgi fragmentation cui and wang ) . additionally, hyperactivation of golgi-associated myosins, including myo a that directly binds to golph to promote golgi dispersal (dippold et al. ; allan et al. ) , is detected in many aggressive cancers. golgi fragmentation is one of the essential and earliest events in apoptosis, where several golgins and grasp are cleaved by activated caspases, as described in previous sections. several membrane structures including the golgi are used by viruses as viral factories to replicate, concentrate, and assemble the viral genome and proteins into viral particles (miller and krijnse-locker ; netherton et al. ; salonen et al. ) . as a highly dynamic organelle, golgi serves as a membrane scaffold for multiple viruses, including infectious hepatitis c virus, enteroviruses, poliovirus, foot-and-mouth-disease virus, dengue virus, coronavirus, kunjin virus, tick-borne encephalitis virus, rubella virus, and bunyamwera virus (miller and krijnse-locker ; harak and lohmann ; risco et al. ; salanueva et al. ; delgui et al. ; westerbeck and machamer ) , and is frequently fragmented after infection (campadelli et al. ; salanueva et al. ; yadav et al. ; avitabile et al. ; lavi et al. ; hansen et al. ; rebmann et al. ) . viruses use golgi membranes directly and/or hijack master controllers of golgi biogenesis and trafficking to generate vesicles that are used as the site of viral rna replication (quiner and jackson ; hansen et al. ; short et al. ) , wrapping (sivan et al. ; alzhanova and hruby ; alzhanova and hruby ; nanbo et al. ; lundu et al. ; procter et al. ) , intracellular transduction (nonnenmacher et al. ) , and secretion ). viral infection triggers golgi fragmentation via diverse mechanisms, ranging from phosphorylating key golgi structural proteins such as grasp (rebmann et al. ) , activating the src kinase to phosphorylate the dynamin gtpase (martin et al. ) , targeting the immunity-related gtpase m (irgm) to the golgi to induce gbf phosphorylation (hansen et al. ) , modulating vesicular trafficking (yadav et al. ; johns et al. ) , to impeding the major histocompatibility complex (mhc) class i trafficking, antigen presentation, and/or cytokine secretion rohde et al. ). the golgi is the central hub in the secretory pathway, where proteins and lipids are processed, sorted, and dispatched to distinct destinations. as a well-organized polarized membrane structure, golgi function is tightly related to its structural integrity. thus, the first key question in golgi biology concerns how the stacked golgi structure forms. during the past decades, proteins with a variety of functions have been identified in the maintenance of golgi structure and regulation of golgi function, including but not limited to grasps, golgins, kinases, phosphatases, ubiquitin e ligases, and deubiquitinases, as summarized above. more detailed investigations need to be done to investigate how golgi structural proteins and their interacting molecules cooperate together to form the stacked golgi structure. golgi structural and functional defects have been increasingly reported in stress and disease conditions. in addition to its central role in protein sorting and trafficking, the golgi has been more recently recognized as a hub of signaling pathways, which facilitates golgi reaction upon stresses and diseases. thus, the second key question in golgi biology concerns how the golgi structure becomes defective in stress and disease conditions. in this regard, much effort and some progress have been made, such as golgi fragmentation in ad by cdk -mediated grasp phosphorylation as discussed above. however, many questions remain. for example, is there an unfolded protein response (upr)-like mechanism at the golgi to cope with different stresses? is there a common stress sensor on the golgi? how do the signaling pathways on the golgi sense and transduce stress signals? apparently, a more systematic analysis of golgi response to different stressors is necessary. gene expression profile and posttranslational modification analysis of golgi structural proteins, golgi enzymes, and signaling molecules will fast forward the field and shed light on new directions. considering lipid organization and modification are important for golgi function and certain lipids can work as signaling molecules, more attention may be put on lipids at the golgi, in addition to proteins. the third key question in golgi biology concerns how golgi structure alteration affects its function in trafficking, glycosylation, and sorting. the consequence of golgi fragmentation in different diseases is likely different, but it has been reported that golgi cisternal unstacking by depleting grasp proteins enhances protein trafficking. this, however, impairs accurate glycosylation and causes missorting of lysosomal enzymes to the extracellular space (xiang et al. ; zhang and wang ; bekier et al. ; wang et al. ) . consistently, golgi fragmentation in ad enhances app trafficking and aβ production, while rescue of the golgi causes app accumulation in the golgi and reduces aβ secretion (joshi et al. ) . most recently, we have obtained evidence that golgi structure disassembly by grasp depletion reduces cell attachment and migration while accelerating cell growth and cell cycle progression (ahat et al. ). it will be interesting to investigate how golgi fragmentation enhances cancer cell proliferation and metastasis in the future. future efforts may also aim at developing small chemicals or molecular tools to rescue the golgi structure in diseases, which may delay the disease development. grasp depletion-mediated golgi destruction decreases cell adhesion and migration via the reduction of α β integrin small gtpase rab b and its specific binding protein golgiassociated rab b interactor-like (gari-l ) regulate golgi morphology a novel missense mutation in scyl bp produces geroderma osteodysplastica phenotype indistinguishable from that caused by nullimorphic mutations rab recruitment of p into a cis-snare complex: programming budding copii vesicles for fusion motoring around the golgi the p -interactive proteins gm and giantin participate in endoplasmic reticulum-golgi traffic a trans-golgi network resident protein, golgin- , accumulates in viral factories and incorporates into virions during poxvirus infection a host cell membrane protein, golgin- , is essential for poxvirus morphogenesis the golgin protein coy functions in intra-golgi retrograde transport and interacts with the cog complex and golgi snares differential expression of a novel ankyrin containing e ubiquitin-protein ligase, hace , in sporadic wilms' tumor versus normal kidney traffic jam: a compendium of human diseases that affect intracellular transport processes death by committee: organellar trafficking and communication in apoptosis mutant sod inhibits er-golgi transport in amyotrophic lateral sclerosis redistribution of microtubules and golgi apparatus in herpes simplex virus-infected cells and their role in viral exocytosis alterations of golgi organization in alzheimer's disease: a cause or a consequence? golgi integral membrane protein manipulates cellular proliferation, apoptosis, and cell cycle in human head and neck cancer purification and functional interactions of grasp with rab grasp , a protein involved in the stacking of golgi cisternae mapping the interaction between grasp and gm , components of a protein complex involved in the stacking of golgi cisternae golgi matrix proteins interact with p cargo receptors and aid their efficient retention in the golgi apparatus aurora-a recruitment and centrosomal maturation are regulated by a golgi-activated pool of src during g knockout of the golgi stacking proteins grasp and grasp impairs golgi structure and function stathmin / -triggered microtubule loss mediates golgi fragmentation in mutant sod motor neurons the golgi matrix protein giantin is required for normal cilia function in zebrafish role of phosphatidylinositol -phosphate (pi p) and its binding protein golph in hepatitis c virus secretion selective export of human gpi-anchored proteins from the endoplasmic reticulum membrane targeting of p phosphorylation mutants and their effects on golgi integrity and secretory traffic characterization of pskh , a novel human protein serine kinase with centrosomal, golgi, and nuclear localization mutants of the protein serine kinase pskh disassemble the golgi apparatus involvement of golgin- in cell surface transport of renal romk channel: co-expression of golgin- increases romk currents rab and arl gtpase family members cooperate in the localization of the golgin gcc the golph pathway regulates golgi shape and function and is activated by dna damage fragmentation and dispersal of golgi proteins and redistribution of glycoproteins and glycolipids processed through the golgi apparatus after infection with herpes simplex virus the aaa-atpase cdc /p regulates spindle disassembly at the end of mitosis making the auroras glow: regulation of aurora a and b kinase function by interacting proteins aurrand-lions m ( ) genetic, structural, and chemical insights into the dual function of grasp in germ cell golgi remodeling and jam-c polarized localization during spermatogenesis jnk controls fragmentation of the golgi complex and the g /m transition through phosphorylation of grasp thyroid hormone, t -dependent phosphorylation and translocation of trip from the golgi complex to the nucleus caspase cleavage of the golgi stacking factor grasp is required for fas/cd -mediated apoptosis erk is a negative regulator of o-galnac glycosylation and cell migration p -activated protein kinase is overexpressed in hepatocellular carcinoma and enhances cancer metastasis involving c-jun nh -terminal kinase activation and paxillin phosphorylation a caspase cleavage fragment of p induces fragmentation of the golgi apparatus and apoptosis zfpl , a novel ring finger protein required for cis-golgi integrity and efficient er-to-golgi transport adhesion of golgi cisternae by proteinaceous interactions: intercisternal bridges as putative adhesive structures the golgi mitotic checkpoint is controlled by bars-dependent fission of the golgi ribbon into separate stacks in g rab a over-expression prevents golgi apparatus fragmentation and partially corrects motor deficits in an alpha-synuclein based rat model of parkinson's disease identification and characterization of two novel (neuro)endocrine long coiled-coil proteins hace (hect domain and ankyrin repeat containing e ubiquitin protein ligase ) grasp and grasp sequentially promote the transport of c-terminal valine-bearing cargos to and through the golgi complex dvc (c orf ) recruits the p protein segregase to sites of dna damage rab and rab have key roles in er-golgi trafficking the endosomal pathway and the golgi complex are involved in the infectious bursal disease virus life cycle pentameric assembly of potassium channel tetramerization domain-containing protein new insights into membrane trafficking and protein sorting the trans-golgi network golgin, gcc , is required for endosome-to-golgi transport and maintenance of golgi structure the coiled-coil membrane protein golgin- is a novel rab effector required for golgi ribbon formation coordination of golgin tethering and snare assembly: gm binds syntaxin in a p -regulated manner golph bridges phosphatidylinositol- -phosphate and actomyosin to stretch and shape the golgi to promote budding phosphorylation of the vesicletethering protein p by a casein kinase ii-like enzyme is required for golgi reassembly from isolated mitotic fragments a general amphipathic alpha-helical motif for sensing membrane curvature asymmetric tethering of flat and curved lipid membranes by a golgin autophagy-based unconventional secretory pathway for extracellular delivery of il- beta the role of grasp in golgi fragmentation and entry of cells into mitosis asymmetric clasp-dependent nucleation of noncentrosomal microtubules at the trans-golgi network how accelerated golgi trafficking may drive alzheimer's disease (comment on human autoantibodies to a novel golgi protein golgin- : high similarity with golgin- /gm autoantigen dna damage triggers golgi dispersal via dna-pk and golph signalling to and from the secretory pathway grasp regulates golgi ribbon formation the golgin gmap /trip anchors ift to the golgi complex neuropathology of parkinson's disease inter-and intracisternal elements of the golgi apparatus. a system of membrane-to-membrane cross-links tmf is a golgin that binds rab and influences golgi morphology cdc p is required for the cell cycle commitment point at start via degradation of the g -cdk inhibitor far p parkinson's diseaseassociated mutant lrrk phosphorylates rab l and modifies trans-golgi morphology fragmentation of golgi apparatus of nigral neurons with alpha-synuclein-positive inclusions in patients with parkinson's disease anterior horn cells with abnormal tdp- immunoreactivities show fragmentation of the golgi apparatus in als a functional deficiency of tera/vcp/p contributes to impaired dna repair in multiple polyglutamine diseases a syntaxin -snare complex distinguishes two distinct transport routes from endosomes to the trans-golgi in human cells the er-golgi intermediate compartment is a key membrane source for the lc lipidation step of autophagosome biogenesis rescue of deltaf -cftr trafficking via a grasp-dependent unconventional secretion pathway the golgi as a potential membrane source for autophagy post-golgi sec proteins are required for autophagy in saccharomyces cerevisiae the atpase vcp/p functions as a disaggregase against toxic huntingtin-exon aggregates three basic residues of intracellular loop of the beta- adrenergic receptor are required for golgin- -dependent trafficking finding the golgi: golgin coiled-coil proteins show the way the gtpase arf p and the er to golgi cargo receptor erv p cooperate to recruit the golgin rud p to the cis-golgi a central role for vesicle trafficking in epithelial neoplasia: intracellular highways to carcinogenesis golgi fragmentation occurs in the cells with prefibrillar alpha-synuclein aggregates and precedes the formation of fibrillar inclusion the golgi ribbon in mammalian cells negatively regulates autophagy by modulating mtor activity small gtp-binding protein associated with golgi cisternae rab proteins as major determinants of the golgi complex structure the a protein from multiple picornaviruses utilizes the golgi adaptor protein acbd to recruit pi kiiibeta acbd interaction with tbc domain protein is differentially affected by enteroviral and kobuviral a protein binding a new inhibitor of apoptosis from vaccinia virus and eukaryotes ap is essential for generation of autophagosomes from the trans-golgi network hepatitis c virus triggers golgi fragmentation and autophagy through the immunity-related gtpase m a non-canonical role of the p complex in rig-i antiviral signaling ultrastructure of the replication sites of positive-strand rna viruses aaa atpase p /vcp is essential for trim -mediated virus neutralization multiple rab gtpase binding sites in gcc suggest a model for vesicle tethering at the trans-golgi myristoylation restricts orientation of the grasp domain on membranes and promotes membrane tethering gerodermia osteodysplastica is caused by mutations in scyl bp , a rab- interacting golgin distinct aaa-atpase p complexes function in discrete steps of nuclear assembly the nh -terminal domain of golgin- contains both golgi and nuclear targeting information golgi structure in stress sensing and apoptosis mitotic golgi partitioning is driven by the membrane-fissioning protein ctbp /bars mammalian golgi-associated bicaudal-d functions in the dynein-dynactin pathway by interacting with these complexes tethering function of the caspase cleavage fragment of golgi protein p promotes apoptosis via a p -dependent pathway isolation and characterization of ara as the first androgen receptor n-terminal-associated coactivator in human prostate cells scyl binding protein promotes the ubiquitin-dependent degradation of pirh and has tumorsuppressive function in the development of hepatocellular carcinoma structural basis for the interaction between the golgi reassembly-stacking protein grasp and the golgi matrix protein gm golgi structure formation, function, and post-translational modifications in mammalian cells monoubiquitination of syntaxin regulates golgi membrane dynamics during the cell cycle biogenesis of neurosecretory vesicles identification and characterization of a novel golgi protein, golgin- the role of grasps in morphological alterations of golgi apparatus: mechanisms and effects golgin- -associated golgi fragmentation triggers tau hyperphosphorylation by activation of cyclin-dependent kinase- and extracellular signalregulated kinase fip /rcp binding to golgin- regulates retrograde transport from recycling endosomes to the trans-golgi network rab dependent post-golgi trafficking of hsv envelope proteins to sites of virus envelopment golgi defects enhance app amyloidogenic processing in alzheimer's disease abeta-induced golgi fragmentation in alzheimer's disease enhances abeta production caspase-mediated cleavage of the stacking protein grasp is required for golgi fragmentation during apoptosis rescuing defective vesicular trafficking protects against alphasynuclein toxicity in cellular and animal models of parkinson's disease syncytia formation induced by coronavirus infection is associated with fragmentation and rearrangement of the golgi apparatus potential role for protein kinases in regulation of bidirectional endoplasmic reticulum-to-golgi transport revealed by protein kinase inhibitor h protein sialylation by sialyltransferase involves radiation resistance golph mediated golgi stress response in modulating n a cell death upon oxygen-glucose deprivation and reoxygenation injury golgi phosphoprotein inhibits the apoptosis of human glioma cells in part by downregulating n-myc downstream regulated gene dja maintains golgi integrity via interaction with grasp the golgin gcc is required for efficient retrograde transport of cargo from the early endosomes to the trans-golgi network peripheral golgi protein grasp is a target of mitotic polo-like kinase (plk) and cdc arl a acts with gcc to modulate golgi complex organization lrrk regulates the dynamic profile of dendritic golgi outposts through the golgin lava lamp binding relationships of membrane tethering components. the giantin n terminus and the gm n terminus compete for binding to the p c terminus how rab proteins determine golgi structure rab is a novel regulator of golgi apparatus organization that is needed for er-to-golgi trafficking and cell growth rab a mediates proinsulin to insulin conversion in beta-cells by maintaining golgi stability through interactions with golgin- loss of the golgin gm causes golgi disruption, purkinje neuron loss, and ataxia in mice e-cadherin transport from the trans-golgi network in tubulovesicular carriers is selectively regulated by golgin- cdc kinase directly phosphorylates the cis-golgi matrix protein gm and is required for golgi fragmentation in mitosis caspase-mediated cleavage of syntaxin and giantin accompanies inhibition of secretory traffic during apoptosis interaction of arl -gtp with grip domains recruits autoantigens golgin- and golgin- /p onto the golgi autoantigen golgin- , an effector of arl gtpase, participates in traffic from the endosome to the trans-golgi network paclitaxel induces apoptosis through activation of nuclear protein kinase c-delta and subsequent activation of golgi associated cdk in human hormone refractory prostate cancer grip domain-mediated targeting of two new coiled-coil proteins, gcc and gcc , to subcompartments of the trans-golgi network the trans-golgi network grip-domain proteins form alpha-helical homodimers targeting of severe fever with thrombocytopenia syndrome virus structural proteins to the ergic (endoplasmic reticulum golgi intermediate compartment) and golgi complex p a, a type i transmembrane protein, controls arf -dependent resensitization of protease-activated receptor- by influence on receptor trafficking caspase-resistant golgin- disrupts apoptosis induced by secretory pathway stress and ligation of death receptors golgi disassembly in apoptosis: cause or effect? the golgi complex in stress and death a filter at the entrance of the golgi that selects vesicles according to size and bulk lipid composition intersectin- interacts with the golgin, gcc , to couple the actin network and golgi architecture golgin tethers define subpopulations of copi vesicles caspase- is localized at the golgi complex and cleaves golgin- during apoptosis herpes simplex virus type neuronal infection perturbs golgi apparatus integrity through activation of src tyrosine kinase and dyn- gtpase gtp-bound forms of rab induce the redistribution of golgi proteins into the endoplasmic reticulum dominant spinal muscular atrophy is caused by mutations in bicd , an important golgin protein genomewide screen identifies a novel p /cdc- -dependent pathway regulating er-stress-induced gene transcription bicaudal-d regulates copiindependent golgi-er transport by recruiting the dynein-dynactin motor complex the golgi ribbon and the function of the golgins signaling at the golgi the ubiquitin-selective segregase vcp/p orchestrates the response to dna double-strand breaks direct binding of ubiquitin conjugates by the mammalian p adaptor complexes, p and ufd -npl modification of intracellular membrane structures for virus replication molecular characterization of gcp , a -kda protein associated with the cytoplasmic face of the golgi membrane an essential cytoplasmic domain for the golgi localization of coiled-coil proteins with a cooh-terminal membrane anchor inhibition of the secretory pathway by foot-and-mouth disease virus bc protein is reproduced by coexpression of b with c, and the site of inhibition is determined by the subcellular location of c an intercisternal structure in the golgi apparatus a putative nuclear receptor coactivator (tmf/ara ) associates with hbrm/hsnf alpha and brg- /hsnf beta and localizes in the golgi apparatus rab interaction with a gm effector complex regulates copii vesicle cis-golgi tethering nuclear import is required for the pro-apoptotic function of the golgi protein p fragmentation of the golgi apparatus: an early apoptotic event independent of the cytoskeleton the golgin coiled-coil proteins of the golgi apparatus characterization of a cis-golgi matrix protein, gm the vesicle docking protein p binds gm , a cis-golgi matrix protein, in a mitotically regulated manner epstein-barr virus acquires its final envelope on intracellular compartments with golgi markers loss of soluble n-ethylmaleimide-sensitive factor attachment protein alpha (alphasnap) induces epithelial cell apoptosis via down-regulation of bcl- expression and disruption of the golgi a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication mutations in bicd , which encodes a golgin and important motor adaptor, cause congenital autosomal-dominant spinal muscular atrophy golph l antagonizes golph to determine golgi morphology caspases: killer proteases aurora a kinase (aurka) in normal and pathological cell division homologs from caenorhabditis elegans, cdc- . and cdc- . , suppress the aggregate formation of huntingtin exon containing expanded polyq repeat centrosome-targeting region of cg-nap causes centrosome amplification by recruiting cyclin e-cdk complex syntaxin -dependent retrograde transport to the trans-golgi network is required for adeno-associated virus transduction fragmentation of golgi complex and golgi autoantigens during apoptosis and necrosis identification and characterization of gcp , a novel acylated golgi protein that interacts with gcp dynamic and transient interactions of atg with autophagosomes, but not membrane integration, are required for autophagy stacks off tracks: a role for the golgin atcasp in plant endoplasmic reticulum-golgi apparatus tethering the arf-like gtpases arl p and arl p act in a pathway that interacts with vesicle-tethering factors at the golgi apparatus vcp/p cooperates with yod , ubxd and plaa to drive clearance of ruptured lysosomes by autophagy the golgin lava lamp mediates dynein-based golgi movements during drosophila cellularization cdc /p segregase is modulated by cyclindependent kinase to determine cyclin fate during g progression sialylation of epidermal growth factor receptor regulates receptor activity and chemosensitivity to gefitinib in colon cancer cells dna damage modulates nucleolar interaction of the werner protein with the aaa atpase p /vcp common variants in or near znrf , colec , scyl bp and api are associated with diabetic retinopathy in chinese patients with type diabetes tmf/ara is a bc-box-containing protein that mediates the degradation of stat onco-golgi: is fragmentation a gate to cancer progression? restoration of compact golgi morphology in advanced prostate cancer enhances susceptibility to galectin- -induced apoptosis by modifying mucin o-glycan synthesis sec a is critical for both conventional and unconventional secretion of cftr ysk is activated by the golgi matrix protein gm and plays a role in cell migration through its substrate - - {zeta} plk docking to grasp phosphorylated by cdk suggests a mechanism for golgi checkpoint signalling the hcmv assembly compartment is a dynamic golgi-derived mtoc that controls nuclear rotation and virus spread gm and grasp -dependent lateral cisternal fusion allows uniform golgi-enzyme distribution fragmentation of the golgi apparatus provides replication membranes for human rhinovirus a golgi ribbon unlinking: an organelle-based g /m checkpoint grasp: a multitasking tether an nsf-like atpase, p , and nsf mediate cisternal regrowth from mitotic golgi fragments cdc /p promotes reformation of the nucleus by extracting the kinase aurora b from chromatin golgins and grasps: holding the golgi together a novel -kda golgin protein induced by brain ischemia and phosphorylated by akt protects against apoptosis env p associates with the golgin protein imh at the trans-golgi network in candida albicans phosphorylation of golgi peripheral membrane protein grasp is an integral step in the formation of the human cytomegalovirus cytoplasmic assembly compartment a functional role for the gcc golgin in mannose -phosphate receptor recycling rab and rab regulate chlamydia trachomatis development and golgin- -dependent golgi fragmentation golgi fragmentation is rab and snare dependent in cellular models of parkinson's disease gmap- recruits gammatubulin complexes to cis-golgi membranes and is required for golgi ribbon formation structural maturation of rubella virus in the golgi complex toxicity and aggregation of the polyglutamine disease protein, ataxin- is regulated by its binding to vcp/p in drosophila melanogaster microtubule nucleation at the cis-side of the golgi apparatus requires akap and gm orf virus interferes with mhc class i surface expression by targeting vesicular transport and golgi giantin interacts with both the small gtpase rab and rab polymorphism and structural maturation of bunyamwera virus in golgi and post-golgi compartments viral rna replication in association with cellular membranes human and viral golgi anti-apoptotic proteins (gaaps) oligomerize via different mechanisms and monomeric gaap inhibits apoptosis and modulates calcium acbd -mediated recruitment of pi kb to picornavirus rna replication sites golgin- is a rab binding partner involved in golgi structure identification of a redox-sensitive cysteine in gcp that regulates its interaction with golgin- gcp preferentially interacts with a caspase-generated golgin- fragment regulation of the metastatic cell phenotype by sialylated glycans golph modulates mtor signalling and rapamycin sensitivity in cancer the role of the tethering proteins p and gm in transport through the golgi apparatus in vivo cohen syndromeassociated protein, coh , is a novel, giant golgi matrix protein required for golgi integrity the amyloid hypothesis of alzheimer's disease at years mitotic inhibition of grasp organelle tethering involves pololike kinase (plk ) phosphorylation proximate to an internal pdz ligand tbc d is a bridging factor for endosomal vesicle capture by golgins at the trans-golgi author correction: tbc d is a bridging factor for endosomal vesicle capture by golgins at the trans-golgi vamp is required to maintain the ribbon structure of the golgi apparatus a grasp -rab effector complex linking golgi structure to membrane traffic providence virus (family: carmotetraviridae) replicates vrna in association with the golgi apparatus and secretory vesicles a role for the vesicle tethering protein, p , in the post-mitotic stacking of reassembling golgi cisternae in a cell-free system golgi architecture and inheritance grasp , a second mammalian grasp protein involved in the stacking of golgi cisternae in a cell-free system sequential tethering of golgins and catalysis of snarepin assembly by the vesicle-tethering protein p centrosomal anchoring of the protein kinase ck delta mediated by attachment to the large, coiled-coil scaffolding protein cg-nap/akap golgi coiled-coil proteins contain multiple binding sites for rab family g proteins retrograde transport from early endosomes to the trans-golgi network enables membrane wrapping and egress of vaccinia virus virions isolation of a matrix that binds medial golgi enzymes identification and characterization of a novel golgi protein, gcp , that interacts with the integral membrane protein giantin interaction of golgin- with the cog complex mediates the intra-golgi retrograde transport trans-golgi protein p /golgin- is involved in phagophore formation characterization of the aggregation-prevention activity of p / valosin-containing protein a role for giantin in docking copi vesicles to golgi membranes rab -dependent er-golgi transport dysfunction is a common pathogenic mechanism in sod , tdp- and fus-associated als vesicle-associated membrane protein is implicated in trans-golgi network vesicle trafficking novel genetic tools reveal cdk 's major role in golgi fragmentation in alzheimer's disease golgi-snare gs potentiates cisplatin-induced apoptosis by forming gs -mdm -p complexes and by preventing the ubiquitination and degradation of p mitotic golgi vesiculation involves mechanisms independent of ser phosphorylation of gm golgi fragmentation in amyotrophic lateral sclerosis, an overview of possible triggers and consequences the golgi-associated protein grasp regulates spindle dynamics and is essential for cell division characterization of a novel giant scaffolding protein, cg-nap, that anchors multiple signaling enzymes to centrosome and the golgi apparatus centrosomal proteins cg-nap and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex cell cycle regulation of golgi membrane dynamics molecular mechanism of mitotic golgi disassembly and reassembly revealed by a defined reconstitution assay reconstitution of the cell cycle-regulated golgi disassembly and reassembly in a cell-free system the role of grasp in golgi cisternal stacking and cell cycle progression the ubiquitin ligase hace regulates golgi membrane dynamics during the cell cycle sequential phosphorylation of grasp during mitotic golgi disassembly mena-grasp interaction couples actin polymerization to golgi ribbon linking cyclin-dependent kinase -mediated bcl-xl/bcl- phosphorylation acts as a functional link coupling mitotic arrest and apoptosis role of microtubules in the organization of the golgi complex identification and characterization of rat -kda golgi-associated protein recognized by autoantibodies from a patient with rheumatoid arthritis allosteric regulation of grasp protein-dependent golgi membrane tethering by mitotic phosphorylation structural and functional analysis of a novel coiled-coil protein involved in ypt gtpase-regulated protein transport in yeast the localization and phosphorylation of p are important for golgi disassembly-assembly during the cell cycle is a p adaptor required for golgi and er biogenesis in interphase and at the end of mitosis vcp/p extracts sterically trapped ku / rings from dna in double-strand break repair golgi fragmentation precedes neuromuscular denervation and is associated with endosome abnormalities in sod -als mouse motor neurons adp-ribosylation factors (arfs) and arf-like (arl ) have both specific and shared effectors: characterizing arl -binding proteins direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping the multiple facets of the golgi reassembly stacking proteins accumulation of alpha-synuclein/nacp is a cytopathological feature common to lewy body disease and multiple system atrophy golgi fragmentation during fas-mediated apoptosis is associated with the rapid loss of gm sod mutations causing familial amyotrophic lateral sclerosis induce toxicity in astrocytes: evidence for bystander effects in a continuum of astrogliosis the golgi apparatus. state of the art years after camillo golgi's discovery a direct role for grasp as a mitotically regulated golgi stacking factor vcip acts as a deubiquitinating enzyme during p -p -mediated reassembly of mitotic golgi fragments mapping the functional domains of the golgi stacking factor grasp golgi cisternal unstacking stimulates copi vesicle budding and protein transport interactions: a dynamic cycle of p binding monomeric snare motifs and releasing assembled bundles a role for the rab b bicaudal-d interaction in retrograde transport in neuronal cells intracellular membrane morphology unraveling the golgi ribbon golgi ribbon disassembly during mitosis, differentiation and disease progression the golgi matrix protein gm : a specific interacting partner of the small gtpase rab b src kinase regulates the integrity and function of the golgi apparatus via activation of dynamin a coronavirus e protein is present in two distinct pools with different effects on assembly and the secretory pathway golgin- is required for the golgi membrane sorting of the insulin-responsive glucose transporter glut in adipocytes the golgin family of coiled-coil tethering proteins carminomycin i is an apoptosis inducer that targets the golgi complex in clear cell renal carcinoma cells structural basis for recruitment of grip domain golgin- by small gtpase arl grasp and grasp play complementary and essential roles in golgi cisternal stacking new components of the golgi matrix regulation of protein glycosylation and sorting by the golgi matrix proteins grasp / golgi positioning influenza infection modulates vesicular trafficking and induces golgi complex disruption endoplasmic reticulum (er) chaperone regulation and survival of cells compensating for deficiency in the er stress response kinase, perk atg vesicles are an important membrane source during early steps of autophagosome formation functional involvement of tmf/ara in rab -dependent retrograde membrane traffic aggregation of a hepatitis c virus replicase module induced by ablation of p /vcp peroxisome proliferator-activated receptor delta regulation of mir- a in ischemia-induced cerebral vascular endothelial injury convergence of cell cycle regulation and growth factor signals on grasp acbd functions as a scaffold to organize the golgi stacking proteins and a rab b-gap overexpression of golph promotes proliferation and tumorigenicity in breast cancer via suppression of the foxo transcription factor cell cycle regulation of vcip deubiquitinase activity and function in p /p -mediated golgi reassembly grasps in golgi structure and function glycosylation quality control by the golgi structure the golgi stacking protein gorasp /grasp serves as an energy sensor to promote autophagosome maturation under glucose starvation grasp facilitates autophagosome maturation under glucose deprivation phosphorylation regulates vcip function in golgi membrane fusion during the cell cycle scy -like -binding protein (scyl bp ) suppressed sciatic nerve regeneration by enhancing the rhoa pathway golgi protein facilitates the interaction of hepatitis c virus ns a with apolipoprotein e to promote viral particle secretion grasp senses glucose deprivation through o-glcnacylation to promote autophagosome-lysosome fusion structural basis for the interaction between golgi reassembly-stacking protein grasp and golgin the mammalian golgi regulates numb signaling in asymmetric cell division by releasing acbd during mitosis regulates golgi apparatus structure through methylation of the golgin gm golgi as an mtoc: making microtubules for its own good acknowledgment we thank members of the wang lab for stimulating discussions. this work was supported by the national institutes of health (grants gm and gm ), mcubed, and the fast forward protein folding disease initiative of the university of michigan to y. wang, and a university of michigan rackham predoctoral fellowship to e. ahat. key: cord- -z bdiuvx authors: ulasli, mustafa; verheije, monique h.; de haan, cornelis a. m.; reggiori, fulvio title: qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus date: - - journal: cell microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: z bdiuvx coronaviruses (cov) are enveloped positive‐strand rna viruses that induce different membrane rearrangements in infected cells in order to efficiently replicate and assemble. the origin, the protein composition and the function of these structures are not well established. to shed further light on these structures, we have performed a time‐course experiment in which the mouse hepatitis virus (mhv)‐induced membrane rearrangements were examined qualitatively and quantitatively by (immuno)‐electron microscopy. with our approach we were able to confirm the appearance of , previously reported, membranous structures during the course of a complete infection cycle. these structures include the well‐characterized double‐membrane vesicles (dmvs), convoluted membranes (cms) and virions but also the more enigmatic large virion‐containing vacuoles (lvcvs), tubular bodies (tbs) and cubic membrane structures (cmss). we have characterized the lvcvs, tbs and cmss, and found that the cov‐induced structures appear in a strict order. by combining these data with quantitative analyses on viral rna, protein synthesis and virion release, this study generates an integrated molecular and ultrastructural overview of cov infection. in particular, it provides insights in the role of each cov‐induced structure and reveals that lvcvs are ergic/golgi compartments that expand to accommodate an increasing production of viral particles. compartments that expand to accommodate an increasing production of viral particles. viruses require cellular membranes in one or more steps of their infection cycle for replication, assembly and/or release, and therefore they have developed sophisticated mechanisms to opportunistically rearrange host membranes for their needs. for example, a common feature among positive (+) strand rna viruses is the assembly of their replication-transcription complexes (rtcs) in association with cytoplasmic membranes (salonen et al., ; miller and krijnse-locker, ) . the potential benefit of anchoring the rtcs to lipid bilayers is still unclear, but it may facilitate and co-ordinate different steps of the viral life cycle, and/or delay induction of the host immune response (ahlquist, ; haller et al., ) . enveloped viruses are another example; they generate their new virions by budding through cellular membranes (garoff et al., ) . cov are enveloped (+) strand rna viruses (weiss and navas-martin, ; gorbalenya et al., ) . they are pathogens of veterinary importance, but the relevance of this family of viruses has increased considerably due to the recent emergence of new human viruses such as the cov nl and the severe acute respiratory syndrome-cov (sars-cov). these viruses cause severe respiratory tract diseases and patients often have evidence of other organ dysfunctions (godfraind and coutelier, ; peiris et al., ; saif, ) . after fusion of the viral and cellular membranes, cov disassemble thereby releasing their genomic rna, which allows the production of non-structural proteins (nsp's) (brian and baric, ; sawicki et al., ) . these nsp's collectively form the rtcs and induce the formation of cytoplasmic dmvs into which the rtcs are anchored (ziebuhr et al., ) . the mechanism underlying the biogenesis of cov-induced dmvs is completely unknown even if experimental evidences indicate an endoplasmic reticulum (er) origin (harcourt et al., ; kanjanahaluethai et al., ; oostra et al., ; knoops et al., ) . the cov genomes encode for a common set of four structural proteins: the envelope (e), the membrane (m), the nucleocapsid (n) and the spike (s) proteins . e, m and s proteins are integral membrane components and after insertion in the er limiting membrane, they are transported to the er-to-golgi intermediate compartment (ergic), where together with the n protein and the genomic rna, they assemble into virions by inducing the invagination and luminal pinching off of the limiting membrane of this organelle (tooze et al., ; ng et al., ; goldsmith et al., ; stertz et al., ) . the resulting luminal virions subsequently reach the extracellular environment following the conventional secretory pathway (tooze et al., ) . the first ultrastructural analyses of cov-infected cells by electron microscopy (em) were already preformed in the s (svoboda et al., ; david-ferreira and manaker, ; ruebner et al., ) . the presence of dmvs and virions was the obvious morphological changes described at that time. these initial observations have successively been corroborated by numerous studies. more recently, other membranous rearrangements have been described. an electron tomography analysis of sars-cov-infected cells has confirmed the presence of reticular inclusions in between dmvs (knoops et al., ) , which were already reported in one of the original works (david-ferreira and manaker, ) . the recent study has proposed that these reticular inclusions, re-named cms, are the precursors of dmvs (knoops et al., ) . in addition to cms, it has been shown that the cov triggers the formation of highly organized crystalloid conformations, tubular rearrangements and vacuoles enclosing viral particles that have been named cmss, tbs and lvcvs respectively (david-ferreira and manaker, ; ruebner et al., ; tooze et al., ; ng et al., ; goldsmith et al., ; almsherqi et al., ; knoops et al., ) . the function of cms, cmss, tbs and lvcvs in cov infection is largely unknown. a major difficulty in understanding the role of these different structures has been the absence of a quantitative and qualitative em analyses over time that could help ordering them during the infection cycle. we have now filled this gap by performing a time-course em and immunoelectron microscopy (iem) examination of mhv-infected cells. by combining these qualitative and quantitative data with the measurement of viral rna synthesis, viral protein production and progeny virus release, we have, for the first time, integrated ultrastructural analyses with molecular information. this approach has allowed us to establish that mhv induces the formation of six membranous rearrangements in the following order: dmvs, cms, virions, lvcvs, tbs and cmss. importantly, we were able to show that most membrane rearrangements (lvcvs, tbs, cmss and possibly cms) observed in addition to the key structures in the infection (dmvs and virions) actually appear to be the consequence of a massive synthesis of viral proteins. in particular, lvcvs are ergic/golgi compartments that expand to accommodate an increasing production of virions. all together, our study provides an overall comprehensive picture of the ultrastructural events taking place inside a cell in the course of a cov infection. to understand the relationship between the different membranous structures induced by mhv and their role during the infection, we infected the cells at high multiplicity of infection (moi) and we analysed, in a timecourse manner, their ultrastructure by em as well as various other infection parameters during a period of h as described under experimental procedures. in order to be able to correlate our em and iem analyses with the progression of a cov infection inside the host cells, we first measured important known parameters that reflect the cov life cycle: viral rna replication/transcription, viral protein synthesis and secretion of progeny virus. to study the rna replication/transcription rate during mhv infection, the amount of genomic rna (grna) and that of subgenomic rna encoding for the n protein (sgrna n) was determined at each time point by rt-pcr as described in experimental procedures. both grna and sgrna n were already detected at h post infection (p.i.; fig. a ). their amount gradually increased until the h time point, after which the levels remained constant, resulting from a decline in rna synthesis (sawicki et al., ) . at each time, the amount of sgrna was about -fold higher than that of grna. this analysis allowed us to ascertain that the observed changes in the levels of grna and sgrna synthesis are identical to those measured using similar or different assays in various cell lines infected with diverse cov (sethna and brian, ; versteeg et al., ; sawicki et al., ) . next, we analysed the synthesis of the structural proteins during the infection. to this end, we measured the production rate of the m protein at the different time points by short pulse radio-labelling experiments followed by sds-page analysis of the crude cell lysates. the synthesis of the m protein was already detectable at h p.i. and continued to increase until h p.i. with the main increase occurring between and h p.i. (fig. b) . essentially, identical results were obtained when we analysed the production of the structural proteins s and n in the same way (data not shown). this correlates perfectly with the synthesis kinetics of the sgrna (fig. a) and consequently indicates that these mrnas are immediately available for translation. after h of infection, we observed a drop in the production of the m protein (fig. b) . these results again correspond with those of others (rottier et al., ; hilton et al., ; tahara et al., ) . to monitor the assembly and release of mhv over time, the infectivity levels in the culture supernatants collected at the different p.i. times were determined. secretion of mhv virions was first detected at h p.i. and increased until h p.i. before slowing down (fig. c ). this observation matches with the analysis of the viral rna and structural protein production ( fig. a and b) because as expected, it shows that mhv assembly and release are processes that follow intracellular mhv replication. all together, these measurements demonstrate that mhv infection in hela-ceacam a cells progresses following the typical, established dynamics, thereby validating the use of this cell line. in addition, they also show that the h time window used in our time-course analysis comprises all the phases of a cov infection and therefore our examinations allow obtaining a complete overview of a cov life cycle. as a first ultrastructural analysis, we compared the morphology of cells at h p.i. with that of those at h p.i. by em in order to make a repertoire of all the membranous rearrangements that mhv induces. we identified six different structures. the most abundant of them were large vesicles with an average diameter of - nm, which are limited by a double-membrane and often were clustered together ( fig. a and b; and fig. s a, arrows) . these are the characteristic dmvs induced by cov (svoboda et al., ; david-ferreira and manaker, ; ruebner et al., ; pedersen et al., ; gosert et al., ; snijder et al., ; knoops et al., ) . surprisingly, most of the observed dmvs appeared to have an invagination, which, from time to time, was associated to what looked as a small vesicle with a diameter of - nm (fig. s b, arrow) . this structural peculiarity is cell type-specific because it was also observed in mhv-a -infected mouse embryonic fibroblasts (data not shown) but not in mouse lr cells (knoops et al., ) . in the centre of the dmv clusters, we frequently observed a small network of membranes with a diameter varying from to nm, which have recently been described in sars-cov-infected cells and called cms [ fig. a and b, arrowheads; (knoops et al., ) . the cms were often in close proximity of the er (< - nm distance), and sometimes appeared to be connected with this organelle (fig. s c , arrow). virions were the third structure that we identified. these dark circular structures with a diameter of - nm were found in the lumen of either a stack of adjacent cisternae, very likely the golgi, or what appeared to be secretory vesicles (fig. c ) as well as extracellularly ( fig. s d ) in complete agreement with their known assembly and secretion mechanisms. the virions were also observed inside large circular organelles with a diameter of approximately - nm (fig. d, arrow) . interestingly, we observed virion particles assembling by invagination at the limiting membrane of these compartments through a process identical to the one occurring at the ergic (fig. d, arrow) . because similar organelles have previously been described (ng et al., ; goldsmith et al., ; knoops et al., ) , we called them in the same way: the lvcvs. the fifth conformation that we detected was a condensed rearrangement of membranes with a diameter of approximately - nm but without an apparent ultrastructural organization, which seemed to be connected to the er (fig. e ). these structures have already been described a long time ago and called tbs ( david-ferreira and manaker, ) . the sixth classified structure was a rectangular, extended (up to nm in length) and highly organized membranous conformation always continuous to what appeared to be a swollen er cisterna (fig. f , arrow; the arrowhead indicates the swollen er). a morphologically similar subcellular arrangement has been observed in sars-cov infected cell sections and consequently, we also called these structures cms (almsherqi et al., ; almsherqi et al., ) . to see whether the changes observed at the ultrastructural level correlate with the other measured infection parameters ( fig. ) , we first morphologically determined the number of cell sections at each time point that demonstrate visible signs of infection. to this end, the number of cell sections demonstrating at least one of the six structures induced by the mhv was determined at each time point. at h p.i., % of the cell sections showed visible signs of infection and this percentage gradually increased during time until reaching % at h p.i. (fig. a) . importantly, the percentages of cell sections with visible signs of infection as determined with the em analysis were very similar