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Cell Vol 116 153 166 January 23 2004 Copyright 2004 by Cell Press The Mechanisms of Vesicle Budding and Fusion Juan 5 Bonifacino vquot and Benjamin S Glick2 Cell Biology and Metabolism Branch National Institute of Child Health and Human Develo ment National Institutes of Health Bethesda Maryland 20892 2Department of Molecular Genetics and Cell Biology The University of Chicago 920 East 58th Street Chicago Illinois 60637 Genetic and biochemical analyses of the secretory pathway have produced a detailed picture of the mo lecular mechanisms involved in selective cargo trans port between organelles This transport occurs by means of vesicular intermediates that bud from a do nor compartment and fuse with an acceptor compart ment Vesicle budding and cargo selection are medi ated by protein coats while vesicle targeting and proteins Precise regulation of these two aspects of vesicular transport ensures efficient cargo transfer while preserving organelle identity Like other landmark papers the articles by Novick et al 1980 and Balch et al 1984 featured in the supple ment to this 30th Anniversary issue of Cell introduce themselves right off the library shelves The bound vol umes spontaneously open to the right pages which are halfdetached tattered and inscribed with pencil markings testaments to the countless times that these articles have been read and copied Naturally these papers have already been the subject of many reviews Herein we will once more attempt to convey the enor mous influence that these studies have had on the field of intracellular protein trafficking this time by focusing on how they helped to bring about the current under standing of the molecular mechanisms of vesicle bud ding and fusion The Vesicular Transport Hypothesis The stage for the discoveries discussed here was set over 30 years ago that is BC or before Cell by the work of George Palade and colleagues on protein secretion Palade 1975 This work established that newly synthesized secretory proteins pass through a series of membraneenclosed organelles including the endoplasmic reticulum ER the Golgi complex and se cretory granules on their way to the extracellular space Figure 1 Proteins destined for residence at the plasma membrane endosomes or Iysosomes share the early stations of this pathway Ge the ER and the Golgi com plex with secretory proteins Importantly the secretory proteins are often found within small membrane enclosed vesicles interspersed among the major or Correspondence juanhelixnihgov Review ganelles of the pathway Such observations inspired the vesicular transport hypothesis which states that the transfer of cargo molecules between organelles of the secretory pathway is mediated by shuttling transport vesicles According to this hypothesis vesicles bud m a donor compartment vesicle budding by a process that allows selective incorporation of cargo into the forming vesicles while retaining resident proteins in the donor compartment protein sorting Thevesicles I largete r quot39 ccepturquot com partment vesicle targeting into which they unload their cargo upon fusion of their limiting membranes vesicle fusion An updated representation of the steps of vesicular transport is shown in Figure 2 The pro cesses of budding and fusion are iterated at the consec utive transport steps until the cargo reaches its final destination within or outside the cell To balance this forward movement of cargo organelle homeostasis re quires the retrieval of transport machinery components and escaped resident proteins from the acceptor com partments back to the corresponding donor compart ments retrograde transport a process that is also proposed to occur by vesicular transport All of these steps are tightly regulated and balanced so that a large amount of cargo can flow through the secretory pathway without compromising the integrity and steadystate composition of the constituent organelles Genetic and Biochemical Dissection of the Secretory Pathway In his 1974 Nobel Prize lecture Palade stated Further understanding of the secretory process is now becom ing dependent on adequate information on the chemis try of these membranes and on the reactions involved in their interactions Palade 1975 The challenge to find this information was taken on by Randy Schekman and Jim Rothman who in the late 1970s independently set out to elucidate the molecular mechanisms that un derlie vesicular transport Inspired by Arthur Kornberg s molecular dissection of DNA replication Schekman and Rothman embarked on the task of reducing vesicular transport to a set of elementary biochemical reactions Toward this goal each investigator initially pursued a different approach Schekman and colleagues had the foresight to choose for their studies the yeast Saccharomyces cerevisiae at a time when it was not yet clear that yeast and mam mals had similar secretory apparatuses The ease of 39 39 39 yea ldllUWeu 39 I set were defective in protein secretion Twentythree comple mentation groups each corresponding to a different gene were identified in the study by Novick et al 1980 Strikingly electron microscopy of the sec mutants at accumulation of various types of membraneenclosed structures Figure 3 Depending on the mutant strain these structures appeared as 1 small vesicles of 60 100 nm diameter that presumably corresponded to the Cell 154 Cfmolgllex O Lysosome p trans 0 Plasma 4a DiS TGN Membrane Late Endosome Multivesicular Body OO COPI V 0 Early Endosome WV Nucleus h ERGIC b Recycling Clmhnn ER Exit it Endosome k lt Immature Secretory Secretory Granule Ie Nuclear Envelope Q Granu V t v The scheme depicts the compartments of the secretory lysosomalvacuolar and endocytic pathways Transport steps are indicated by arrows Colors indicate the known or presumed locations of COPII blue COPI red and clathrin orange Clathrin coats are heterogeneous 1 x n ED 4 1 Figure 1 Intracellular Transport Pathways associated clathrin in endocytosis are known with certainty Less well understood are the exact functions of COPI at the ERGIC and Golgi complex and of clathrin at the TGN early endosomes and immature secretory granules The pathway of transport through the Golgi stack is still being investigated but is generally believed to involve a combination of COPImediated vesicular transport and cisternal maturation Pelham and Rothman 2000 Additional coats or coatlike complexes exist but are not represented in this figure transport carriers 2 an enlarged ER network or 3 a complex Schekman and colleagues quickly recognized cupshaped e anous organelle the Berkeley that each of these structures represented an exagger body which was later identified as an abnormal Golgi ated secretory pathway intermediate that had accumu Donor Cytosol Acceptor 9 Uncoaling Tethering gt gt Docg Budding Q I o Soluble 39 F Cargo I Protein 9 Coat Proteins T Initiation MW Transmembrane Cargo Protein 39 r vSNARE Figure 2 Steps of Vesicle Budding and Fusion 1 Initiation of coat assembly The membraneproximal coat components blue are recruited to the donor compartment by binding to a membraneassociated GTPase red andor to a specific phosphoinositide Transmem rane cargo proteins and SNAREs begin to gather at the assembling coat 2 Budding The membranedistal coat components green are added and polymerize into a meshlike structure Cargo L quotand L 39 39 39 u i I and theuunui 39 iulel 39 L at luya p t in 4Uncoating n of the small GTPase phosphoinositide hydrolysis and the action of quot g f quot 39 no a rounds of vesicle budding 5 Tethering The naked vesicle moves to the acceptor compartment possibly guided by the cytoskeleton and becomes tethered to the acceptor compartment by the combination of a GTP bound Rab and a tethering factor 6 Docking The v and t quotF quot39 39 f h quot h quot39 I This quot promotes fusion of the vesicle and acceptorlipid bilayers Cargo is transferred to the acceptor compartment and the SNAREs are recycled as shown in Figure 7B quot uuiliunal Review 155 Figure 3 Electron Micrographs of S cerevisiae sec Mutants lncubated at the Nonpermissive Temperature of 37 C The mutant strains sh own in this figure illustrate the accumulation of transport vesicles A the expansion of the ER B and the expansion of the Golgi complex Berkeley bodyquot C Symbols ves vesIcles va vacuole er endoplasmic reticulum n nucleus nm nuclear membrane np nuclear pore Bb Berkeley body Pictures reprinted from Novick et al 1980 lated due to a specific block in protein transport Subse quent identification of the genes that were defective in these sec mutants revealed a fascinating array of novel proteins involved at multiple stages of the secretory pathway a list of the 23 original SEC genes and their protein products is shown in Table 1 of Schekman and Novick 2004 Rothman and colleagues addressed the same prob lem using a completely different strategy Few had con templated studying cell biological processes by in vitro reconstitution but these researchers devised an inge nious cellfree assay to measure protein transport be 39 L quot