to those as evaluated by if (data not shown) and correlated well with the rest of the measured parameters, demonstrating that this is a reliable alternative approach to follow the mhv infection. to understand the role of the six mhv-induced structures during an infection and to unravel their relationship, we the quantitatively analysed the em sections obtained at the different p.i. time points. two values were calculated: (i) the percentage of cell profiles containing a specific structure and (ii) the average number of a given structure per cell section. our analysis revealed that the dmvs are the first membrane rearrangement to be detected in the infected cells. dmvs were already observed at h p.i. in about % of the cell sections and the number of cell sections positive for these vesicles gradually increased over time (fig. b ). the average number of dmvs per cell section reached a maximum, e.g. dmvs/cell section, at h p.i. (fig. c ). interestingly, the localization and morphology of the dmvs changed during the infection. at early time points, from to h p.i., dmvs were small ( - nm diameters) with a regular circular shape and distributed throughout the cytoplasm. from h onwards, dmvs organized in clusters mostly found in the perinuclear region of the cell. the dmv invaginations became more pronounced at h p.i. after which the small vesicles located in their interior also became more prominent (fig. s b , arrow). two hours later, the shape of the dmvs started to change acquiring a less circular form and with protuberances emerging from their surface that entered invaginations of adjacent dmvs (fig. s e , arrows). the next structures to be detected during the mhv infection were the cms, which became apparent at h p.i. the cms were always found in close proximity to at least one dmv (< - nm distance). at the early infection time points, e.g. - h p.i., the cms had small sizes ( - nm diameters) and were present in only % of the cells (fig. d ). their number per cell and their size, however, increased during the progression of the infection reaching a plateau at h p.i. (fig. e ; cm/cell section, - nm in diameter). a dramatic change in the percentage of cms was observed at h p.i. when the number of cm positive cells had increased from % to %. overall, these data suggested that the cms are structures that are functionally connected with dmvs as suggested (knoops et al., ) . virions appeared at h p.i. and their intracellular number became constant ( virions/cell section) already at h p.i., probably upon reaching an equilibrium between synthesis and secretion ( fig. f and g). the virions were mostly observed in the golgi cisternae at h p.i. in agreement with the fact that mhv particles assemble at the ergic and are released into the extracellular space by passing through the secretory pathway (ng et al., ; stertz et al., ) . lvcvs became detectable at h p.i. (fig. h ) in coincidence with a more than twofold increase in the number of virions per cell profile (fig. i ). this observation suggested that the formation of lvcvs is probably induced by a higher production of virions in the cells. the tbs also became visible at h p.i. (fig. j ). the number of tb-positive cell sections increased during time as well as the size of these structures (fig. k) , with an average diameter of - nm at h p.i. to one of about nm at h p.i. initially, each infected cell profile contained only one tb but, after h p.i., we occasionally observed more than one tb per cell section. the cmss, in contrast, were even more rare (observed in only % of the cells) and only detectable after h p.i.; hence a significant statistical analysis could not be performed. we concluded that the tbs and the cmss are not required for the early steps of the mhv infection cycle but rather the result of an advanced infection. to further understand the role of the mhv-induced structures, we explored by iem the presence or absence of viral non-structural and structural proteins in the six identified structures. the antibodies used were recognizing either nsp /nsp , nsp , nsp , the n, the m or the e proteins. we immunolabelled cryo-sections obtained from cells fixed at and h p.i. to be able to detect compositional changes. however, the labelling profiles at these two infection time points were identical; the only major difference was the higher immunoreactivity of the h p.i. samples due to the higher amounts of viral proteins. as expected (gosert et al., ; snijder et al., ; stertz et al., ) , the nsp /nsp , nsp and nsp proteins were decorating the surface of the dmvs (fig. a-c) . importantly, we discovered that these nsp's were also present in the cms but not in the other mhvinduced structures (fig. a -c and fig. s a -h). the n protein was also distributed on both dmvs and cms (fig. d ) suggesting a possible direct relationship between these two structures. like the two other struc-tural proteins, i.e. m and e, the n protein was found in virions present in the golgi complex and lvcvs ( fig. e-h) . the m and e proteins were additionally observed in the limiting membrane of the golgi cisternae but not on other mhv induced membranous rearrangements (fig. s ) . interestingly, we discovered that the tbs contain the e protein but are negative for the other tested viral proteins (fig. i, fig. s a and c) . this result, plus the fact that the tbs appear at the late stage of the mhv infection ( fig. j and k) , indicates that they could be generated by self-assembly, possibly in the er, of high levels of e protein. this hypothesis is sustained by the observation that the individual expression of the e protein induces the formation of a complex of tubular and smooth membranes with morphology reminiscent to that of the tbs (raamsman et al., ) . none of the tested antibodies labelled the cmss. because these structures appear at the late stage of the infection (data not shown), we speculate that they are induced by aggregation of the s protein for which we do not have an antibody compatible with our iem procedure. to acquire information about the host organelle origins of the mhv-induced structures, cryosections were obtained from cells harvested at and h p.i., and labelled with antibodies recognizing the protein disulfide isomerize pdi (er), ergic (ergic), gm (cis-golgi), tgn- (trans-golgi network, tgn) and lamp (late endosomes and lysosomes). these antibodies localized to the expected compartments in non-infected cells (fig. s ) . none of the employed organelle protein markers labelled the dmvs or the cms (data not shown), even though the latter ones were often observed in proximity of the er (fig. s c) . as previously reported, forming and completed virions were observed in the ergic and golgi cisternae ( fig. a and data not shown respectively). importantly, ergic , gm and tgn were found on the lvcvs as well, albeit at very low levels ( fig. b-d) . notably, despite its close proximity and almost clear continuity with the er, tbs were not positive for pdi (fig. e ). in contrast, the cmss contained this er protein marker (fig. f ) in agreement with morphological connection with this organelle (fig. f ). the low labelling of lvcvs with antibodies against ergic , gm and tgn- indicated that these organelles are derived from the ergic and/or golgi complex. it has been shown that golgi cisternae can increase in size in order to accommodate large luminal cargo proteins such as collagen (bonfanti et al., ) . therefore, we hypothesized that lvcvs are golgi cisternae that have expanded to increase their capacity to contain a higher number of viral proteins and/or forming virions. to sustain this notion, we first examined if the golgi changes its organization and subcellular distribution during the course of an mhv infection. we took advantage of the hela-galnact -gfp stable cell line (storrie et al., ) , which expresses the fluorescent golgi protein marker galnactt -gfp, and inoculated these cells with mhv-srec before analysing them by if at , and h p.i. the presence of viral proteins and virions in the golgi was assessed using anti-m protein antibodies. in non-infected cells, the golgi appeared as a juxtanuclear concentration of ribbon-like structures as expected (fig. a , upper panel; storrie et al., ) . the mhv infection caused three major changes, which were already detectable at h p.i. but became more prominent at h p.i. (fig. a, middle and lower panels) . first, the golgi lost its compact organization; the organelle was scattered throughout the cytoplasm. second, the intensity of the galnact -gfp signal in the golgi decreased. third, galnact -gfp appeared to partially localize to the er. to unravel the effects of mhv infection on the golgi at the ultrastructural level, the same samples were also processed in parallel for iem and immunolabelled with anti-gfp antibodies. at h p.i the galnact -gfp was exclusively concentrated in the golgi complex, where it distributes into several cisternae (fig. b ). in contrast, at h p.i., the labelling was found onto two different types of structures. the first were fragmented golgi complexes (fig. c ). this morphological change during an mhv infection phenomenon has previously been reported (lavi et al., ) . the second types of labelled structures were lvcvs, in keeping with our notion that these compartments have an ergic/golgi origin (fig. d) . to substantiate that lvcvs derive from the golgi, we statistically evaluated whether there is a numeric relationship between these two compartments and whether the appearance of lvcvs correlates with a decrease of golgi complexes. to this end, we determined the number of lvcvs and golgi complexes per cell profile at the different p.i. time points by counting these two organelles in the em preparations of the mhv infection time-course experiment. as shown in fig. e , the lvcvs were first detected at h p.i. and their number subsequently increased concomitant with a reduction of golgi complexes. all together, our data strongly suggest that lvcvs are ergic/golgi cisternae that expand as a consequence of a large local production of virions. -caplen et al., ; david-ferreira and manaker, ; ruebner et al., ; salanueva et al., ; escorcia et al., ; gosert et al., ; snijder et al., ; stertz et al., ; knoops et al., ; banacha et al. ), cms, lvcvs and cmss have exclusively been described in sars-cov-infected cells (goldsmith et al., ; almsherqi et al., ; knoops et al., ) and (table ). this apparent discrepancy is probably due to various reasons, including the rarity of some of these structures, the examination of a single infection time point and the use of em procedures lacking high resolution. in our study, we have detected all these types of membrane rearrangements during the course of an mhv infection because we performed a time-course ultrastructural analysis using state-of-the-art em and iem procedures. combining our data with the previous ones, we can assert that sars-cov is inducing the same type of structures and therefore we hypothesize that these membrane rearrangements are also formed during infections with other cov. as a result, the data presented in this study generate a model that could be applicable to all cov, even if some minor differences could exist. a major difficulty in understanding and studying the role of dmvs, cms, virions, lvcvs, tbs and cmss in the cov life cycle has been the absence of information concerning their appearance and fate in the course of an infection. another obstacle has also been the very limited characterization of lvcvs, tbs and cmss but also in part of cms. our time-course qualitative and quantitative examination of the mhv infection fills these two gaps (table ) , and thus generates a more comprehensive chronological picture of the ultrastructural transformations occurring in the host cells that are induced by this virus. mhv replication (fig. a) double-membrane vesicles were the first structure (knoops et al., ) that we observed after inoculating the cells with mhv ( h p.i.; fig. b and c). these vesicles are known to play a crucial role in viral rna synthesis (gosert et al., ; prentice et al., ; ahlquist, ; haller et al., ; sawicki et al., ) . in agreement with this notion but also with previous studies (gosert et al., ; snijder et al., ; stertz et al., ; knoops et al., ) , we found that the tested components of the rtcs, i.e. nsp , nsp , nsp and nsp , localize to dmvs (fig. ) and that viral rna production becomes detectable simultaneous with the appearance of dmvs ( h p.i.; fig. a) . moreover, the rna levels correlate with the number of dmvs throughout the course of the mhv infection (fig. s ) . a particularly interesting observation was that the production of rna reaches a steady-state coincident with a diminution in the generation of dmvs (fig. a, fig. c and d, fig. s ). this observation could support a new concept in which viral rna synthesis is dictated by the number of dmvs (and thus rtcs) rather than by, or in addition to, the regulation of the rate of rna replication and transcription. alternatively, depletion of one or more host factors required for dmv biogenesis could lead to an identical outcome. obviously, future studies are necessary to address this issue. a remarkable new feature that we observed while studying dmvs generated in hela cells was the presence of an invagination, which often contained a small vesicle ( fig. a and b, fig. s b ). this characteristic has not previously been documented perhaps because it is cell type specific. yet, it may provide valuable information about the dmv biogenesis. the small vesicles could carry newly synthesized viral proteins and/or host components to dmvs. alternatively; these profiles may also represent the fusion events between dmvs described for the sars-cov (knoops et al., ) . unfortunately, the tokuyasu cryosectioning technique that we have used for our iem analyses does not allow the preservation of inner content of the dmvs and the small vesicles intimately associated with them (fig. d, e and i, fig. s b and h, fig. s b , c and e; gosert et al., ; snijder et al., ; stertz et al., ) . hence, we could not determine the presence of viral proteins and double-stranded rna (dsrna) in these small vesicles. it has recently been proposed that cms are the site of generation of dmvs (knoops et al., ) . our data showing that the cms have the same viral protein composition as the dmvs may support this hypothesis ( fig. a-e, table ). in addition, they reinforce the model (knoops et al., ) that cms are also involved in the overview of the mhv-induced structure composition established by iem. the presence (+) or absence (-) of nsp's, the various structural proteins and the analysed organelle protein markers in the dmvs, cms, virions, lvcvs, tbs and cmss is illustrated. the p.i. appearance time of these mhv-induced structures is also indicated. replication and transcription of the viral rna. as already observed (knoops et al., ) , however, the cms appear after dmv formation ( fig. b and d) . because of this observation, we favour the idea that cms are unlikely to be the precursor structure of dmvs. potentially, they could originate from the dmvs but we never observed continuity between these two structures. another possibility is that cms are structures generated either for nsp's storage or by accumulation of excess nsp's that cannot be incorporated into dmvs. the possibility that the dmv biogenesis could require host components makes it possible that depletion of these components causes an accumulation of the newly synthesized transmembrane nsp's in the er (harcourt et al., ; kanjanahaluethai et al., ; oostra et al., ; oostra et al., ) , which leads to a clustering of rtcs at this site that results in the formation of cms. in accordance with this hypothesis, cms are frequently connected with the er (fig. s c ; knoops et al., ) . in addition, their size increases over time especially after h p.i. when dmv formation slows down. it remains to be investigated whether cms are able to synthesize viral rna and to eventually pack dsrna into their interior, which seems not to be the case so far (knoops et al., ) . virion assembly and release (fig. b) as expected, the virions were positive for the structural proteins, m, e and n, but not for the nsp's ( fig. and fig. s , table ). their immunolabelling with the anti-e protein antiserum, however, was weak, consistent with the low number of e proteins per virion (vennema et al., ; de haan and rottier, ) . in agreement with the previous literature about their formation and release, virions were seen assembling in the ergic/golgi (fig. c) and complete viral particles were present in these organelles but also in secretory vesicles and the extracellular space (fig. g, fig. a, fig. c, fig. s d ). these observations were first recorded at h p.i. (fig. f and g). from h p.i., virions were also forming and contained in the lvcvs (fig. d, fig. h and i, fig. e, f and h, fig. b-d, fig. d, fig. s a and c, table ). lvcvs have already been described in sarsinfected cells (ng et al., ; goldsmith et al., ; knoops et al., ) and the observation that viral particles assemble at the limiting membrane of these compartments has led us to hypothesize and to show that they have a golgi origin (fig. b-d and fig. ). the enlargement of the ergic/golgi compartments that leads to the formation of the lvcvs is probably a consequence of the accumulation of massive amounts of viral proteins and/or luminal virions that require an expansion of this organelle to accommodate them. this phenomenon has already been reported in the case of large cargo molecules passing through the golgi (bonfanti et al., ) . it cannot be excluded a priori, however, that an overloading in viral proteins causes a fusion of the golgi cisternae, which results in the lvcv formation. the fact that the secretory pathway is flooded by viral components is also emphasized by the formation of virions in the er from h p.i. (fig. s ) . the labelling efficiency of lvcvs for ergic and golgi protein markers, however, was reduced as compared with that of the same organelles in non-infected cells ( fig. b and fig. s b-d) . these observations indicate that some of the standard golgi functions of this compartment are probably altered by the high content of viral components. this is exemplified by the observation that the golgi cisternae fragment and the golgi protein marker galnact -gfp partially localizes to the er during the course of an mhv infection ( fig. a and c) . in addition, the secretion of gaussia luciferase is reduced during mhv infection (verheije et al., ) . nevertheless, the lvcvs are functional structures because the extracellular release of mhv is still effective at the advanced stages of the mhv infection when numerous lvcvs are present in each cell (fig. c, fig. h and i). lvcvs are probably not an organelle exclusively induced by covs, as lvcv-like structures have also been observed in cells infected with the (+) rna rubellavirus (risco et al., ; novoa et al., ) , which also assembles its progeny virions in association with the golgi compartment. our data show that tbs and cmss are infrequent structures ( fig. j and data not shown), that they appear at the late stages of the cov infection ( fig. j and k, and data not shown) and that they probably contain a single viral protein (fig. i, table ). these observations indicate that they are the result of the massive production of viral proteins that cannot be incorporated into the virions (fig. b ). the tbs are only positive for the e protein but not for the nsp's, the n and the m protein ( fig. s d -e, fig. s a and c; table ). we cannot formally exclude that they also contain the s protein but it is unlikely because the individual expression of the e protein is sufficient to induce the formation of a complex of smooth tubular membranes with morphology reminiscent to that of the tbs (raamsman et al., ) . the e protein is a transmembrane component that is inserted in the er before being transported to the ergic/golgi (lim and liu, ; corse and machamer, ) that is also able to selfinteract (raamsman et al., ; von brunn et al., ) . importantly, even if not positive for the er resident chaperone pdi, the tbs have clear membrane continuity with the er (fig. e, fig. e, fig. s d and f) . consequently, a very likely scenario is that the tbs are generated by excess e protein that self-aggregates in the er. the cmss were negative for any of the tested viral proteins ( fig. s g and h, fig. s d and f, table ), and therefore we speculate that they are induced by the s protein, for which we do not have an antibody compatible with our iem procedure. the cmss are clearly connected with er and positive for pdi ( fig. f and fig. f ). interestingly, it has previously been shown that overexpression of transmembrane proteins that have the capacity to self-interact can lead to the formation of geometrical and crystalloid conformations in the er that have been called either 'cubic membranes' or 'organised smooth er', which resemble the cmss (snapp et al., ; almsherqi et al., ; . therefore, the simplest model that we propose is that cmss are formed by the self-assembly of excess s protein in the er. in conclusion, our study has ordered and characterized the different membranous rearrangements induced during a cov infection. this information paves the way for future investigations about the biogenesis and the function of these structures, which is crucial to understanding the cov life cycle and eventually for the development of more effective therapies against these pathogens. hela-ceacam a cells (verheije et al., ) , the hela-galnact -gfp stable cell line (storrie et al., ) and lr cells, used to propagate and titrate mhv-a and mhv-srec, were maintained in dulbecco's modified eagle medium (dmem; cambrex bioscience, walkersville, md, usa) containing % fetal calf serum (bodinco, alkmaar, the netherlands), iu of penicillin·per millilitre and mg ml - of streptomycin (both from life technologies, rochester, ny, usa). hela cells expressing the mouse ceacam a receptor (hela-ceacam a cells), which allows infecting these human cells with mhv-a (verheije et al., ) , were used for the time-course mhv infection. we decided to employ hela cells because many antibodies against human proteins are available and those cells have previously been successfully used for the study of cov replication (gosert et al., ) . the hela-ceacam a cells were inoculated with mhv-a at an moi of tissue culture infection dose (tcid ) as determined on lr cells, in phosphate-buffered saline (pbs) containing mg ml - diethylaminoethyl-dextran (pbs-deae). after min, hela-ceacam a cells were washed and maintained in complete dmem containing mm of the fusion inhibitor mhr peptide (genscript, piscataway, nj; bosch et al., ) . the mhr peptide prevents fusion of adjacent cells upon interaction of expressed s protein and mceacam a receptor as well as additional infection by the residual inoculum in the medium or, at later times, secreted by the infected cells (bosch et al., ) . as a result, the mhv infection is synchronized. subsequently, aliquots of infected cells and culture supernatants were collected for analysis at , , , , , , , , and h p.i. the hela-galnact -gfp stable cell line was infected with mhv-srec, a recombinant mhv strain with an extended host range (de haan et al., ) . these cells were then fixed at , and h p.i. before being processed for if and iem. the total rna was isolated from the infected cells using the trizol reagent (invitrogen, san diego, ca, usa). rna was further purified using the rneasy mini-kit (qiagen, hilden, germany) according to the manufacturer instructions with subsequent dnasei treatment on the column. rna integrity was determined by spectrometry using a uv-mini device (shimadzu, kyoto, japan). taqman single-tube reverse transcription-pcr (rt-pcr) assay (pe biosystems, foster city, ca) was performed as described (de haan et al., ) and used to analyse both the orf b region (de haan et al., ) and the n protein-encoding region (raaben et al., ) of the viral genome. the reactions were performed in triplicate according to the manufacturer's instructions using an abi prism sequence detector (foster city, ca, usa). thirty minutes before the indicated time points, cells were starved for min in cysteine-and methionine-free modified eagle's medium containing % fetal calf serum and mm hepes, ph . . cells were subsequently radiolabelled in the same medium containing mci of s in vitro cell-labelling mixture (amersham pharmacia biotech, freiburg, germany) for min and lysed in the tesv lysis buffer ( mm tris-hcl, ph . , mm edta, mm nacl, mm pmsf, % triton x- ). the lysates were analysed by sds-page. due to the preferential and sustained synthesis of viral proteins during the course of an infection, the radioactive bands corresponding to the m protein ( - kda) and the structural proteins s and n were the most prominent on the autoradiographs. the m protein was quantified using a phosphorimager system (molecular dynamics, uppsala, sweden). the amount of virions present in the culture supernatants was determined by end-point dilutions on lr cells and then calculating the tcid values. cells were fixed with karnovsky ( % para-formaldehyde, . % glutaraldehyde, mm cacl , mm mgcl in . m sodium cacodylate ph . ), first at room temperature for min and then overnight at °c. cell pellets were then post fixed with % oso / . then % kcnfe in . m cacodylate buffer for h on ice and dehydrated stepwise with increasing concentrations of ethanol. the dehydrated pellets were rinsed with propylene oxide at room temperature and then embedded in epon (degtyarev et al., ) . after resin polymerization, - nm sections were cut using an ultra-microtome (leica) and contrasted with uranyl acetate and lead citrate before being viewed in a jeol or a jeol electron microscope (jeol, tokyo, japan). for the quantitative analyses, cell profiles were randomly selected at each p.i. time point and three different quantifications were performed. first, the number of cells containing at least one of the six mhv-induced structures was counted to determine morphologically the proportion of infected cells. second, we calculated the percentage of cell sections positive for each mhvinduced structure. third, we determined the average number of each structure per cell section and calculated the standard deviation. the standard deviation values were subsequently used to perform the t-test, which revealed the significance of the data about dmvs (p = . ), cms (p = . ), virions (p = . ), lvcvs (p = . ) and golgi (p = - ) but not for the tb (p = . ). the calculation of the standard deviation and the t-test could not be performed for the cmss because of the rarity of these structures. cells were directly fixed by adding double-strength fixative ( % para-formaldehyde/ . % glutaraldehyde or % paraformaldehyde, both in . m phosphate buffer, ph . ) to the culture for min at room temperature. this fixative was replaced by fresh standard strength fixative ( % paraformaldehyde/ . % glutaraldehyde or % para-formaldehyde, both in . m phosphate buffer, ph . ) and incubated overnight at °c. fixation was subsequently continued for h at room temperature before washing the cells times with . m phosphate buffer, ph . and scraping them from the plate in the same buffer containing % gelatin. cells were successively centrifuged and embedded in % gelatin (slot and geuze, ) . the obtained pellets were cut into small cubes, which were cryo-protected in . m sucrose and subsequently frozen in liquid nitrogen. finally, after trimming to a suitable block shape, - nm ultrathin sections were cut at - °c on dry diamond knives (diatome ag, biel, switzerland) using either an uc or an uct ultra-microtome (leica). cryo-sections were labelled with rabbit antisera recognizing nsp and nsp [a kind gift of s. baker (schiller et al., ) ], nsp [a kind gift from s. baker (schiller et al., ) ], nsp [a kind gift of m. denison (lu et al., ) ], m protein (krijnse-locker et al., ) , e protein (de haan et al., ) , gm [a kind gift of e.s. sztul (styers et al., ) ], gfp (abcam, cambridge, uk) or mouse monoclonal antibodies against the nucleocapsid n (a kind gift from stuart siddell), pdi (stressgen, ann arbor, mi, usa), ergic (alexis biochemicals, lausen, switzerland) or sheep anti-tgn- (serotec, oxford, uk) antiserum. the specificity of the antibodies against viral proteins was tested on mockinfected cells (data not shown). antibodies were visualized with or nm gold particles conjugated to protein a (slot and geuze, ) . rabbit antibodies were directly detected with protein a-gold particles whereas mouse and sheep antibodies where bridged with polyclonal anti-mouse (dako, carpinteria, ca, usa) and anti-sheep (nordic, tady, sweden) igg antibodies. labelled cryosections were finally contrasted with an uranyl acetate-uranyl acetate methyl cellulose mixture. all specimens were imaged as described for the conventional em. e and f. the e protein is not distributed on the dmvs, cms and cmss. arrows and arrowheads indicate tbs and cmss, respectively, whereas asterisks mark the dmvs. cm, convoluted membranes; er, endoplasmic reticulum; g, golgi; l, lysosome; lvcv, large virion-containing vacuole; m, mitochondria. white bar, nm; black bar, nm. fig. s . specific immunolabelling of intracellular organelles. mock-infected cells were fixed and processed with antibodies against (a) pdi (er), (b) ergic (ergic), (c) gm (cis-golgi cisternae), (d) tgn (trans-golgi) and (e) lamp (late endosomes and lysosomes). er, endoplasmic reticulum; l, lysosome; m, mitochondria; n, nucleus; pm, plasma membrane. white bar, nm; black bar, nm. fig. s . correlation between the viral rna synthesis and dmv biogenesis. the number of dmvs/ cells at each time point of the mhv infection was calculated by multiplying the percentage of dmv-containing cells with the average number of dmvs per cell. results are plotted on a log graph together with the grna and sgrna n amounts expressed using arbitrary units. fig. s . virion assembly can occur in the er. immunolabelling of hela-ceacam a infected cells with anti-pdi antibodies at h p.i. assembly of virions (arrows) can be observed in the er. er, endoplasmic reticulum; m, mitochondria. bar, nm. please note: wiley-blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses direct template matching reveals a host subcellular membrane gyroid cubic structure that is associated with sars virus cubic membranes: a legend beyond the flatland* of cell membrane organization 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associated with the severe acute respiratory syndrome coronavirus replication complex the intracellular sites of early replication and budding of sarscoronavirus recycling of golgi-resident glycosyltransferases through the er reveals a novel pathway and provides an explanation for nocodazoleinduced golgi scattering depletion of beta-cop reveals a role for cop-i in compartmentalization of secretory compartments and in biosynthetic transport of caveolin- an electron microscopic study of viral hepatitis in mice coronavirus translational regulation: leader affects mrna efficiency replication of coronavirus mhv-a in sac-cells: determination of the first site of budding of progeny virions sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes mouse hepatitis coronavirus rna replication depends on gbf -mediated arf activation redirecting coronavirus to a nonnative receptor through a virusencoded targeting adapter transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus virusencoded proteinases and proteolytic processing in the nidovirales the authors thank s. baker, m. denison, t. nilsson, s. siddell and e.s. sztul for reagents, matthijs raaben and eddie te lintelo for technical assistance, peter rottier and judith klumperman for the critical reading of the manuscript, and marc van peski and rené scriwanek for assistance with the preparation of the figures. f.r. and c.a.m.h. are supported by the utrecht university (high potential grant). additional supporting information may be found in the online version of this article: b. dmvs with invaginations that appear to contain a small vesicle. c. a cm (arrow) that appears to be connected with the er. d. virions (arrows) released in the extracellular space. e. dmvs with protuberances emerging from their surface entering the invagination of an adjacent dmv (arrows). er, endoplasmic reticulum; m, mitochondria; n, nucleus; pm, plasma membrane. white bar, nm; black bar, nm. fig. s . mhv-induced structures negative for non-structural proteins. hela-ceacam a cells infected with mhv-a were fixed at h p.i. before being processed for iem and immunolabelled as described in experimental procedures. a-c. nsp /nsp , nsp and nsp do not localize to the lvcvs (arrows). d-f. nsp /nsp , nsp and nsp are not present in the tbs (arrow). g and h. the cmcs (arrows) are not positive for nsp and nsp . asterisks mark the dmvs. m, mitochondria; n, nucleus; pm, plasma membrane. bar, nm. fig. s . mhv-induced structures negative for structural proteins. hela-ceacam a cells inoculated with mhv-a were fixed at h p.i. before being processed for iem and immunolabelled as described in fig. . a. the n protein does not localize to the tbs. b. the m protein is not present on both dmvs and cms. c and d. the tbs and the cmss are not positive for the m protein. key: cord- -hduv ur authors: nan title: sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-golgi network of att cells date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: hduv ur murine hepatitis virus (strain a ), (mhv-a ) is a coronavirus that buds into pre-golgi compartments and then exploits the exocytic pathway of the host cell to reach the exterior. the fibroblastic cells in which replication of this virus is usually studied have only a constitutive exocytic pathway that the virus uses. mhv-a also infects, albeit inefficiently, att cells, murine pituitary tumor cells with a regulated as well as a constitutive exocytic pathway. here we examine att cells at early times after the infection, when the golgi apparatus retains its morphological and biochemical integrity. we observe that progeny coronavirus and secretory protein destined for the secretory granules of the regulated exocytic pathway traverse the same golgi stacks and accumulate in the trans-golgi network. their pathways diverge at this site, the condensed secretory proteins including the acth going to the secretory granules and the coronavirus to post-golgi transport vesicles devoid of acth. on very rare occasions there is missorting such that aggregates of condensed secretory proteins and viruses occur together in post-golgi vesicles. we conclude that the constitutive and regulated exocytic pathways, identified respectively by the progeny virions and the secretory protein acth, diverge at the exit from the trans-golgi network. i n cells of the anterior pituitary tumor cell line art there are two exocytic pathways, one is constitutive and the other is regulated, such that secretion is enhanced by seeretagogues (gumbiner and kelly, ) . the major envelope glycoprotein of an endogenous murine leukemia virus (gumbiner and kelly, ) and the secreted glycoprotein laminin (burgess et al., ) are examples of proteins that are exported via the constitutive pathway. on the other hand, part of the proopiomelanocortin made in these calls follows the regulated pathway in which it is cleaved proteolyrically to yield acth and is stored in secretory granules (gumbiner and kelly, ; moore and kelly, ) . foreign peptide hormones, specified by cloned genes from various endocrine and exocrine cells transfected into att cells, are sorted into the secretory granules of the regulated pathway (reviewed in kelly, ) . condensed secretory proteins first accumulate in the trans-most golgi compartment of att cells. this extreme trans-compartment, which lies beyond the tppase-positive cisternae of the golgi stack and has been termed the trans-golgi network or trans-golgi reticulum (griffiths and simons, ) , is the site of formation of the secretory granules of the regulated pathway . the corona~arus mouse hepatitis virus strain a (mhv-a ) i infects and replicates efficiently in some mouse . abbre~ations used in this paper: ecm, extracellular matrix; mhv-a , mouse hepatitis virus a ; pi, protease inhibitor cocktail ( ig/ml leupeptin fibroblastic cell lines in which the maximum rate of release of progeny virions occurs well before cell lysis. the virus, which buds into pre-golgi compartments, moves through the golgi cisternae and exploits vesicles of the constitutive exocytic pathway of these fibroblastic hosts, which lack a regulated pathway, to reach the cell surface; the post-golgi vesicles filled with progeny virions that can be seen near the cell surface apparently fuse with the plasma membrane to release the virus into the medium (for review see dubois-dalcq et al., ) . mhv-a can, however, also infect att cells inefficiently (tooze and tooze, ) . in att cells, as in fibroblastic cells, the virus buds into pre-golgi compartments. the progeny virions then traverse the golgi stacks and are transported thence to the cell surface. assuming that mhv-a utilizes the constitutive pathway in att cells, as it does in fibroblasts, we can use the viral particles as a morphological marker for that pathway and use condensing secretory proteins, including acth, as a morphological marker for the regulated pathway. we can then ask at what level do the two pathways diverge? which is the last compartment to contain both virions and condensing secretory in distilled hzo, .tg/ml antipain in distilled h , p.g/ml pepstatin in h , mm bertzamidine in dimethylsulfoxide, gtg/ml phenylmethylsulfonyl fluoride in anhydrous ethanol, and u/ml trasylol); ripa, immunoprecipitation buffer ( % [wt/vol] tx- , . % lwt/vol] sds, . m nac , % [wt/vol] sodium dodeeylate, ram tris, ph . ); vsv, vesicular stomatitis virus. proteins and are the two markers segregated into different populations of transport vesicles at the exit from this compartment? we report here our attempts to answer these questions by electron microscopy and immunocytochemistry. our observations indicate that the constitutive and regulated pathways in att cells do indeed diverge at the exit from the trans-golgi network. art d v cells were propagated at ~ in a % coz incubator as previously described (tooze and tooze, ) in dme supplemented with % horse serum and . g/liter of glucose. when propagated on normal tissue culture plastic surfaces or glass att cells form dense clumps many ceils deep. when plated on plastic or glass surfaces coated with extracellular matrix (ecm; international bio-technologies ltd., jerusalem, israel), the att cells adhere and flatten within h, and grow as a monolayer. this greatly facilitates optical microscopy. in addition we have found that art cells growing on ecm am about five times more susceptible to infection with the coronavirus mhv-a than are parallel cultures growing on plastic surfaces. only between and % of the cells on plastic can be infected, whereas - % of the cells on ecm are infected. this effect is specific for mhv-a . vesicular stomatitis virus (vsv) infects essentially all the art cells in cultures growing on plastic or on ecm. we assume that when art cells grow flattened on ecm, either the numbers or the accessibility of the plasma membrane receptors exploited by the virus increases. but whatever the mechanism the increased susceptibility of the cells to infection facilitated several experiments reported here. coronavirus mhv-a , propagated on sac-cells as previously described (tooze et al, ) , was used to infect art cells at a multiplicity of infection of - pfu/cell. the virus was applied in rrd of culture medium per -ram dish for - h at ~ in a % co incubator. a stock of vesicular stomatitis virus (indiana serotype) was grown and titered on baby hamster kidney cells (bhk- ) as described in fuller et al. ( ) . infection of art cells on plastic or ecm was performed by rinsing the cell monolayer with eagle's medium supplemented with . % bsa, mm hepes, mm glutamine, tig/ml penicillin, and gg/ml streptomycin, and then applying the virus at a multiplicity of infection of pill/ cell in a volume of . ml per -mm dish. the monolayers were returned to the ~ % co incubator for h, rinsed with the supplemented eagle's medium to remove unbound virus, and then with growth medium. the cells were returned to the incubator in growth medium for the remainder of the incubation period. cells growing on glass coverslips coated with ecm were infected with mhv-a and subsequently superinfected with vsv as detailed in results. the cells were then fixed in % paraformaldehyde in pbs containing i mm mgc and i mm cach and labeled for indirect immunofluorescence microscopy using a procedure based on that of ash et al. ( ) . briefly the fixed cells were first incubated with a rabbit antiserum against vsv-g protein, generously provided by dr. b. burke (european molecular biological laboratory, heidelberg), and then with rhodamine-conjugated sheep anti-rabbit antibody. after this labeling the cells were permeabilized with . % triton x- and then incubated with an affinity-purified mouse monoelonai antibody against the e glycoprotein of mhv-a followed by fluoresceln-conjugated sheep anti-mouse antiserum. all incubations with antibodies were at room temperature for rain. the double-labeled cells were examined in a zeiss photomicroscope hi with a planapo x oil immersion objective and appropriate filters. cells grown on plastic petri dishes were fixed for h with modified karnowsky's fixative containing % glutaraldehyde. fixed cultures were postfixed in % osmium tetroxide in . m cacndylate buffer ph . for i h and then dehydrated, removed from the dishes with propylene oxide, and embedded in epon. thin sections were viewed in phuips electron microscopes after contrasting with uranyl acetate and lead citrate. we followed the procedure given by reggio et al. ( ) . the fixative used was % paraformaldehyde and . % glutaraldehydr in . m sodium phosphate, ph . . we used an affinity-purified antibody against aci~ kindly provided by dr. r. kelly (university of california, san francisco) and an aflinity-purified antiserum against the carboxy-terminal domain of the e glycoprotein mhv-a ('iboze and stanley, ) . for some of the immunoperoxidase labeling experiments the cells were grown on plastic dishes coated with ecm. after immunoperoxidase labeling the cells were dehydrated, removed from the dishes with propylene oxide, embedded in epon, and thin sectioned for electron microscopy. at -h postinfection five -mm dishes of att cells infected with mhv-a were washed three times with methionine-free modified eagle's medium (mem-met) containing . gm/liter sodium bicarbonate, and labeled with . i~ci/gl of [ s]methionlne in mem-met containing % dialyzed fcs, mm hepes, mm glutamine, gg/ml penicillin, and ~tg/ml streptomycin for rain. the incorporation of [~s]methionine was stopped by adding ml of growth medium per dish supplemented with lo times the normal amount of methionine ( . mg/ml) for the chase, or by placing the cells on an aluminium slab in ice and washing them three times with calcium and magnesium-free pbs at ~ for the zero time point. each -ram dish of cells was solubilized in ripa buffer ( % (wt/vol) tx- , . % (wt/vol) sds, . m nac , % (wt/vol) sodium dodecylate, mm tris, ph . ) containing protease inhibitors (pi) ( ig/ml leupeptin in distilled h , ~g/ml antipain in distilled h , ~g/ml pepstatin in h , mm benzamidine in dimethylsulfoxide, gg/ml phenylmethylsulfouyl fluoride pmsf in anhydrous ethanol, and u/ml trasylol) at ~ the insoluble debris was pelleted in an eppendorf centrifuge (brinkmmm instruments, inc., westhury, ny) for min at top speed at ~ the supernatant was preprecipitated with an equal volume of washed pansorbin (calbiochem, frankfurt, federal republic of germany) in ripa/pi ( % wt/vol) (see below) for rain at room temperature. the pansorbin was pelleted for rain at room temperature at , rpm in an eppendorf centrifuge, and the clarified supernatant was removed. to each sample gg of an affinity-purified antibody against the el glycoprotein of mhv-a (tooze and stanley, ) was added, and the mixture was rotated at ~ overnight. to the immune complexes, gl of washed pansorbin in ripa]pi ( % wt/vol) was added and the mixture was incubated at ~ for rain and pelleted. the pellet was then washed three times with ripa/pi buffer; once with mm tris-hcl, ph . , . % sds, mm edta plus pi; once with mm tris-hcl, ph . , . % sds, mm edta plus pi; and once with . m naci, mm tris-hcl, ph . , mm edta. the final wash was with mm tris-hc , ph . , and mm edta. all washes were for min at , rpm at room temperature. the samples were divided in two during the final wash. one-half was then resuspended in gl of sample buffer containing . m tris-hci ph ~ , mm edta, m sucrose, % (wt/vol), % (wt/vol) sds, bromopbenol blue, and mm dithiothreitol by shaking at ~ for min, followed by freezing at - ~ for min, followed by an additional -min incubation at ~ with shaking. the insoluble material was pelleted in an eppendorf centrifuge (bdnkmann instruments, inc.) for rain at top speed at room temperature, and prepared for sds-page as previously described (green et ai., ) . the other half was washed twice and resuspended in gl of mm sodium acetate, ph . and mm cac at ~ then gl of mu/gl neuraminidase from clostridium perfringens (sigma chemical co., munich, frg) in mm sodium acetate, ph . , mm caci was added, and the volume was adjusted to i~l with the same buffer. after a -h incubation rotating end over end at ~ the pansorbin was pelleted, solubilized in sample buffer to release the digested e glycoprotein, and run on a % sds-polyacrylamide gel. the pulse chase to evaluate the extent of n-glycosylatiou of vesicular stomatitis g protein in cells infected with the corouavirus mhv-a was performed essentially as described by fuller et al. ( ) . infected cells were pulsed with [~ss]methionine ( i~ci in . ml of mem lacking methionine and supplemented with . % bsa per -ram dish) for min and then chased with ml of the same medium containing . mg/ml methionine for various times. the cells were harvested by cooling on ice, rinsing twice with ice-cold pbs, and then lysed with . ml of % triton x-i in pbs cor~nining mm phenylmethylsulfouyl fluoride. the cells were scraped from the dish with a plastic pipette tip and the lysate spun at g for rain at ~ to remove nuclei. the supernatant was then subjected to two rounds of phase partitioning bordier, ) to enrich for the g protein. equal numbers of counts (corresponding to approximately one-twentieth of the total lysate) were then prepared for sds-page on a - % polyacrylamide gel. replication of mhv-a in att cells ultimately results in disruption of the golgi apparatus and death of the cells. it was necessary, therefore, to establish that at early times postinfection the golgi apparatus maintains its structure and function and is not yet disrupted by the cytopathic effects of viral replication. until at least h after infection of att cells with mhv-a the golgi apparatus maintains its stacked cistemal structure. progeny virus particles, budded into pre-golgi compartments, can be seen in the dilated rims of the golgi cisternae and accumulating in dilations of the trans-golgi network (figs. and ). from the trans-golgi network virions are transported, packed into spherical vesicles, to the cell surface. at these early times postinfection the morphology of the golgi stack and trans-golgi network is normal and, except for the presence of virions, identical to that in uninfected cells (see also figs. and ) . it is noteworthy that in att cells and also fibroblastic host cells the coronavirions invariably occur in the dilated rims of golgi cistemae and not in the comparatively flat stacked central regions of the cisternae. as figs. , , and show, virions in the trans-golgi network are often kidney shaped and have a fairly uniform and high electron density. by contrast the virions in the golgi cisternae and pre-golgi compartments are spherical structures with ribonucleoprotein core material immediately below the viral envelope and an "empty" center. this morphological maturation of the virus, which also occurs in other host-cell types (for review see dubois-dalcq et al., ) , is particularly pronounced in att cells, and at early times postinfection is strictly correlated with delivery of the viruses to the trans-golgi network. virions with the mature morphology are, therefore, a convenient and reliable morphological marker of transand post-golgi compartments. to establish that at between - h postinfection the golgi apparatus is still biochemically competent, as well as mor-phologicauy intact, we assayed its ability to o-glycosylate the e glycoprotein of mhv-a . the only known posttranslational modification of the transmembrane e glycoprotein is the addition of o-linked glycans during transit of the golgi apparatus (niemann and klenk, ) . in fibroblasts these are added posttranslationally to serine and threonine residues at the amino terminus of the protein (niemann et al., ) , which is exposed on the lumenal surface of intracel- att cells. infected cells were pulsed for rain with [ s]methionine and then chased for the times indicated, up to min. the e glycoprotein of mhv-a was then immunoprecipitated from detergent-solubilized cells and analyzed by sds-page. during the -min pulse, some of the el becomes partially glyeosylated (first lane). by min of chase virtually all of the primary translation product has chased into the two higher molecular weight forms. as shown for the -min sample, digestion with neumminidase converts the highest molecular weight form to an intermediate form, indicating that the former is the sialylated or fully glycosylated form of the protein. at -h postinfection att cells were pulsed for min with [ s]methionine and then chased for up to min. fig. shows the result. after the -min labeling the e glycoprotein is resolved on polyacrylamide gels into two bands. after a -min chase a third higher molecular weight band is resolved. these data indicate that, as in fibroblastic saccells (tooze, s. a., j. tooze, and g. warren, manuscript in preparation), the o-glycosylation of e occurs in at least two steps and that some e molecules are partially glycosylated by the end of a -min pulse. by counting the radioactivity in the three bands during the chase we found that within min of its synthesis, - % of the e is found in the highest molecular weight form and presumably is fully glycosylated. about the same percentage has been partially glycosylated. to establish that the highest molecular weight species had terminal sialic residues on the sugar chains, and therefore was fully glycosylated, an aliquot of e was taken after the -min chase. one-half was digested with neuraminidase