r I 39 IRnInh 3 et al 1984 Balch 2004 Figure 4 This assay consisted of incubating 1 a donor Golgi fraction derived from Vesicular Stomatitis Virus VSVinfected cells lacking labeled substrate for GlcNAc transferase l 4 cytosol and 5 ATP Transport between the donor and acceptor lated by immunoprecipitation Combining this cellfree assay 39k I 39 I A 39 39l39 139 led h ill nti fication of various components involved in vesicle bud ding and fusion Strikingly these disparate methodologies converged with the discovery that two of the genetically identified Sec proteins were orthologous to biochemically identi fied proteins required for intra Golgi transport Wilson et al 1989 Griff et al 1992 The implication of this finding was profound yeast and mammals share a con served vesicular transport machinery which can be dis Apo erful synergy developed from the combined use of these approaches in many laboratories The results have pro duced a detailed molecular picture of the mechanisms of trafficking in the secretory pathway and the related endocytic and vacuolarlysosomal targeting pathways Central to these mechanisms are the two most critical events in the lifetime of a transport vesicle namely bud ding and fusion Role of Protein Coats in Vesicle Budding and Cargo Selection The budding of transport vesicles and the selective in corporation of cargo into the forming vesicles are both mediated by protein coats Kirchhausen 2000 Bonifac ino and LippincottSchwartz 2003 Figure 2 These Table 1 Components of the COPII ER Export Machinery Yeast Proteins Human Orthologs Functions andor Properties Small GTP binding protein of the Ras superfamily Sar1pGTP binding subunit GTPaseactivating protein GAP for Sar1p Component of the membranedistal layer of COPII coat probably contains Bpropeller Component of the membranedistal layer of COPII coat probably contains Bpropeller a Guanine nucleotide exchange factor GEF for Sar1p type II transmembrane protein Sar1p Sar1a Sar1b Sec23p Sec23A Sec23B Sec24p Sec24A Cargo binding subunit Lst1p Sec24B lss1p Sec24C Sec2 Sec13p Sec13 omaIn Sec31p Sec31A Sec31B domaIn Sec16p Unknown Scaffold 39 FD associated with the ER membr ne Sec12p Sec12 PREB with probable Bpropeller domaIn Sed4p Unknown Sec12p homolog devoid of Sar1pGEF activity putative Sar1p GAP inhibitor Cell 1 56 quotDONORquot GOLGlCONTAINING FRACTION FROM VSVINFECTED 158 MUTANT DONOR COMPARTMENT quotACCEPTORquot GOLGICONTAINING FRACTION FROM UNINFECTED WILDTYPE CELLS ACCEPTOR COMPARTMENT Figure 4 Schematic Representation of the In Vitro Assay for Protein Transport between Donor and Acceptor Golgi Compartments See text for details Reprinted from Balch et al 1984 coats are supramolecular assemblies of proteins that are recruited from the cytosol to the nascent vesicles The coats deform flat membrane patches into round buds eventually leading to the release of coated trans port vesicles The coats also participate in cargo selec tion by recognizing sorting signals present in the cyto solic domains of transmembrane cargo proteins Vesicle budding and cargo selection at different stages of the exocytic and endocytic pathways are mediated by dif ferent coats and sorting signals The first coats to be identified and characterized contained a scaffold pro tein clathrin as their main constituent Roth and Porter 1964 Pearse 1975 Clathrin coats were initially as sumed to participate in most if not all vesicular trans port steps within the cell However later studies demon strated that the function of these coats was restricted to postGolgi locations including the plasma membrane the transGolgi network T GN and endosomes A major discovery by the Rothman and Schekman labs was the existence of nonclathrin coats that mediate vesicular transport in the early secretory pathway Maters et al 1991 Barlowe et al 1994 One of these coats COPII is now known to mediate export from the ER to either the ERGolgi intermediate compartment ERGIC or the Golgi complex Barlowe et al 1994 while another coat COPI is involved in intra Golgi transport and retrograde transport from the Golgi to the ER Letourneur et al 1994 Of the various protein coats that have been identi fied to date COPII is one of the best understood and the one that we will use as an example in our discussion of vesicle budding Composition of COPII The identification and characterization of COPII are among the greatest achievements to emerge from the sec mutant screen In S cerevisiae the core COPII components are the small Raslike GTPase Sar1 p the Sec23pSec24p subcomplex and the