while the other was similarly incubated but without neuraminidase. this digestion converted the highest molecular weight form to an intermediate form (fig. ) . these results show that at - -h postinfection the golgi apparatus is biochemically, as well as morphologically, intact and adds o-linked sugar chains terminating in sialic acid residues to the viral e glycoprotein. to provide further evidence that the constitutive pathway functions normally at early times after infection with mhv-a we performed the following double-infection experiment. art cells growing on ecm-coated glass coverslips were infected with mhv-a for h at "/~ the cells were then washed and incubated at c for . h until . -h postinfection. the cultures were then superinfected with vsv for i h and incubated for a further . h at ~ they were then fixed, h after infection with mhv-a and . h after infection with vsv, and processed for immunofluorescence microscopy using a double-labeling procedure with a rabbit antiserum against vsv-g protein and an affinity-purified mouse monoclonal antibody against e of mhv-a . all of the cells were infected by the vsv and expressed g protein at their cell surfaces, including the - % of cells that had been infected by mhv-a and therefore contained the coronaviral e glycoprotein accumulated in the golgi region (fig. ) . this establishes that between . -and -h postinfection with the coronavirus the constitutive exocytic pathway of att cells continues to function and can transport vsv-g protein to the plasma membrane. the same is true for cells of the murine fibroblastic line sac-(data not shown). we used the well characterized n-glycosylation of vsv g protein to test further the integrity of its transport to the plasma membrane surface in mhv-infected cells (fig. ) . knipe et al. ( ) originally showed that the increase in apparent molecular weight of the g protein marked the acquisition of terminal sialic acid, the last identified step in the golgi processing. there were no detectable differences in the kinetics of this apparent molecular weight shift in the presence or the absence of mhv infection implying that neither the rate nor extent of sialylation are affected (fig. ) . hence the processing of a plasma membrane glycoprotein seems to be unaffected by up to h of mhv-a infection. using an antibody specific for the carboxy-terminal domain of the glycoprotein e of mhv-a (tooze and stanley, ) we labeled mhv-a -infected att cells using the immunoperoxidase procedure. as fig. shows, membranes of the stacked golgi cisternae were heavily labeled on their the journal of cell biology, volume , cytoplasmic face; by contrast membranes of the trans-golgi network, identified by the presence of mature virions as well as extensive surface coats, were unlabeled. post-golgi vesicles transporting progeny virions to the cell surface and secretory granules were also unlabeled (not shown). this establishes that, at - -h postinfection, the e glycoprotein in att cells enters the exocytic pathway and reaches the membranes of the stacked golgi cisternae where it accumulates and is apparently retained, perhaps being responsible for the eventual vesicularization of the golgi cisternae. e is not, thereafter, transported as an integral protein of cellular membranes to the trans-golgi network and beyond. this pattern of selective e transport is also seen in infected sacfibroblastic cells (tooze and stanley, ) and provides further evidence that the golgi apparatus retains its functional integrity at early times postinfection. budded virions are not labeled presumably because the carboxy-terminal epitopes of e recognized by the antibody are masked by association with nucleocapsid protein and genomic rna on the inner face of the virion's envelope (tooze and stanley, ) . condensation of secretory proteins and the formation of secretory granules of the regulated exocytic pathway occurs exclusively in the trans-golgi network in uninfected att cells . progeny coronavirions that have budded in pre-golgi compartments traverse the golgi stacks and are thence transported to the plasma membrane. we therefore examined thin sections of infected att cells under the electron microscope to determine which compartments contained both condensing secretory proteins and coronavirus. in many infected cells with progeny virions in the golgi stack there was no evidence of condensing secretory proteins in the trans-golgi network (fig. ) ; since mhv-a infections are asynchronous, in some cells viral protein synthesis may have competed with cellular protein synthesis. in other cells virions and condensing secretory proteins were in different dilations of the complex trans-golgi network (fig. ) . in a third class, however, clumps of condensing secretory proteins and progeny virions occurred in the same regions of the trans-golgi network (fig. , a and b ). note that although they are in proximity in the same compartment, the virions do not occur embedded in or surrounded by condensing secretory proteins. this separation is maintained as the secretory proteins condense further into secretory granule cores. fig. b, inset shows two morphologically mature cores and two virions in the same trans-golgi compartment. as we have recently shown in art cells, detachment of secretory granules from the trans-golgi network occurs at a late stage in tooze et infection. aft cells were infected with mhv-a for . h and then with vsv for a further . h. after infection with vsg the ceils were pulse-chased with [ ss]methionine (see materials and methods) and the g protein of vsv and the e protein of mhv-a were then enriched by tx-u phase partition as described in materials and methods. the increase in apparent molecular weight of g protein, which indicates its sialylation, was first detected between and min and occurs with the same kinetics in the presence or absence of mhv-a infection. the mhv-a e protein is seen in the infected cells (+ lanes) in a longer exposure of the bottom portion of the gel. the e is the middle band of the three bands within the bracket. this establishes that the e protein of mhv-a was being synthesized at the same time as the vsv g protein. the maturation of the cores, after they have become condensed and have assumed a spherical or ovoid shape with a well-defined surface. the two cores shown in fig. b , inset are at a stage at which they detach from the trans-golgi network. note also that there are no virions in the secretory granule close to the golgi stack shown in fig. . we have never seen virions embedded in secretory granule cores either in the golgi region or at the cell periphery, where the mature granules accumulate. fig. shows condensing secretory protein in the trans-golgi network of uninfected att cells. comparison of fig. , a and b with fig. shows that the only detectable change at these early times after infection with mhv-a is the presence of virions in the golgi complex. clathrin is a marker for the trans-side of the golgi apparatus (for review see griffiths and simons, ) and, as we have previously shown by inununocytochemistry , in uninfected att ceils the trans-golgi network and immature secretory granules in the golgi region often have clathrin coats on parts of their surface (fig. ) , and are the site of budding or fusion of clathrin-coated vesicles. coats that resemble clathrin in morphology, but were not positively identified as such by immunocytochemistry, were also often present in infected cells on the cytoplasmic face of parts of the trans-golgi network containing virions and on peri-golgi vesicles with virions (figs. a and ). the observations presented above indicate: (a) that virions and secretory proteins traverse the same golgi stacks and accumulate in the trans-golgi network; we have shown previously by immunocytochemistry that acth is present in all cisternae of the golgi stacks of att cells (tooze and tooze, note that only morphologically mature virions are found on the trans-side of the golgi complex. many dilations of the trans-golgi network contain either condensing secretory proteins (arrowheads) or condensed virions (/arge arrows). when both virions and condensing secretory proteins share the same dilation, they are well separated (lower lej? and inset). we have previously shown that the coat on the cytoplasmic surface of the trans-golgi network (between the small arrows) is clathrin . note the coated vesicle budding from or fusing with the trans-golgi network (curved arrow). (b) in this example the trans-golgi network contains condensing secretory protein (arrows) and one dilation also contains virions (open arrow). the virions have the mature morphology and are well separated from the condensing secretory protein. note that the latter is more uniformly compact than in a, and is beginning to assume the form of a secretory granule core. (insert) part of the trans-golgi network in another infected cell in which two mature virions (arrow) and two aggregates of secretory proteins that have condensed into ovoid granule core structures (arrowheads) are in the same compartment but still quite separate in the sense that there are no virions within the cores. bars, . lun. figures - . (fig. ) condensing secretory proteins in the trans-golgi network of an uninfected att cell. the condensing secretory protein (open arrows) is within two dilations of the trans-golgi network. note the clathrin coat on parts of the membrane (between the arrows) and also coated vesicles. interestingly, on the cis-side of the golgi stack a sheet of rough endoplasmic reticulum is differentiated in one region into annulate lamellae (al) and in another into a transitional element (te). apparently two morphological differentiations of the rough endoplasmic reticulum can occur in very close proximity in these tumor cells. bar, . inn. (fig. ) condensed virions in the trans-golgi network. note the prominent coat on the cytoplasmic face of one of the compartments enclosing a mature virion (arrow). the coat has the characteristic morphology of clathrin. bar, . ima. (fig. ) part of an infected cell exposed to i~m chloroquine from . -to -h postinfection. note the large accumulations of condensed virions in the trans-golgi network (arrows) and immature uncondensed virions in the golgi cisternae (arrowheads). post-golgi transport vesicles packed with mature virions can be seen below the plasma membrane (open arrows). bar, . ~tm. figure . post-golgi transport vesicles and secretory granules close to the lower surface of infected att cells, growing on extracellular matrix, after immunoperoxidase labeling with anti-acth antibody. in a note that the secretory granules are heavily labeled for acth (arrows). a completely unlabeled transport vesicle packed with virions (arrowhead) is close to the lower cell surface, which lies on the layer of extra~llular matrix (ecm). b shows, near the lower surface of a cell growing on ecm, one of the extremely rare classes of post-golgi transport vesicles which contain both virions (arrows) and a clump of condensed secretory protein that labels heavily with anti-acth antibody (arrowhead). some of the immunoperoxidase reaction product has diffused to coat the virions and the membrane of the vesicle. bars, . lain. ). (b) that within the trans-golgi network secretory proteins condense into granule cores that exclude virions. (c) beyond the trans-golgi network virions are packed in vesicles lacking condensed secretory proteins. we attempted to confirm this sorting ofvirions from secretory proteins entering granules of the regulated exocytic pathway. infected cultures were labeled using an affinity-purified antiserum against acrh by the immunoperoxidase method, and the post-golgi exocytic compartments were examined. this labeling reveals acth in all compartments of the exocytic pathway from the rough endoplasmic reticulum onwards . the post-golgi compartments that occurred below the plasma membrane at the cell periphery and lacked coats on their cytoplasmic surface fell into three categories: (a) labeled secretory granules that did not contain coronavirions and were identical to secretory granules in uninfected cells (fig. a ) ; (b) post-golgi vesicles packed with many virions but either not labeled for acth (fig. a) or with traces of reaction product on the vesicles inner surface (in this class of vesicles there was no evidence of clumps of condensed secretory proteins); (c) extremely rare vesicles containing both virions and clumps of condensed secretory proteins (fig. u b) that resemble in their labeling for acth the core material of secretory granules in both infected and uninfected cells (compare fig. a and b) . this third class comprised considerably < % of the total post-golgi vesicles containing mature virions. the fact that we have never observed post-golgi vesicles containing virions and aggregates of condensed secretory proteins in thin sections of conventionally fixed and embedded cells is further evidence of their rarity. their detection is dependent upon labeling the cells for acth by the immunoperoxidase procedure. the extreme trans-golgi cisterna is the most acidic compartment of the golgi apparatus (anderson and pathak, ) , but its ph is believed for a variety of reasons not to be below (anderson and pathak, ; griffiths and simons, ) . in att cells, as we have recently shown (unpublished results), the trans-golgi network is much less heavily labeled with -( , -dinitroanilino- "amino-n-methyldipropylamine (anderson et al., ) than are the secretory granules whose ph we estimate to be between . and . . to test whether the lower ph of this trans-most compartment of the golgi apparatus is necessary for the morphological maturation of coronavirus particles, we exposed infected cultures to ttm chloroquine from . - -h postinfection and then fixed and processed them for electron microscopy. as fig. shows, although exposure to chloroquine causes dilation of the trans-golgi network, the virions that accumulated in this compartment in the presence of this weak base were as compact and electron dense as those in untreated cells. we obtained the same result when infected cultures were exposed from . - -h postinfection to mm ni-i c (data not shown). it is also noteworthy that ~tm chloroquine did not inhibit the formation of the post-golgi vesicles packed with mature virus particles that occur close to the plasma membrane (fig. ) . however, in both infected and uninfected cells in the same cultures exposed for . h to either ttm chloroquine or d mm ni-i c there was little if any condensing secretory protein in the trans-golgi network of the many sections we examined (data not shown). exposure of the cells to these lysomotrophic agents apparently, therefore, had a differential effect inhibiting condensation of secretory proteins but not inhibiting the morphological maturation of the coronavirions. viral infection is a convenient way in which to introduce foreign genes into cells to study the synthesis, intracellular transport, and sorting of foreign proteins. the approach has one chief disadvantage, namely the cytopathic effects that accompany viral replication. this problem can, however, be largely avoided by studying the cells early after infection before the onset of the cytopathic changes. in the case of att ceils infected by mhv-a we believe that at - -h postinfection the exocytic pathways, and in particular the golgi apparatus, still function normally for the following reasons: (a) the golgi stack has a normal morphology. (b) the golgi enzymes responsible for o-linked glycosylation are functional. (c) vsv-g protein is n-glycosylated in the same way in both uninfected and mhv-a -infected att cells. furthermore the g protein is transported to the plasma membrane in both uninfected and mhv-a -infected cells. therefore the constitutive exocytic pathway is fully functional after infection with the coronavirus. (d) in infected cells, as in uninfected cells , the first compartment in which condensed secretory proteins occur is the trans-golgi network, indicating that secretory proteins of the regulated exocytic pathway are being synthesized, transported, and condensed normally. (e) the e envelope glycoprotein of the virus is not transported beyond the golgi stack, except as an integral component of the envelopes of progeny virions (fig. ) . by contrast the second viral envelope glycoprotein e is transported to the cell surface, as we have previously shown (tooze and tooze, ) . therefore the golgi apparatus in mi-iv-a -infected ceils at - -h postinfection can sort one coronaviral glycoprotein from the other. by these five criteria the golgi apparatus maintains its structural and functional integrity until at least -h postinfection with mhv-a . in this context it is interesting to note that in cells infected with uukuniemi virus (a bunyavirus) the constitutive exocytic pathway also continues to transport semliki forest virus glycoprotein to the plasma membrane even after the golgi stack has vacuolized as a result of the uukuniemi virus infection (gahmberg et al., ) . in this case the biochemical integrity of the golgi complex survives longer than its morphological integrity. unlike many other viruses, mhv-a does not, early in infection, efficiently inhibit cellular protein synthesis. there is nevertheless, during coronavirus infections, a progressive competitive inhibition of host-protein synthesis (tooze, s. a., unpublished data) . since the early stages of mhv-a infections are by no means synchronous, in any one culture at - -h post infection the extent of virus budding varies considerably from infected cell to cell. however, in most in-fected cells the number of virions increases rapidly after h of infection. concomittantly the number of infected cells with condensing secretory proteins in their trans-golgi network rapidly declines and acth can no longer be detected by the immunoperoxidase method in cisternae of the endoplasmic reticulum (data not shown). our observations depended, therefore, on examining the cells during the relatively short time after the budding of progeny virions had begun and before cellular protein synthesis was inhibited. in the pulse-chase experiment reported here, two glycosylated forms of the mhv-a e glycoprotein were resolved (fig. ) . however, the simplicity of the gel electrophoretic pattern of glycosylated e molecules from att cells contrasts with the ladder of variously glycosylated molecules obtained by gel electrophoresis of e isolated from infected murine fibroblasts (niemann et al., ; tooze, s. a., j. tooze, and g. warren, manuscript in preparation) . in murine fibroblasts the o-glycan chain added to e has the composition n-acetylgalactosamine-galactose-sialic acid and in ~o % of these chains an additional sialic acid residue is added to the n-acetylgalactosamine residue; moreover an average three of the four amino-terminal acceptor amino acids (nh -ser-ser-thr-thr) are glycosylated (niemann et al., ) . our data indicate that in att cells the o-glycosylation of e is considerably simpler than in fibroblastic cells. different patterns of o-glycosylation of the same protein in different cell types is, however, not unprecedented. cummings et al. ( ) have shown that the low density lipoprotein receptor is differently o-glycosylated in different cells. we have now shown the same to be true of the e glycoprotein of mhv-a . in att cells, at - -h postinfection with mhv-a , both progeny virions and condensing secretory proteins accumulate in the trans-golgi network. within that compartment condensed secretory proteins, perhaps by a phase separation process, form the secretory granule cores that exclude virions. on the other hand, the post-golgi vesicles transporting mature virions were either not labeled or only very weakly labeled by the immunoperoxidase reaction using antibody that recognizes both acth and its precursor, proopiomelanocortin. whether or not there exists in infected cells a further class of vesicles that constitutively transport uncleaved proopiomelanocortin but not virions is an open question. furthermore chloroquine, at lxm, does not block the formation of the vesicles that transport virions to the cell surface. these properties are consistent with the interpretation that post-golgi vacuoles transporting progeny mhv-a are part of the constitutive exocytic pathway. using the presence of virions as a morphological marker of the constitutive pathway in infected att cells, we can conclude that the trans-golgi network is the compartment from which the constitutive and regulated pathways diverge in these cells. the fact that extremely rare post-golgi vesicles located at the cell periphery and packed with virions also contain clumps of condensed secretory protein indicates that although sorting of condensed secretory proteins into the cores of secretory granules of the regulated pathway is efficient, it is not absolutely rigorous. a number of groups have invested a great deal of effort in attempting to identify the site of divergence of intracellular transport pathways to the cell surface. this compartment will be the major site for the sorting of newly synthesized plasma membrane and secretory proteins. the evidence accumulated so far has narrowed down but not identified the site of sorting either for components destined for the constitutive and regulated pathways of secretion or for membrane proteins destined for different plasma membrane domains (reviewed in griffiths and simons, ) . biochemical and morphological studies (rindler et al., ) indicate that proteins destined for separate cell surfaces colocalize throughout the golgi stack and that they separate before reaching the plasma membrane (kelly, ; marlin and simons, ; misek et al., ; pfeiffer et al., ) . griffiths and simons ( ) proposed that the sorting of these components occurs in the trans-golgi network since components to be sorted are in contact during the last identifable steps of golgi processing . a critical test of this hypothesis has been difficult since it requires simultaneous observation of components destined for the two pathways. the experiment must also be done with sufficient time resolution to rule out the possibility of transient formation of a compartment beyond the trans-golgi network which fissions to produce sorted transport vesicles. the system we use here fulfills these requirements because it allows us to observe directly the separation of the constitutive and regulated exocytic pathways. as far as we are aware, these observations are the first direct visualization of the sorting at the exit of the trans-golgi network of components taking different routes to the cell surface. they rely on having readily distinguishable morphological markers for each pathway, and they establish for this system that there is no sorting compartment beyond the trans-golgi network. in both att cells and in fibroblastic host cells (our unpublished observations) the post-golgi vesicles that apparently transport progeny coronavirus to the cell surface are large structures, holding several virions each ~ , a in diameter. these vesicles occur close to the plasma membrane and lack clathrin coats on their cytoplasmic surface. in uninfected cells there are no post-golgi vesicles of this size; no doubt the vesicles responsible for the constitutive transport of endogenous proteins are much smaller. with the exception of hepatocytes, which exocytose v. low density lipoprotein particles, mammalian cells normally never transport large particles along the entire exocytic pathway but only membrane proteins and soluble proteins. however, the very fact that cells of both epithelial (att ) and fibroblastic (sac-, cll cells) origin can accomodate to the transport of coronavirions from pre-golgi compartments to the cell surface proves that the transport vesicles at all stages of the exocytic pathway have the capacity to enlarge to carry large particulate macromolecular assemblies such as virions, even though normally they are never called upon to do so. it would be of interest to determine by immunocytochemical means whether or not the constitutive pathway vesicles that transport progeny virions from the trans-golgi network to the cell surface in either sac-or att cells also contain constitutively secreted cellular proteins such as laminin. such experiments are planned. the striking and irreversible morphological maturation of coronavirions, which takes place during their transit of the golgi apparatus in several types of host cells (see dubois-dalcq et al., ) , occurs in art cells at early times after infection exclusively in the trans-golgi network. this morphological maturation is not, however, inhibited by ~tm chloroquine, or by the presence of mm ni-hc . it cannot, therefore, be dependent on the lower ph in the transmost cisterna of the golgi apparatus compared with the earlier compartments (anderson and pathak, ) . by contrast, condensation of secretory proteins and their packaging into secretory granules in these cells seems to be ph dependent, since moore et al. ( ) established that ~tm chloroquine diverts acth from the regulated to the constitutive pathway. consistent with these results we observed little, if any, condensing secretory proteins in the trans-golgi network of either uninfected or infected art cells after their exposure to ~tm chloroquine or mm ni-hci for . h (data not shown). in conclusion, the trans-golgi network of att cells appears to be the site of sorting of material destined for the constitutive and regulated exocytic pathways, which diverge at the exit from this compartment. visualization of acidic organelles in intact cells by electron microscopy vesicles and cisternae in the trans golgi apparatus of human fibroblasts are acidic compartments antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts phase separation of integral membrane proteins in triton x- solution the exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer biosynthesis of n-and o-linked oligosaccharides of the low density lipoprotein receptor assembly of the coronvidrae an enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell share the same late golgi compartments vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line efficient transport of semliki forest virus glycoproteins through a golgi complex morphologically altered by uukuniemi virus glycoproteins passage of viral membrane proteins through the golgi complex the trans golgi network; sorting at the exit site of the golgi complex two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells pathways of protein secretion in eukaryotes localization of two intracellular forms of the vesicular stomatitis viral glycoprotein sorting of a plasma membrane protein occurs before it reaches the cell surface in cultured epithelial cells biogenesis of epithelial cell polarity: intracellular sorting and vectorial exocytosis of an apical plasma membrane glycoprotein chloroquine diverts acth from a regulated to a constitutive pathway in art cells secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways the carbohydrates of the o-glycosidically linked oligosaccharides of glycoprotein el coronavirus glycoprotein el, a new type of viral glycoprotein intracellular sorting and basolateral appearance of the g protein of vesicular stomatitis virus in mdck cells use of immunocytocbemical techniques in studying the biogenesis of cell surfaces in polarised epithelia viral glycoprotein destined for apical or basolateral plasma membrane domains traverse the same golgi apparatus during their intracellufar transport in doubly infected mdck cells identification of two epitopes in the carboxy terminal amino acids of the el glycoprotein of murine hepatitis virus a by using hybrid proteins infection of art murine pituitary tumour cells by mouse hepatitis virus strain a : virus budding is restricted to the golgi region clathrin-coated vesicular transport of secretory proteins during the formation of acth-containing secretory granules in art cells replication of coronavirus mhv-a in sac-cells: determination of the first site of budding of progeny virus we thank our colleagues stella hurtiey, jean gruenberg, and gareth griffiths (european molecular biological laboratory, heidelberg) for their constructive comments on the manuscript, and ines benner who patiently and skillfully typed this manuscript.received for publication march , and in revised form may . key: cord- -ky y xjt authors: füllekrug, joachim; nilsson, tommy title: protein sorting in the golgi complex date: - - journal: biochim biophys acta mol cell res doi: . /s - ( ) - sha: doc_id: cord_uid: ky y xjt even after one hundred years, the golgi apparatus remains a major challenge in the field of cell biology. this is particularly true in terms of transport and of protein sorting. for example, the question how cargo proteins are transported through this organelle is still a matter of debate. emphasis has been put on the role of anterograde and retrograde transport vesicles. these have been proposed to carry cargo from cisterna to cisterna and to recycle components needed for further rounds of transport. alternatively, anterograde movement of cargo takes place in cisternal membranes rather than transport vesicles. these membranes assemble and mature in a cis to trans direction. in this case, retrograde transport vesicles need to recycle all components of the golgi apparatus and this demands a highly dynamic and efficient sorting machinery. here we will discuss possible mechanisms for protein sorting in the context of cisternal maturation and propose that a common mechanism is sufficient to explain both transport of cargo and sorting of resident proteins. the main function of the exocytic pathway is to modify and deliver newly synthesised lipids and proteins to the cell surface. this forward movement of cargo takes place against gradients of resident proteins which occupy the pathway. these are enzymes which catalyse a variety of secondary modi¢cations such as processing of n-and o-linked oligosaccharides, addition of m- -p residues to lysosomal proteins, sulfation, phosphorylation or acetylation. in addition, proteins that confer structure and promote motility, docking and fusion of transport vesicles or membranes all need to be targeted or recruited to their appropriate location. in this review, we will discuss sorting of resident proteins in the golgi complex mainly in the context of cisternal maturation [ , ] . this model (see fig. ), also mentioned elsewhere in this issue, provides an alternative to the current textbook model of vesicular transport between (sub-)compartments de¢ned by function and composition. several excellent reviews discussing vesicular transport and protein sorting have been published over the years (e.g. [ , ] ) and though much attention has been given to this model, the cisternal maturation model has remained a good alternative reconciling many observations incompatible with the vesicular transport model. for example, macromolecules too large to enter transport vesicles still move through the secretory pathway and are readily found in cisternal membranes of golgi stacks. this is most obvious in some algae where a gradient of macromolecular (scale) assembly is revealed in a cis to trans direction strongly suggestive of a maturation process (reviewed by [ ] ). morphologically, the golgi apparatus also exhibits the structural characteristics of an organelle in transit, that is, one which is assembled at the cis side and taken apart at the trans side (for review, see [ ] ). one of the attractive features of the cisternal maturation model is that there are no pre-existing compartments apart from the er. this is in contrast to the vesicular transport model where the pathway is divided into several (sub-)compartments; the er, the er to golgi intermediate compartment (ergic), cis golgi network (cgn), cis, medial, trans cisternae and the trans golgi network (tgn). these are read- fig. . how cisternal maturation would work. importantly, this drawing depicts not spatial but time resolution. a given population of cargo molecules moves over time t ^t through the secretory pathway. t : cargo is selected and concentrated at er exit sites with the help of cop ii components. t : cop ii vesicles shed their coat and fuse with retrograde cop i vesicles carrying cis golgi proteins, forming the ¢rst cisterna of the golgi apparatus. t : cargo is modi¢ed by cis golgi proteins, and these enzymes are recycling to fuse with cop ii vesicles; at the same time, retrograde vesicles containing medial golgi enzymes start to join the cargo containing cisterna. t : cargo is modi¢ed by medial golgi proteins, and these enzymes are recycling to fuse with cis golgi cisternae; at the same time, retrograde vesicles containing trans golgi enzymes start to join the cargo containing cisterna. t : cargo is modi¢ed by trans golgi enzymes, and these enzymes are recycling to fuse with medial cisternae; sorting and budding of vesicles result ultimately in the consumption of the trans cisterna. t : vesicles move to lysosomes, secretory granules or the plasma membrane. the grey bars at the cytosolic face of the individual cisternae correspond to the intracisternal matrix. if cargo molecules have a certain a¤nity for retrograde vesicles, they would undergo more than one round of maturation thus explaining di¡erent kinetics for transport from er to plasma membrane. an essential feature of this maturation model is the prediction that golgi resident proteins are more concentrated in retrograde vesicles than they are in the cisternae of the golgi apparatus. furthermore, since new cisternae are only assembled at the er-golgi intermediate compartment, every cisterna has to move forward to provide space for newly forming cisternae. ily discernible morphologically and therefore serve as useful names. however, in the cisternal maturation model, they re£ect early to late maturation stages of the cisternal cargo carriers. these form near er exit sites and move towards the microtubule organising center (mtoc) where they are seen as stacks assembled laterally into a golgi ribbon. the number of cisternae in a given stack is usually ^ but higher numbers have been observed (for example, in nurse cells of the insect oniscus, the golgi stack comprises up to cisternae [ ] ). such variation is permitted in the cisternal maturation model as new cisternae are constantly being formed and old ones disassembled at the level of the tgn. thus the number of cisternae re£ects the number of cargo carriers put through the pathway at the same time. as cargo containing cisternae mature forward, resident proteins need to move in the opposite direction. this is mediated by retrograde transport carriers (rtcs) and requires a highly e¤cient sorting machinery. as there is no need to preserve and maintain compartmental boundaries (there are no pre-existing compartments) a simpler and more dynamic sorting process can be envisaged compared to sorting in the vesicular transport model. however, it is the process of protein sorting which drives cisternal maturation making this a fundamental one. as residents in the cisternal maturation model rely on constant recycling, they would be predicted to display gradient-like distributions when observed at steady state. historically, it was assumed that residents occupy particular (sub-)compartments of the pathway and do not exhibit such gradients (for review, see [ ] ). this dates back almost years when, in , gersh [ ] suggested that`it may be possible to conceive of the golgi apparatus as a framework whose structure is of such nature that it may accommodate certain enzymes or other activities in an orderly manner'. this was based on the fundamental observation that the golgi apparatus is directly involved in the processing of carbohydrates (con¢rmed shortly after by leblond, [ ] ). this was done well before the use of electron microscopy revealing the cisternal-like structures of the golgi stack. nevertheless, it set the stage for a view which prevails even today that cisternae contain separate sets of modifying enzymes. as golgi cisternae di¡er slightly in density, from earlier ones being heavier to later ones being lighter, subcellular fractionation did indeed con¢rm this, demonstrating that enzymatic activities could be separated from each other (although only partially; [ , ] ). this was also shown at the ultrastructural level revealing either the location of a particular enzyme or its product (using lectins) (for a comprehensive review, see [ ] ). the ¢rst resident of the golgi apparatus to be mapped at this level was the glycosylation enzyme l , -galactosyltransferase (galt) which was shown to reside in the trans cisternae [ ] and this was followed by the mapping of k , -sialyltransferase (sia-lylt) to the tgn [ ] . as both galt and sialylt add terminal monosaccharides to glycoproteins with complex n-linked structures, there was clearly a correlation between function and localisation. two other enzymes, l , -n-acetylglucosaminyltransferase i (nagt i) and k , - , mannosidase ii (mann ii), both acting earlier, were subsequently found in medial cisternae [ , ] . as these enzymes were found in more than one cisterna, this was taken as evidence for cisternal duplication. the ¢nding that nagt i and galt were present together in one cisterna but separately in adjacent ones argued that rather than having unique sets of enzymes, cisternae contained unique mixtures [ ] . this observation was later extended to include all four enzymes, nagt i, mann ii, galt and sialylt demonstrating overlapping distributions for all four enzymes [ ] . formation of gradients was also suggested from low but signi¢cant amounts of enzymes in more distal cisternae. this was more obvious when examining the distribution of three enzymes involved in initiation of o-linked glycosylation. n-acetylgalactosaminyltransferase-t , -t and -t were all found throughout the golgi stack as distinct gradients [ ] . this shows that golgi resident enzymes do form gradients across the pathway and that these are unique to the particular protein. the constant recycling of residents via rtcs ensures that these are maintained in the pathway. however, it does not explain how residents exhibit di¡erential gradients in such a way that enzymes such as galt are found later than for example nagt i. to explain this, glick and co-workers [ ] proposed an elegant yet simple hypothesis suggesting that residents have di¡erential abilities to enter rtcs. furthermore, they showed that if this was the case, competition between di¡erent residents is su¤cient to establish and maintain gradients in the context of cisternal maturation. residents with a high ability to enter rtcs would be found in early compartments whereas those with a lower ability would be found in later ones. we suggest that such di¡erential ability is achieved through a combination of two mechanisms: the ¢rst envisages a direct interaction between residents and coat components forming the rtc. this ensures and allows for their incorporation. the second envisages a mechanism which is milieu induced promoting incorporation of residents into rtcs. both mechanisms are related to current sorting models for er and golgi residents and we will discuss these below in the context of cisternal maturation. the surprising ¢nding that the membrane spanning domain (msd) of golgi residents su¤ced to localise reporter molecules to the appropriate part of the pathway suggested that this domain harboured important sorting information (for review, see [ ] ). attention was focussed on this domain and di¡erent models were put forward explaining how an msd would mediate protein sorting (for review, see [ ] ). in one model, bretscher and munro argue that membrane thickness determines how far resident proteins would travel into the pathway. they showed that, on average, resident proteins of the exocytic pathway have signi¢cantly shorter membrane spanning domains compared to those found on the plasma membrane [ ] . furthermore, munro [ , ] showed that the amino acid composition of the msd of golgi residents could be altered to poly-leucins without changing their intracellular distribution but that extending their lengths resulted in plasma membrane localisation. this suggested that length rather than amino acid composition is important. the notion that polar residues were more common in msds of golgi residents further argued that their hydrophobic stretches were shorter than those of plasma membrane proteins. a model was proposed based on a gradual increase of membrane thickness from the er towards the plasma membrane [ ] . such a membrane thickness gradient would be due to a gradual increase in cholesterol and sphingomyelin concentration. indeed, early work suggested the presence of a cholesterol gradient across the exocytic pathway [ ] . in a vesicular transport model, this gradient would be maintained by selective sampling of cholesterol into forward transport vesicles but not into rtcs. in the cisternal maturation model, the gradient would be maintained by exclusion of cholesterol and sphingomyelin from budding rtcs. however, in the context of the latter model, sorting through membrane thickness would not prevent residents to enter rtcs prematurely, and could therefore not explain observed gradients of residents in later parts of the pathway. so far, no correlation has been observed between the length of msds of golgi resident glycosylation enzymes and their corresponding intra golgi localisation (for discussion, see [ ] ). furthermore, upon shortening the cytoplasmic domain of galt, this molecule is subsequently found on the plasma membrane showing that its msd is compatible with this membrane [ ] . also, the high degree of conserved amino acids observed in msds of golgi residents would not be predicted if they merely serve as membrane anchors with a speci¢ed length. rather, as proposed earlier, msds aid in the formation of oligomeric protein complexes [ , ] . this could be possible either through a direct interaction with spe-ci¢c lipids or lipid domains or through protein-protein interaction. arguments in favour of such a scenario come from the notion that golgi residents may be isolated as detergent insoluble complexes, in vitro (see below). however, such complexes do not £oat when subjected to gradient centrifugation. rather, they are readily sedimentable suggestive of complexes with a low ratio of lipid to protein held together by protein-protein interactions. that golgi residents are capable of mediating direct protein-protein interaction comes from several lines of evidence. first, an msd of the cis golgi resident m protein of avian coronavirus infectious bronchitis virus has been shown to directly promote oligomerisation as well as localisation [ , ] . this domain su¤ces to localise the g protein of vesicular stomatitis virus to the cis golgi when expressed as a hybrid protein. when polar residues facing one side of the putative k-helix were mutated, it resulted not only in a loss of intracellular localisation but also in a marked decrease in oligomeric properties of the mutated hybrid protein. this showed, for the ¢rst time, a direct role of msds in oligomerisation of golgi residents and that loss of this property leads to mislocalisation. second, as mentioned above, golgi residents can be readily isolated as detergent insoluble complexes [ , ] . these highly oligomeric structures can be disassembled/reassembled by the addition/removal of salt [ ] . also, full-length galt can be crosslinked into high molecular complexes [ ] . third, the medial enzymes, nagt i and mann ii form hetero oligomers (or kin oligomers) in vivo [ ] . the domain su¤cient in mediating this hetero interaction was shown to reside in the stalk region of nagt i [ ] . similarly, a family of small membrane proteins, the p s, cycling between the cgn and the er, can be isolated as detergent insoluble complexes and as with nagt i and mann ii, they also oligomerise in vivo [ , ] . in the context of cisternal maturation, we suggest that oligomerisation directly promotes incorporation of residents into rtcs and that this event is milieu induced. this would provide an explanation for individual gradients exhibited by di¡erent residents along the pathway. there is a ph as well as a lipid gradient (see above) in the golgi apparatus and these would over time trigger oligomerisation and subsequent incorporation of residents into rtcs. we further suggest that such oligomerisation results in an increased ability of the residents to bind coat components needed to form an rtc through cooperative binding. thus, residents would be distilled at various levels of the pathway depending on their particular ability to oligomerise and directly bind coat components. that such direct binding is possible has been shown for several residents although mainly in the early parts of the pathway (see below). following the`distillation hypothesis' which postulated selective retrieval of residents against a forward £ow of cargo [ ] , er localisation signals were identi¢ed in both soluble [ ] as well as membrane proteins [ , ] . these were shown to confer er localisation when appended to reporter molecules acting as retrieval signals [ , ] . for lumenal er residents, a carboxyterminal kdel motif was shown to bind a receptor [ ] localised to the early part of the golgi apparatus [ , ] . likewise, for type i membrane proteins, the retrieval motif k(x)kxx was thought to bind a receptor oriented towards the cytoplasm. then, a groundbreaking discovery by cosson and co-workers showed that the k(x)kxx motif interacts with coat components of the cop i coatomer directly [ ] . moreover, genetic evidence cemented this ¢nding demonstrating that cop i coatomer functionally mediates retrieval [ ] . as this form of coatomer is found on golgi associated transport vesicles [ ] , cop i vesicles constitute rtcs. the presence of k(x)kxx or related signals on membrane proteins other than er residents allows for the possibility that this motif also acts later in the pathway. both ergic- [ ] and some members of the p protein family display k(x)kxx retrieval motifs and reside in the interface between the er and the golgi apparatus [ , , ] . surprisingly, both ergic- and the p s not only use k(x)kxx related signals for their steady state localisation, but further dissection of their cytoplasmic domains revealed a signal which acts in the opposite direction. both ergic- and p family members interact directly with sec , a component of the cop ii coat involved in budding from the er [ , ] . this suggests that their cytoplasmic domains contain a positive export signal and mutation of this leads to accumulation in the er. thus, the combination of an export and a retrieval signal ensures that ergic- and the p s localise to the interface between er and golgi apparatus through constant cycling. do other golgi resident proteins display similar signals? in terms of er export signals, it appears that golgi glycosylation enzymes do [ ] . the cytoplasmic tails of these proteins also bound sec , probably ensuring their rapid export from the er after synthesis. there is also evidence for retrieval [ ^ ] though future analysis will reveal whether this is mediated by cop i vesicles. . recycling through the er as golgi residents redistribute to the er upon brefeldin a treatment, it was proposed that this fungal metabolite highlights an already existing pathway of limited fusion between the golgi apparatus and the er [ ] . this would allow golgi residents to enter directly into the er and subsequently appear at er exit sites. such a recycling model has also been proposed to explain how golgi redistributes upon depolymerisation of microtubules or mitosis (see above and storrie and yang, this issue). it is di¤cult to envisage how this could account for observed gradients of late glycosylation enzymes, particularly in the context of cisternal maturation. if the steady state distributions of nagt i, mann ii, galt and sialylt are the consequence of constant recycling through the er, their distributions would be expected to appear £atter than their observed sharp gradients. moreover, if glycosylation enzymes were to travel through the er, unless they were inactive, they would act on er resident glycoproteins. as this appears not to be the case, it is hard to reconcile this with a continuous £ow of late golgi residents through the er [ ] . in summary, we favour the model that steady state distribution of resident proteins along the exocytic pathway relies on a combination of oligomerisation and coat binding. this allows for a machinery maintaining residents at various levels of the pathway whilst moving cargo forward in a vectorial manner. international cell biology protein sorting by transport vesicles coat proteins and vesicle budding scale formation in algae three-dimensional electron microscopy: structure of the golgi apparatus the cell ( nd edn progress in unraveling pathways of golgi tra¤c a protein component of the golgi apparatus distribution of periodic acid-reactive carbohydrates in the adult rat early and late functions associated with the golgi apparatus reside in distinct compartments compartmentation of asparagine-linked oligosaccharide processing in the golgi apparatus subcellular organization of glycosylation in mammalian cells immunocytochemical localization of galactosyltransferase in hela cells: codistribution with thiamine pyrophosphatase in trans-golgi cisternae paulson, demonstration of an extensive trans-tubular network continuous with the golgi apparatus stack that may function in glycosylation attachment of terminal n-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the golgi stack cell type-dependent variations in the subcellular distribution of alpha-mannosidase i and ii overlapping distribution of two glycosyltransferases in the golgi apparatus of hela cells mapping the distribution of golgi enzymes involved in the construction of complex oligosaccharides localization of three human polypeptide galnactransferases in hela cells suggests initiation of o-linked glycosylation throughout the golgi apparatus a cisternal maturation mechanism can explain the asymmetry of the golgi stack targeting and retention of golgi membrane proteins golgi localization of glycosyltransferases: more questions than answers cholesterol and the golgi apparatus sequences within and adjacent to the transmembrane segment of k- , -sialyltransferase specify golgi retention an investigation of the role of transmembrane domains in golgi protein retention heterogeneous distribution of ¢lipin^cholesterol complexes across the cisternae of the golgi apparatus membrane protein assembly the membrane spanning domain of l- , -galactosyltransferase speci-¢es trans golgi localization a golgi retention signal in a membrane-spanning domain of coronavirus e protein oligomerization of a membrane protein correlates with its retention in the golgi complex retention of a cis golgi protein requires polar residues on one face of a predicted k-helix in the transmembrane domain isolation of a matrix that binds medial golgi enzymes nilsson, gp l/emp /p protein family members of the cis golgi network bind both cop i and ii coatomer post-translational modi¢cations distinguish cell surface from golgi-retained l , galactosyltransferase molecules. golgi localization involves active retention kinrecognition between medial golgi enzymes in hela cells the role of the membrane-spanning domain and stalk region of n acetylglucosaminyltransferase i in kin retention, kin recognition and structural maintainence of the golgi apparatus in hela cells erv p, a component of cop iicoated vesicles, forms a complex with emp p that is required for e¤cient endoplasmic reticulum to golgi transport the golgi apparatus: two organelles in tandem a c-terminal signal prevents secretion of luminal er proteins short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum identi¢cation of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum recycling of proteins from the golgi compartment to the er in yeast retrieval of transmembrane proteins to the endoplasmic reticulum erd , a yeast gene required for the retention of luminal endoplasmic reticulum proteins, a¡ects glycoprotein processing in the golgi apparatus localization of sed , a putative vesicle targeting molecule, to the cis-golgi network involves both its transmembrane and cytoplasmic domains localization of the lys, asp, glu, leu tetrapeptide receptor to the golgi complex and the intermediate compartment in mammalian cells coatomer interaction with di-lysine endoplasmic reticulum retention motifs coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum rothman, puri¢cation of a novel class of coated vesicles mediating biosynthetic protein transport through the golgi stack ergic- , a membrane protein of the er-golgi intermediate compartment, carries an er retention motif a major transmembrane protein of golgi-derived copi-coated vesicles involved in coatomer binding involvement of the transmembrane protein p in biosynthetic protein transport the recycling of ergic- in the early secretory pathway. ergic- carries a cytosolic endoplasmic reticulum determinant interacting with cop ii a hypothesis on the tra¤c of mg , a medial golgi sialoglycoprotein, from the trans-golgi network to the golgi cisternae warren, evidence for recycling of the resident medial/trans golgi enzyme, n-acetylglucosaminyltransferase i, in ldld cells localization of a yeast early golgi mannosyltransferase, och p, involves retrograde transport rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er retention of membrane proteins by the endoplasmic reticulum key: cord- -qy vj uu authors: pierini, roberto; cottam, eleanor; roberts, rebecca; wileman, thomas title: modulation of membrane traffic between endoplasmic reticulum, ergic and golgi to generate compartments for the replication of bacteria and viruses date: - - journal: semin cell dev biol doi: . /j.semcdb. . . sha: doc_id: cord_uid: qy vj uu several bacteria and viruses remodel cellular membranes to form compartments specialised for replication. bacteria replicate within inclusions which recruit membrane vesicles from the secretory pathway to provide nutrients for microbial growth and division. viruses generate densely packed membrane vesicles called viroplasm which provide a platform to recruit host and viral proteins necessary for replication. this review describes examples where both intracellular bacteria (salmonella, chlamydia and legionella) and viruses (picornaviruses and hepatitis c) recruit membrane vesicles to sites of replication by modulating proteins that control the secretory pathway. in many cases this involves modulation of rab and arf gtpases. many intracellular pathogens replicate in specialised compartments in cells. for bacteria these are generally described as vacuoles or inclusions, and for viruses they are called virus factories or viroplasm (fig. ) . the formation of replication sites can involve extensive modification of membrane compartments. early changes probably reflect the mobilisation of cellular defences against infection such as phagocytosis and autophagy [ , ] while later stages indicate the steps taken by the pathogen to avoid cellular defences * corresponding author. tel.: + . e-mail address: t.wileman@uea.ac.uk (t. wileman). and generate a compartment specialised for replication. intracellular bacteria remain within membrane-bound vacuoles to avoid delivery to lysosomes and then recruit vesicles from the secretory pathway to provide nutrients necessary for microbial growth and cell division. many viruses generate densely packed membrane vesicles to shield them from recognition by cellular defence pathways that recognise double-stranded rna, and at the same time the membranes provide a platform to recruit viral and host proteins required for replication [ ] [ ] [ ] . this review describes how recent work on intracellular bacteria such as salmonella, chlamydia and leigionella draws parallels with studies on (+) strand rna viruses where microbial proteins recruit membranes by modulating proteins that control the secretory pathway. in many cases this involves modulation of rab and arf gtpases. after reaching a peri-golgi position, the vacuoles generate long membrane filaments (sifs) and initiate replication. legionella and chlamydia escape interaction with the endosomal/lysosomal pathway. chlamydia inclusions migrate along microtubules and replicate next to a fragmented golgi. legionella-containing vacuoles attach to er and golgi-derived vesicles and replication begins following fusion of vacuoles with the er. picornaviruses and hepatitis c replicate within perinuclear arrays of densely packed membrane vesicles called viroplasm. er: endoplasmic reticulum; mvb: multivesicular bodies; scvs: salmonella-containing vacuoles; sif: salmonella induced filaments; sp: spacious phagosome; lcv: legionella-containing vacuole. the rab and arf families of small gtpases act as molecular switches which regulate the formation, docking and fusion of membrane vesicles that carry cargo from one membrane compartment to another (fig. ) . vectorial transport through the secretory pathway is maintained because different organelles recruit different gtpases and specific tethering proteins able to direct interactions between transport vesicles and acceptor membranes. vesicle fusion is consolidated by pairing between snare proteins [ , ] . the arf gtpase regulates the formation of copi coated vesicles which form at golgi membranes to carry cargo back to the er. copii coated vesicles assemble at the endoplasmic reticulum (er) and carry proteins and lipids to the ergic and golgi where docking and fusion are con-trolled by rab and golgi tethering proteins such as p , gm and golgin- . the functions of other rab proteins are described when they appear in the text. salmonella enterica enters the cell within a membrane compartment known as the spacious phagosome (sp), which then shrinks to enclose one or two bacteria within salmonella-containing vacuoles (scvs) [ ] [ ] [ ] . early sp and scv maturation result in recruitment of rab and several rab effector proteins which normally con- translocation to membranes requires membrane-bound gdi displacement factors (gdfs) which release the gtpase and expose a c-terminal lipid group for membrane binding. guanine nucleotide exchange factors (gefs) exchange gdp for gtp and allow the activated gtpase to bind effector proteins which facilitate vesicle budding and fusion. the cycle is completed by gtp-hydrolysis, which requires gtpase activating proteins (gaps), followed by extraction of the gdp-bound enzyme from the membrane by gdi. trol endocytosis. sorting nexins may facilitate shrinkage of the sp [ ] during formation of the scv, while rab effectors such as early endosome antigen prime the scv for progress along the endocytic pathway. scv lose rab and accumulate rab , lamp and acidify. rab recruits rab -interacting lysosomal protein (rilp) which links the scv to the dynein motor protein allowing movement to the golgi and mtoc [ ] . this, coupled with acidification of the vacuole, activates secretion of bacterial salmonella pathogenicity island (spi- ) proteins which are needed for continued golgi positioning and replication. the pip b and sifa proteins recruit host kinesin interacting protein skip to the scv to displace kinesin and inhibit outward movement [ , ] . sifa may also impair the rab -rilp interaction to release dynein [ ] . ssef and sseg locate to the surface of the vacuole where they may tether the vacuoles to the golgi [ , ] . golgi positioning may also involve sipa and sifa proteins, actin [ ] , and recruitment of myosin ii [ ] . golgi positioning appears to be crucial for replication [ ] and is thought to facilitate the supply of nutrients from golgi-derived vesicles. the exact pathway followed by the vesicles remains to be determined. vacuoles recruit different rabgtpases during maturation [ ] showing complex interaction with the secretory pathway. vesicles leaving the trans-golgi network (tgn) accumulate next to the scv, but do not appear to fuse [ ] , making it unclear whether they deliver nutrients. interestingly, long thin tubules of membrane called salmonella induced filaments (sifs) containing endosomal and lysosomal markers extend from the scvs soon after positioning at the golgi. sifs require secretion of spi- proteins, ssef and sseg, and extend towards the cell periphery along microubules where they recruit rab and rab and take up fluid phase endocytic markers [ ] [ ] [ ] [ ] [ ] . sifs may provide nutrients to bacteria from outside the cell by fusing with vesicles trafficking between late endosomes and the golgi. chlamydia invades the cell as an infectious, but replication defective, elementary body (eb) and differentiates into a replicative form called the reticulate body (rb), within a membrane compartment called an "inclusion". chlamydia does not interact with the endosomal/lysosomal pathway [ ] but, as seen for salmonella, the inclusions recruit dynein and migrate along microtubules to a peri-golgi position [ ] , and again peri-golgi positioning at the mtoc is required for replication. inclusions receive sphingomyelin and cholesterol from exocytic vesicles that would normally deliver lipids from the tgn to the plasma membrane [ ] [ ] [ ] [ ] and can also gain lipids directly from lipid storage organelles called lipid droplets. this involves the bacterial lda protein which may link lipid droplets to the outer surface of the inclusion [ ] . chlamydia infection also results in proteolysis of tethering protein golgin by caspases leading to fragmentation of the golgi into ministacks. inhibition of golgi fragmentation reduces replication and also prevents the delivery of lipids to the inclusion suggesting that fragmented golgi ministacks provide vesicles able to fuse with the vacuole [ ] . non-lipid nutrients can also be delivered to chlamydia inclusion by endocytosis and fusion with multivesicular bodies (mvb) [ ] . chlamydia effector proteins called 'integral inclusion membrane' (inc) proteins recruit several rabgtpases to the inclusions [ ] . mature inclusions recruit rab and rab [ , ] , which are generally present in recycling endosomes and could interact with incoming mvbs. inclusions recruit rab , which may activate tethering and subsequent fusion with golgi-derived vesicles or ministacks. rab and rab , which are involved in golgi trafficking, are associated with inclusions in a species-dependent manner [ ] . rab recruitment requires the gtp-bound form suggesting that the inc proteins act as rab effector proteins. inc protein cpn , has coiled coil domains that share sequence similarity with rab effectors such as the tethering proteins golgin and golgi matrix protein gm , and interacts with rab , rab and rab [ ] . whether cpn plays a role in golgi fragmentation seen during infection has not been tested. inca proteins such as ct have snare motifs and interact directly with host snares that facilitate vesicle fusion [ ] . the glutamate residue in the snare-like motif of inca suggests similarity with q-snares that would interact with r-snares. this is consistent with co-precipitation of vamp , vamp and vamp with inca, and location of vamp in the inclusion membrane. the precise role the snare homologues play in inclusion formation is not known. they interact with snares involved in endosome trafficking and may aid recruitment of mvbs to the inclusion. legionella pneunomophila is an intracellular bacterium of amoeba but can infect humans following inhalation of aerosols from contaminated water and air cooling systems. inhaled bacteria infect alveolar macrophages and become surrounded by a membrane compartment called the legionella-containing vacuole (lcv), which avoids interactions with the endosomal/lysosomal pathway [ ] . the lcvs quickly recruit arf and rab gtpases, and associate with vesicles containing er and ergic markers, suggesting recruitment of vesicles trafficking between er and golgi compartments. membrane vesicles remain associated with the lcv for several hours during which time the lcvs lose rab and arf [ , ] . the vesicles tethered to the lcv [ ] eventually fuse with the vacuole and deliver er markers, and this is coincident with the onset of replication. delivery is inhibited by brefeldin-a, and a dominant negative sar [h g] gtpase slows bacterial replication, suggesting involvement of copi and copii transport vesicles [ ] . rabs are recruited to the lcv by bacterial effector proteins drra (also called sidm) and lepb [ ] . drra first acts as a rab specific gdf recruiting rab onto the cytosolic face of the lcv membrane [ , ] . drra then acts as a rab-gef protein promoting gdp-gtp exchange to preserve rab in its active form [ , ] . the lepb protein binds the lcv at later times and is also present on mature lcvs that have acquired er markers. lepb binds activated rab -gtp and acts as gap [ ] leading to release of rab from the vacuole and this is coincident with fusion of the vacuole with er-derived vesicles. drra and lepb show exclusive binding to rab , but dot/icm effector lida binds rab , rab and rab [ ] . lida does not have gap or gef activity and cannot affect gtp hydrolysis, but works with drra/sidm to recruit rab to vacuoles [ ] . the presence of arf and rab could facilitate fusion with both er and golgi-derived membranes. rab may interact with effector proteins such as p or the gm /grasp complex would normally tether copi and copii vesicles to the cis-golgi. silencing experiments suggest that the trapp tethering complex, which facilitates docking of copii vesicles to ergic and golgi membranes, and sec b, which is a v-snare found in er to golgi transport vesicles are both required for replication [ ] [ ] [ ] . these results suggest that copii vesicles deliver the host factors required to sustain microbial growth and cell division within the er-derived vacuole. the (+) strand rna viruses produce replicase proteins with rna-dependent rna polymerase and helicase activity which are targeted to the cytoplasmic face of the er by non-structural proteins with membrane binding motifs. this invariably results in the generation of densely packed membrane vesicles called viroplasm. work in this area has concentrated on understanding how these vesicles are formed, and how their use as replication sites impacts on the secretory pathway. multiple interactions between replicase proteins probably induce the membrane curvature required to generate vesicles in a way that parallels virus budding from membranes [ , ] . virus replication can, however, slow secretion suggesting that the formation of viroplasm may divert vesicles normally involved transport between the er and golgi into the replication complex. as described above for bacteria, this can involve modulation of small gtpases. a search for a link between virus replication and membrane trafficking proteins was stimulated by the observation that bfa blocks replication of enteroviruses such as poliovirus and coxsackieviruses [ ] . bfa inhibits arf-gtp exchange proteins (arf-gefs) and reduces the arf -gtp needed to generate copi coats in the golgi. inhibition of replication by bfa has led to a model where viral activation of arf-gefs would recruit arf , and arf effector proteins, needed to recruit membranes into replication compartments [ ] . this is supported by genome-wide interference screens where copi proteins are important for replication of both animal and plant picornaviruses [ , ] , and is consistent with the increase in arf-gtp levels seen in cells following poliovirus infection. moreover, arf- and arf-gef proteins gbf and big / are recruited into replication complexes [ , ] , and poliovirus replication is inhibited by sirna for gbf [ ] . sequestration of arf- into the replication complex would also explain the block in secretion seen in infected cells [ ] . for poliovirus, secretion is blocked by the a protein which tethers the replicase to er membranes. a does not have intrinsic arf-gef activity but the a protein recruits arf-gefs such as gbf and big / to membranes, and a activates arf-gef activity 'in vitro' [ ] . this provides an explanation for inhibition of poliovirus replication by bfa, and how the sequestration of arf and arf-gefs into replication sites, could block secretion. evidence that this generates densely packed vesicles is less clear. a alone cannot generate the vesicles, and recruitment of arf-gefs by a is not essential for replication [ ] . moreover, bfa does not prevent formation of densely packed vesicles by poliovirus, so vesicle formation does not require activated arf-gef proteins [ ] . an alternative mechanism of action for a is provided from coxsackievirus, which is also a bfa-sensitive enterovirus. the coxsackievirus a protein inhibits arf-gef activity, and reduces arf-gtp [ ] [ ] [ ] and may inhibit er to golgi transport by stabilizing arf -gdp-gbf complexes which would in turn reduce the supply of arf -gtp for copi coats. a may also slow secretion by inactivating lis , a component of the dynein-dynactin motor complex [ ] and contribute to microtubule-dependent disruption of the golgi apparatus seen during poliovirus infection [ ] . these experiments explain how a would slow secretion, but again do not provide a mechanism for generation of the densely packed vesicles, or paradoxically, explain why the virus would be sensitive to bfa. several picornaviruses families are resistant to bfa, and their a proteins do not slow er to golgi transport [ , , ] . for these viruses copii coated vesicles may provide membranes for replication [ ] [ ] [ ] [ ] while other studies implicate a role for autophagosomes [ ] . in the case of foot-and-mouth disease virus, secretion is blocked by the er-tethered bc protein [ , ] , rather than a, and although less well studied, the b and bc of enteroviruses also slow secretion when expressed alone in cells [ , ] . these proteins can therefore affect membrane traffic and for c this may involve binding to er membrane protein reticulon , which is involved in er to golgi trafficking and is required for replication [ ] . hepatitis c virus replication occurs within a web of er membranes. the ns a protein is anchored to the cytoplasmic face of the web and recruits the rna-dependent rna polymerase, ns b. both ns a and ns b bind vamp associated proteins (vaps) [ , ] . ns b binds to a coiled coil domain (ccd) in vap that resembles the ccd of snare proteins. silencing vap-b reduces the stability of ns b, and depletion of vap-b from cell free assays reduces rna replication. current models suggest that a vapa/b heterodimer forms a scaffold that supports the replication complex. vap proteins also bind to snares involved in er to golgi transport, making it is possible that the vap scaffold also facilitates membrane fusion to generate the membrane web from the er. this may be true for other (+) strand rna viruses such as norwalk virus where membranebound nsp also binds vap-a [ ] . ns a also binds tbc d , a rab -gap protein, and both tbc d and ns a co-localise with er markers. tbc d activates rab gtp hydrolysis, and since depletion of tbc d and rab greatly reduce hepc replication [ , ] it is likely that activation of rab -gtp is in some way involved in virus infection. local blockade of rab could, for example direct transport vesicles to make membrane webs. rab is also recruited to the replication complex [ ] by binding to ns b, and virus-induced membranes recruit rab effectors eea , rabaptin and rab suggesting the web may fuse with endosomes. a functional role for rab in viral genome replication is suggested because dominant negative rab reduces replication of replicons, and partial silencing of rab reduces synthesis of viral rna. the studies described above set the stage or further work where it will be important to analyse interactions between host and microbial proteins at a structural level, and see how these interactions contribute to virulence and/or immune evasion. the work also provides a precedent for studies on other pathogens that use membrane compartments for replication. brucella for example, traffic through lysosomes before fusing with the er [ ] , and coxiella replicate in a spaceous parasitophorous vacuole formed from secondary lysosomes [ ] . recent work on coronaviruses draws parallels with poliovirus where sars-cov replicates within densely packed membranes [ ] , and mouse hepatitis virus shows sensitivity to bfa and requires gbf mediated arf activation [ , ] . disruption or use of the golgi is not restricted to the (+) strand rna viruses. the negative strand rna bunyaviruses replicate in the golgi apparatus where they form tubes that contain replicase proteins [ ] . the large dna viruses such as fowlpox encode homologues of snap proteins involved in snare pairing [ ] , while african swine fever virus replicates at the mtoc, slows secretion, and causes golgi fragmentation and microtubule-dependent loss of the tgn [ ] . asfv also encodes a multigene family of proteins with c-terminal kedl sequences able to interfere with kdel-receptor mediated er to golgi trafficking and redistribute er proteins to the ergic [ ] . in all these cases the consequences of modulating the early secretory pathway in the context of virus survival and/or pathogenesis remain to be discovered. cellular autophagy: surrender, avoidance and subversion by microorganisms aggresomes and autophagy generate sites for virus replication a guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication modification of intracellular membrane structures for virus replication wrapping things up about virus replication rabs and their effectors: achieving specificity in membrane traffic coats, tethers, rabs, and snares work together to mediate the intracellular destination of a transport vesicle membrane dynamics and spatial distribution of salmonella-containing vacuoles salmonella-containing vacuoles: directing traffic and nesting to grow the salmonella-containing vacuole: moving with the times sorting nexin- defines an early phase of salmonella-containing vacuole-remodelling during salmonella infection salmonella impairs rilp recruitment to rab during maturation of invasion vacuoles the intracellular fate of salmonella depends on the recruitment of kinesin the salmonella effector protein pipb is a linker for kinesin- functional dissection of ssef, a type iii effector protein involved in positioning the salmonella-containing vacuole the spi- type iii secretion system restricts motility of salmonella-containing vacuoles salmonella spi effector sipa persists after entry and cooperates with spi effector to regulate phagosome maturation and intracellular replication role for myosin ii in regulating positioning of salmonella-containing vacuoles and intracellular replication sseg, a virulence protein that targets salmonella to the golgi network a network of rab gtpases controls phagosome maturation and is modulated by salmonella enteritica serovar typhimurium intracellular salmonella enterica redirect exocytic transport processes in a salmonella pathogenicity island -dependent manner salmonella trafficking is defined by continuous dynamic interactions with the endolysosome system dynamic behaviour of salmonella-induced membrane tubules in epithelial cells dynamic remodelling of the endosomal system during formation of salmonella-induced filaments by intracellular salmonella enterica restricted fusion of chlamydia trachomatis vesicles with endocytic compartments during the initial stages of infection chlamydia trachomatis uses host cell dynein to traffic to the microtubule-organizing center in a p dynamitinindependent process lipid metabolism in chlamydia trachomatis-infected cells: directed trafficking of golgi-derived sphingolipids to the chlamydial inclusion the chlamydial inclusion: escape from the endocytic pathway golgi-dependent transport of cholesterol to the chlamydia trachomatis inclusion the chlamydial inclusion preferentially intercepts basolaterally directed sphingomyelin-containing exocytic vacuoles cytoplsmic lipid droplets are translocated into the lumen of the chlamydia trachomatis parisitophorous vacuole chlamydia causes fragmentation of the golgi compartmenmt to ensure reproduction trafficking from cd -positive late endocytic multivesicular bodies is essential for intracellular development of chlamydia trachomatis chlamydia effector proteins and new insights into chlamydial cellular microbiology rab gtpases are recruited to chlamydial inclusions in both a species-dependent and speciesindependent manner the gtpase rab interacts with chlamydia trachomatis inclusion membrane protein ct chlamydia pneumoniae inclusion membrane protein cpn interacts with multiple rab gtpases snare protein mimicry by an intracellular bacterium legionella pneumophila dota protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites a bacterial guanine nucleotide exchange factor activates arf on legionella phagosomes attachment and fusion of endoplasmic reticulum with vacuoles containing leigonella pneumophila targeting of host rab gtpase function by the intravacuolar pathogen legionella pneumophila legionella pneumophila proteins that regulate rab membrane cycling a bifunctional bacterial protein links gdi displacement to rab activation the legionella pneumophila effector protein drra is a rab guanine nucleotideexchange factor legionella subvert the functions of rab and sec b to create a replicative organelle rna interference analysis of legionella in drosophila cells: exploitation of early secretory apparatus dynamics legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system parallels among positive strand rna viruses, reverse transcribing viruses and double strand rna viruses inhibition of poliovirus rna synthesis by brefeldin a involvement of cellular membrane traffic proteins in poliovirus replication copi activity coupled with fatty acid biosynthesis is required for viral replication identification of essential host factors affecting tombusvirus rna replication based on the yeast tet promoters hughes collection poliovirus proteins induce membrane association of gtpase adp ribosylation factor hijacking components of the cellular secretory pathway for replication of poliovirus rna a critical role of a cellular membrane traffic protein in poliovirus rna replication inhibition of cellular protein secretion by poliovirus proteins b and a a viral protein that blocks arf- mediated cop-i assembly by inhibiting the guanine nucleotide exchange factor gbf effects of picornavirus a proteins on protein transport and gbfidependent cop-i recruitment molecular determinants of the interaction between coxsackievirus protein a and guanine nucleotide exchange factor gbf poliovirus protein a binds and inactivates lis , causing block of membrane protein trafficking and deregulation of cell division poliovirus infection blocks ergic-to-golgi trafficking and induces microtubule-dependent disruption of the golgi complex inhibition of cellular protein secretion by picornaviral a proteins effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: bc but not a blocks endoplasmic reticulum-to-golgi transport differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae cellular copii proteins are involved in production of the vesicles that form the poliovirus replication complex intracellular location and translocation of silent and active poliovirus replication complexes biogenesis of cytoplasmic membranous vesicles for plant potyvirus replication occurs at endoplasmic reticulum exit sites in a copi-and copii-dependent manner subversion of cellular autophagosomal machinery by rna viruses inhibition of the secretory pathways by foot-and-mouth disease virus bc protein is reproduced by coexpression of b with c, and the site of inhibition is determined by the subcellular localisation of c inhibition of protein trafficking by coxsackievirus b : multiple viral proteins target a single organelle reticulon binds the c protein of enterovirus and is required for viral replication hepatitis c virus rna polymerase and ns a complex with a snare-like protein human vap-b is involved in hepatitis c virus replication through interaction with ns a and ns b norwalk virus nonstructural protein p forms a complex with the snare regulator vap-a and prevents cell surface expression of vesicular stomatitis virus g protein tbc d is a rab gtpase-activating protein that mediates hepatitis c virus replication a rab-gap tbc domain protein binds hepatitis c virus ns a and mediates virus replication participation of rab , an early endosome protein, in hepatitis c virus rna replication machinery brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment lounging in a lysosome: the intracellular lifestyle of coxiella burnetii sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum mouse hepatitis coronavirus rna replication depends on gbf -mediated arf activation localization and membrane topology of coronavirus nonstructural protein : involvement of the early secretory pathway in replication the unique architecture of bunyamwera virus factories around the golgi complex fowlpox virus encodes nonessential homologs of cellular alpha-snap, pc- , and an orphan human homolog of a secreted nematode protein african swine fever virus causes microtubule-dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane the subcellular distribution of multigene family proteins of african swine fever virus is determined by differences in c-terminal kdel endoplasmic reticulum retention motifs key: cord- - p rsf authors: de haan, cornelis a.m.; rottier, peter j.m. title: molecular interactions in the assembly of coronaviruses date: - - journal: adv virus res doi: . /s - ( ) - sha: doc_id: cord_uid: p rsf this chapter describes the interactions between the different structural components of the viruses and discusses their relevance for the process of virion formation. two key factors determine the efficiency of the assembly process: intracellular transport and molecular interactions. many viruses have evolved elaborate strategies to ensure the swift and accurate delivery of the virion components to the cellular compartment(s) where they must meet and form (sub) structures. assembly of viruses starts in the nucleus by the encapsidation of viral dna, using cytoplasmically synthesized capsid proteins; nucleocapsids then migrate to the cytosol, by budding at the inner nuclear membrane followed by deenvelopment, to pick up the tegument proteins. viruses are multimolecular assemblies that range from small, regular, and simple to large, pleiomorphic, and complex. they consist of virus-specified proteins and nucleic acids and, in the case of enveloped viruses, of host-derived lipids. in infected cells the assembly of these different components into virions occurs with high precision amidst a huge background of tens of thousands of host compounds. two key factors determine the efficiency of the assembly process: intracellular transport and molecular interactions. directional transport ensures the swift and accurate delivery of the virion components to the cellular compartment(s) where they must meet and form (sub)structures. some viruses achieve this goal relatively simply when genome production occurs in close proximity to the virion assembly site (e.g., picornaviruses). many viruses, however, have evolved more elaborate strategies. this is illustrated, for instance, by the a-herpesviruses. assembly of these viruses starts in the nucleus by the encapsidation of viral dna, using cytoplasmically synthesized capsid proteins; nucleocapsids then migrate to the cytosol, by budding at the inner nuclear membrane followed by deenvelopment, to pick up the tegument proteins. subsequently, the tegumented capsids obtain their final envelope by budding into vesicles of the trans-golgi network (tgn), where the viral envelope proteins have congregated after their synthesis in the endoplasmic reticulum; the assembled viral particles are finally released by fusion of the virion-containing vesicles with the plasma membrane. to achieve their transport goals viruses provide their components with address labels that can be read by the transport machinery of the cell. once brought together, formation of the viral (sub)structures is governed and driven by their interactions. whereas the assembly of nonenveloped viruses is generally restricted to the cell cytoplasm, although often in association with membranes, that of enveloped viruses involves multiple cellular compartments, as exemplified already for herpesviruses. this review deals with the assembly of coronaviruses. we first describe what is known about the structure of the coronavirion and about the relevant properties of the structural components. we summarize the limited ultrastructural information about coronavirus assembly and budding. the main body of the review describes the interactions between the different structural components of the viruses and discusses their relevance for the process of virion formation. this review has a limited scope; for further information about other aspects of coronavirus biology the reader is referred to other reviews (de vries et al., ; enjuanes et al., ; gallagher and buchmeier, ; holmes, ; holmes et al., ; lai, ; lai and cavanagh, ; lai et al., ; masters, ; perlman, ; rossen et al., ; sawicki and sawicki, ; siddell, ; ziebuhr et al., ) . coronaviruses are a group of enveloped, plus-stranded rna viruses presently classified as a genus, which, together with the genus torovirus, constitutes the family coronaviridae. these viruses are grouped with two other families, the arteriviridae and the roniviridae, into the order nidovirales. this classification is not based on structural similarities-in fact, structure and composition of the viruses from the different families differ significantly-but on common features of genome organization and gene expression (de vries et al., ; lai and cavanagh, ) . coronaviruses infect a wide variety of mammals as well as avian species (table i ). in general they cause respiratory or intestinal infections, but some coronaviruses can also infect other organs (liver, kidney, and brain). until recently, these viruses were mainly of veterinary importance. this situation has changed quite dramatically because of the emergence of severe acute respiratory syndrome- (sars-cov) in late , which emphasized the potential relevance of coronaviruses for humans. on the basis of antigenic and genetic relationships the coronaviruses have been subdivided into three groups (table i) ; the taxonomic position of sars-cov has not been formally assigned. coronavirus particles have a typical appearance under the electron microscope. by the characteristic, approximately -nm-long spikes that emanate from their envelope the viruses acquire the solar image to which they owe their name (fig. ) . the -to -nm virions have a pleiomorphic appearance that, whether artifact or real, reflects a pliable constellation, a feature that has severely hampered the ultrastructural analysis of these viruses. hence, our knowledge about the structure of coronaviruses is still rudimentary. the schematic representation of the current model of the coronavirion drawn in fig. is based on morphological and biochemical fig . electron micrographs of mouse hepatitis virus strain a (mhv-a ) virions without (a) and with (b) the hemagglutinin-esterase (he) envelope protein (viruses kindly provided by r. de groot, virology division, utrecht university, the netherlands; image courtesy of j. lepault, vms-cnrs, gif-sur-yvette, france). large, club-shaped protrusions consisting of spike (s) protein trimers give the viruses their corona solis-like appearance. viruses containing the he protein display a second, shorter fringe of surface projections in addition to the spikes. (c) schematic representation of the coronavirion. the viral rna is encapsidated by the nucleocapsid (n) protein forming a helical ribonucleoprotein (rnp), which is in turn part of a structure with spherical, probably icosahedral, configuration. the nucleocapsid is surrounded by a lipid bilayer in which the s protein, the membrane glycoprotein (m), and the envelope protein (e) are anchored. in addition, some group coronaviruses contain the he protein in their lipid envelope as illustrated on the right side of the particle. observations. as this picture illustrates, the particle consists of a nucleocapsid or core structure that is surrounded by a lipid envelope. anchored in this envelope are the three canonical coronavirus membrane proteins: the membrane (m) protein, the envelope (e) protein, and the spike (s) protein. viruses from group have an additional, fourth membrane protein, the hemagglutinin-esterase (he) protein. as a consequence these viruses display a second, shorter ( nm) fringe of surface projections in addition to the spikes (fig. b) (bridger et al., ; king et al., ; sugiyama and amano, ) . the ribonucleoprotein (rnp) core contains one copy of the viral genomic rna. this rna is packaged into a helical structure by multiple copies of nucleocapsid protein (n). size estimations of the flexible cylindrical structures varied quite considerably, ranging between and nm in diameter and up to . mm in length (see laude and masters, ) . the ribonucleoprotein helix appears in turn to be contained within a spherical, probably icosahedral, configuration as indicated by various ultrastructural approaches using purified transmissible gastroenteritis virus (tgev) and mouse hepatitis virus (mhv) (risco et al., (risco et al., , . the molar ratio of the major structural proteins, s:n:m, has been variously estimated to be approximately : : (sturman et al., ) , : : (cavanagh, a) , : : (hogue and brian, ) , and : : (liu and inglis, ) , although an m:n molar ratio of has also been reported (escors et al., a) . the s:he molar ratio was estimated to be (hogue and brian, ) . the e protein is only a minor virion component and was calculated to occur in infectious bronchitis virus (ibv), tgev, and mhv virions at a rate of approximately , , and molecules per particle, respectively (godet et al., ; liu and inglis, ; vennema et al., ) . the lipid composition of coronaviral envelopes has been studied only to a limited extent. comparison of the phospholipid composition of mhv with that of its host cell showed increased levels of sphingomyelin, phosphatidylserine, and phosphatidylinositol and a decrease in the level of phosphatidylethanolamine (van genderen et al., ) . whether the lipid composition of mhv is an accurate reflection of its budding compartment or whether certain lipids become enriched in the virus during budding is not known. what follows is a general description of the individual virion components and their properties. this description is by no means complete as it is restricted to the information that is of relevance to the main topic of this review. for a schematic representation of the coronavirus life cycle see fig. . the coronavirus life cycle. the replication cycle starts with attachment of the virion by its s protein, that is, through the s subunit thereof, to the receptors on the host cell. this interaction leads to fusion of the virus envelope with a cellular membrane, coronaviruses contain a single-stranded positive-sense rna genome of some to kilobases, the largest nonsegmented viral rna genomes known. the rna has a -terminal cap and a terminal poly(a) tract. both genomic termini contain untranslated regions (utrs) of some - nucleotides that harbor several cis-acting sequences and structural elements functioning in viral replication and transcription. coronaviruses have a typical genome organization characterized by the occurrence of a distinctive set of genes that are essential for viability and occur in a fixed order: -polymerase ( pol)-s-e-m-n- (fig. ) . the pol gene comprises approximately twothirds of the genome, from which it is translated directly. it encodes two large precursors (pol a and pol ab), the many functional cleavage products of which are collectively responsible for rna replication and transcription (for reviews on coronavirus transcription and replication see de vries et al., ; lai, ; lai and cavanagh, ; lai et al., ; sawicki and sawicki, ; ziebuhr et al., ) . the more downstream pol b gene is translated by translational readthrough, for which the s subunit is responsible. from the genomic rna that is released by disassembly of the incoming particle the pol a and pol b genes are translated, resulting in the production of two large precursors (pol a and pol ab), the many cleavage products of which collectively constitute the functional replication-transcription complex. genes located downstream of the pol b gene are expressed from a -coterminal nested set of subgenomic (sg) mrnas, each of which additionally contains a short leader sequence derived from the end of the genome (shown in red). transcription regulatory sequences (trss) located upstream of each gene serve as signals for the transcription of the sgrnas. the leader sequence is joined at a trs to all genomic sequence distal to that trs by discontinuous transcription, most likely during the synthesis of negative-strand sgrnas. in most cases, only the -most gene of each sgrna is translated. multiple copies of the n protein package the genomic rna into a helical structure in the cytoplasm. the structural proteins s, m, and e are inserted into the membrane of the rough endoplasmic reticulum (rer), from where they are transported to the er-to-golgi intermediate compartment (ergic) to meet the nucleocapsid and assemble into particles by budding. the m protein plays a central role in this process through interactions with all viral assembly partners. it gives rise to the formation of the basic matrix of the viral envelope generated by homotypic, lateral interactions between m molecules, and it interacts with the envelope proteins e, s, and he (if present), as well as with the nucleocapsid, thereby directing the assembly of the virion. virions are transported through the constitutive secretory pathway out of the cell-the glycoproteins on their way being modified in their sugar moieties, whereas the s proteins of some but not all coronaviruses are cleaved into two subunits by furin-like enzymes (see text for references). using a ribosomal frameshift mechanism for which a "slippery" sequence and a pseudoknot structure are required. the genes located downstream of pol b are expressed from a -coterminal nested set of subgenomic (sg) rnas, each of which additionally contains a short leader sequence derived from the end of the genome. transcription regulatory sequences (trss) located upstream of each gene serve as signals for transcription of the sgrnas. the leader sequence is joined at a trs to all genomic sequence distal to that trs by discontinuous transcription, most likely during the synthesis of negative-strand sgrnas (sawicki and sawicki, ) . besides the characteristic genes encoding the replicative and structural functions, coronaviruses have a more variable collection of additional genes that are located in two clusters in the -terminal onethird of the genome. the genes differ distinctly in their nature and genomic position among the coronavirus groups, but they are specific for each group. these so-called group-specific genes appear not to be essential as shown by the occurrence of natural mutants defective in some of them (brown and brierley, ; herrewegh et al., ; kennedy et al., ; luytjes, ; shen et al., ; vennema, ; vennema et al., ; woods, ) and by the observed viability of engineered deletion mutants lacking some or all of these genes (de haan et al., b; fischer et al., ; haijema et al., ; ortego et al., ; sola et al., ) . except for the group -specific he protein and, possibly, the poorly characterized i protein (fischer et al., ; senanayake et al., ) , the latter encoded by an open reading frame completely contained within the n gene, the group- the single-stranded, positive-sense rna genome contains -and -terminal untranslated regions (utrs) with a -terminal cap and a -terminal poly(a) tract. the leader sequence (l) in the utr is indicated. all coronaviruses have their essential genes in the order -pol-s-e-m-n- . the pol a and pol b genes comprise approximately twothirds of the genome. the more downstream pol b gene is translated by translational readthrough, using a ribosomal frameshift mechanism. transcription regulatory sequences (trss) located upstream of each gene, which serve as signals for the transcription of the subgenomic (sg) rnas, are indicated by circles. the genes encoding the structural proteins he, s, e, m, and n are specified. gray boxes indicate the accessory, group-specific genes, in the case of group coronaviruses genes a, he, , a, and i. specific proteins do not appear to occur in virions. although their functions have not yet been resolved, mutant studies indicate that they play important roles in the interaction of coronaviruses with their host (de haan et al., b; fischer et al., ; haijema et al., ; ortego et al., ) . the n protein is the most abundantly expressed viral protein in infected cells (for a review, see laude and masters, ) . its size varies considerably between viruses from different groups ( - amino acids, i.e., molecular masses ranging between and kda), n proteins from group coronaviruses (table i) being the largest. whereas the amino acid sequences of n proteins are quite similar within the groups, the homology between proteins from different coronavirus groups is rather limited ( - %). an exception is a region spanning about residues within the amino-terminal one-third of the n molecule, where high sequence identity has been conserved across the different groups. despite the overall sequence variation the n proteins have a number of common characteristics. consistent with their role as nucleic acidbinding proteins they are all highly basic because of the abundance of arginine and lysine residues. these are clustered mainly in two nearby regions in the middle of the molecules. the abundance of basic residues is reflected in the calculated overall isoelectric points of the n proteins, the values of which are in the range of . - . . these numbers are the more significant in view of the acidic nature of the very carboxyterminal domain; pi values ranging from . to . were calculated for the terminal residues (parker and masters, ) . another general characteristic of the n proteins is their high content ( - %) of serine residues, which are potential targets for phosphorylation. although these residues occur all over the n molecule, their relative abundance within the first of the two basic regions is notable. little is known about the three-dimensional structure of the n protein. of the sars-cov n protein the amino-terminal domain (residues - ) was analyzed by nuclear magnetic resonance spectroscopy. it appeared to consist of a five-stranded b sheet with a folding distinct from that of other rna-binding proteins (huang et al., ) . in coronavirus-infected cells the n protein can often be detected as one major and several minor forms, the latter polypeptides having a slightly lower molecular weight. the major species appeared to comigrate in gels with the n protein observed in virions, indicating that only the full-len gth n spec ies is incorp orated in to partic les. how th e minor n spec ies ar ise and whet her they are of partic ular significan ce for infection is unc lear. they are most likely derived by proteoly tic proc essing from the major n species. this is supp orted by stud ies from eleo uet et al. ( ) , who show ed the tge v n prot ein to be cleav ed by casp ases. cas pase cleava ge sites wer e also predicted in the carboxy terminus of several other coronavirus n proteins (eleouet et al., ; ying et al., ) . these features are in agreement with observations showing that antibodies directed against the carboxy terminus of the mhv and tgev n proteins were not reactive with the faster migrating electrophoretic forms. furthermore, these smaller n protein forms appeared to be derived from the major species as judged from pulse-chase analyses (for a review see laude and masters, ) . the n protein is the only coronavirus structural protein known to become phosphorylated (for references see laude and masters, ) . both the major and minor n species appear to be phosphorylated as shown for mhv-a in sac(À) cells (rottier et al., b) and for tgev in llc-pk cells (garwes et al., ) . of the many potential target serines only a few are actually modified in the case of mhv (stohlman and lai, ; wilbur et al., ) . n protein phosphorylation does not seem to play a critical role in the regulation of virus assembly. in contrast, it has been hypothesized that dephosphorylation of the protein might facilitate disassembly during mhv cell entry (kalicharran et al., ; mohandas and dales, ) . immunofluorescence microscopy has shown the n protein to be localized in a particulate manner throughout the cytoplasm of coronavirus-infected cells. although the protein lacks a membrane-spanning domain it was found in association with membranes (anderson and wong, ; sims et al., ; stohlman et al., ) . for mhv, the n protein was found to colocalize partly with the membrane-associated viral replication complexes van der meer et al., ) . in addition to its cytoplasmic localization, the n proteins of ibv, mhv, and tgev have also been demonstrated to localize to the nucleolus both in coronavirus-infected cells and when expressed independently wurm et al., ) . putative nuclear localization signals were identified in these proteins. the ibv n protein was found to interact with nucleolar antigens, which appeared to occur more efficiently when the n protein was phosphorylated, and to affect the cell cycle (chen et al., ) . however, because mhv is able to replicate in enucleated cells (brayton et al., ; wilhelmsen et al., ) the nucleolar localization of the n protein does not appear an essential step during infection. although the primary function of the n protein is the formation of the viral ribonucleoprotein complex, several studies indicate the protein to be multifunctional. as indicated by its intracellular localization, the n protein is a likely component of the coronavirus replication and transcription complex. its presence is not an absolute requirement for replication and transcription because a human coronavirus (hcov) rna vector containing the complete pol ab gene appeared to be functional in the absence of the n protein (thiel et al., ) . however, the efficiency of the system was much enhanced when the protein was present. furthermore, using an in vitro system, it was demonstrated that antibodies to the n protein, but not those against the s and m proteins, inhibited viral rna synthesis by % (compton et al., ) . interactions that have been observed between the n protein and leader/trs sequences nelson et al., ; stohlman et al., ) and between n protein and the utr (zhou et al., ) suggest a role for the n protein in the discontinuous transcription process. furthermore, the n protein was also shown to interact with cellular proteins that play a role in coronavirus rna replication and transcription (choi et al., ; shi et al., ) . in addition, the n protein was reported to function as a translational enhancer of mhv sgrnas (tahara et al., ) . the m protein (previously known as e protein) is the most abundant envelope protein. it is the "building block" of the coronavirion and has been shown to interact with virtually every other virion component, as detailed in section iv. the m protein is - residues in length, except for the group m proteins, of which the amino terminus is about residues longer. despite large differences in primary sequences between m proteins from different antigenic groups, their hydropathicity profiles are remarkably similar. the m protein is highly hydrophobic. it has three hydrophobic domains alternating with short hydrophilic regions in the amino-terminal half of the protein, with the exception of the aforementioned group m proteins, which have at their amino terminus a fourth hydrophobic domain that functions as a cleavable signal peptide. the carboxy-terminal half of the protein is amphipathic, with a short hydrophilic domain at the carboxy-terminal end (fig. ) . in the center of the protein, directly adjacent to the third hydrophobic domain, is a stretch of eight amino acids that is well conserved (swwsfnpe). the conservation of the overall chemical features suggests that there are rigid structural constraints on the m protein as a result of functional requirements (for a review on the m protein, see rottier, ) . biochemical and theoretical studies led to a topological model for the mhv m protein rottier et al., rottier et al., , , in which the polypeptide spans the lipid bilayer three times, leaving a small amino-terminal domain ( - residues) in the lumen of intracellular organelles (or outside the virus), whereas the carboxyterminal half of the protein is located on the cytoplasmic side of the membrane (or inside the virion). the lumenal domain and the hydrophilic carboxy terminus are susceptible to protease digestion and are thus exposed. the bulk of the carboxy-terminal half of the m protein is protease resistant, indicating that the amphipathic part of the protein is either folded tightly or embedded in the polar surface of the membrane. indeed, a mutant lacking all three transmembrane domains was found to be associated with membranes (mayer et al., ) . the model for the disposition of the m protein in the membrane was confirm ed for ibv (cavanag h et al., ) . interest ingly, the fig . membrane topology of the coronavirus envelope proteins. the he and s proteins are both type i membrane proteins, with short carboxy-terminal cytoplasmic tails. the he protein forms disulfide-linked homodimers, whereas the s protein forms noncovalently linked homotrimers. the s subunits presumably constitute the globular head, whereas the s subunits form the stalk-like region of the spike. the m protein spans the lipid bilayer three times, leaving a small amino-terminal domain in the lumen of intracellular organelles (or on the outside of the virion), whereas the carboxy-terminal half of the protein is located on the cytoplasmic side of the membrane (or inside the virion). in tgev virions some of the m proteins have their cytoplasmic tail exposed on the outside (not shown). the m protein is glycosylated at its amino terminus (indicated by a diamond). the amphipathic domain of the m protein is represented by an oval. the hydrophilic carboxy terminus of the e protein is exposed on the cytoplasmic side of cellular membranes or on the inside of the virion. the e protein may span the bilayer once (b) or twice (a). m protein of tgev was shown to adopt an additional conformation. in virions about one-third of the m molecules have their carboxy terminus exposed on the virus surface rather than buried inside the particle (escors et al., a; risco et al., ) . this appears to have immunological consequences (risco et al., ) but the real significance of the dual topology is unclear. in mhv the m protein was found to assume only one defined membrane topology . the coronavirus m protein is almost invariably glycosylated in its exposed amino-terminal domain. this provides the virion with a diffuse, hydrophilic cover on its outer surface. whereas the group and coronaviruses and sars-cov all contain m proteins with only n-linked sugars, the m proteins of group coronaviruses are o-glycosylated (for a review see rottier, ). an exception is mhv- , the m protein of which carries both o-and n-linked sugars (yamada et al., ) . n-glycosylation is initiated in the endoplasmic reticulum by the cotranslational linkage of a large oligosaccharide structure to the polypeptide at asparagine residues within the consensus sequence nxs/t (where x is any amino acid). in contrast, mucin-type o-glycosylation starts posttranslationally with the addition of an n-acetylgalactosamine (galnac) monosaccharide to a hydroxylamino acid. o-glycosylation is subsequently completed by stepwise addition of other monosaccharides such as galactose, n-acetylglucosamine, fucose, and sialic acid. mhv m proteins carry a well-conserved ss(x)ttxxp sequence at their extreme amino terminus. despite the apparent presence of multiple hydroxylamino acids as potential oligosaccharide acceptor sites the m protein of mhv-a was found to be modified by the addition of only a single oligosaccharide side chain (de haan et al., b) . this side chain, when studied in ost - cells, appeared to be attached to the threonine at position . mutation studies, however, revealed that alternative acceptor sites can also be used. no unique sequence motifs for o-glycosylation of mhv m could be identified, which is probably related to the occurrence in cells of multiple galnac transferases (de haan et al., b) . as the expression of these enzymes varies in cells, conservation of the ss(x)ttxxp motif in mhv m protein may serve to increase opportunities for the protein to become glycosylated in different cell types. the distinct conservation of n-and o-glycosylation among the m proteins of the different groups of coronaviruses suggests that the presence and the particular type of carbohydrates are somehow beneficial to the virus, most likely in its interaction with the host. glycosylation of the m protein appeared not to be required for envelope assembly (de haan et al., a,) or for interaction with the s protein (de haan et al., ) , nor did it influence virus replication in vitro (de haan et al., a laude et al., ) . coronaviruses are able to induce interferon a (ifn-a) by their glycoproteins (baudoux et al., a,b) . for tgev (charley and laude, ; laude et al., ) and mhv (de haan et al., ) , the oligosaccharides linked to the m protein were demonstrated to be important for efficient ifn induction in vitro. the glycosylation status of the mhv m protein was found to influence the ability of the virus to replicate in the liver but not in the brain . thus, viruses with n-glycosylated m proteins replicated to a significantly higher extent in liver than otherwise identical viruses carrying o-glycosylated m proteins. mhv with unglycosylated m proteins replicated to the lowest extent. the mechanism behind these observations remains to be elucidated. when expressed in cells independently from the other viral proteins, the m proteins of mhv, ibv, tgev, and feline coronavirus (fcov) accumulate in the golgi compartment, that is, beyond the site of virus budding locker et al., ; machamer and rose, ; machamer et al., ; rottier and rose, ) , which is the intermediate compartment between the er and the golgi (ergic). the fine localization of the different proteins is, however, not the same. for instance, whereas the mhv m protein is concentrated in the trans-most golgi compartments, the ibv m protein localizes to the cis side of the golgi complex. signals for localization appear to reside in the hydrophilic part of the cytoplasmic tail and in the transmembrane domains. the extreme carboxy-terminal tail of mhv m was shown to be necessary, although not sufficient, for golgi localization (armstrong and patel, ; locker et al., ) . mutant proteins lacking this domain were transported to the plasma membrane. also, mutation of a single tyrosine in this domain, which occurs in the context of a potential internalization signal, resulted in plasma membrane localization (c. a. m. de haan and p. j. m. rottier, unpublished results). the first transmembrane domain of the ibv m protein was shown to be required and sufficient for localization to the cis-golgi region (machamer and rose, ; machamer et al., machamer et al., , swift and machamer, ) . this is not the case for the mhv m protein of which mutants with only the first transmembrane domain did not leave the er (armstrong et al., ; locker et al., ; rottier et al., ) . moreover, insertion of the first transmembrane domain of mhv m into a reporter protein resulted in a chimeric protein that was transported to the cell surface (armstrong and patel, ; machamer et al., ) , unlike a similar chimeric protein containing the first transmembrane domain of ibv that was retained in the golgi compartment . other mhv m mutant proteins lacking the first and second transmembrane domains were also not efficiently retained in the golgi compartment and were diverted to endosomal structures (armstrong et al., ; locker et al., ) . the mechanism by which golgi retention of m proteins is regulated has not yet been resolved. however, oligomerization of the proteins, mediated by the transmembrane domains, seems to play an important role, perhaps in combination with retrieval mechanisms maceyka and machamer, ) . formation of oligomeric complexes has been demonstrated to correlate with golgi retention of a reporter protein containing the first transmembrane domain of ibv m (weisz et al., ) while also the golgi-resident mhv m protein was found to occur in large, homomeric complexes (locker et al., ) . the lumenal domain of the m protein does not appear to contribute to localization; its deletion from mhv m did not affect the intracellular destination of the protein (mayer et al., ; rottier et al., ) . in infected cells the m proteins of ibv and mhv were observed to occur in the membranes of the budding compartment as well as in the golgi compartment. under these conditions their cis-trans distribution in the golgi compartment was the same as when these proteins were expressed independently machamer et al., ) . the e protein (previously known as sm protein) is a small protein ( - residues) and a minor component of the coronaviral envelope. although the primary structures of e proteins are quite conserved within the different coronavirus groups, they share little homology between the groups. however, the proteins have several structural features in common. the e protein contains a relatively large hydrophobic region in its amino-terminal half, followed by a cysteine-rich region, an absolutely conserved proline residue, and a hydrophilic tail. e is an integral membrane protein, which is assembled in membranes without the involvement of a cleaved signal peptide . its membrane topology has not been firmly established. although the opposite was proposed initially for the tgev e protein (godet et al., ) , there seems to be consensus about the hydrophilic carboxy terminus being exposed on the cytoplasmic side in cells or on the inside of the virion (corse and machamer, ; raamsman et al., ) . the amino terminus of the mhv e protein was not detectably present on the virion outside but appeared to be exposed cytoplasmically when it was extended with an amino-terminal epitope tag (maeda et al., ) , consistent with a topological model in which the hydrophobic domain spans the bilayer twice. for the ibv e protein evidence was provided indicating that the amino terminus is exposed lumenally in cells, consistent with a single spanning topology (corse and machamer, ) (fig. ) . the e protein is not glycosylated but appears to become palmitoylated. this was shown most convincingly for the ibv e protein by labeling with [ h]palmitate (corse and machamer, ) , both in ibv-infected cells and when the protein was expressed. mutagenesis revealed that one or both of the two conserved cysteines became modified. the result is consistent with the observed increase in electrophoretic mobility of the mhv e protein after treatment with hydroxylamine, an agent that cleaves thioester-linked acyl chains (yu et al., ) . others, however, were not able to confirm this posttranslational modification (godet et al., ; raamsman et al., ) . in coronavirus-infected cells the e protein has been observed by immunofluorescence studies to occur at intracellular membranes as well as at the cell surface (godet et al., ; smith et al., ; tung et al., ; yu et al., ) . when expressed exogenously from cdna, the e protein was detected only in intracellular organelles, although at different locations. the mhv e protein localized to pre-golgi membrane compartments, as was demonstrated by its colocalization with rab- , a marker for the endoplasmic reticulum and the ergic, by electron microscopy . the ibv e protein, tagged at its amino terminus with an epitope, was also localized to pre-golgi compartments (lim and liu, ) . in another study, however, the ibv e protein was shown to accumulate in the golgi apparatus, being distributed throughout the complex machamer, , ) . while the former study identified an ertargeting signal in the extreme carboxy terminus of the e protein (lim and liu, ) , the latter studies, using carboxy-terminal truncations, mapped the golgi-targeting information to a region between tail residues and (corse and machamer, ) . in addition, these authors showed the ibv e cytoplasmic tail to be necessary and sufficient for golgi targeting. the e protein was identified as a virion component relatively late, due to its low abundance and its small size (godet et al., ; liu and inglis, ; yu et al., ) . it was estimated to occur in ibv, tgev, and mhv virions at a rate of about , , and molecules per particle, respectively (godet et al., ; liu and inglis, ; vennema et al., ) . because of its low abundance the e protein may not have a genuine structural function in the virion envelope. rather, it may have a morphogenetic function by taking strategic positions within the m protein lattice to generate the required membrane curvature. alternatively, it may serve to close the neck of the budding particle as it pinches off the membrane (vennema et al., ) . expression of the e protein alone induced the formation of characteristic membrane structures also observed in infected cells, which apparently consist of masses of tubular, smooth convoluted membranes (david-ferreira and manaker, ; raamsman et al., ) . in addition, it resulted in the formation of vesicles containing the e protein, shown to be released from the cells (corse and machamer, ; maeda et al., ) . mhv infection induces caspase-dependent apoptosis in some, but not all, cells. by expressing the viral structural proteins separately in cells, the activity could be attributed to the e protein . apoptosis induction has not been reported for e proteins from other coronaviruses. coronavirus e proteins share structural similarities with small hydrophobic membrane proteins found in other enveloped viruses. examples are the vpu protein of hiv- , the k protein of alphaviruses, and the m protein of influenza virus. these proteins, also known as viroporins (gonzalez and carrasco, ) , were demonstrated to modify membrane permeability and to help the efficient release of progeny virus. the s protein (previously known as e ) constitutes the spikes, the hallmark of coronaviruses under the electron microscope. it is the major determinant of host range, tissue tropism, pathogenesis, and virulence. it is a relatively large, -to -amino acid-long type i glycoprotein with a cleavable n-terminal signal sequence and a membrane-anchoring sequence followed by a short hydrophilic carboxy-terminal tail of about residues (fig. ) . when comparing primary sequences, the s protein shows two faces: an amino-terminal half with hardly any sequence similarities and a carboxy-terminal half in which regions with significant conservation can be observed (de groot et al., a,b ; for a review see cavanagh, ) , consistent with the distinctive functions of these domains (see later). the s protein is synthesized as a heavily glycosylated polypeptide as demonstrated by the susceptibility of the glycans to endoglycosidases and by the dramatic effect of the n-glycosylation inhibitor tunicamycin. the number of potential n-glycosylation sites ranges from (mhv) to [feline infectious peritonitis virus (fipv)]. the s protein has not been reported to contain o-linked sugars. cotranslational n-glycosylation is an essential requirement for proper folding, oligomerization, and transport of the s protein, as has also been shown for other (viral) glycoproteins (doms et al., ) . growth of coronaviruses in the presence of tunicamycin resulted in the production of spikeless, noninfectious particles mounir and talbot, ; rottier et al., a; stern and sefton, ) . these particles were devoid of s protein, which was found to aggregate in the endoplasmic reticulum when glycosylation was inhibited (delmas and laude, ) . folding of the s protein is a relatively slow process. besides the addition of oligosaccharides it involves the formation and rearrangement of many intramolecular disulfide bonds. for the s protein of mhv-a , the lumenal domain of which contains cysteine residues, the major conformational events appear to take about min during which the protein passes through a continuous spectrum of folding intermediates (opstelten et al., a) . folding of s is probably the rate-limiting step in the process of oligomerization. sufficiently folded s protein monomers associate in the endoplasmic reticulum to form trimers (delmas and laude, ; lin et al., ) , with a half-time of approximately h (delmas and laude, ; vennema et al., a,b) . trimerization is likely to be required for export out of the endoplasmic reticulum. in infected cells s protein trimers interact with m protein and perhaps also with e protein, and migrate to the virus assembly site. a fraction of the s protein is transported to the plasma membrane where it can cause cell-cell fusion, a feature formally attributed to the s protein by its individual expression in cells (de groot et al., ; pfleiderer et al., ) . under such expression conditions the bulk of the s protein remains intracellularly (vennema et al., a) in the endoplasmic reticulum . retrieval signals have been identified in the cytoplasmic tail of the s proteins from coronavirus groups and as well as in the tail of the sars-cov s protein, but not in the group mhv s protein (lontok et al., ) . during its transport to the cell surface, either alone or as part of virions, the s protein undergoes further modifications. the n-linked sugars are modified and become mature during passage through the golgi complex. the mhv s protein was shown to become palmitoylated, a modification that may already take place in the endoplasmic reticulum (van berlo et al., ) . as a late step the s protein can be cleaved. a basic amino acid sequence resembling the furin consensus sequence motif (rxr/kr) occurs approximately in the middle of the protein and was shown to be the target of a furin-like enzyme in the case of mhv-a (de haan et al., ) . cleavage has been demonstrated for s proteins from coronavirus groups and , but not for s proteins from group viruses (cavanagh, ) or from sars-cov (bisht et al., ) . the resulting amino-terminal s subunit and the membrane-anchored s subunit remain noncovalently linked. it has been suggested that the s subunit constitutes the globular head, whereas the s subunit forms the stalk-like region of the spike (cavanagh, b; de groot et al., a,b) . the coronavirus s protein has two functions, which appear to be spatially separated. the s subunit (or the equivalent part in viruses with uncleaved s protein) is responsible for receptor binding, and the s subunit is responsible for membrane fusion. for several coronaviruses the receptor-binding site in s has been mapped. for mhv strain jhm (mhv-jhm), for instance, it was located in the domain composed of the amino-terminal residues of the s molecule (kubo et al., ) , residues - and - being particularly important (saeki et al., ; suzuki and taguchi, ) . this amino-terminal domain also determined ceacam receptor specificity of various mhv strains (tsai et al., ) . for tgev (godet et al., ) , hcov- e breslin et al., ) , and sars-cov (babcock et al., ; wong et al., ) the receptor-binding domains have also been mapped to the s subunit, although in different regions. in several cases neutralizing antibodies were demonstrated to bind the receptor-binding domains and to prevent the interaction with the receptor (godet et al., ; kubo et al., ; sui et al., ) . the interaction between the s protein and its receptor is the major determinant for virus entry and host range restriction. nonpermissive cell lines can be rendered susceptible by making them express the receptor (see later references). coronaviruses can also be retargeted to specific cells by exchanging the ectodomain of the s protein for that of an appropriate other coronavirus, as was demonstrated for mhv (kuo et al., ) and fipv (haijema et al., ) . receptors have so far been identified for the group coronavirus mhv (ceacam; dveksler et al., dveksler et al., , williams et al., ) ; the group coronaviruses tgev and porcine respiratory coronavirus (prcov) (papn; delmas et al., t resnan et al., ) , and hcov- e (hapn; yeager et al., ) ; and for sars-cov (ace ; li et al., ) . the s proteins of the group coronaviruses have been observed to exhibit hemagglutinating activities. although for bovine coronavirus (bcov), hcov-oc , and hemagglutinating encephalomyelitis virus (hev) -o-acetylated sialic acids were identified as a receptor determinant (krempl et al., ; kunkel and herrler, ; schultze and herrler, ; schultze et al., a; vlasak et al., b) , specific receptors for these viruses have not been identified. also, the mhv s protein appears to bind sialic acid derivatives in addition to its specific receptor ceacam, which may suggest that sialic acids function as an additional receptor determinant for mhv-like coronaviruses (wurzer et al., ) . the ectodomain of the s subunit, which is involved in the fusion process, contains two heptad repeat (hr) regions (de groot et al., a,b) , a sequence motif characteristic of coiled coils. mutations in the first (i.e., membrane-distal) hr region of the mhv s protein resulted in fusion-negative phenotypes (luo and weiss, ) or in a low-ph dependence for fusion (gallagher et al., ) , whereas mutations in the second hr region caused defects in s protein oligomerization and fusion ability (luo et al., ) . a fusion peptide has not yet been identified in any of the coronavirus spike proteins, but is predicted to be located at (bosch et al., b; chambers et al., ) or within (luo and weiss, ) the amino terminus of the first hr region. binding of the s subunit to the (soluble) receptor, or exposure to c and an elevated ph, has been shown to trigger conformational changes that are supposed to facilitate virus entry by activation of the fusion function of the s subunit (breslin et al., ; gallagher, ; lewicki and gallagher, ; matsuyama and taguchi, ; miura et al., ; sturman et al., ; taguchi and matsuyama, ; zelus et al., ) . this conformational change is thought to lead to exposure of the fusion peptide and its interaction with the target membrane, further changes resulting in the formation of a heterotrimeric six-helix bundle, characteristic of class i viral fusion proteins, during the membrane fusion process. indeed, peptides corresponding to the hr regions of mhv (bosch et al., ; xu et al., ) and sars-cov (bosch et al., b; ingallinella et al., ; liu et al., ; tripet et al., ; zhu et al., ) were found to assemble into stable oligomeric complexes in an antiparallel manner, which in the natural situation would result in the close colocation of the fusion peptide and the transmembrane domain. these peptides were further shown to be inhibitors for viral entry (bosch et al., (bosch et al., , b liu et al., ; yuan et al., ; zhu et al., ) . besides the hr regions, other parts of the s protein are also likely to be important for the fusion process. all coronavirus s proteins contain a highly conserved region (de groot et al., a) , rich in aromatic residues, downstream of the second hr region, part of which may form the start of the transmembrane domain. the function of this domain is unknown, but a similar region in the hiv- env protein was demonstrated to be important for viral fusion and env incorporation into virions (salzwedel et al., ) . immediately downstream of the transmembrane domain all s proteins contain a cysteine-rich region (de groot et al., a) . using a mutational approach including deletions, insertions, and substitutions, both the transmembrane domain and the cysteine-rich region immediately downstream thereof, but not the carboxy-terminal part of the cytoplasmic tail, were shown to be important for mhv s protein-induced cell-cell fusion (bos et al., ; chang and gombold, ; chang et al., ) the cleavage requirements of the s proteins for the biological activities of the coronavirus spike remain enigmatic. whereas the s proteins of group coronaviruses, such as fipv (vennema et al., a) , are not cleaved, those of other coronaviruses, particularly of groups and , are cleaved to variable extents, depending on the viral strain and the cell type in which the viruses are grown (frana et al., ; reviewed by cavanagh, ) . cleavage of the s proteins is not required to expose the internal fusion peptide. whereas cleavage of the mhv s protein generally correlates strongly with cell-cell fusion (cavanagh, ) , virus-cell fusion appeared not to be affected by preventing s protein cleavage, indicating that these fusion events have different requirements (de haan et al., ) . similarly, whereas trypsin activation of sars-cov s protein was required for cell-cell fusion, it did not enhance the infectivity of cell-free pseudovirions (simmons et al., ) . for mhv- , the spikes of which are able to initiate fusion without prior interaction with the primary mhv receptor (gallagher et al., ) , the stability of the s -s heterodimers after s protein cleavage is low, allowing receptor-independent fusion. during cell culture adaptation, however, selected mutant viruses carried deletions in the s subunit, downstream of the receptor-binding domain, which resulted in stabilized s -s heterodimers and receptor-dependent fusion activity (krueger et al., ) . virions of group coronaviruses generally contain a fringe of shorter surface projections in addition to the characteristic spikes (bridger et al., ; king et al., ; sugiyama and amano, ) . these viruses express and incorporate into their particles an additional membrane protein, he (for a review see brian et al., ) . although all group viruses contain an he gene, the protein is not expressed by all mhv strains (luytjes et al., ; yokomori et al., ) , indicating that he is a nonessential protein also in these viruses. the he gene encodes a type i membrane protein of - residues that contains a cleavable signal peptide at its amino terminus (hogue et al., ; kienzle et al., ) and a transmembrane domain close to its carboxy terminus, leaving a short cytoplasmic tail of about residues (fig. ) . the ectodomain contains - putative n-linked glycosylation sites. the putative esterase active site (fgds) is located near the (signal-cleaved) he amino terminus. the coronavirus he protein has % amino acid identity with the he- subunit of the he fusion protein of influenza c virus and the he protein of torovirus (cornelissen et al., ) . it has been suggested that coronaviruses have captured their he module from influenza c virus or a related virus (luytjes et al., ) . however, influenza c virus, toroviruses, and coronaviruses may well have acquired their he sequences independently, not from each other but from yet another source (cornelissen et al., ) . the he protein becomes cotranslationally n-glycosylated when expressed in cells, giving rise to a polypeptide of approximately - kda that rapidly forms disulfide-linked dimers (hogue et al., ; kienzle et al., ; king et al., ; parker et al., ; yokomori et al., ; yoo et al., ) . the he dimers (or a higher order structure thereof) become incorporated into virions, while a proportion is transported to the cell surface (kienzle et al., ; pfleiderer et al., ) . little is still known about the function(s) of the coronavirus he protein. the protein contains hemagglutinin and acetyl esterase activities (brian et al., ) . while the he proteins of bcov, hev, and hcov-oc hydrolyze the -o-acetyl group of sialic acid and therefore appear to function as receptor-destroying enzymes (schultze et al., b; vlasak et al., a) , the he proteins of mhvlike coronaviruses function as sialate- -o-acetylesterases (klausegger et al., ; regl et al., ; wurzer et al., ) . although inhibition of the esterase activity of bcov resulted in a -to -fold reduction in viral infectivity (vlasak et al., a) , it was shown both for bcov and for an mhv strain expressing an he gene that the s protein is required and sufficient for infection (gagneten et al., ; popova and zhang, ) . in view of these results it has been proposed that the he protein might play a role at an even earlier step and may mediate viral adherence to the intestinal wall through the specific yet reversible binding to mucopolysaccharides. the process of binding to sialic acid receptors followed by cleavage and rebinding to intact receptors could theoretically result in virus motility and even allow migration through the mucus layer covering the epithelial target cells in the respiratory and enteric tracts (cornelissen et al., ) . several studies have indicated the he protein to play a role in pathogenicity. the he protein of bcov (deregt and babiuk, ) , but not that of mhv, was able to induce neutralizing antibodies. however, passive immunization of mice with nonneutralizing, mhv he-specific antibodies protected the animals against a lethal mhv infection (yokomori et al., ) . furthermore, intracerebral expression of the he protein in mice was found to affect the neuropathogenicity of mhv (yokomori et al., ; zhang et al., ) . strikingly, he protein-defective mhv mutants were rapidly selected during viral infection in the mouse brain (yokomori et al., ) , which may suggest that the he protein plays a more critical role during the infection of other tissues. early electron microscopic studies demonstrated that coronavirus morphogenesis takes place at intracellular membranes and identified the cisternae of the endoplasmic reticulum as the site of budding of ibv and hcov- e (becker et al., ; chasey and alexander, ; hamre et al., ; oshiro et al., ) . later studies revealed that early in infection particle formation occurs predominantly at smoothwalled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and the golgi complex (ergic). this so-called intermediate compartment was shown to be used as the early budding compartment by mhv, ibv, fipv, tgev, and sars-cov (goldsmith et al., ; klumperman et al., ; tooze et al., ) . at later times during infection the rough endoplasmic reticulum was seen to gradually become the major site of mhv budding in fibroblasts (tooze et al., ) . as already mentioned, ultrastructural studies localized the mhv and ibv m proteins in the budding compartment(s) but also in the golgi complex, that is, beyond the site of budding tooze et al., ) . apparently, accumulation of m protein alone is not sufficient to determine the site of budding; other viral and/or cellular factors are required as well. for mhv the e protein, which was found to localize to the intermediate compartment by immunoelectron microscopy (immuno-em), was suggested to be such a candidate , but more players are likely to be involved. whether the helical nucleocapsids, visible as electron-dense cytoplasmic elements adjacent to budding profiles (david-ferreira and manaker, ; dubois-dalcq et al., ; massalski et al., ; risco et al., ; and references given previously), are a determining factor is unclear. in this respect, knowledge about the budding location of coronavirus-like particles (see later) might be informative. coronavirions are subject to an intracellular postbudding maturation process that occurs while they are on their way through the constitutive exocytic pathway by which they are exported out of the cell (risco et al., ; salanueva et al., ; tooze et al., ) . indications of this had already been noticed in early morphological studies with hcov- e (becker et al., ; chasey and alexander, ; hamre et al., ; oshiro et al., ) and mhv (holmes and behnke, ; , but were described in somewhat more detail for mhv by tooze and coworkers ( ) . the pictures show "immature" virions in pre-golgi compartments and golgi cisternae that appear as spherical structures with the ribonucleoprotein core immediately below the viral envelope and with an "empty" center. by contrast, virions in the trans-golgi network and beyond have the mature morphology showing a fairly uniform, high internal electron density. an extensive analysis of the structural maturation of coronavirions was reported for tgev (risco et al., ; salanueva et al., ) . budding was shown to yield relatively large virions with an annular, electron-dense internal periphery and a clear central area. smaller particles, with the characteristic morphology of extracellular virions, that is, having a compact, dense inner core with polygonal contours, were seen to accumulate in secretory vesicles in the periphery of the infected cell. both types of particles appeared to coexist in the golgi complex (fig. ) . obviously, the larger particles are the precursors of the smaller mature virions (salanueva et al., ) and probably undergo their morphological maturation during their transport through the golgi complex. the reorganization of the particle gives rise to the supposedly icosahedral core shell and is accompanied by a dramatic, approximately % reduction of the particle volume. it is presently unknown what triggers the morphological reorganization in the golgi complex. application of drugs affecting the state of the organelle did not give clues. as virions encounter an increasingly acidic ph on passage through the golgi stack, studies addressing this parameter were done with lysosomotropic agents. thus, chloroquine and nh cl were applied to mhv-infected cells to elevate the ph at the trans side of the golgi complex, but no effect on the maturation of mhv was observed (tooze et al., ) . monensin, a drug that reversibly disorganizes the golgi complex and blocks transport along the exocytic pathway, led to the accumulation of the large, annular tgev virions; after reversal of the blockade formation of the small, compact particles was again restored (salanueva et al., ) . these observations confirm that the golgi complex is necessary for tgev structural transformation. nocodazole treatment of cells causes a reversible fragmentation of the golgi complex. under these conditions tgev virions were still able to undergo normal structural maturation. in contrast, still another golgi-disrupting compound, brefeldin a, prevented their maturation (risco et al., ) . this compound leads (risco et al., ) . (b) for a direct comparison of size and morphology a small, dense particle and a large particle are shown (salanueva et al., ) . pictures were kindly provided by c. risco. to a redistribution of golgi membranes to the er, leaving no definable golgi system. interestingly, mhv particles accumulated in infected cells under these conditions appeared to be infectious when liberated by sonication (j. meertens and p. j. m. rottier, unpublished results), leaving us with an intriguing question about the function of the maturation process. a. nucleocapsid assembly helical nucleocapsids are assembled in the cytoplasm of coronavirus-infected cells. they have been recognized by their tubular appearance in electron microscopy studies with several viruses including ibv, hcov- e, tgev, and mhv (becker et al., ; chasey and alexander, ; david-ferreira and manaker, ; dubois-dalcq et al., ; hamre et al., ; massalski et al., ; oshiro et al., ; risco et al., ) . large inclusions of nucleocapsids were seen to accumulate late in the infection of cells with hcov (caul and egglestone, ) and mhv-jhm (dubois- dalcq et al., ) . the structure of the nucleocapsid as it occurs in infected cells has not been studied in any detail. ribonucleoprotein particles supposed to represent nucleocapsids have been isolated from mhv-infected cells and were shown to consist of genomic rna and n protein (perlman et al., ; robb and bond, ; spaan et al., ) . the particles sedimented as edta-resistant structures of - s in sucrose gradients. during the active phase of viral replication the majority ( %) of the intracellular genome-size rna was found in these structures (spaan et al., ) . ultrastructural studies of nucleocapsids derived from purified virion preparations have shown quite a variety of helical structures, depending on the virus and the experimental conditions used. the overall feature, however, was that of a thread-like coil, sometimes appearing to be hollow, with a diameter varying between and nm and a length ranging from . mm up to mm (caul et al., ; davies et al., ; kennedy and johnson-lussenburg, ; macnaughton and davies, ) biochemical analysis of nucleocapsids prepared by detergent disruption of purified coronaviruses revealed the presence of genomic rna and n protein. interestingly, however, particles obtained by treatment of virions with nonidet p- appeared to be spherical when viewed under the electron microscope and, in addition, to contain m protein, as was observed with tgev (garwes et al., ) , hev (pocock and garwes, ) , and mhv-jhm (wege et al., ) . the presence of the m protein was found for mhv-a to depend on the preparation conditions. whereas the protein was absent when the virions had been disrupted with nonidet p- at c, solubilization at c resulted in copurification of m protein with the nucleocapsid (sturman et al., ) . the higher temperature was found to cause a conformational change in the m protein, leading to its aggregation and association with the viral rna in the nucleocapsid. similarly, nucleocapsid structures essentially lacking the m protein were also reported for ibv when virions were treated with detergent at low temperature (davies et al., ) . although there is no direct evidence yet, it seems reasonable to assume that the helical nucleocapsids seen accumulating in the cytosol of infected cells constitute the reservoir that feeds into the viral budding system. the location where these nucleocapsids are assembled has not been defined. their production may take place either free in the cytoplasm, where the n protein is synthesized, or, alternatively, in association with the membrane-bound structures where genomic rna is produced. the observed colocalization of n protein with the replication complexes (bost et al., (bost et al., , denison et al., ; van der meer et al., ) is consistent with the latter possibility. coronavirus replication appears to occur on double-membrane vesicles (gosert et al., ) , which utilize components of the cellular autophagy pathway (prentice et al., ) . whereas early in infection the replication complexes were shown to be almost entirely discrete from sites of m protein accumulation, at later times of infection helicase and n proteins appeared to colocalize with the m protein (bost et al., (bost et al., , . it was proposed that the translocation of helicase-n protein complexes to sites of virus assembly may serve as a mechanism to deliver the newly synthesized rna and nucleocapsids and to facilitate the retention of the m protein in the intermediate compartment. encapsidation of genomic rna into a nucleocapsid is presumably initiated by an interaction of the n protein with a specific nucleotide sequence, the packaging signal, which is subsequently followed by the polymerization of n proteins around the rna molecule in a nonsequence-specific manner. the selective incorporation of genomic rna into virions would predict the packaging signal to be located in sequences unique to this rna, that is, within the approximately -kb region comprising the utr and open reading frame (orf ) with the exception of the leader sequence. although the data obtained so far support this prediction, no consistent picture has emerged yet. the approach generally used to map the packaging signal involved the study of helper virus-assisted encapsidation of natural and artificially obtained defective rna genomes. thus, a -nucleotide region located at the end of the pol b gene was initially identified for mhv (van der most et al., ) , which was subsequently narrowed to an area of nucleotides (fosmire et al., ) . within this area a stable stem-loop of nucleotides was predicted. mutation studies revealed that the integrity of this secondary structure was important and that the sequence of the packaging signal could be trimmed further to a minimum stretch of nucleotides (fosmire et al., ) . the signal appeared to be sufficient for rna packaging as its inclusion allowed a synthetic subgenomic mrna of mhv-a to be packaged specifically; the encapsidation efficiency of the mrna was, however, significantly lower than that of the defective genomic rna from which it was transcribed . even a nonviral rna was found to be packaged into mhv particles when provided with the packaging signal (woo et al., ) . buoyant density analysis of the particles revealed that the rna was not assembled separately but copackaged with helper virus rna. studies of the corresponding pol b region of another group coronavirus, bcov, indicated that within this group the packaging signal is structurally and functionally conserved. a -nucleotide sequence with significant homology ( %) to that of mhv was identified within a cloned -nucleotide segment sharing % homology overall . when this segment was fused to a noncoronavirus reporter gene sequence, the resulting rna appeared to be packaged not only by the homologous helper virus bcov but also by mhv. conversely, when the mhv packaging signal was fused to the reporter gene sequence, the rna was found to be encapsidated also in the context of a bcov-infected cell . mapping studies of packaging signals in the genomes of group and group coronaviruses have yielded quite different results. for ibv, deletion mutagenesis of a defective rna led to the conclusion that only the sequences in the utr and/or a region of the utr were specifically required for packaging, although parts of the pol b sequence, but not any part in particular, also enhanced the efficiency (dalton et al., ) . somewhat similar conclusions could be drawn from a study with tgev (izeta et al., ) . by comparing packaging efficiencies of different defective genomes it was inferred that information for packaging was present both at the genomic end (about . kb) and in parts of pol b. a packaging signal was subsequently mapped to a fragment representing the terminal nucleotides of the genome by inserting a series of overlapping segments covering a stretch of about nucleotides from the end of the genome into an mrna reporter expression construct contained within a defective genome (escors et al., ) ; only the terminal sequence conferred to the mrna the ability to become packaged by helper virus. it is too early to conclude that the apparently contrasting results reflect true, fundamental differences in encapsidation strategies between the different (groups of ) coronaviruses. although the overview may suggest the existence of different cis-acting signals, the data still allow a scenario in which multiple domains in the genome are involved cooperatively, each one contributing differently to (the efficiency of) the encapsidation process. such contributions would not necessarily concern n protein binding only; the exceptional complexity of the coronaviral genome might call for additional provisions, related perhaps to the structuring of the encapsidation complex. several observations indeed imply the involvement of multiple domains. the efficient rescue, for instance, of a bcov defective genome (drep) that completely lacks the putative -nucleotide packaging signal entails the participation of additional sequence(s) (chang and brian, ; . another example is the strongly increased rescue of an otherwise poorly packaged defective tgev genome (m ) due to the presence of about . kb of sequences derived from the pol b gene (m ) (izeta et al., ) . there is no direct evidence yet for the actual functioning of the presumed packaging signals in the initiation of nucleocapsid assembly. binding of n protein to these signals, the first step in the process, has so far been addressed only for the -nucleotide sequence of mhv. specific binding to rna transcripts containing this sequence was indeed demonstrated biochemically with mhv n protein derived from infected cells, from virions, and from cells expressing the protein (molenkamp and spaan, ) . the binding efficiency, however, appeared to be relatively weak as was shown by comparing n protein binding to different parts of a packageable defective genome (midi-c) . the highest binding efficiency was observed with an rna transcript representing about kb from the end of pol a. remarkably, not even removal of the packaging signal from midi-c rna affected binding to the n protein as measured in the filter-binding assay used. the observations indicate that the domain containing the -nucleotide sequence does not function as a packaging signal in the conventional sense, adding further support to the notion that the intricacies of coronaviral nucleocapsid assembly are complex. apart from the studies mentioned, the occurrence of n-rna interactions has been amply documented. this is not surprising as the n protein has been implicated in several other processes that involve interaction with rna, such as replication, transcription, and translation (lai and cavanagh, ; see also section ii.c). as the relevance of these interactions for viral assembly is generally unclear, a brief survey of the available information is included here. in addition, an overview of data on the mapping of rna interactions on the n polypeptide is schematically presented in fig. . a high-affinity interaction between the n protein and the leader was demonstrated for mhv-a . using an rna overlay protein blot assay and various in vitro rna transcripts, the binding of n protein was localized to a stretch of nucleotides (nucleotides - ) at the end of the leader . the stretch included the pentanucleotide repeat ucuaa now known to be critical for transcription. biochemical analyses of the interaction measured a dissociation constant (k d ) of nm for bacterially expressed mhv n protein to the leader rna (nelson et al., ) . consistent with the presence of a leader at the end of all viral rnas, an n protein-specific monoclonal antibody coimmunoprecipitated genomic rna as well as the subgenomic rnas from mhv-infected cells . similar observations were made for bcov . packaging of subgenomic rnas has been reported for tgev (sethna et al., ) , bcov (hofmann et al., ) , and ibv (zhao et al., ) but not for mhv, suggesting that their incorporation is not mediated by n protein-leader interaction. whereas for tgev and ibv the relative packaging of subgenomic rnas was found to be inefficient, genomic rna appearing in virions at a more than -fold molar excess over any subgenomic rna species, the bcov subgenomic n and m mrnas appeared to be packaged as abundantly as the genome (hofmann et al., ) . a reevaluation for tgev revealed that the detection of subgenomic rnas in virions was related to the purity of virus preparations, indicating that mrnas were not specifically encapsidated (escors et al., ) . besides the leader, the n protein has been found to bind to other parts of the coronaviral genome. in addition to the binding site in the mhv fig . structural organization of the coronavirus n protein. common features and their distribution along the polypeptide chain are shown schematically. the hatched box indicates the most conserved part of the n protein, with a high proportion of aromatic residues. the n protein contains many basic residues throughout the polypeptide, but with particular clustering in two regions (þþþ). the upstream cluster contains a serine/arginine-rich region (s/r). the carboxy terminus, which contains a high proportion of acidic residues, is also indicated (ÀÀÀ). the bars indicate parts of the n protein that have been implicated in n-n and n-rna interaction. furthermore, the location of the deletion in the mhv-a mutant alb (d) is indicated as well as the domain where second-site mutations in revertant viruses of alb are mapped. finally, the parts of the n protein that could not be transferred from bcv into the mhv genome are marked. references are included in the figure. pol a gene mentioned previously, a high-efficiency binding site was identified by the same authors in the half of the n gene of both mhv and bcov . the ibv n protein was shown to bind sequences in the terminal utr of the genome (zhou et al., ) . coronavirus n proteins do not contain sequence motifs typically found in other rna-binding proteins. they appear to bind rna both nonspecifically (masters, ; robbins et al., ) and in a sequencespecific way nelson and stohlman, ; nelson et al., ; stohlman et al., ) . non-sequence-specific rna binding has been mapped to a large central domain of the mhv n molecule (fig. ) (masters, ) . also, the leader-binding property was assigned to this domain; this activity was initially mapped to the area containing the two highly basic regions (nelson and stohlman, ) , but was later narrowed to a -residue segment containing the serine/ arginine-rich basic region (nelson et al., ) . interestingly, this particular region could not be interchanged with its bcov counterpart in a study on the functional equivalence of the n proteins from these related viruses (peng et al., b) . another domain in the mhv n protein implicated in viral rna binding was mapped to an area that partly overlaps with the second basic region. the assignment was based on an analysis of second-site revertants of mhv mutant alb , the virions of which are extremely thermolabile because of a -residue deletion located between the central and carboxy-terminal domain of the n protein (koetzner et al., ) . the reverting mutations correlated with restoration of the disturbed rna-binding capacity of the mhv n protein and were found clustered close to the basic region some residues on the amino side of the deletion (fig. ) (peng et al., a) . although all these studies consistently attribute a major role in rna binding to the central portion of the coronaviral n protein, the interaction of the ibv n protein with the utr of ibv rna mentioned previously was mapped to the amino-and carboxy-terminal domains of the molecule (zhou and collisson, ) . utr rna-binding activity was also assigned to the amino-terminal domain of the sars-cov n protein on the basis of studies using nuclear magnetic resonance spectroscopy (huang et al., ) . it is obvious that the wrapping of the -kb coronaviral genome into the compact helical nucleocapsid is largely driven by n protein interactions. as there are no indications for packaging of the rna into a preformed capsid, these interactions can be described by the following model. packaging is initiated by binding of the n protein, either as a monomer or in a multimeric form, to the rna. by analogy to other rna viruses, this sequence-specific interaction may induce a conformational change in the n protein, thereby creating a nucleation site for the cooperative stacking of n protein units along the entire length of the rna, now in a non-sequence-specific way. these n units can again be monomeric or consist of defined multimers. finally, helix formation is driven by interactions between n molecules separated along the ribonucleoprotein chain but that become adjacent in neighboring helices. this model predicts multiple nonequivalent interactions between n molecules. n-n interactions have been experimentally demonstrated for mhv, bcov, and hcov-oc . high molecular weight species of the n protein, possibly trimers, were detected by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) of virion preparations under nonreducing (but not under reducing) conditions, which is indicative of intermolecular disulfide bonds between the n subunits (hogue et al., ; narayanan et al., b; robbins et al., ) . these complexes are likely to be additionally stabilized by noncovalent interactions as coronavirus n protein cysteines are not well conserved, the sars-cov n protein even lacking any cysteine residues. both monomeric and oligomeric n species were able to bind rna (robbins et al., ) . multimeric forms of the n protein were also found in association with intracellular genomic rna in mhv-infected cells as shown after the selective isolation of this ribonucleoprotein through coimmunoprecipitation with the m protein (narayanan et al., b) . high molecular weight forms of the n protein corresponding to dimers and trimers were also demonstrated in vitro after ultraviolet (uv) cross-linking of bcov n protein to rnas . few studies have addressed the identification of the n-n interaction domains. the results so far are inconsistent (fig. ), but this might as well reflect the predicted occurrence of nonequivalent interactions. interaction sites were mapped to the amino-terminal part of the mhv n protein (wang and zhang, ) . using an in vitro binding assay in which the full-length n protein was incubated with bacterially expressed fusion proteins containing different segments of the n protein, interaction was observed with a polypeptide derived from the amino-terminal one-third (residues - ) of the protein and with a polypeptide representing the central part (residues - ). the latter domain contains the serine/arginine-rich region implicated in the binding to the leader/trs-specific sequences (nelson et al., ) . this domain could not be replaced by the corresponding domain from bcov without loss of viral viability, from which the authors indeed inferred an involvement in protein-protein interactions (peng et al., b) . the same domain was also shown to be essential for the homotypic association of the sars-cov n protein . using a mammalian two-hybrid approach, the n-n interaction appeared to be abolished completely when the serine/arginine-rich region had been deleted. however, in another study using the yeast two-hybrid system this interaction was not confirmed. a polypeptide consisting of the amino-terminal two-thirds of the sars-cov n protein, that contains the serine/arginine-rich region, exhibited no association with the full-length protein (surjit et al., ) . rather, self-association was attributed to the carboxy-terminal residues of the molecule, which lacks the motif. unlike most other enveloped viruses, coronaviruses have the remarkable feature of being able to independently assemble their envelope. indications for this were already noticed in early electron microscopy studies of viral preparations and infected cells showing the occurrence of apparently "empty" particles (afzelius, ; chasey and alexander, ; macnaughton and davies, ) . incomplete virions with the typical coronavirus morphology but lacking the n protein and the genome could indeed be separated from normal ibv particles by their lower density in sucrose gradients (macnaughton and davies, ) . the definition of virus-like particles (vlps) and the requirements for their formation were established by the coexpression of the coronaviral structural proteins in mammalian cells (vennema et al., ) . membranous particles were assembled when the mhv envelope proteins m, e, and s were coexpressed, without the need for an n protein or genomic rna. the particles were released from the cells and, when examined under the electron microscope, appeared to be morphologically indistinguishable from authentic virions, that is, they had the characteristic shape and dimensions of normal virions. also, their membrane protein composition was similar to that of mhv, with a high abundance of m protein and only trace amounts of e protein. quite surprisingly, only the m and e proteins were required for particle assembly. both s and n proteins were dispensable for particle formation but, whereas the s protein became incorporated when present, this was not the case for the n protein (vennema et al., ) , except in combination with (defective) genomic rna, in which case a nucleocapsid was coassembled (bos et al., ; kim et al., ) . individual expression of the m or the e protein in cells did not give rise to formation of vlps although e protein synthesis by itself led to the secretion of e-containing vesicles (corse and machamer, ; maeda et al., ) . the nature of these particles has not been characterized in much detail; the vesicles induced by mhv e sedimented slightly slower than virions (maeda et al., ) whereas the particles obtained with ibv e had about the same density as virions (corse and machamer, ) . besides for mhv and ibv, vlps have so far been described for bcov (baudoux et al., b) , fipv , and tgev (baudoux et al., b) . the observations demonstrate the unique budding mechanism of coronaviruses, which is dependent solely on the envelope proteins m and e but independent of a nucleocapsid. somewhat similar observations have been described for the flavivirus tick-borne encephalitis virus and for hepatitis b virus, which also produce proteolipid particles on expression of their envelope proteins prem and e (allison et al., ; mason et al., ) and s (patzer et al., ; simon et al., ) , respectively, but these particles are much smaller than the corresponding virions. in contrast, particles with the typical, large size of coronaviruses are acquired by the concerted action of just the proteins m and e (baudoux et al., b; vennema et al., ) . budding of enveloped viruses generally requires a nucleocapsid (for a review see garoff et al., ) . for retroviruses the gag protein, the precursor to the nucleocapsid, is all that is needed to obtain particles resembling immature virions; the env protein is dispensable. budding of alphaviruses, on the other hand, requires both the envelope proteins and the nucleocapsid. interestingly, the same appears to hold true for arteriviruses (r. wieringa, a. a. f. de vries, and p. j. m. rottier, unpublished observations), which are closely related to the coronaviruses, share with them a triple-spanning envelope protein, and bud into early membranes of the secretory pathway, like coronaviruses but unlike alphaviruses. it is unknown how the coronavirus m and e proteins cooperate in budding. as the extensive electron microscopy work with m proteins from various coronaviruses gave no indications that this protein causes membrane bulging by itself, it is believed that the function of the e protein in coronavirus budding is in the induction of curvature in the m protein lattice (see later) and the subsequent budding of the membrane (vennema et al., ) . by its low abundance in the virion, the e protein does not seem to serve a genuine structural function in that it occupies frequent, regular positions in the m protein framework. consistent with an important role of the e protein in particle morphogenesis, mutations in its hydrophilic carboxy-terminal part, introduced by targeted recombination into the mhv genome, yielded thermolabile viruses one of which showed aberrant virion morphology with pinched and elongated shapes when viewed in the electron microscope (fischer et al., ) . revertant analyses revealed that a single second-site amino acid change within the e protein was able to reverse the phenotypic effect of the original mutations, providing support for possible interactions between e protein monomers during budding (fischer et al., ) . unexpectedly, complete deletion of the e gene from the coronaviral genome does not abolish virion formation, demonstrating that the protein is not essential for budding. whereas this deletion dramatically (at least -fold) reduced the release of infectivity from infected cells in the case of mhv-a , knockout of the tgev e gene resulted in a lethal phenotype (curtis et al., ; ortego et al., ) . remarkably, however, in the latter case virions still assembled but these appeared to be unable to leave the cells (j. ortego and l. enjuanes, personal communication). the vlp system offers a convenient assay to study many aspects of coronavirus envelope assembly. it was thus used to analyze the primary structure requirements of the m and e proteins for particle formation. for the m protein such studies demonstrated each of its different domains to be important. in general, mutations (deletions, insertions, and point mutations) in the lumenal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain of the mhv m protein strongly affected its ability to form vlps (de haan et al., a) . the assembly process was particularly sensitive to changes in the carboxy terminus of the protein. truncation by only one residue reduced the efficiency severely whereas removal of two residues fully abolished particle formation. these effects appeared to be less severe in the context of a normal coronaviral infection, probably because additional interactions can compensate. the single-residue deletion, when introduced into the mhv genome, was without measurable phenotype and also a mutant virus with a truncation of two residues could be obtained, although with difficulty, as it was severely affected in its growth (de haan et al., a; kuo and masters, ) . the importance of the m protein cytoplasmic and transmembrane domains was confirmed by vlp studies in the ibv system; mutant proteins lacking portions of either of these domains were unable to support particle assembly (corse and machamer, ) . studies of the primary structure requirements of the e protein for vlp formation revealed that the sequence of its hydrophobic domain was not critical. the assembly capacity of the protein was maintained when its transmembrane region was partly or completely replaced by the corresponding domain of the vesicular stomatitis virus (vsv) g protein (corse and machamer, ) . however, when its small aminoterminal ectodomain was additionally replaced by the large vsv g protein counterpart, the chimeric protein became nonfunctional. deletion of the transmembrane, but not the amino-terminal, domain rendered the e protein essentially assembly incompetent (lim and liu, ) . deletions in the cytoplasmic carboxy-terminal half of the e protein mapped the cysteine-rich region as the most important part for vlp assembly (lim and liu, ) . although the interaction between m and e proteins is amply demonstrated by their interdependence for vlp formation, direct evidence for their interaction has actually been provided only for the ibv proteins (corse and machamer, ; lim and liu, ) . the two proteins could be cross-linked to each other in ibv-infected cells and in cells coexpressing the m and e genes (corse and machamer, ) . it appeared that the cytoplasmic tails of both proteins were required, suggesting they are involved in the interaction. in another study m-e interaction was demonstrated by a coimmunoprecipitation assay. also in this assay the cytoplasmic domain of the e protein, comprising the cysteine-rich region, was found to be important as its deletion affected m-e interaction to the greatest extent when compared with other deletion mutant e proteins (lim and liu, ). the results from both studies also showed that the ability of mutant e or m proteins to interact did not correlate with their assembly competence. apparently, other requirements such as homotypic e or m interactions or interactions with host cell components must be met. the specificity of the interaction between the m and e proteins during particle assembly was further demonstrated by the poorly successful attempts to generate chimeric vlps. no particles were observed when heterologous combinations of tgev and bcov m and e proteins were coexpressed (baudoux et al., b) and the same was true for heterologous combinations of fipv and mhv m and e proteins (h. vennema and p. j. m. rottier, unpublished results) . in both studies chimeric m and e proteins were also tested, demonstrating that, except in one case, exchanges between corresponding domains rendered the proteins assembly incompetent. only when the tgev m protein amino-terminal ectodomain was replaced with that of bcov did the chimeric polypeptide support vlp formation in combination with the tgev e protein; vlp formations was also supported, but to different extents, with tgev/bcov chimeric e proteins and-poorly, however-with the bcov e protein (baudoux et al., b) . the reciprocal m construct, the bcov m protein carrying the tgev ectodomain, was nonfunctional, even in combination with tgev e protein. consistently, replacement of the ectodomain with that of fipv m also abolished the productive partnership of mhv m protein with mhv e (de haan et al., ) . as the disproportionate amounts of m and e proteins in vlps already imply, homotypic interactions between m molecules must constitute the energetic basis underlying the formation of the coronaviral envelope. in mhv-based vlps generated by coexpression of m and e proteins, for instance, the sheer excess of m protein-the relative molar presence of e in the particles is less than %-is evidence for the strong interactive forces between m molecules. hence, envelope assembly is thought to be driven primarily by laterally interacting m molecules that form a two-dimensional lattice in intracellular membranes (opstelten et al., b . large multimeric complexes of m protein have indeed been demonstrated biochemically after individual expression of the mhv protein in cells. when the association of the m molecules was maintained by the careful selection of cell lysis conditions, sucrose gradient analysis revealed the existence of large heterogeneous (up to about molecules) complexes, which accumulated in the golgi compartment (locker et al., ) . somewhat smaller complexes were obtained when the cytoplasmic tail of the protein was removed; these complexes were no longer retained in the golgi apparatus but transported to the cell surface. apparently, the tail domain is not essential for the lateral interactions between m proteins, but it is critically required for budding (de haan et al., a . similar higher order complexes of the m protein have also been demonstrated in mhv-infected cells as well as in mhv virions (opstelten et al., b . further support for the existence of homotypic m protein interactions and additional insight into the domains involved in these interactions came from work with mutant m proteins that are unable to assemble into vlps. in these studies mhv m proteins with deletions in either the transmembrane regions, the amphipathic domain, or the extreme carboxy terminus or with substitutions of the lumenal domain were tested for their ability to associate with other m proteins and to be rescued into vlps formed by assembly-competent m proteins (de haan et al., a . it appeared that the mutant proteins maintained these biological activities despite the often severe alterations; actually, the only mutant protein that had lost these abilities was one in which all three transmembrane domains had been replaced by a heterologous transmembrane domain. it was concluded that m protein molecules interact with each other through multiple contact sites, particularly at the transmembrane level. it was furthermore hypothesized that the full complement of interactions between the m molecules is required for efficient particle formation; possibly, all these interactions are required to provide the free energy to generate and stabilize the budding envelope. the failure of m protein mutants capable of associating with assembly-competent m protein to assemble into vlps by themselves (de haan et al., a indicates that additional interactions with viral (e) and/or host proteins is required. in this respect it is of note that the ifn-inducing capacity of the m protein, demonstrated for tgev and bcov, also requires the presence of the e protein, which suggests that the induction of ifn is dependent on a specific, probably regularly organized structure of the m protein (baudoux et al., a,b) . it is unclear how the presence of the e protein alters the m protein lattice to achieve this effect. coronavirus envelope assembly is not dependent on the s protein or the he protein. this is obvious from work with vlps as well as with viruses showing that bona fide particles were produced when these proteins were either simply absent or unavailable for assembly. availability can be compromised under conditions in which proper folding of the proteins is affected. inhibition of n-glycosylation by the drug tunicamycin, for instance, can lead to aggregation and retention of membrane proteins in the endoplasmic reticulum and has been shown to prevent the incorporation into virions of both the s protein mounir and talbot, ; rottier et al., a; stern and sefton, ) and the he protein (mounir and talbot, ) . the same effect has been observed with temperature-sensitive mhv mutants carrying defects in their s gene, which, when grown at the restrictive temperature, gave rise to spikeless particles ricard et al., ) . both s and he proteins are assembled into the coronaviral envelope through interactions with the m protein. such interactions have been demonstrated for mhv and bcov m and s proteins and for bcov m and he proteins, in infected cells, in cells coexpressing the proteins, and in virions (nguyen and hogue, ; opstelten et al., ) . complexes of the proteins were shown by coimmunoprecipitation and cosedimentation analyses as well as by immunofluorescence studies in which the intracellular transport of s and he proteins to the plasma membrane was found to be inhibited by coexpressed m protein, the proteins being retained in the golgi apparatus, the natural residence of the m protein. the kinetics with which the proteins engage in heteromeric complex formation appeared to be different for the different proteins. this effect is due to their different rates of folding and oligomerization. for the s protein these rates are low, involving the formation of multiple intramolecular disulfide bonds and the addition of numerous oligosaccharide side chains (delmas and laude, ; opstelten et al., a; vennema et al., a,b) . in contrast, folding of the mhv and bcov m proteins is independent of disulfide bonds and glycosylation, as a result of which they are, for instance, swiftly transported out of the endoplasmic reticulum (opstelten et al., a) . as a consequence, m molecules enter into m-s and m-he complexes immediately after their synthesis whereas for newly synthesized s and he molecules it took - min before they started to appear in these heterocomplexes (nguyen and hogue, ; opstelten et al., ) . the importance of folding as a major rate-limiting step was illustrated by the inability of the s protein to interact with m protein when its folding had been inhibited by in vivo reduction; only completely oxidized s molecules were association competent (opstelten et al., a . whether the m protein interacts with s and he proteins while they are still in their monomeric form or only after their oligomerization remains to be elucidated. it is, however, clear that the proteins engage in interaction with each other in early compartments, most likely the endoplasmic reticulum, as judged from the oligosaccharide maturation states of freshly formed protein complexes (de haan et al., ; nguyen and hogue, ; opstelten et al., ) . only dimers of he were associated with he-m-s complexes that were observed in bcov-infected cells; because the appearance of he in these complexes correlated with the kinetics of he dimerization it was concluded that proper oligomerization is most likely a requirement for its association (nguyen and hogue, ) . interestingly, such heterotrimeric complexes were not observed on coexpression of the three proteins in cells. under these conditions only the heterodimeric m-s and m-he associations were detected. the structural domains of m and s proteins that are involved in the formation and stabilization of their complex have been identified. using the coimmunoprecipitation and colocalization assays referred to previously, the essential domains in the mhv m protein were mapped by a mutag enetic appro ach (de ha an et al ., ) . it appear ed tha t m-s complex formation was sensitive to changes in all membrane-associated parts of the m molecule. interactions between m and s proteins were found to occur at the level of the transmembrane domains and of the amphipathic domain, which is located on the cytoplasmic face of cellular membranes. in contrast, neither the lumenally exposed amino terminus nor the hydrophilic cytoplasmic tail of the m protein was required; even the deletion of these parts-known to abrogate the ability of the protein to form vlps-did not prevent association with the s protein. chimeric s proteins were used to show that the large ectodomains of the spikes are not involved in interaction with m proteins. such chimeric proteins were constructed from the mhv and fipv s proteins and consisted of the ecto-or lumenal domain from the one and the transmembrane plus endodomain from the other. these proteins, which seemed biologically fit as they were still fusion active, were initially tested in coexpression studies with the m and e proteins from either virus for their ability to be incorporated into vlps. they were found to assemble only into viral particles of the species from which their carboxy-terminal domain originated . the chimeric s genes were subsequently incorporated into the proper coronavirus genomic background, creating the chimeric viruses fmhv and mfipv, the spike ectodomains of which are from the feline and murine coronavirus, respectively; these studies provided the basis for the development of a novel targeted recombination system for reverse genetics of coronaviruses (haijema et al., ; kuo et al., ) . further fine mapping of the carboxy-terminal parts of the s protein involved in m-s protein interaction revealed the importance of the cytoplasmic tail. again using coimmunoprecipitation and vlp incorporation assays, it appeared that increasing truncations gradually abolished the association with the m protein (b. j. bosch, c. a. m. de haan, and p. j. m. rottier, unpublished results) . the significance of the tail domain was demonstrated most convincingly by showing the coimmunoprecipitation and vlp assembly of a chimeric vsv g protein the cytoplasmic tail of which had been replaced by that of mhv s. tail truncations were tolerated in the context of the coronavirus; recombinant mhvs were generated that lacked or (but not ) residues from the s protein carboxy terminus, but their growth was impaired by about -and -fold, respectively. also, tail extensions were tolerated, allowing the construction of a recombinant mhv with a spike protein extended at its carboxy terminus by the green fluorescent protein (gfp), yielding green fluorescent virions (bosch et al., a) . the extension was, however, lost quite rapidly on serial passaging of the virus. molecular details of the interaction of m and he proteins and the requirements of he for incorporation into viral particles have not been described. one study reported that he protein mutants lacking part of their ectodomain were not assembled into particles (liao et al., ) . most likely, however, this observation was due to folding or maturation defects of the mutant proteins. in another study the bcov s and he proteins were shown to be incorporated into mhv particles when coexpressed in mhv-infected cells. apparently, homology between the proteins of these related group coronaviruses is sufficiently high for heterologous m-s and m-he interactions to occur (popova and zhang, ) . numerous electron microscopy studies have pictured the process of virion assembly in the coronavirus-infected cell. they show the close apposition of-presumably preassembled-tubular nucleocapsids to intracellular membranes, the appearance of membrane curvature at the contact sites, the "growth" of these buds into particle-sized vesicles, and the ultimate detachment of virions from the membranes by pinching off. it has become clear that the m protein is the central player, which, through its interactions with every known component of the virion, orchestrates the entire assembly process (see fig. ). in the process two levels of interaction can be distinguished. one is the level of the membrane where, as detailed in the previous section, the m protein interacts ( ) with itself, to generate the basic molecular framework of the viral envelope, ( ) with the e protein, to induce curving and budding of the m protein-modified membrane, and ( ) with s and he, to coassemble these spikes into the viral envelope. the other level at which the m protein operates involves the incorporation of the nucleocapsid into the virion. here, two types of interactions have been described: interactions of the m protein with the n protein and with the viral genome. an instrumental role of the m protein in drawing the nucleocapsid into the budding particle is indicated by their demonstrated interaction in studies with virion preparations. the m protein has been shown to remain associated with subviral particles obtained after treatment of virions with detergent that removes the spikes (escors et al., a,b; garwes et al., ; lancer and howard, ; wege et al., ) . the association was shown for mhv to be temperature dependent (sturman et al., ) . in the case of tgev the association of m with the spherical structure, termed the core, was stabilized by basic ph and divalent cations but lost at high salt concentration, resulting in disruption of the core structure and release of the helical nucleocapsid (escors et al., a,b; risco et al., ) . in an elegant study of the binding of in vitro-translated m polypeptides to purified nucleocapsids the ionic interaction was mapped to a -residue sequence in the hydrophilic carboxy-terminal tail domain (escors et al., b) . also in infected cells, interaction of the m protein with ribonucleoprotein structures, presumably nucleocapsids, has been demonstrated. using m-specific antibodies, structures containing both n protein and genomic rna were coimmunoprecipitated with m protein from mhv-infected cell lysates (narayanan et al., ) . conversely, m protein was coprecipitated when an n-specific antibody was used, while in this case all viral mrnas copurified because of their known leader-mediated affinity for the n protein. these interactions did not require an s or an e protein (narayanan et al., ) . although interactions between the m and n proteins might intuitively be expected to drive the process of attachment of the nucleocapsid to the intracellular target membrane, direct experimental evidence for this interaction is strikingly lacking. significantly, mhv m and n proteins coexpressed in cells were found not to interact (narayanan et al., ) nor did purified tgev n protein interact with in vitrosynthesized m protein (escors et al., b) . because the n protein occurs in coronavirus-infected cells in various configurations-as a free protein and in association with an array of partners including the viral genome-it is obvious that a selection mechanism must act to ensure that only nucleocapsids are assembled into particles. consistently, coexpressed n protein is not incorporated into vlps, but its inclusion depends on the presence of viral rna (bos et al., ; vennema et al., ) . thus, unless the selection process is performed by a mechanism not involving the n protein, its association with genomic rna to form a nucleocapsid seems required to generate the unique conformation that enables it to interact with the m protein. the only evidence to date for an interaction between m and n proteins is indirect and comes from genetic studies. analysis of second-site revertants of a constructed mhv-a mutant virus lacking the two carboxyterminal residues of its m protein revealed that the highly defective growth phenotype of this virus could be restored, among others, by mutations in the carboxy-terminal domain of the n protein (kuo and masters, ) . in two independently obtained revertants the n protein had lost residues of this-among different strains of mhv highly conserved-domain because of a frameshifting -nucleotide deletion. the results argue strongly for a direct cooperation of the carboxy-terminal regions of the m and n proteins during virion formation. other indications supporting the occurrence of m-n interactions come from studies of complexes of m protein with ribonucleoprotein from mhv-infected cells and with tgev cores (escors et al., b; narayanan et al., ) . when such complexes were treated with rnase the association of m and n proteins was not destroyed, suggesting a direct interaction. however, the presence of short rnas inaccessible to the rnase but sufficient to bridge the m-n interaction could not be excluded. the most unusual interaction that the coronaviral m protein seems to engage in involves genomic rna. this interaction has so far been reported only for mhv, by makino and co-workers. these workers had shown earlier that the -nucleotide packaging signal located in the pol b gene could mediate the incorporation into virions of rnas of even noncoronaviral origin (woo et al., ) . they subsequently showed that this incorporation is most likely effected by a direct and specific interaction of the signal with the m protein. when defective genomic rnas or nonviral rnas were introduced into helper mhv-infected cells, they could subsequently be isolated as ribonucleoproteins from lysates of the cells by immunoprecipitation with an m-specific antibody, but only if the rnas contained the packaging signal (narayanan and makino, ) . coexpression experiments using noncoronaviral vectors showed the interaction to be independent of the n protein. a reporter gene transcript generated in cells expressing the m protein could be coimmunoprecipitated with an anti-m monoclonal antibody provided that the rna carried the packaging signal (narayanan et al., a) . moreover, when the e protein was additionally coexpressed, the signal-containing rna-but not an identical rna lacking this sequence-was found to be coincorporated into vlps, irrespective of the presence of the n protein. altogether these observations reveal a hitherto unknown type of interaction between a viral envelope protein and genomic rna. although its significance remains to be further established the m-rna interaction seems to provide additional selectivity to the assembly of the coronaviral nucleocapsid. assembly of viruses is a process of generally high specificity. directed by specific targeting signals, the viral structural components colocalize to distinct places in the cell where unique and complex molecular interactions control their assembly. these rules hold particularly for naked viruses and many of the smaller enveloped viruses; there are, however, many examples where the process is considerably less selective and where "nonself " (host or viral) components are coassembled (see, e.g., garoff et al., ) . interestingly, formation of the large, pleiomorphic coronaviruses appears to combine aspects of both great selectivity and extreme flexibility. with the m and e proteins as the fixed minimal requirement, coronaviral particles appear to tolerate the presence of all other viral components in practically every possible combination. a nucleocapsid is not required but, if available, it can take almost any length as defective (including chimeric) genomes of largely varying sizes have been accommodated. rnas need not necessarily be packaged into a nucleocapsid; whether of viral or nonviral origin, if provided with the proper packaging signal they can be taken in even in the absence of an n protein. also in the composition of their viral envelope these viruses are highly flexible. spikes seem to be incorporated in variable numbers depending on availability. they tolerate severe manipulation, both of their ectodomain and of their endodomain. thus, swapping of ectodomains between unrelated coronaviruses (i.e., from different groups) creates viable chimeric viruses whereas a foreign protein such as gfp appended to the s protein endodomain is accommodated in the particle, although reluctantly. some coronaviruses have an he protein but continue growing well if for any reason the gene is not (properly) expressed as happens among different mhvs. consistently, s and he proteins are incorporated independent from each other. direct interactions between s and he were not observed when the proteins were coexpressed in cells (nguyen and hogue, ) . this result is consistent with the idea that these proteins are separately drawn into the m protein lattice by their distinctive interactions with m molecules. it is also consistent with the concept that these proteins assume different positions within this lattice, a hypothesis based on the presumed different geometric requirements for the incorporation of trimeric and di-or tetrameric s and he complexes, respectively. how, in the face of this enormous flexibility in accommodating all these various numbers and combinations of viral components, do coronaviruses manage to maintain specificity? host proteins have not been noticed to occur in virions, although this may simply not have been looked at carefully enough. by probing the specificity, using viral and nonviral membrane proteins, it appeared that foreign proteins are effectively excluded from coronaviral particles . however, some missorting was found to occur, consistent with earlier observations (yoshikura and taguchi, ) . the picture of coronaviral envelope formation is one that is directed entirely by lateral interactions between the envelope proteins. in infected cells, membrane proteins-viral and cellular-are sampled for fit into the lattice formed by m molecules. the specificity of the molecular interactions acts as a quality control system to warrant the formation of the two-dimensional assemblies that contain the full complement of viral membrane proteins but from which cellular proteins are segregated. for each cellular protein the efficiency of this exclusion process is determined by its lack of interaction with the m protein, its lack of fit in the m protein framework, and its success in competing with the s and he oligomers for the (geometrically different) vacancies within this framework. the precise location of coronavirus budding and the factors that govern it have not been established. although it is clear that particle formation occurs at membranes early in the secretory pathway, up to the cis-golgi compartment, the precise site has not been identified for any coronavirus. several considerations may explain this lack of knowledge. one is that these early compartments are themselves rather complex and highly dynamic and have hence been difficult to define structurally. another is the possible alteration of the structural integrity of these compartments by infection; studies of these effects have not been described. a third complication may be that coronaviruses do not behave uniformly, different viruses possibly preferring different membranes for budding. in this respect it may be of note that differences have been observed, for instance, in the intrinsic localization of the m proteins from ibv and mhv; they appeared to accumulate on the cis and trans side of the golgi apparatus, respectively machamer et al., ) . it has long been assumed that the m protein determines the site of coronavirus budding. when, however, this protein appeared to localize beyond this site, the idea became attractive that the association of the envelope proteins may create the novel targeting signals that direct these multimeric complexes to the budding site. in support of such a notion is the fact that the s and he proteins, when coexpressed with the m protein, are retained in the golgi apparatus rather than being transported to the plasma membrane. the critical question now is whether and how the e protein affects the localization of the m protein. as the e protein by itself does not seem to localize to the virion budding site it will be of great interest to determine the membranes at which vlps assemble. as the envelope proteins can direct particle formation by themselves, it may seem that the nucleocapsid is not leading the assembly process. still, besides giving rise to virions rather than vlps, its involvement in assembly might have important consequences. first of all, nucleocapsids may enhance the efficiency of the budding process. the physical yields of vlps obtained by coexpression of the envelope proteins in cells are generally poor. although there may be many reasons for this, in infected cells the availability of nucleocapsids is likely to facilitate particle production. empty particles, considered to be vlps, have nevertheless been observed during natural infection (afzelius, ; macnaughton and davies, ) . their formation might simply serve as a means to dispose of excess viral membrane proteins from infected cells if required. another effect of the nucleocapsid could involve the localization of budding. it is conceivable that, unless a defined budding station is created by a specific interplay between viral and host proteins (for which no indications yet exist), preassembled nucleocapsids dock at those intracellular membrane sites where sufficiently sized patches of m protein-based envelope structure have accumulated. early in infection such patches might start to form only after the envelope proteins have left the endoplasmic reticulum and become concentrated in intermediate membranes on their way to the golgi complex. later, when viral protein synthesis increases, this density might be reached earlier, perhaps explaining the observed late budding in the endoplasmic reticulum (goldsmith et al., ; klumperman et al., ; tooze et al., ) . it will again be interesting to learn where vlps independently bud and how this relates to the local density of the envelope proteins because this information will shed light on the role of the nucleocapsid in the localization of virion assembly. the picture of coronavirus assembly that the available literature allows us to draw in this review is still a rough draft. we know the identity and some characteristics of the key elements of the picture, we know the relative positions and orientations of most of them, but we are unable to fit them all into a sensible composition. although this may seem like a discontented retrospective, it certainly is not. in the years that the senior author has been in coronavirology research, enormous progress has been made in practically all its aspects including virion assembly. however, the rewarding act of compiling and ordering the available information and trying to abstract from it actual knowledge was at the same time a sharp and recurrent confrontation with the unknown. we want to conclude this work by summarizing what in our opinion will be the main issues for the near future. with the obstacle of reverse genetics technology solved, "structure" will be the dominating issue of the next decade. biology has taught that molecular insight into processes will eventually depend critically on detailed structural information. for coronavirus assembly this means data on the individual structural components and, particularly, on the virion. with respect to the former, this will be most challenging for the membrane proteins, notably m and e. virions, by their apparent elasticity, have eluded structural analysis. here, despite the still limited resolution to be expected, cryoelectron microscopy should provide the urgently required insight into the structural organization of the particle. another issue will be the cell biology of assembly. this actually refers to a number of poignant problems at every stage of the process. starting with nucleocapsid formation we must admit that we know practically nothing. by which interactions and where on the genome the packaging is initiated, how the wrapping of the rna proceeds, how the condensation of the ribonucleoprotein structure takes place, and where in the cell these activities take place are all unresolved questions. although we seem to know more about the budding process, several fundamental issues are still unresolved. obvious issues are the site of budding and the determinants of its location, and the inclusion of the nucleocapsid into the budding particle. an intriguing issue is the budding mechanism itself: how is membrane curvature generated and, particularly, how is the directionality determined. coronaviruses, like other intracellularly budding viruses, direct their particles out of the cytoplasm into the organelles, that is, opposite to the natural direction of cellular vesicle budding. once again, simply nothing is known about the governing principles. a third field of research that has yet to open is the contribution of host cellular factors to the assembly process. work has so far been concentrating on the viral components and their interplay. although there have been incidental indications, studies on the specific involvement of host proteins apparently had to await the development of appropriate technologies and these are now becoming available. although the serious health threat caused by the - epidemic of sars apparently has waned, 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induced at c either by soluble murine ceacam receptors or by ph expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective-interfering rna alters viral pathogenesis presence of subgenomic mrnas in virions of coronavirus ibv the amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with genomic rna the infectious bronchitis virus nucleocapsid protein binds rna sequences in the terminus of the genome following the rule: formation of the -helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors virus-encoded proteinases and proteolytic processing in the nidovirales we thank henk halsema for making most of the drawings. we are grateful to cristina risco for providing the electron micrographs demonstrating the structural maturation of virions; to raoul de groot, berend jan bosch, and jean lepault for their combined efforts in providing the electron micrographs of the purified virions; and to raoul de groot for critical reading of the manuscript. key: cord- -wbfnhedy authors: smith, sylvia b.; st jules, robert s.; o'brien, paul j. title: transient hyperglycosylation of rhodopsin with galactose date: - - journal: experimental eye research doi: . / - ( ) -j sha: doc_id: cord_uid: wbfnhedy abstract rhodopsin's oligosaccharide chains contain predominantly two types of sugar residues: mannose and n-acetylglucosamine. in the present work, bovine and rat rhodopsin were analysed biochemically for the presence of a third sugar, galactose. treatment of bovine rod outer segments (ros) with galactose oxidase followed by reduction with tritium-labeled sodium borohydride revealed the presence of existing molecules of galactose on rhodopsin. rats injected intravitreally with [ h]galactose and [ c]leucine and maintained in darkness were killed hr, hr, , , or days following the injection. retinas were collected for subcellular fractionation and rhodopsin from each of the fractions was purified by cona sepharose chromatography and sds-page. during the first hr, galactose selectively labeled rhodopsin in the golgi-enriched fraction resulting in increased [ h] [ c] ratios in both golgi and ros. the data suggested that trimming was occurring at the transition from golgi to ros. furthermore, a decrease in isotope ratio in the ros between hr and day suggested further trimming of rhodopsin after membrane assembly in the ros. additional in vivo experiments demonstrated existing molecules of galactose on rhodopsin's oligosaccharide chain using lectin affinity chromatography. rats injected intravitreally with [ s]methionine were dark-adapted for hr. following subcellular fractionation of retinas, cona purified rhodopsin from ros was applied to one of two additional lectin columns: ricinus communis agglutinin (rca) or griffonia simplicifolia i (gsa). eight to nine per cent of the labeled rhodopsin was bound to and eluted from rca, whereas none bound to gsa, indicating the presence of a β-galactoside. the rca agarose eluted protein co-electrophoresed with a rhodopsin standard and was light sensitive. galactose was shown to be the terminal sugar on this subset of rhodopsin and was not capped by neuraminic acid. binding of rhodopsin's oligosaccharide to rca was abolished by pre-treatment with β-galactosidase. decreased binding of rhodopsin to rca was observed following intravitreal injection of castanospermine but not swainsonine. of those two inhibitors of glycoprotein trimming, only castanospermine would be expected to prevent the addition of galactose to the oligosaccharide. the association of galactose with rat rhodopsin appeared to be a transient one. at hr, – % of rhodopsin contained galactose, at hr only · % had galactose and by hr less than % did. the galactose was trimmed from rhodopsin's oligosaccharide presumably after its role was complete. separation of rhodopsin of the plasma membranes from rhodopsin of discs indicated that % of the galactose-containing rhodopsin was in the plasma membrane and only % was in the discs. these findings suggested a possible role for galactose in new disc formation with subsequent removal after the discs are sealed. rhodopsin contains two oligosaccharide chains linked to asn, and asn,, (hargrave, ) . for the most part, these chains contain only two types of sugar residues : mannose and n-acetylglucosamine (heller, ; shichi et al., ; heller and lawrence, : plantner and kean, ) . the synthesis of asparagine-linked glycoproteins typically involves the rough endoplasmic reticulum for the initiation of oligosaccharide synthesis and both the rough endoplasmic reticulum and golgi complex for modification of the oligosaccharide chain (kornfeld and kornfeld, in more complex glycoproteins prior to the addition of a terminal trisaccharide consisting of n-acetylglucosamine, galactose and neuraminic acid. rhodopsin actually contains the first sugar of the terminal trisaccharide as demonstrated by glucosamine incorporation studies (o'brien and muellenberg, ; bok, hall and o'brien, ) . the remaining sugars, galactose and neuraminic acid have not usually been detected in the rhodopsin oligosaccharide chain. however, o'brien ( ) demonstrated the direct transfer of galactose in vitro to bovine rhodopsin and showed chromatographically that the radiolabeledgalactose was incorporated as galactose since the radioactivity coincided with a galactose standard and was not converted to some other sugar (such as mannose). more recently, galactose was successfully incorporated into rat rhodopsin in vitro following incubation of whole retinas with [ h] galactose (st jules, smith and o'brien, ) . fukuda. papermaster and hargrave ( ) analysed bovine rhodopsin's academic press limited amino terminal glycopeptide using gas chromatography and mass spectrometry and reported that in addition to the major oligosaccharide structure which contained three n-acetylglucosamine residues and three mannose residues, a minor variation of rhodopsin contained . mol galactose per three nacetylglucosamine residues and three mannose residues. these data suggested that up to % of rhodopsin contained galactose. morphologic studies of carbohydrate-containing molecules in photoreceptor cells have invoked a different methodology utilizing lectins for detecting the presence of specific sugars. ricinus communis agglutinin (rca) is a lectin that specifically recognizes terminal galactose moieties associated with either glucosamine or n-acetylglucosamine in a / - linkage (baenziger and fiete, ) . transmission electron microscopic studies of frog, bovine, monkey and rat retinas have shown binding of rca to rod outer segments and in some cases rod inner segments (nir and hall, ; hicks and molday, ; koide et al., i : hicks and barnstable, ) . likewise, similar results were obtained in fluorescent microscopic studies detecting rca binding in frog, monkey, mouse, human, rabbit, and fish retinas (bridges and fong, ; bridges, ; uehara et al., ; blanks and johnson, ) . of great interest was the work by hicks and molday ( ) who published photomicroscopic evidence of post-embedding, direct labeling by rca in the basal area of the ros. in the present study, we used biochemical techniques to characterize the subset of rhodopsin which appears to contain galactose. we present evidence to confirm the incorporation of galactose into rhodopsin in in vivo experiments. additionally. we demonstrate the presence of galactose in bovine rhodopsin by radiolabeling existing moieties. finally, using lectin affinity chromatography, an analytical procedure which is rapid, reliable and highly sensitive (cummings, merkle and stults, ) , we are able to separate the galactose-containing species of rhodopsin and study its transient association with the oligosaccharide chain. for most experiments, sprague-dawley rats, -l g (taconic farms, germantown, ny) were used. one set of experiments utilized bovine retinas dissected from bovine eyes obtained fresh from a slaughterhouse. for studies of in vivo labeling of rhodopsin with galactose, rats were anesthetized with ether and injected intravitreally with /ai per eye of a saline solution containing pci ['"clleucine and . ,uci jq-s-'h]galactose ( . ci mmol '). in other in vivu experiments, eyes were injected with ,li ]-?i)leucine ( ci mmol ') or ,&i [" s]nlethionine ( ci mmol ') or i/ci ["%]methionine ;ind //ci ]:sh]glucosamine ( .x ci mmol ). the rats m'ere maintained in darkness and at various intervals were killed with co,. the retinas were removed by proptosing the eye using a curved forceps, the prongs of which were ensheathed in polyethylene tubing. the cornea was slit with a scalpel blade causing the lens and vitreous humor to be extruded. the retina was lifted free of any other ocular material simply by squeezing and lifting the forceps upwards. the sclera remained attached to the optic nerve. this procedure takes only -l s and the retinas are removed intact and ready for biochemical workup. no elaborate dissection is required. ["h]galactose, ]"h]glucosamine, ["hlleucine and [' 'clleucine were purchased from new england nuclear: [""slmethionine was purchased from american radiolabeled chemicals, inc. isolated retinas were fractionated in dim red light by a method described previously (st jules, smith and o'brien, ) . four to six retinas were suspended in ml % sucrose containing mm nacl, rnlv mgci,, and mm tris acetate ph . . rod outer segments were broken off with -set bursts of a vortex mixer. after a s-min centrifugation at g the floating ros were removed with the supernatant and the retinal debris was treated again as above. the pooled crude ros suspension was diluted with volumes of buffered % sucrose, or in some cases . % nacl, and were sedimented by centrifugation at g for min. both the ros and retinal debris were separately homogenized in ml % sucrose made up in mm nacl, mm mgcl,. and mm tris acetate ph - using seven strokes of a dounce tissue grinder (wheaton ml. b pestle with . - . s-inch clearance). these homogenates were layered over separate linear .- % sucrose gradients made up in the same buffer. after centrifugation for hr in a spinco sw rotor at rpm, bands were removed with a pasteur pipet. diluted with loo/, sucrose and sedimented by centrifugation for min at g. two bands of unsealed and sealed ros were near the top of the ros gradient (godchaux and zimmerman. ) . below these was a zone of fine particles, enriched in golgi. in the retinal debris gradient was a trace of the golgi-enriched fraction and a prominent zone of coarse particles, including the rough endoplasmic reticulum, nuclei and synaptosomes. these fractions are described in more detail in st jules and o'brien ( ) . with the bovine retinas, discontinuous gradients were prepared by overlaying the bovine ros sus-pension with the following sucrose solutions: ml of d . , ml of dl. and ml of dl. . the sucrose solutions were made up in mm tris acetate, ph . , containing . mm mgcl,. after centrifugation for min at rpm in a spinco sw ti rotor, the ros were removed from the interface at dl. lldl. with a pasteur pipette, diluted with % sucrose and sedimented by centrifugation for min at g. rhodopsin-containing membranes from four to six rat retinas were extracted in dim red light for hr at °c with ml % emulphogene bc (general aniline and film) in mm tris acetate buffer, ph . , containing mm mgcl, and mm cacl,. the extract was clarified by centrifugation at g for min and applied to a . ml column of cona sepharose (pharmacia). after washing with ml . % emulphogene in the same buffer, the rhodopsin was eluted with eight applications of ~ aliquots of . m amethylmannoside (sigma) also made up in the same buffer with . % emulphogene. twenty microliters of each -~ fraction were sampled for radioactivity. peak fractions were pooled and aliquots were adjusted to % sodium dodecyl sulfate (sds) and mm edta. samples were incubated at room temperature ( °c) for hr in dim red light. after the addition of one half volume of % glycerol containing . y bromophenol blue (biorad) the samples were applied to % polyacrylamide disc gels with % stacking gels for electrophoresis by the method of laemmli ( ) . gels were stained with coomassie brilliant blue (biorad) and cut into . mm slices with a hoefer gel slicer and the slices containing the stained opsin were noted. slices were digested overnight at °c in ncs protein solubilizer (amersham) with . % water. slices were counted (two per vial) in econofluor- (new england nuclear, boston, ma) using a beckman ls scintillation counter. the mobility of radioactive opsin could be compared directly with that of the coomassie blue stained mature opsin in the same cylindrical gel. those experiments utilizing two isotopes (i.e. ["hlgalactose and [xlleucine) permitted determination of the ratio of these isotopes in the opsin band of the gels. to radiolabel existing galactose moieties on rhodopsin, bovine ros purified by a discontinuous sucrose density gradient procedure, were subjected to enzymatic treatment with galactose oxidase followed by a reduction with tritium labeled sodium borohydride. in these experiments no external source of radioactive galactose was provided. ros from two bovine retinas were washed by centrifugation with pbs for min at g. they were suspended in ml pbs, an additional ml of pbs containing u of galactose oxidase (worthington) was added to one ros sample and ml pbs without the enzyme was added to a second ros sample as a control. five microliters . m pmsf in ethanol were added to each sample. ros were incubated at room temperature with occasional swirling for min in the dark. ros were centrifuged at g for min. then resuspended in pbs and centrifuged for the same length of time and speed. ros were suspended in pbs. to each sample was added . mci of tritium labeled sodium borohydride (new england nuclear) in . n naoh made up in ml pbs. after min at room temperature, additional pbs was added and ros were centrifuged at g for min. subsequently, ros were washed twice by centrifugation in pbs. in a repeat of this procedure, one sample received pretreatment with u neuraminidase from clostridium perfringens (sigma) at ph . for hr at 'c and the subsequent galactose oxidase incubation time was doubled. the ros were solubilized and rhodopsin was analysed by sds-page as described above. to further characterize the subset of rhodopsin which contains galactose (without introducing an external source of galactose) lectin affinity chromatography was used. in this method, cona purified rhodopsin or in some cases the clarified extract of ros from six rat retinas labeled with [ s]methionine was applied to columns of either ricinus communis agglutinin i (rca) or grifsonia simplicijolia i (gsa), specific for p-linked or a-linked galactose residues, respectively. rca agarose was purchased from u.s. biochemical corp., cleveland, oh and gsa agarose was purchased from biocarb chemicals, lund, sweden. columns were washed with . % emulphogene in mm tris acetate buffer, ph . , containing mm mgcl, and itim cacl,. samples were eluted from rca agarose or gsa agarose columns with ,ul aliquots of either . m / -methyl-n-galactopyranoside or amethyl-n-galactopyranoside (u.s. biochemical corp.), respectively. aliquots ( ~ ) were analysed for radioactivity using a beckman ls scintillation counter. aliquots of the peak fractions were applied to sds-polyacrylamide gels and analysed for radioactivity of opsin as described above. to determine if the galactose of rhodopsin was capped with a neuraminic acid, rat ros labeled with [ s]methionine were incubated with u neuraminidase from vibrio cholerae (boehringer-mannheim) at °c for hr. a second ros sample was incubated with no enzyme. the incubated suspension was centrifuged for min at g. the ros were extracted for hr and the clarified extracts were applied to kca agarose columns. aliquots of the peak fractions were applied to sds-polyacrylamide gels and analysed for radioactivity of opsin as described above. cona purified rhodopsin labeled with [%]methionine and ["hlglucosamine was incubated at °c for hr with it of n-glycanase (genzyme, boston, ma) in . % sds, mm /$mercaptoethanol, ,llm pmsf. mm edta, . % np- . ph . . at the end of the incubation the ph of the sample was adjusted to . by the addition of acetic acid. the solution was buffered with mes (sigma). b-galactosidase ( mu) from diplococcus pneumoniae (boehringer-mannheim) were added to half of the sample. both the enzyme treated sample and the control were incubated at °c for hr. the samples were applied to separate rca agarose columns and subsequently eluted with -,ll~ aliquots of p-methyl-ogalactopyranoside as described above. the amount of radioactivity eluted from rca after /%galactosidase treatment was compared to that eluted from rca without the enzyme treatment. to determine the consequences of the interruption of glycoprotein processing on the galactosylation of rhodopsin, two inhibitors of the glycoprotein processing pathway, castanospermine and swainsonine (boehringer-mannheim) were used in in vivo experiments. the concentration of the stock solution of castanospermine was mg ml-'. it was prepared by dissolving mg of the inhibitor in ~ dimethyl sulfoxide (dmso, sigma). the concentration of the stock solution of swainsonine was mg ml-l. it was prepared by dissolving ,ug of the inhibitor in ~ water. rats were injected intravitreally with [ s]methionine as a control or [" s]methionine and either castanospermine ( ,ug per eye) or swainsonine ( . ,ug per eye). since the castanospermine preparation utilized dmso at a final concentration of . ,ul per eye the same amount of dmso was also injected into the eyes of all other rats to control for any effects of dmso on the galactosylation of rhodopsin. after hr of dark adaptation rats were killed, retinas were collected for subcellular fractionation. rhodopsin was purified from the ros fraction and the golgienriched fraction by cona sepharose chromatography. a loo-~ fraction of the cona eluate was saved for sds-page and the remainder was applied to rca agarose columns for elution with /&methyl-ngalactopyranoside as described above. the rca eluates were then applied to sds-page gels. the percentage of cona purified rhodopsin which contained galactose was determined from the radioactivity of the rhodopsin band of the gels of the rca eluate as c.ompared to the cona gels for each treatment group. the data from the three repetitions of this experiment were analysed via a one-way analysis of variance for the ros samples and for the golgi samples. the test of significance used was tukey's multiple comparison procedure. l&jht-sensitivity experiments ros labeled with [ %]methionine and purified on a linear sucrose gradient were extracted with . % emulphogene as described above. prior to purification of rhodopsin over cona sepharose, half of the clarified extract was exposed to light for min at °c. each sample was then applied to a cona sepharose column and chromatographed as described above. the eluted samples were each applied to separate rca agarose columns and eluted according to the previously described procedures. selected eluted fractions were applied to sds polyacrylamide gels along with a ["hlrhodopsin standard for determination of the amount of radioactivity present in opsin under the conditions of light exposure and darkness. in order to determine whether the galactosecontaining rhodopsin was present in photoreceptor discs or plasma membrane a modification of the method of molday and molday ( ) was used. [ "s]methionine-labeled ros from rats were prepared using the linear sucrose gradient procedure described previously. ros were suspended in ml homogenizing buffer consisting of mm tris acetate, ph . . mm mgcl,, % sucrose. neuraminidase ( . u) from anthrobacter ureufaciens (boehringer-mannheim) was added to the ros suspension and incubated for hr at °c. after centrifugation at g for min. the pellet was suspended in ml of homogenizing buffer to which ricin agarose (u.s. biochemical corp.) was added. this mixture was incubated for hr at °c. ricin agarose was used rather than ricin-golddextran because it is sufficiently dense to pellet through the sucrose gradient (boesze-battaglia and albert, ). the ros were pelleted in homogenizing buffer by centrifugation at g for min, hypotonically lysed for min in . m tris buffer, ph . , and washed twice with . m tris buffer ph . (containing mm edta) by centrifugation at g for min. the sample was treated with trypsin (boehringer-mannheim) for min at °c at a final concentration . pg ml -i, soybean trypsin inhibitor (boehringer-mannheim) was then added to a final concentration of pg ml-'. after two additional min washings with . m tris buffer at g, the ros membranes were layered on linear sucrose gradients consisting of ml of a - % (w/v) sucrose underlaid with i ml hoy, (w/v) sucrose. all sucrose solutions were made up in mm nacl, mm mgcl,, and mm tris acetate ph . . after centrifugation for hr in a spinco sw rotor at rpm, the band of discs was drawn off using a pasteur pipet and the plasma membranes at the bottom of the tube were also removed. these two fractions were separately centrifuged at g for min followed by the addition of . % emulphogene to each pellet to extract rhodopsin. extracts were applied to rca agarose columns, washed with . % emulphogene, and eluted according to the previously described procedures. selected eluates were applied to sds-polyacrylamide gels and the amount of radioactivity present in rhodopsin was determined for the discs and plasma membrane. injection, four retinas were collected for subcellular fractionation. rhodopsin from each of the fractions was purified by cona sepharose chromatography and sds-page. table ii provides the ratios of [ h] derived from galactose to [l*c]leucine in rhodopsin from the subcellular fractions enriched in either rough endoplasmic reticulum, golgi or ros. the labeling pattern was complex. galactose was probably converted to glucose and mannose residues of the core . results in the experiments using bovine retinas, purified ros were treated with galactose oxidase to oxidize the sixth carbon hydroxyl group of galactose to a carbonyl group. this treatment was followed by exposure to tritium labeled sodium borohydride which chemically reduced the carbonyl group of galactose back to a hydroxyl with the introduction of a tritium atom (gahmberg and hakomori, ; carubelli and wen, ) . figure shows that without galactose oxidase treatment there was tritium incorporation into rhodopsin. there was a twofold increase in label following the galactose oxidase treatment. the galactose oxidase-dependent label tended to be in a species that migrated slower than rhodopsin as is indicated in the figure by the dashed line representing the difference between the two treatment groups. table i illustrates another experiment in which the galactose oxidase incubation time was doubled and the amount of radioactivity in the galactose oxidase treated ros was . -fold greater (over dpm) than the samples not treated with the enzyme. pretreatment with neuraminidase did not increase the labeling. the non-specific labeling of opsin in the samples not treated with the enzyme was due perhaps to reductions of the retinyl-lysine schiff s base linkage. these experiments demonstrate the presence of galactose in the oligosaccharide chains of native rhodopsin in ros plasma membranes and basal folds. since the enzyme does not have access to the carbohydrate chains within the sealed discs, it is not known whether there are any galactose residues in the disc membrane rhodopsin. of sodium dodecyl sulfate-polyacrylamide (sds-page) gels of bovine ros. the ros were purified by discontinuous sucrose density gradients and were incubated min with ( ) or without (m) galactose oxidase. both samples were then incubated with tritium labeled sodium borohydride. aliquots representing the ros of . retinas were solubilized and applied to gels for electrophoresis. although there was incorporation of tritium into rhodopsin without galactose oxidase treatment, there was a considerable increase in label following the galactose oxidase treatment. the galactose oxidase-dependent label tended to be in a species that migrated slower than rhodopsin as indicated by the dashed line (a) representing the difference between the two treatment groups. the coomassie blue-stained opsin band on a parallel gel coincided with the radioactive profile of the untreated sample. or gsa (@-- ). six rats were injected intravitreally with [?!i]methionine and were killed hr later at which time retinas were collected for subcellular fractionation and rhodopsin purification by cona sepharose chromatography. half of the sample was applied to the rca agarose column and half to the gsa agarose column. the washed rca agarose column was eluted with . m /?-methyl-o-galactopyranoside and the washed gsa agarose column was eluted to . m cc methyl-o-galactopyranoside beginning at fraction in both cases. at hr post-injection. - . x of the labeled rhodopsin bound to and was eluted from the rca column, whereas none bound to gsa. oligosaccharide in the rough endoplasmic reticulum. during the first hr. however, galactose selectively labeled rhodopsin in the golgi-enriched fraction resulting in increased [ h]/[ %] ratios in both golgi and ros. by day the galactose pool was exhausted and carbohydrate labeling that occurred in the rough endoplasmic reticulum-enriched fraction was probably due to glycogen turnover. the decreasing isotope ratios at each time point suggest that trimming was occurring at the transition from golgi to ros. furthermore, the decrease in isotope ratio in the ros between hr and day suggests further trimming of coomassie blue and cut into . mm slices for counting. migration was from left to right. the radioactivity profiles were slightly to the left of the coomassie blue-stained opsin band. as expected (see fig. ) of an opsin having a larger oligosaccharide. rhodopsin after membrane assembly in the ros. some of this lost carbohydrate may have been galactose, particularly in the ros. studies to determine the nature of the linkage of galuctose to rhodopsin rats injected intravitreally with [ s]methionine were maintained in darkness for hr at which time retinas were collected for subcellular fractionation and rhodopsin purification by cona sepharose chromatography. the cona-purified rhodopsin was then applied to one of two lectin columns: rca or gsa. rca specifically recognizes ,/i-linked galactose moieties (baenziger and fiete, ) and gsa is specific for a-linked galactose residues (blake and goldstein, ) . at hr, s- . % of the labeled rhodopsin bound to and was eluted from the rca column, whereas none bound to gsa, thus indicating the presence of a /j'-galactoside (fig. ) . the protein eluted from rca was shown to be rhodopsin by coelectrophoresis with rhodopsin that had been labeled with [ h]leucine in vivo for hr (fig. ) . rats injected intravitreally with [ s]methionine were maintained in darkness for and hr at which time retinas were collected for subcellular fractionation. ros were incubated with and without neuraminidase. the amount of rhodopsin bound to and eluted from the rca agarose columns was not increased by treatment with neuraminidase. the total dpm in the opsin region of sds-polyacrylamide gels for the two conditions and time points are shown in two groups of six rats were injected intravitreally with [%imethionine and killed at the indicated times. ros were prepared and half of each sample incubated with neuraminidase. after extraction, the rhodopsin samples were purified on cona sepharose and subsequently chromatographed on ricin agarose. table iii . these data suggest that although complex asparagine-linked oligosaccharides often have a terminal trisaccharide containing n-acetylglucosamine linked to galactose and capped with neuraminic acid (kornfeld and kornfeld, ) rhodopsin lacks neuraminic acid. demonstration that binding of the rhodopsin oligosaccharide to rca can be decreased with p-galactosidase treatment rats were injected intravitreally with [ s]methionine and [ h]glucosamine to provide markers for the polypeptide and the oligosaccharide, respectively. they were maintained in darkness for hr at which time retinas were collected for subcellular fractionation and rhodopsin purification by cona sepharose chromatography. the cona-purified rhodopsin was treated initially with n-glycanase to hydrolyze the asparagine-linked oligosaccharides from rhodopsin. this treatment was followed by incubation of half of the rhodopsin was incubated at °c for hr with nglycanase to hydrolyze the h-labeled oligosaccharide. p-galactosidase was added to half of the sample. both the / galactosidase treated sample and the control were incubated at °c for hr. each sample was applied to an rca agarose column which was washed and subsequently eluted with p-methyl-u-galactopyranoside beginning at fraction . the amount of radioactivity (dpm h) eluted from rca agarose after ,&galactosidase treatment was significantly less ( . %) than that of the sample eluted from rca agarose without the enzyme treatment ( . %). the sample with p-galactosidase which specifically hydrolyses terminal galactose residues that are pl- linked to n-acetylglucosamine. as shown in fig. , the amount of radioactivity eluted from rca after / galactosidase treatment was significantly less than that of the sample eluted from rca without the enzyme treatment. these same results were obtained in repetitions of this experiment and suggest that removal of galactose from the rhodopsin oligosac- rhodopsin was labeled for hr following simultaneous intravitreal injection of [%]methionine and swainsonine or castanospermine, or neither in three groups of six rats. * data are expressed as the mean percentages of three experiments + s.d. one-way analysis of variance for the ros and golgi samples indicated that there was a significant difference among the three groups (ros: f = . , p = . ; golgi: f = . , p = . ). tukey's paired comparisons test confirmed that the percentages obtained for the castanospermine treated group differed significantly from the control or swainsonine treated group, but these latter two groups did not differ significantly (p < . ). methionine were maintained in darkness for hr at which time retinas were collected for subcellular fractionation. half of a sample of detergent extracted purified ros labeled in vivo with [yg]methionine was exposed to light for min while the remainder of the sample was kept dark. each sample was chromatographed on cona sepharose and eluted with cc-methyl mannoside. the cona sepharose column eluates were applied to rca agarose which was washed with buffer and then eluted with p-methyl-ngalactopyranoside beginning at fraction . as would be expected, the dark sample bound to and was eluted from cona sepharose. . % of that sample was bound to and eluted from rca agarose. in contrast, considerably less radioactivity was eluted when the light-exposed sample was chromatographed on cona. what little was eluted did not bind to rca. ( -e) dark: ( -o) light. charide using this highly specific enzyme dramatically reduces the binding of the oligosaccharide to rca. these data provide further support for the presence of galactose on the oligosaccharide of rhodopsin. rats in groups of six injected intravitreally with [ s]methionine only or [? ]methionine and either catanospermine or swainsonine were dark adapted for hr at which time retinas were collected for subcellular fractionation. rhodopsin was purified from the ros-and golgi-enriched fractions by cona sepharose chromatography and the eluates were applied to kca agarose columns. both the kc'a agarose eluates and a sraction of the cona sepharose eluates were applied to sds-page gels. the data in table iv . representing three replicates of this experiment. show that the mean percentage of cona purified rhodopsin from ros which bound to rc. was greatly reduced in the castanospermine exposed group. a similar effect was seen in the golgi-enriched fraction. the statistical analysis of these data indicated that the percentages obtained in the castanospermine treated ros and golgi samples were significantly different from the control or swainsonine treated group. these data demonstrate that castanospermine. which inhibits glucosidase i, disrupted the binding of rhodopsin to rca, whereas swainsonine, which inhibits golgi mannosidase ii. did not. since rca is specific for p-galactose residues, it appears likely that in the presence of castanospermine. galactose was never linked to the oligosaccharide chain of rhodopsin. rats injected intravitreally with ["%]methionine were maintained in darkness for hr at which time retinas were collected for subcellular fractionation. half of a sample of detergent extracted purified ros labeled in vivo with [ s]methionine was exposed to light while the remainder of the sample was kept dark. each sample was chromatographed on cona sepharose and eluted with z-methyl mannoside. when rhodopsin is bleached it binds to cona. but is not eluted from the lectin by a-methyl mannoside. the cona sepharose column eluates were applied to rca agarose columns and eluted with p-methyl-d-galactopyranoside. the elution patterns from the cona sepharose columns and the rca agarose columns are shown in [fig. (a) and (b)], respectively. as would be expected, the dark sample bound to and was eluted from cona. furthermore. . % of that sample was bound to and eluted from rca. in contrast, much less radioactivity was eluted when the light-exposed sample was chromatographed on cona. what little was eluted did not bind to rca. sds-polyacrylamide gels of the material eluted from rca indicated that the dark sample coincided with a rhodopsin standard labeled with [ h]leucine. whereas there was virtually no radioactivity detected on the gel of the light exposed sample (fig. ) . these data indicate that the cona purified material that binds to rca is light sensitive and is therefore rhodopsin. compartments have galactose-containing rhodopsin n-linked oligosaccharides are typically assembled in the endoplasmic reticulum on dolichylphosphate and are then transported by means of vesicles to the golgi membrane (kornfeld and kornfeld, ) . in the trans golgi. galactose may be added to a terminal n- fig. s(b) ]. the dark sample coincided with a rhodopsin standard labeled in vivo with [ h]leucine, whereas there was virtually no radioactivity detected on the gel of the light-exposed sample. as before (fig. ) the radioactive opsins migrated slightly slower than the coomassie blue-stained mature opsin. . elution profiles from rca agarose columns of bands separated from a linear so- % sucrose gradient over which had been layered a crude ros suspension. the ros suspension was prepared from retinas of six rats which had been intravitreally injected with [%]methionine and dark adapted for hr. this procedure typically yields four bands on the gradient: a faint uppermost band - . cm from the top of the gradient, two bands of unsealed and sealed ros ( and . respectively) and a zone of fine particles, enriched in golgi. pellets of each of these bands were detergent extracted and applied to separate rca agarose columns which were then washed. as shown, the golgi-enriched fraction and both ros fractions (ros and ros ) bound to and were eluted from rca with ,&methyla-galactopyranoside. the uppermost band did not demonstrate rca binding. thus rhodopsin appears to have acquired galactose residues before leaving the golgi. acetylglucosamine of the oligosaccharide chain. in an effort to determine if the golgi apparatus, as well as the ros, of rod photoreceptor cells contained galactose, subcellular fractionation of ros was performed. rats were injected intravitreally with [ s]methionine and were maintained in darkness for hr at which time retinas were collected and crude ros preparations were made using the vortexing procedure described. linear - % sucrose gradients of crude ros suspensions which have been centrifuged for hr typically have four bands. a very faint uppermost band is approximately . - . cm from the top of the gradient. two bands of unsealed and sealed ros are below the . cm band (godchaux and zimmerman, ) . below these is a zone of fine particles, enriched in golgi. pellets of each of these bands were detergent extracted and applied to separate rca columns. as shown in fig. , the golgi-enriched fraction and both ros fractions (ros and ros ) bound to and were eluted from rca. the uppermost band did not demonstrate rca binding. sds-page of these fractions showed that the golgi and ros fractions coelectrophoresed with a rhodopsin standard. although the golgi-enriched fraction is not entirely free of ros, the data suggest that the galactose residue of rhodopsin is present on the oligosaccharide in the golgi as it is in other glycoproteins containing n-linked oligosaccharides. the results of the in vivo experiments in which [ h]galactose was used to label rhodopsin over several days (described above) suggested that galactose might be trimmed from the oligosaccharide of rhodopsin in the transition from golgi to ros as well as in ros over time. to test this, a time course experiment was conducted. rats were injected with [ s]methionine and [ h]galactose and groups of animals were killed , and hr following injection. retinas were collected for subcellular fractionation, ros were extracted and rhodopsin was purified by cona sepharose chromatography. a small fraction of the cona eluate was applied to sds-polyacrylamide gels and the remainder was applied to rca. table v provides the data obtained from gel electrophoresis of the cona-and rca-eluted samples. electrophoresis of the rca agarose eluates revealed that the percentage of the cona-purified rhodopsin that bound to rca decreased over time. at hr, . % of the rhodopsin bound to rca, after hr . % was bound and by hr only . % was bound. electrophoresis of the cona-purified rhodopsin showed that the ratio of the isotopes (that is the ratio of [ h]galactose to [ s]methionine) decreased over tie from . at hr to . by hr. clearly, trimming of galactose from rhodopsin had occurred suggesting that galactose may be an early component of rhodopsin, but as the molecule progresses through the ros the galactose appears to be lost. in contrast, the ratio of the isotopes ([ h]galactose to [ s]methionine) in the rhodopsin that bound to rca was higher than in the total pool eluted from cona and did not change significantly over time. thus, the ratio changes were due to removal of galactose residues. retinas were collected at the indicated times. ros were prepared and rhodopsin was purified on cona sepharose. aliquots were analysed by sds-page and the rest chromatographed on ricin agarose. ricin eluates were also analysed by sds-page. it was of interest to determine whether the galactose-containing rhodopsin was present in photoreceptor discs or plasma membrane. a modification of the method of molday and molday ( ) was used in which nine rats were injected intravitreally with [ s]methionine and were maintained in darkness for hr, at which time ros were prepared using the linear sucrose gradient procedure described previously. after treatment with neuraminidase ros were incubated with ricin-agarose. subsequent to lysis and trypsin treatment, the ros membranes were layered on linear - x sucrose gradients underlaid with % sucrose. following centrifugation, discs and the plasma membranes were removed. detergent extracts of these two components were applied to rca agarose columns. elution patterns from rca revealed significant binding of plasma membrane extracts and a small amount of binding of disc extracts. sdspolyacrylamide gels of the eluates showed that the total amount of radioactivity present in rhodopsin of the plasma membrane ( dpm) was . times greater than the total amount of radioactivity present in disks ( dpm). from these experiments it appears that at hr post-injection, . % of the labeled galactose-containing rhodopsin is in the plasma membrane and . % is in discs. although rhodopsin's oligosaccharide chains have been thought to contain mainly two types of sugar residues [mannose and n-acetylglucosamine (heller, yhx ; shichi et al., : heller and lawrence. : plantner and kean. ) ], there is evidence that a small fraction of rhodopsin also may contain galactose. fukuda et al. ( ) found that % of the oligosaccharides in bovine rhodopsin contained galactose. furthermore, o'brien ( i ) showed that bovine ros preparations support the transfer of galactose from ijdp-galactose to rhodopsin. st jules et al. ( i demonstrated the in vitro incorporation of galactose into rhodopsin and its subsequent removal after rhodopsin had reached the outer segment. these findings suggested that galactose may in fact be associated with rhodopsin of the ros. if only for a short period of time. perhaps the transient nature of its association explains its rather elusive detection by some biochemical techniques. in the current experiments, efforts were focused on demonstrating the presence of galactose by methods which could detect existing molecules, rather than solely by methods to show incorporation of exogenous galactose. (;alactose molecules were shown to exist on bovine rhodopsin using the galactose oxidase-sodium borohydride treatment. the results clearly demonstrated that there were galactose molecules present on rhodopsin which were altered by the highly specific galactose oxidase treatment and were then reduced by the sodium borohydride with the introduction of radiolabel. the bovine model does not permit in vivo studies of oligosaccharide synthesis, however. so it became necessary to investigate the incorporation of galactose in the rat model. the studies in which galactose was injected intravitreally in rats and monitored over several days demonstrated a selective labeling during the first hr in the golgi-enriched and the ros fractions. thus, even though conversion of galactose to other sugars commonly found on rhodopsin's oligosaccharide chain probably occurred, the initial pulse of galactose was apparently incorporated as galactose in the golgi where galactosyl transferase is known to be localized. the results strongly suggested that the galactose had been added to rhodopsin and then trimmed over time. the need to demonstrate the presence of galactose without directly incorporating it into rhodopsin lead to the use of lectin affinity chromatography. this powerful technique allows for the separation of glycoproteins based on the affinity for certain sugar moieties (cummings et al., ) . cona. which has an affinity for mannose residues. has long been used in the purification of rhodopsin. in the present work. a portion of the cona-purified rhodopsin was shown to bind to the lectin rca which specifically recognizes terminal galactose moieties associated with either glucosamine or n-acetylglucosamine in a pl- linkage (baenziger and fiete, ) . whereas no rhodopsin bound to gsa which has an affinity for xlinked galactose (blake and goldstein, ) . methionine was injected intravitreally into rats in order to label the polypeptide chain of rhodopsin. that the radiolabeled protein which bound to rca was indeed rhodopsin was verified by light sensitivity experiments. in addition, the binding of the rhodopsin oligosaccharide to rca could be decreased significantly by cleaving the galactose moiety with the highly specific enzyme / -galactosidase. on the other hand, treatment with neuraminidase, which specifically hydrolyses neuraminic acid from oligosaccharide chains, neither increased nor decreased the binding of rhodopsin to rca. these experiments indicated that the galactose of rhodopsin was not capped with neuraminic acid as is often the case with complex asparagine-linked oligosaccharides whose terminal trisaccharides contain n-acetylglucosamine linked to galactose capped with neuraminic acid (kornfeld and kornfeld, ) . the galactose of terminal trisaccharides of n-linked oligosaccharides are typically added to n-acetylglucosamine in the trans-golgi (kornfeld and kornfeld, ) . in the present work, it was of interest to determine if galactose was associated with rhodopsin purified from the golgi-enriched fraction as well as ros. it was determined by subcellular fractionation and subsequent rca chromatography that the rhodopsin of the golgi-enriched fraction did indeed contain galactose. furthermore, it was shown that the addition of galactose to the n-acetylglucosamine could be inhibited by in vivo exposure to castanospermine which specifically inhibits glucosidase i. glucosidase i is a rough endoplasmic reticulum enzyme that cleaves the terminal glucose from the glc,man,glcnac, (repp et al., ) . with this early step in the processing of the oligosaccharide disrupted, the subsequent steps in the processing pathway could not continue. the addition of galactose to n-acetylglucosamine was prevented because the oligosaccharide chain was never processed to the point where galactose is typically added to n-acetylglucosamine. by way of contrast, the addition of galactose to n-acetylglucosamine was not affected by swainsonine which inhibits the golgi mannosidase ii. this enzyme typically cleaves two mannose residues from one of the branches of the oligosaccharide chain (elbein, ) . the processing of the other branch of the oligosaccharide chain can continue normally. it is to this other chain, which contains a mannose and an nacetylglucosamine, that galactose is typically added. however, it should be noted that a negative result with swainsonine is of limited significance since there is no assurance that it reached and inhibited the target enzyme. to the best of our knowledge, these experiments provide the first evidence in a mammalian system that glycoprotein processing can be modified in rhodopsin following in vivo exposure to the appropriate inhibitory agent. chambers et al. ( ) reported that intraocular injection of frogs with up to ,ug of castanospermine and swainsonine resulted in neither a decrease in rhodopsin content nor a change in the length of photoreceptor outer segments. fliesler. rayborn and hollyfield ( a) demonstrated that in vitro exposure of xenopus retinas to castanospermine resulted in the hyperglycosylation of opsin. the opsin underwent normal intracellular transport and was utilized for the biogenesis of ros membranes having normal disc morphology. the hr to day in vivo galactose labeling studies of rhodopsin purified from rough endoplasmic reticulum-, golgi-enriched fractions and ros, suggested that the amount of galactose-derived label associated with rhodopsin was not constant. it appeared from this work that trimming had occurred in the ros over the course of several hours and also had occurred in transit from one subcellular compartment to another, namely, the golgi-enriched fraction to ros. we chose to investigate the trimming phenomenon which was apparently occurring in the ros using lectin affinity chromatography. two hours after the injection, approximately - % of the labeled rhodopsin bound to rca suggesting that about s- % of newly synthesized rhodopsin contains a / -galactose residue. the percentage of rhodopsin which contained galactose decreased by more than half within hr and by hr of the injection less than % of rhodopsin contained galactose. one might surmise that if the galactose concentration is greatest within hr of injection, perhaps it constitutes a transient component of rhodopsin's oligosaccharide chain. one possibility is that it is associated with the rhodopsin of the plasma membrane and basal folds of the ros and is removed as discs are sealed and move apically. the work of hicks and molday ( ) emphasized the presence at the basal area of the outer segments of ricin-binding compounds. the possible function of a galactose on rhodopsin at this one area is open to speculation. it has been shown that the oligosaccharide of rhodopsin or some other ros protein such as peripherin is essential for the process of disc morphogenesis by fliesler and basinger ( ) and by fliesler, rayborn and hollyfield ( ) who successfully disrupted disc formation with tunicamycin, an inhibitor of oligosaccharide synthesis. mechanisms have been proposed (fliesler, rayborn and hollyfield, b; fliesler, ) showing how the oligosaccharide of rhodopsin could play an important role in disc formation. as new discs are formed, presumably the opposite faces of a newly forming disc must be aligned and brought into close apposition. it might be that the oligosaccharide plays some role in the process in much the same way that a lectin binds a ligand or a hydrolase interacts with an oligosaccharide substrate. if there were an enzymesubstrate interaction, perhaps a carbohydrate residue such as galactose on one surface might be cleaved by a hydrolase such as a galactosidase on the opposing surface. during this process the two membrane surfaces would come into close apposition with the exclusion of extracellular matrix allowing fusion of the new disc. this hypothesis led to the experiment in which ros plasma membranes were separated from discs for the purpose of determining in which compartment the galactose-containing rhodopsin was most prevalent. the experiments showed that the greatest preponderance of the galactose-containing rhodopsin was in the plasma membrane component. this finding lends support to the hypothesis that a transiently present galactose is important in new disc formation in bovine and rat retinas because the galactose appears to be associated with the plasma membrane until the discs are finally fused. this proposed mechanism would not specifically require galactose as the substrate, only that the components of the oligosaccharide, however they may vary among species, be the substrates of the appropriate hydrolases. in the summary, the present work has shown through biochemical techniques that a subset of rhodopsin contains galactose. tn fact, shortly after it is synthesized, approximately - "/( of the rhodopsin in the ros contains this sugar residue. the galactose appears to be added in the golgi complex in the same way that a terminal trisaccharide is assembled on a complex oligosaccharide chain. the galactose is not capped, however, with neuraminic acid. we presented evidence to confirm the incorporation of galactose into rhodopsin in in vivo experiments. additionally, we demonstrated the presence of galactose in bovine rhodopsin by radiolabeling existing moieties. finally, using lectin affinity chromatography, we were able to separate the galactose-containing species of rhodopsin and study its transient association with the oligosaccharide chain. we determined that galactose is trimmed from rhodopsin over the course of about hr. presumably after it has served its function which may be related to new disc formation. structural determinants of ricinus communis agglutinin and toxin specificity for oligosaccharides. resolution of nucleotide sugars and oligosaccharides by lectin affinity chromatography specific binding of peanut lectin to a class of retinal photoreceptor cells: a species comparison fatty acid composition of bovine rod outer segment plasma membrane the biosynthesis of rhodopsin as studied by membrane renewal of rod outer segments lectin receptors of rods and cones : visualization by fluorescent label different distribution of receptors for peanut and ricin agglutinins between inner and outer segments of rod cells in vitro radiolabeling of galactosyl and n-acetylgalactosaminyl moieties ofglycoproteins with carbon-l effects of glycosylation inhibitors on the frog retina separation and analysis of glycoprotein oiigosaccharides y ). inhibitors of glycoprotein synthesis retinal rod outer segment membrane assembly: studies with inhibitors of enzymes involved in n-linked oligosaccharide biosynthesis and processing tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin i y a). inhibition of oligosaccharide processing and membrane morphogenesis in retinal rod photoreceptor cells protein-bound carbohydrate involvement in plasma membrane assembly : the retinal rod photoreceptor cell as a model rhodopsin carbohydrate : structure of small oligosaccharides attached at two sites near the nh, terminus external labeling of cell surface galactose and galactosamine in glycolipid and glycoprotein of human erythrocytes soluble proteins of intact bovine rod cell outer segments the amino-terminal tryptic peptide of bovine rhodopsin. a glycopeptide containing two sites of oligosaccharide attachment structure of visual pigments i. purification, molecular weight and composition of bovine visual pigmew structure of the glycopeptide from bovine visual pigment localization of lectin receptors on bovine photoreceptor cells using dextrangold markers lectin and antibody labelling of developing rat photoreceptor cells: an electron microscope immunocytochemical study i:ltrastructural localization of lectin receptors in the monkey retinal photoreceptors and pigment epithelium : application of lectin-gold complexes on thin sections. f;xp assembly of asparagine-linked oligosaccharides cleavage of structural proteins during the assembly of the head of bacteriophage t differences in protein composition of bovine retinal rod outer segments disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method. ultrastructural localization of lectms binding sites on the surface of retinal photoreceptors and pigment epithelium rhodopsin as a glycoprotein : a possible role for the oligosaccharide in phagocytosis the biosynthesis of rhodopsin in vitro acylation of bovine rhodopsin by [ h]palmitic acid carbohydrate composition of bovine rhodopsin the effects of processing inhibitors of n-linked oligosaccharides on the intracellular migration of glycoprotein e of mouse hepatitus virus and the maturation of coronavirus particles biochemistry of visual pigments i. purification and properties of bovine rhodopsin the acylation of rat rhodopsin in vitro and in vivo the localization and timing of post-translational modifications of rat rhodopsin localization of fluorescent-labeled lectin binding sites on photoreceptor cells of the monkey retina key: cord- -ra actc authors: nan title: retention of p in an er-golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers date: - - journal: j cell biol doi: nan sha: doc_id: cord_uid: ra actc the type ii membrane protein p is a resident protein of a membrane network interposed between rough er and golgi apparatus. to study the retention of p , mutant forms were expressed in cos cells and the intracellular distribution determined by immunofluorescence microscopy. investigation of chimeric constructs between p and the plasma membrane protein dipeptidylpeptidase iv showed that protein sequences from all three domains of the p protein are required to achieve complete intracellular retention. mutational analysis of the -amino acid cytoplasmic tail of p revealed that the nh -terminal amino acids are necessary for retention. when p was solubilized with triton x- and subjected to centrifugation at , g, it formed large, insoluble oligomers, particularly at neutral ph and below. a comparison of the behavior of wildtype and mutant p proteins in this assay revealed a perfect correlation between the formation of large oligomers and correct intracellular retention. these results suggest that self- association may be a major mechanism by which p is retained between the rough er and the golgi apparatus. dent protein of a membrane network interposed between rough er and golgi apparatus. to study the retention of p , mutant forms were expressed in cos cells and the intracellular distribution determined by immunofluorescence microscopy. investigation of chimeric constructs between p and the plasma membrane protein dipeptidylpeptidase iv showed that protein sequences from all three domains of the p protein are required to achieve complete intracellular retention. mutational analysis of the -amino acid cytoplasmic tail of p revealed that the nh -terminal amino acids are necessary for retention. when igi was solubilized with triton x- and subjected to centrifugation at , g, it formed large, insoluble oligomers, particularly at neutral ph and below. a comparison of the behavior of wildtype and mutant p proteins in this assay revealed a perfect correlation between the formation of large oligomers and correct intracellular retention. these results suggest that self-association may be a major mechanism by which p is retained between the rough er and the golgi apparatus. t he secretory pathway of eukaryotic cells is composed of a series of different compartments through which newly synthesized proteins are transported en route to their final destination in the cell (paiade, ) . each of the subcellular organelles along this pathway contains a characteristic set of proteins that maintain their structural and functionai integrity. a challenging question in contemporary cell biology is to understand how resident components, unlike itinerant proteins, achieve specific localization in a given intracellular compartment. it is currently believed that secretory proteins are transported by a default pathway while resident proteins are selectively retained in individual organelles by means of specific signals (pfeffer and rothman, ; wieland et al., ; karrenbauer et al., ) . two types of signais, which mediate either the retention or the retrieval of proteins, have been identified to date. a variety of lumenal er proteins are retained in the cell by means of a cooh-terminal tetrapeptide (kdel) or related sequence (munro and pelham, ; reviewed by pelham, pelham, , ) that allows retrievai of the proteins from a post-er site by means of kdel receptors pelham, , ) . a group of type i transmembrane proteins of the er are also retained in this or-ganelle by a retrieval mechanism (jackson et al., ) . these proteins contain cytoplasmic sequences consisting of two lysine residues positioned three and four or five residues from the cooh terminus (kkxx or kxkxx, where x can be almost any amino acid) (jackson et ai., ; shin et ai., ) . recycling of these proteins is believed to occur from multiple post-er locations along the secretory pathway (jackson et ai., ) . in addition, retrieval has been suggested to be the mechanism by which the integral membrane protein tgn is targeted to the tgn (bos et ai., ; humphrey et al., ) . tgn possesses a tyrosine-containing motif within its cytoplasmic tail that is both necessary and sufficient for tgn localization (bos et ai., ; humphrey et ai., ; wong and hong, ) . in contrast to these examples, localization of golgi proteins is achieved by retention rather than retrieval. the retention of several golgi proteins has been shown to depend primarily on their single transmembrane segment with some additional contribution to retention by the sequences just adjacent to those membrane anchors (nilsson et ai., ; swift and machamer, ; machamer et al., ) . in the case of the c~ , -siaiyltransferase, the cytoplasmic and lumenal sequences flanking the transmembrane segment appear to be the crucial elements for retention rather than the transmembrane segment itself (munro, ; dahdal and colley, ) . it has been postulated that resident golgi proteins oligomerize in the golgi apparatus upon recognition of identical or related proteins, forming a complex of sufficient size to prevent entry into transport vesicles (machamer, ; nilsson et al., nilsson et al., , nilsson et al., , . recently, oligomerization of a chimeric protein containing the first membrane-spanning domain of the m glycoprotein of avian coronavirus has been correlated to its retention in the golgi apparatus (weisz et al., ) . in contrast to the considerable progress that has been made in understanding protein localization in the er and the golgi apparatus, it is presently unclear how the recently identified er-golgi intermediate compartment (ergic) maintains its resident population of proteins (for a review see hauri and schweizer, ) . the ergic comprises a membrane system interposed between the rough er and the cis side of the golgi apparatus that has been shown to mediate er-to-golgi exocytic protein transport (schweizer et al., ; lotti et al., ) . the ergic may correspond to the "budding compartment" of mouse coronavirus (tooze et al., ; krijnse-locker et al., ) and to the "pre-golgi vacuoles" of semliki forest virus-infected cells (saraste and kuismanen, ) . isolation of the ergic from vero cells indicated that this compartment has unique properties since it does not share major marker proteins with its direct neighbor organelles, the rough er and cis-golgi apparatus (schweizer et al., ) . currently, a limited number of marker proteins are available for the ergic. the ergic was originally defined by a -kd type i transmembrane protein (schweizer et al., ) termed ergic- . ergic- is specifically localized in the ergic at °c but undergoes a temperature-dependent redistribution (lippincott-schwartz et al., ; schweizer et al., ) which suggested that it may follow a recycling pathway (hauri and schweizer, ) . another marker for the ergic is the small gtp-binding protein rab p (chavrier et al., ) . beta-cop, a major component of non-clathrin-coated vesicles, also associates with the ergic at °c (duden et al., ) . the p -positive pre-golgi elements described by saraste and svenson ( ) may also be part of the ergic. the only membrane protein characterized and cloned so far with a stable er-golgi intermediate localization is p (schweizer et al., a,b) . mabs against p recognized an extended er-golgi intermediate membrane structure which indicated that the ergic may be larger than previously suggested by the ergic- analysis. unlike ergig- , the distribution of p was insensitive to organelle perturbants such as low temperature and brefeldin a (schweizer et al., a) . sequence analysis together with biochemical data demonstrated that p is a nonglycosylated, reversibly palmitoylated type ii transmembrane protein with a amino acid nhe-terminal cytosolic tall, a single transmembrane domain, and a large extracytoplasmic domain of amino acids (schweizer et al., a,b) . as a first step toward elucidating the mechanisms that determine protein localization between the rough er and the golgi apparatus, we have studied the retention of the p marker protein. in the present paper we demonstrate that all three domains of p contribute to the proper intracellular localization of this protein. further, the retention of p correlated with the formation of triton x- -insoluble . abbreviations used in this paper: ergic, er-golgi intermediate compartment; dppiv, dipeptidylpeptidase iv; wt, wild type. oligomers, suggesting that self-association of p protein may serve as a major mechanism for retention. enzymes used in molecular cloning were obtained from boehringer mannheim (indianapolis, in), new england biolabs (beverly, ma), or promega (madison, wi). dme ( . g/ glucose) and rpmi- medium were from gibco brl (grand island, ny); fcs from hazleton biologics (lenexa, ks); nusera from collaborative biomedical products (bedford, ma); deae-dextran, chloroqulne, cnbr-activated sepharose b, and protease inhibitors were from sigma chemical co. (st. louis, mo); ecl western blotting reagents from amersham corp. (arlington heights, il); nitrocellulose from schleicher and schuell (keene, nh); protein a-sepharose beads from repligen corporation (cambridge, ma); fitc goat antimouse igg from cappel ovestchester, pa); cell culture dishes from falcon (becton dickinson co., lincoln park, nj); multichamber slides from nunc inc. (naperville, il); and human plasma fibronectin from the new york blood center. oligonucleotides were synthesized with a solid phase synthesizer ( a; applied biosystems, foster city, ca) by the protein chemistry facility of washington university. all basic dna procedures were as described (sambrook et al., ) . p cytoplasmic tail mutants. the p wt cdna was as described previously (schweizer et al., b) and consisted of the ' untranslated region, bpl- , the nucleotide coding region and bp of the ' noncoding sequence. the full-length cdna was inserted into the ecori site in the polylinker of the bluescript sk-or ks-vector (stratagene, la jolla, ca), respectively, with the initiator atg facing the bamhi restriction site of the polylinker. the resulting constructs were designated pbsk-p or pbks-p , respectively. for transient expression in cos cells, the p insert was subcloned into the ecori site of the pece vector (kindly provided by dr. m. spiess, biozentrum, basel, switzerland) (ellis ct al., ) to give plasmid pece-p . the mutant forms of the p gene were created using standard pcr protocols (ho et al., ) . all mutants start at bp of the original wild type (wt) p edna (schweizer et al., b) . the final pcr products were digested with bamhi and used to replace the bamhi-bamhi fragment of pbsk-p . the entire pcr-derived fragment was sequenced by the dideoxy termination procedure (sanger et al., ) as described (schweizer et al., b) . correct clones were subcloned into the ecori site of the pece vector for transient expression in cos cells. to generate the a - construct (p with deletion of amino acids - ), pbsk-p as template together with the ' primer c-cc cgc cat gcg cag gct cgg cag gc~ g-ct caa c, and a ' primer corresponding to nucleotides - of the p wt sequence were used in the pcr reaction. in a subsequent pcr reaction, the final a - construct and the ' primer aag gat ccg aat tcg ccc gcc atg gct gca ctc agg gcg ctc aac tit were used to produce the a - aa mutant. the same strategy as described for the construction of a - was cartied out for the a - mutant (p with a deletion of amino acids to ), except for the ' primer that was gcc cgc cat gcc ctc gc~ caa aca aag ~ ctc caa gc.k cc~ cca cgg cc_~ ccg cag gct cgg cag ~ gct caa. all other mutations were introduced by the overlap extension technique (ho et al., ) using bp - of the p sequence and bp - of the bluescript ks-vector as downstream and upstream flanking primers. an appropriate partial complementary pair of oligonucleotides in which the desired mutation had been incorporated was chosen as internal primers. the final pcr products were digested with bamhi and used to replace the bamhi-bamhi fragment ofpbks-ai - (ai - introduced in ecori site of pbks). sequencing and further subcloning were as described above. alternatively, final pcr products were directly subcloned into the sinai site of the pece vector and sequenced. for the construction of the a - mutant (p with a deletion of amino acids to ), pbks-p a - was used as pcr template. the internal primers were gcc gcg agc ccc tcg gag aag ggt gcc cgc agg ctc gc~ agg gcg ctc aac ttt for the downstream reaction and gcg oc~ acc ctt ctc cga ggg g-ct cgc ~ gcc gtg gcc gcc ctt gga gcc cct for the upstream reaction, respectively. the final plasmid was designated pece-a - . alanine replacements of seven consecutive amino acids in the p cytoplasmic tail were encoded by gct gca gcc gca gct gca gcc and its complementary sequence. for the substitution of positively charged amino acids and/or glycines within amino acids - of the p sequence by alanines, the following codons were used: gca (for amino acids , , and ) and gct (for amino acids , , , and ). to construct fusion genes of p and dipeptidylpeptidase iv (dppiv), a cdna for human dppiv (misumi et ai., ; kindly provided by dr. y. ikehara [fukuoka university, fukuoka, japan]) was used. for the generation of the ppd and a - ppd chimeras (complete or a - cytoplasmic tail of ) , transmembrane domain of p , and lumenal domain of dppiv), the dppiv cdna was subcloned into a bluescript sk-vector that has the kpni site in its polylinker deleted. a kpni site was then introduced between nucleotides and of the dppiv sequence without altering the amino acid sequence. the resulting plasmid was digested with sali and kpni to give a fragment including the complete lumenal domain of dppiv except for the first nucleotides (bp - ). the pece-p or pece-a - plasmid, respectively, were digested with sali and noti, and the corresponding fragments that encode the complete or mutant (a - ) i) cytoplasmic tail plus the first amino acids of the p transmembrane domain, were isolated. two complementary oligonucleotides corresponding to nucleotides - of the p sequence followed by nucleotides - of the dppiv sequence and a kpni site were sequenced and annealed. these components were assembled in a three-part ligation to give plasmids pece-ppd or pece-a - ppd, respectively. all further chimeras were generated by pcr using the overlap extension technique (horton et al., ) . all chimeric constructs were precisely joined at the transitions between two domains. final pcr products were treated as described above for the p cytoplasmic tail mutants, resulting in constructs pece-ddp (cytoplasmic tail of dppiv, transmembrane domain of dppiv, and lumenal domain of p ), pece-dpp (cytoplasmic tail of dppiv, transmembrane domain of p , and lumenal domain of p ), and pece a - pdp (a - cytoplasmic tail of p , transmembrane domain of dppiv, and lumenal domain of p ), respectively. the following templates were used for the pcr reactions: plasmids pbsk-p and pbsk-dppiv (dppiv cdna subcloned as ecori fragment into the bluescript ks-vector) to generate constructs dpp and ddp, and plasmids pbks i) a . - and pece-ddp to construct the a - pdp chimera. upstream and downstream flanking primers were the same as above. appropriate pairs of partially complementary oligonucleotides which encoded the desired fusion were chosen as internal primers. all mutants were verified by sequencing at the level of the final plasmid. cos cells (african green monkey kidney cells, crl ; american type culture collection, rockville, md) were cultured in dme supplemented with % fcs, units/ml penicillin, t~g/ml streptomycin, and fungizone at °c in a humidified % co: atmosphere. for transient tr~ons cos cells were plated at , - , cells per well in an eight-well multichamber slide in which the wells had been coated for at least rain with tlg/ml fibronectin in pbs, or at cells per -mm plate. suboonfluent cells were washed once with tbs and each -ram plate was exposed to /~g of dna in . ml rpmi- medium containing % nnsera, t~g/ml gentumycin, t~m ehloroquiue, and /~g/ml deae-dextran (rnc plus deae-dextran) for . h. for eightwell multichamber slides . /~g dna in /~ rnc plus deae-dextran was used in each well. the dna solution was then replaced with pbs containing % dmso, cells were incubated for rain at room temperature, and washed with pbs before fresh medium was added. cells were maintained at "c and analyzed - h after transfection. mouse mab g / against the p protein has previously been characterized (schweizer et ai., a) . for the detection of dppiv rnab hbb / (hauri et al., ) was used. cos cells were grown in eight-well multichamber slides. the immunofluorescence procedure for permeabilized cells was that of schweizer et al. ( ) . in brief, formaldehyde-fixed and sapenin-permeabilized cells were incubated with mab g / against p or rnab hbb / against dppiv followed by goat anti-mouse fitc. to probe for surface expression of proteins, cos cells were cooled to °c for rain and kept on ice for the subsequent steps. the cells were washed once with pbs- . % bsa, and incubated with mab g / or mab hbb / for rain. after six wash steps with pbs- . % bsa, the cells were fixed with % p-formaldehyde for rain on ice, followed by another rain at room temperature. cell permeabilization and staining with goat anti-mouse fitc was as described (schweizer et ai., ) . the specimens were examined with a nikon fluorescence microscope and photographed with fujichrome film. after two washes with pbs, transfectod cos cells of one -ram plate were scraped into ml pbs and centrifuged for rain at rpm ( giv). the pellet was resuspended in /~ mnt buffer ( m_m mes, mm tris, mm nacl, . m_m edta, and mm egta) at various phs containing % triton x- , mm iodoacetamide, t~g/mi pmsf, and a : dilution of a protease inhibitor cocktail ( mg/mi benzamidine and mg/ml each of pepstatin a, leupeptin, antipain, and chymostatin in % dimedaylsnifoxide, % ethanol) by passing it five times through a -gange needle connected to a -ml syringe. after a -h solubilization step on ice, the cells were centrifuged for rain at , rpm ( , g~v) in a ti rotor (beckman instnmaents inc., paio alto, ca). the resulting supernatants were carefully harvested and proteins were precipitated by the method of wessel and fluegge ( ) . the precipitates were solub'~,ed in laemmli buffer (laemmli, ) . the pellets in the ti tubes following centrifugation were selubilized directly in sample buffer and sonicated. proteins were separated on % sds-polyaerylamide minigels (bio rad laboratories, richmond, ca) using the laemmli ( ) system, and transferred to nitrocellulose membranes according to the method of towbin et al. ( ) . for the immunoreaction, the nitrocellulose sheet was blocked with % nonfat dry milk powder in pbs, incubated with mab g / against p (diluted : , in pbs- % powdered milk) followed by a horseradish peroxidase-conjugated anti-mouse secondary antibody (amersham corp.). for development the ecl detection system (amersham corp.) was used according to the manufacturer's directions. quantification of autoradiograms was carried out by means of a molecular dynamics personal densitometer (sunnyvale, ca). cos cells ( - -mm plates per preparation) transfected with either p wt or a - aa were washed twice with pbs, scraped into ml pbs per plate and centrifuged for min at rpm ( ga~). each pellet was resuspended in /~ of buffer ( mm na hpo , ph . , % triton x- ), containing mm iodoacetamide, /~g/ml pmsf, and a : dilution of the above described protein inhibitor cocktail by passing it five times through a -gang¢ needle connected to a -ml syringe. after a -h solub'dization step on ice, the cells were centrifuged for rain at , rpm ( , gay) in a ti rotor. p proteins were affinity purified from the resulting supernatants on a column of mab g / coupled to cyanogen bromide-activated sepharose b. mab g / had been purified from culture supernatant by ammonium sulfate precipitation (to % saturation) followed by protein a-sepharose chromatography prior to coupling according to the suppliers instructions. p proteins were eluted with . m glycine, ph . , . % triton x- , and immediately adjusted to ph . with m "iris base. after analysis by sds-page, fractions that revealed a single band of appropriate size (schweizer, a., j. rohrer, and s. kornfeld, unpublished data) were pooled and dialyzed against mnt buffer, ph . , containing . % triton x- . protein was determined with the micro bca protein assay (pierce, rockford, il). mnt buffer, ph . , containing % triton x- was added to . /~g of purified p wt or a - aa protein to a final volume of td before one of three incubations was carried out. the first sample was incubated for rain on ice. the second sample was brought to ph . by addition of a pretitrated volume of n hc and then incubated on ice for min. the third sample was shifted to ph . and incubated on ice for rain; the ph was then returned to . by addition of a pretitrated volume of n naoh followed by another roan on ice. all samples were centrifuged for min at , rpm (i , gay) in a ti rotor. the resulting supernatant and pellet fractions were further treated as described under immunoblotting. the endogenous p could be visualized in saponin-permeabilized cos cells by indirect immunofluorescence using a : dilution of mab g / which is specific for this protein. as shown in fig. a, the fluorescence pattern with its extended er-golgi intermediate membrane structure was very similar to that observed previously with vero cells (schweizer et al., a) . while er-like, the pattern differs from that observed with er markers in that it is less reticular and the outer nuclear membrane does not stain (see fig. c for a typical er pattern and schweizer et al., a) . when the mab g / was tested at a dilution of : , , the p staining pattern was very faint (fig. b) . since this dilution of antibody gave a strong signal with p transfected cos cells (fig. a) , it was possible to readily distinguish between endogenous and transfected p . cos cells transfectext with iri wt exhibited the typical extended membrane structure observed when endogenous p was stained ( fig. a, left cell). in addition, some of the cells showed a large tubular network pattern that was not observed in the untransfected cos cells (fig. a, right cell) (schweizer et al., b) . p wt was never detected at the cell surface, even when the expression level was very high (fig. b) . in our attempt to determine the features of p responsible for retention we initially concentrated on the cytoplasmic domain of the protein. the first construct to be prepared was a truncated form of p in which the amino-terminal residues - of the -amino acid-long p tail were deleted (a - , fig. ) . since p has a type ii membrane orientation, the first nh -terminal amino acid representing the initiator methionine could not be removed. when a - was expressed in cos cells, it was clearly present on the plasma membrane in about half of the transfected cells (fig. d) . upon permeabilization, a - was detected primarily in the rough er as indicated by the reticular staining pattern along with labeling of the outer nuclear membrane (fig. c) . occasionally it was found in the golgi region or at the cell surface (see insert of fig. c for an example of surface staining). in the construction of a - , two arginine residues became positioned near the extreme ni-i -terminus due to the deletion of amino acids within the cytoplasmic tail (fig. ) . we therefore considered the possibility that these positively charged amino acids might serve as an ertargeting signal, thereby impairing the normal transport of the protein (jackson et al., ) . these arginine residues (located in position - and - from the nh terminus of a - ) were therefore changed to alanines (a - aa, fig. ) and the effect monitored by immunofluorescence as outlined above. a - aa showed strong cell surface expression over the entire transfected cell population (fig. f) . further, the staining pattern observed in permeabilized cells (fig. e) was typical for a cell surface protein including juxtanuclear golgi and plasma membrane staining. taken together, these data demonstrated that the cytoplasmic domain of p is necessary for correct intracellular localization. to define more precisely the location of the retention information in the p cytoplasmic tail we made a construct (a - , fig. ) in which amino acids - of p wt were added back to the a - deletion mutant. when cells expressing this construct were analyzed by immunofluorescence, an internal staining pattern close to that of p wt was observed except that no cells with the tubular network type pattern were found (fig. a) . labeling of nonpermeabilized cos cells with anti-p mabs revealed that a - is predomi- for detection an mab to p followed by goat anti-mouse fitc was used. in contrast to p wt, constructs a - and a - aa were observed at the plasma membrane. bar, (a and b) /~m; (c-f) /~m. nantly localized intracellularly (fig. b) ; an occasional cell showed weak staining of the plasma membrane (fig. b, inset) . when the nh~-terminal portion of p was extended to include the nh -terminal residues of the p wt sequence (a - , fig. ) , the resultant protein behaved like p wt when analyzed by indirect immunofluorescence after transfection (fig. , c and d) . the mutant protein was completely retained inside the cell (fig. d) . in addition, the a - staining in permeabilized ceils was indistinguishable from that ofp wt (fig. c) , including cells that exhibit the striking tubular network that is typically found in p wttransfected ceils (fig. c, inset) . we conclude from these results that the nh~-terminal amino acid residues of the p cytoplasmic domain are required for proper intracellular localization. deletion of amino acids - of the p tail, on the other hand, had no detectable effect on the distribution of p in the cell. to further define the sorting determinant that is present within amino acids - of p , this region was subjected to extensive site-directed mutagenesis. first, a series of overlapping alanine stretches was created in the a - mutant. as indicated in fig. a, amino acids - (a - , - a), amino acids - (a - , - a), amino acids - (a - , - a), amino acids - (a - , - a), or amino acids - (a - , - a), respectively, were simultaneously changed to alanines using pcr mutagenesis. surprisingly, these substitutions had no detectable effect on the localization of p . immunofluorescence of transfected nonpermeabilized cells showed that none of the mutants were transported to the plasma membrane (for an example see fig. b) . the internal distribution of the constructs was also indistinguishable from that of/, - or p wt (for an example see fig. a) . when a similar type of alanine scan was carried out in the m - background (fig. b) , a different result was obtained. all of the mutant proteins exhibited some degree of increased transport to the cell surface, with the zk - , - a mutant showing the strongest effect (fig. d) . when cos cells transfected with this mutant were permeabilized, the predominant staining pattern was characteristic for the er and the golgi apparatus indicating that/, - , - a had lost p wt localization (fig. c) . these results clearly show that alanine substitutions have an effect when only amino acids - (/, - ) instead of amino acids - (/, - ) of the p cytoplasmic tail are present. one possible explanation for this finding could be the existence of a redundant signal with one of the components being located within residues - . when we carefully inspected the critical p sequence, a repetitive feature consisting of a positively charged amino acid followed by a glycine residue became apparent. the sequence contains an arginine-glycine combination at amino acid positions and , and two pairs of lysine-glycine at positions and , and and , respectively. in addition, there is a single lysine residue present at amino acid position . the distribution of these residues relative to the amino acids altered in the overlapping alanine substitution experiments was consistent with these residues serving as components of a redundant retention signal. to test this possibility, k ~, r , g s, k °, g h, k , and g in the ~ - sequence were simultaneously changed to aianines (z~. - ,ksrtgski°gnk tga -a [mp ]; fig. ). immunofluorescence analysis of this mutant expressed in cos cells revealed that it was highly expressed at the plasma membrane of non-permeab~ transfected cells (fig. b) . furthermore, in permeabilized ceils transfected with mp the staining was also characteristic for a surface protein (fig. a) . the phenotype of mp is therefore very similar to that of the tailminus mutant a - aa. we next tested whether alunine substitutions of only the positively charged amino acids (k s, w, k °, and k (a - ,ksrtk'°ke~-a [mf ]; fig. ) or only the glycine residues (c_j, g n, and cr n) (a - ,cjg"g -a [mp ]; fig. ) present in the k-rg-kg-kg element would also disrupt p localization. both constructs behaved similarly to a - and p wt as indicated by the lack of cell surface staining (fig. , d and f) and the typical internal pattern (fig. , c and e). this demonstrated that changing positive charges or glycines alone does not affect retention of p . an additional construct was created that has the same mutation as mp except for amino acid position where the original lysine was retained (a - , rtg-skt gttk tc_f'-a [mp ]; fig. ). following transient transfection and immunofluorescence microscopy, this mutant showed two different localizations. some of the chimeric protein was consistenfly detected on the cell surface (fig. h) while the rernaining protein was predominantly found in the er (fig. g) . the latter finding suggested that the introduced changes might partly impair proper folding of the mutant protein which may prevent its exit from the er. the presence of mp at the cell surface shows that k cannot restore correct localization of p in the absence of the three positively charged amino acid-glycine combinations. to further analyze the role of the cytoplasmic tail of p in retention and to determine whether the transmembrane and lumenal domains of this protein contribute to proper intracellular localization, we substituted each of these domains with the corresponding domains of the cell surface protein human dppiv (fig. ) . the serine protease dppiv is a type ii integral membrane protein like p and is found on a variety of epithelial, endothelial and lymphocytic cell types doyle, , ; ogata et al., ) . we first analyzed a chimeric protein (dpp; dppiv cytoplasmic, p transmembrane, p lumenal; fig. ) in which the cytoplasmic tall of dppiv was fused to the transmembrane and lumenal domains of p . cos cells were transfected with dppivwt or the chimeric construct and the localization of the proteins was examined by indirect immunofluorescence of permeabilized and nonpermeabilized cells using mabs to a lumenal epitope of each protein. nontransfected control cells did not react with the anti-dppiv mab (data not shown) consistent with evidence that cos cells have tittle or no endogenous activity of dppiv (ogata et ai., ) . as shown in fig. , a and b dppivwt was located on the cell surface as expected. bright cell surface staining was also observed for dpp (fig. d) . however, unlike dppivwt a portion of dpp was also found in the er (fig. c) , indicating that the export of this chimeric protein out of the er is impaired to some extent: very rarely a cell showed a p wt-related staining pattern. a similar result was obtained with a construct that only contained the lumenal domain of p (ddp; dppiv cytoplasmic; dppiv transmembrane, p lumenal; fig. ) except that most of the er staining was now replaced by cell surface expression (data not shown). these results confirm that the p cytoplasmic tail is necessary for proper localization. in the absence of the p cytoplasmic tail, the lumenal p domain alone or in combination with its transmembrane domain is not sufficient to achieve retention. we next asked whether the lumenal domain of p is dispensable for the correct targeting of the protein. to this end, a chimeric construct in which the lumenal domain of p was replaced by that of dppiv (ppd, p cytoplasmic, p transmembrane, dppiv lumenal; fig. ) was created. in a related construct we linked the cytoplasmic and transmembrane domains of a - to the dppiv lumenal domain (a - ppd, p with deletion of amino acids - cyto- plasmic, p transmembrane, dppiv lumenal; fig. ). when the subeellular localization of the chimeric proteins was analyzed in transfected cos cells, a - ppd showed the same distribution as dppivwt. there was strong labeling of the cell surface ( fig. f ) , but no internal staining with a p wt pattern (fig. e) . the same was true for the ppd chimera (fig. , g and h) except that the internal staining pattern included more er staining in addition to the golgi and cell surface staining predominantly found for dppivwt and a - ppd. these data demonstrate that the presence of the lumenal domain of the p molecule is essential for the correct localization of the protein. the results with the ppd and a - ppd constructs together with the mutational analysis of the p cytoplasmic tail establish that the transmembrane domain of p is not sufficient for its localization. to determine whether this domain is a necessary component, we created an additional chimera that combines a p cytoplasmic tall that has amino acids - deleted with the transmembrane domain of dppiv and the lumenal part of p (a - pdp; p with deletion of amino acids - cytoplasmic, dppiv transmembrane, p lumenal; fig. ). immunofluorescence microscopy of transfected cells showed that the internal distribution of a - pdp is very similar to p wt (fig. i) . unlike p wt, however, some a - pdp molecules were detected on the plasma membrane in unpermeabilized cells (fig. j) . the intensity of this cell surface staining was fainter than that of dppivwt or any of the other chimeric constructs. thus, while the transmembrane domain contributes to the complete intracellular retention of p , it appears to be less important for the overall localization than its cytoplasmic and lumenal counterparts. the retention behavior of p described so far suggested that the underlying molecular mechanism might be related to physical properties within the p protein itself. two findings in particular supported this idea. first, overexpression of p did not saturate the retention mechanism. and second, the p localization could not be transferred to another protein. we therefore speculated that individual molecules might interact with each other to form higher order structures. to test this hypothesis p wt was analyzed by centrifugation. in this experiment cos cells transfected with p wt were solubilized with triton x- at different phs ( . , . , . , . , and . ) and then subjected to centrifugation at , g for h. the resulting supernatant and pellet fractions were then assayed for the presence of p by sds-page and immunoblotting with an anti-p mab. as shown in fig. a there was a ph dependent shift of from the soluble fraction to the pellet. at ph . , p was equally distributed between the pellet and supernatant fractions, whereas increasing amounts of p were recovered in the cos cells transfected with p wt were solubilized with triton x- at the indicated phs and then separated by centrifugation at , g into supernatant (s) and pellet (p). proteins of the two fractions were subjected to sds-page ( % gels) and immunoblotring with an anti-p mab. the numbers at the left margin of the blot indicate known molecular mass in kilodalton. (b) samples of affinity purified p wt protein (lanes - ) or a - aa (lanes - ) in mnt ph . -triton x- were incubated for rain (i), adjusted to ph . and then incubated for rain (ii), or shifted to ph . for mill, then returned to ph . and incubated for another rain (iii). separation into supernatant (s) and pellet (p) and further analysis was as described in a. pellet as the ph decreased. when the experiment was performed with nontransfected cos cells at ph . , + % of the endogenous p was recovered in the pellet fraction. this demonstrates that p oligomerization occurs at physiologic protein concentrations, excluding the possibility that this property is a special feature of transfection and/or overexpression. similar results were obtained with affinity-purified p , as shown in fig. b (lanes - ) . in this experiment, purified p kept at ph . remained completely soluble (lanes and ) . when the ph was shifted to . , about half of the proteins formed oligomers that sedimented at , g centrifugation (lanes and ) . this oligomerization was completely reversed by restoring the ph to . (lanes and ). taken together, these results indicate that p forms triton x- -insoluble oligomers, especially at neutral ph and below. we next tested whether the ability of p to oligomerize correlated with retention and proper localization. to address this point, a selected set of p cytoplasmic tall mutants were expressed in cos cells and the transfected ceils were then solubilized with triton x- at ph . and analyzed as described above. the results of a typical experiment are shown in fig. a and the quantitation of multiple experiments is given in fig. b. p wt which served as a control was exclusively found in the pellet fraction ( ± %). a - , mp , and mp which have intraceilular distributions cos ceils were trmsfeoed with wt or mutant p , solubrized with triton x- at ph . and further analyzed as described in fig. . (p) pellet, (s) supematant. the upper band seen in the pellet lanes of p mutants represents endogenous p that is exclusively found in the pellet fractions. (b) the immtmoblots shown in a and those from additional experiments were quantitated by densitometric scanning. for each sample (x-axis) the amount of wt or mutant detected in the supernatant (dotted light bars) and pellet (striated dark bars) fraction, respectively, is plotted as the percentage of p present in both fractions (y-axis). each value is the average of at least four independent experiments. the standard deviations were ,g . %. similar to p wt were also recovered predominantly in the , g pellet ( + %, + %, and ± %, respectively). in contrast, a - aa and mp which are not localized properly, were found mainly in the supernatant fraction ( ± %, and ± %, respectively). the upper band detected in the pellet lane of each of the p mutants represents endogenous p from cos ceils. similar to transfected p wt, endogenous was exclusively found in the pellet fraction regardless of the behavior of the transfected p mutants and therefore served as a useful internal standard for the distribution of the various mutants. affinity-purified a - aa gave similar results to those obtained with homogenates of cos ceils transfected with a - aa (fig. b, lanes - ) . in summary, the data show a strong correlation between retention of p and the formation of triton x- -insoluble complexes. previous studies have established that p is stably retained between the rough er and the golgi apparatus (schweizer et al., a) . in the present study, we have analyzed the contribution of the individual p domains to retention by creating chimeric constructs with the type ii plasma membrane protein dppiv. the role of the cytoplasmic tail was further analyzed by deletions and point mutations within the p protein itself. the appearance of mutant p proteins at the cell surface and the concomitant loss of the characteristic p wt internal staining pattern were used as criteria for loss of proper retention. using these approaches, we have identiffed the domains of p that determine retention and obtained data that are consistent with the notion that oligomerization of p serves as a major retention mechanism. table i presents a summary of all the constructs analyzed in this study. the striking characteristic of p retention is that no single topological domain is sufficient to specify retention. rather, all three domains are required to achieve complete intracellular retention. the cytoplasmic and lumenal domains of p are most important for proper localization with the transmembrane domain playing a lesser role in this process. these findings with p differ from those obtained with most other proteins of the early secretory pathway where one domain has been shown to be sufficient for retention. in the case of resident lumenal er proteins, the four-amino acid sequence kdel at the carboxyl terminus serves as retrieval signal (munro and pelham, ; pelham, pelham, , . similarly, kkxx/kxkxx elements at the carboxyl terminus of the cytoplasmic domains of type i transmembrane proteins function as retrieval signals (nilsson et al., ; jackson et al., jackson et al., , . when transplanted onto marker proteins, these sequences are sufficient to cause retention in the er. the carboxyl-terminal cytoplasmic domain of tgn is also sufficient for localization of chimeric proteins to the tgn (bos et al., ; humphrey et al., ; wong and hong, ) . other golgi proteins are localized by means of their transmembrane domains (n-acetylglucosaminyltransferase i (burke et al., ; tang et al., ) , galactosyltransferase (nilsson et al., ; aoki et al., ; russo et al., ) , and the m glycoprotein of avian coronavirus (swift and machamer, ; machamer et al., ) or in association with the cytoplasmic and lumenal flanking sequences (or , -sialyltransferase (munro, ; dahdal and colley, ) . the e glycoprotein of the mouse coronavirus is an exception among the proteins localized to the golgi apparatus (armstrong and patel, ) . while a short deletion in the cytoplasmic tail destroyed retention, the cytoplasmic segmet~t alone was not sufficient for retention. similar to the finding with p , most of the sequence appeared to be required for proper localization. molecular dissection of the cytoplasmic tail of p identiffed the nh -terminal amino acids as being necessary for retention whereas amirio acids - were dispensable for this function. within the nh -terminal sequence, substitution of three pairs of amino acids each consisting of a positively charged amino acid followed by a glycine residue ~ very rarely a cell showed a p wt-related staining pattern. the intensity of this cell surface staining was fainter than that of dppivwt or other chimeric constructs. (r , c -s, k ~°, g it , k ~, and g ) by alanines (mp ) resulted in transport of the mutant protein to the plasma membrane, indicating the importance of the corresponding amino acids for correct localization of p . interestingly, changing either the positively charged residues or the glycines alone (mp , mp ) did not affect retention of p . further, the dements required for retention appear to be at least partly redundant as revealed by the analysis of the overlapping alanine scan of a - , a similar scan of ai - , and the cytoplasmic tail deletion mutants. what is the role of the cytoplasmic tail in conferring proper intracellular retention of p ? a potential clue emerged when we analyzed the solubility of the various constructs in the nonionic detergent triton x- . when transfected cells were solubilized with triton x- at ph . and then subjected to ultracentrifugation at , g, the proteins that were properly retained (p wt, a - , mp , and mp ) were predominantly found in the pellet fraction whereas the nonretained proteins (a - aa and mp ) were mostly recovered in the supernatant. endogenous p from cos cells was exclusively found in the , g pellet, demonstrating that the property is not a function of transfection and/or overexpression. while the extent of triton x- -insolubility of p wt increased with decreasing ph, most of the protein was insoluble at ph . , which is similar to the ph of the cytoplasm. since highly purified p wt exhibited the same phdependent insolubility in triton x- , we conclude that this behavior is an intrinsic property of the protein. this does not exclude the possibility that p also interacts with other resi-dent proteins of the compartment or with cytoplasmic elements, such as members of the cytoskeleton. the striking correlation between proper intracellular localization and triton x- -insolubility suggests a retention mechanism for p that involves homooligomerization. the model predicts that upon arrival at their final destination, p molecules encounter an environment that leads to self aggregation resulting in the formation of large oligomers. the specific conditions that might trigger the oligomerization of p are unknown, but could include differences in ion composition, lumenal ph or properties of the local membrane. this proposed retention mechanism is compatible with several additional findings. first, high levels of expression do not result in mislocalization of p to the plasma membrane, indicating that the p retention system is not saturable. if p interacted with a specific receptor, overexpression would likely lead to saturation of the retention machinery and the appearance of the protein at the cell surface. second, as mentioned above, all three domains of p are necessary for complete retention, as would be expected if the molecule formed higher order structures. when triton x- -soluble p molecules were examined by sucrose gradient centrifugation or nonreducing sds-page, the majority of the molecules were present as noncovalently linked dimers (schweizer et al., a; schweizer, a., j. rohrer, and s. kornfeld, unpublished data) . interestingly, this dimerization of p is independent on the cytoplasmic tail, in contrast to the formation of higher oligomeric complexes. taken together, the most straightforward interpretation of our results is as follows. the lumenal domains of individual p molecules interact to form noncovalently linked dimers, with the transmembrane domains possibly having an accessory role. these dimers would represent the basic unit from which higher oligomeric structures are built. upon reaching their final destination, larger complexes form, requiring interactions of the cytoplasmic tails as well as the lumenal and transmembrane domains. the amino-terminal residues of the cytoplasmic tails could either be directly involved in the interactions or, more likely, guarantee a conformation of the cytoplasmic tail that favors higher order assembly. the association of p into large complexes could prevent p molecules from entering budding vesicles and thereby provide a mechanism for retention. alternatively, failure of the mutant p molecules to form the higher order complexes would result in leakage of the molecules out of the compartment with eventual delivery to the cell surface. this proposed model for the localization of p involves a retention rather than a retrieval mechanism, which is compatible with previous data suggesting that p is a stably anchored resident protein. oligomerization has also been suggested as a retention mechanism for proteins in the golgi apparatus (machamer ; nilsson et al., nilsson et al., , nilsson et al., , swift and machamer, ) . the only example where retention in this organelle has actually been correlated with oligomerization is a chimeric membrane protein in which the membrane-spanning domain of vsv g protein was replaced with the first membrane-spanning domain of the m glycoprotein of avian coronavirus (weisz et al., ) . however, in contrast to the chimera, oligomerization of the intact m glycoprotein could not be demonstrated. recently, nilsson et al. ( nilsson et al. ( , have proposed a model whereby golgi enzymes form homo-dimers in which the lumenal domains are bound together while the transmembrane domains interact with those of identical or related enzymes of the same golgi cisternae to form hetero-oligomers. the cytoplasmic domains would be attached to an underlying matrix to stabilize larger arrays. although similar in the overall architecture, this golgi model differs from the model supported by the p data in several ways. in particular, the domains responsible for the formation of higher order structures (cytoplasmic tail versus transmembrane domain) and the kind of oligomers formed (homo-versus hetero-oligomers) are distinct. taken together, oligomerization is likely to be a general mechanism that specifies location of proteins in different compartments of the secretory pathway. oligomerization per se, however, seems to be achievable by different mechanisms and forms of interactions. golgi retention of a trans-golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain the golgi sorting domain of eoronavirus e protein tgn is maintained in the trans-golgi network by a tyrosine-eontaining motif in the cytoplasmic domain the transmembrane and flanking sequences of , n-acetylglucosaminyltransferase i specify medial golgi localization localization of low molecular weight gtp-binding proteins to exocytic and endocytie compartments specific sequences in the signal anchor of the/ -galactoside- , sialyltransferase are not essential for golgi localization beta-cop, a kd protein associated with non--clathrin coated vesicles and the oolgi complex, shows homology to beta-adaptin replacement of insulin receptor tyrosine residues and compromises insulin-stimulated kinase activities and uptake of -deoxyglucose expression and intracellular transport of mierovillus membrane hydrolases in human intestinal epithelial cells the er-golgi intermediate compartment site-directed mutagenesis by overlap extension using the polymerase chain reaction edna cloning for a bile canaliculus domainspecific membrane glyeoprotein of rat hepatocytes molecular dissection of the nh -terminal signal anchor sequence of rat dipeptidylpeptidease iv engineering genes without the use of restriction enz~'mes: gene splicing by overlap extension localization of tgn to the trans-golgi network: involvement of a cytoplasmic tyrosine-eontaining sequence identification of a consensus motif of transmembrane proteins in the endoplasmic reticulum retrieval of transmembrane proteins to the er the rate of bulk flow from the golgi to the plasma membrane characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step cleavage of structural proteins during the assembly of the head of bacteriophage t a human homologue of the yeast hdel receptor ligand-induced redistribution of a human kdel receptor from the golgi complex to the endoplasmic reticalure microtubule-dependent retrograde transport of proteins into the er in the presence of brefeldin a reveals an er cycling pathway immunocytochemical analysis of the transfer of vesicular stomatitis virus g glycoprotein from the intermediate compartment to the golgi complex golgi retention signals: do membranes hold the key? retention of a cis-golgi protein requires polar residues on one face of a predicted a-helix in the transmembrane domain molecular cloning and sequence analysis of human dipeptidylpeptidase iv, a serine proteinase on the cell surface a c-terminal signal prevents secretion of lumenal er proteins sequence within and adjacent to the transmembrane segment of alpha- , -sialyltransferase specify golgi retention short cytoplasmic sequences serve as retention signals for transmembrane proteins in the er the membranespanning domain of beta-l, -galactosyltransferase specifies trans-golgi localization kin recognition. a model for the retention of golgi enzymes. febs (fed. eur kin recognition between medial golgi enzymes in hela cells primary structure of rat liver dipeptidylpeptidase iv deduced from its edna and identification of the nh -terminal signal sequence as membrane-anchoring domain identification of the active site residues in dipeptidylpeptidase iv by affinity labeling and site-directed mutagenesis intracellular aspects of the process of protein transport control of protein exit from the er the retention signal for soluble proteins of the endoplasmic reticulum biosynthetic protein transport and sorting by the er and golgi . , -galactosyltransferase: a short nh -terminal fragment that includes the cytoplasmic and transmembrane domain is sufficient for retention molecular cloning: a laboratory manual dna sequencing with chain terminating inhibitors ire-and post-golgi vacuoles operate in the transport of semliki forest virus membrane glycoproteins to the cell surface distribution of the intermediate elements operating in er to golgi transport identification, by a monoclonal antibody, of a kda protein associated with a tubular-vesicular compartment at the cis-side of the golgi apparatus identification of an intermediate compartment involved in protein transport from the er to the golgi apparatus the isolated er-golgi intermediate compartment exhibits properties that are different from er and cis-golgi characterization of a novel kda membrane protein: implications for the organization of the er-to-golgi pathway a reversibly palmitoylated resident protein (p ) of an er-golgi intermediate compartment is related to a circulatory shock resuscitation protein signals for retention of transmembrane proteins in the endoplasmic reticulum studies with cim truncation mutants a golgi retention signal in a membrane-spanning domain of coronavirus el protein the transmembrane domain of n-glucosaminyltransferase i contains a golgi retention signal site of addition of n-acetylgalactosamine to the e glycoprotein of mouse hepatitis virus-a electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications oligomerization of a membrane protein correlates with its retention in the golgi complex a method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids the rate of bulk flow from the er to the cell surface the sxyqrl sequence in the cytoplasmic domain of tgn plays a major role in trans-golgi network localization we thank dr. y. ikehara (fukuoka university, fukuoka, japan) for kindly providing us with a edna encoding human dppiv; and members of the kornfeld laboratory for critical reading of the manuscript.this work was supported by united states public service grant ca . j. rohrer was the recipient of a damon runyon-walter winchell cancer postdoctoral fellowship. a. sehweizer was supported by the swiss national science foundation, the ciba-geigy jubiliiumsstittung, and a w. m. keck fellowship.received for publication february and in revised form april .