Sec13pSec31 p subcomplex Table 1 Barlowe et al 1994 Sar1 p to gether with Sec23pSec24p form the membraneproxi mal layer of the coat while Sec13pSec31p forms a second membranedistal layer Figure 5 Additional regulatory proteins I39 able 1 participate in COPII assem bly including Sec16p a putative scaffold protein Es penshade et al 1995 and Sec12p a guanine nucleo tide exchange factor GEF for Sar1 p Barlowe and Schekman 1993 Sed4p a Sec12p homolog that may function as an inhibitor of GTP hydrolysis by Sar1 p Gi meno et al 1995 SaitoNakano and Nakano 2000 is likely to be specific to S cerevisiae and closely related species Payne et al 2000 Orthologs of the other struc tural and regulatory COPII components exist in higher eukaryotes including mammals Table 1 Book et al 2001 There are two additional paralogs of Sec24p in S cerevisiae Lst1 p and lss1 p and two or more paralogs of Sar1 p Sec23p Sec24p and Sec31 p in humans Table 1 This diversification of COPII subunits likely endows the coat with the ability to sort different cargo proteins and to be differentially regulated Roberg et al 1999 Shimoni et al 2000 Apart from Sar1 p the subunits of the COPII coat are structurally distinct from those of the COPI and clathrin coats The relative simplicity of COPII as well as its unique role in ER export have facilitated the analysis of its assembly and function Coat Assembly The COPII coat assembles by the stepwise deposition of Sar1pGTP Sec23pSec24p and Sec13pSec31 p onto sites where newly synthesized proteins exit from the ER Figure 5 These ER exit sites also known as transitional ER sites are generally devoid of ribosomes and range in complexity from discrete buds on the nuclear enve lope to convoluted networks of tubules and vesicles Bednarek et al 1995 Orci et al 1991 Bannykh and Balch 1997 The more elaborate ER exit sites are long lived membrane subdomains from which COPII vesicle budding occurs repeatedly Hammond and Glick 2000 Stephens et al 2000 At present it is unclear what marks these sites for COPII recruitment A candidate for this role is Sec16p a large peripheral ER membrane protein Espenshade et al 1995 Sec16p interacts with Sec23p Sec24p and Sec31 p via different domains Es penshade et al 1995 Shaywitz et al 1997 and may serve as scaffold for the nucleation or stabilization of the assembling coat Supek et al 2002 It is likely that Sec16p acts in conjunction with the transmembrane protein Sec12p to recruit GTP bound Sar1p to the ER membrane Sar1pGTP associates with the lipid bilayer through a hydrophobic aminoterminal extension and recruits its effector the Sec23pSec24p subcomplex through interactions with two switch regions charac teristic of Ras superfamily proteins Huang et al 2001 Review 157 Cytasal Sar1pGDP Sec24p Sec23p av 339 u R V Sec12p San pGTP QR Cargo Lumen Proteins Figure 5 Assembly of COPII Sec13pSec31 p Cytosolic Sar1pGDP is converted to membrane bound Sar1pGTP by the transmembrane protein Sec12p Sar1pGTP recruits the Sec23po Sec24p subcomplex by binding to Sec23p forming the prebudding complexquot Tr ansmembrane cargo proteins gather at the assembling coat by binding to Sec24p The Sec13poSec31p subcomplex polymerizes onto Sec23poSec24p and crosslinks the prebudding complexes n 5 S A 1 me I u ateu ar1p Sec23p and S Hum 1 of these proteins Bi et al 2002 The Sec13poSec31p complex is represented as an elongated fiveglobular domain structure based on electron microscopy Lederkremer et al 2001 Sec16p and Sed4p also participate in the assembly of COPII but are not represented here because their roles are less well understood See text for additional details Bi et al 2002 The initiation of COPII assembly thus involves both GTPindependent and GTPdependent re actions that cooperate to deposit the coat at ER exit 3 I es Sar1pGTP together with Sec23poSec24p constitute the socalled pre budding complex which has re cently been analyzed by electron microscopy Leder kremer et al 2001 Matsuoka et al 2001 and Xray crystallography Bi et al 2002 This complex has the appearance of a bow tie with one side corresponding to Sec23p and the other to Sec24p Bi et al 2002 Figure 6 Sec23p makes direct contact with Sar1pGTP Bi et al 2002 while Sec24p participates in cargo rec r I 1 I p LI 1 the prebudding complex recruits the Sec13poSec31p subcomplex which consists of two Sec13p and two Sec31p subunits Lederkremer et al 2001 Sec13po Sec31p appears by electron microscopy as a flexible elongated structure that polymerizes to form a mesh ike scaffold Lederkremer et al 2001 Matsuoka et al 2001 Sec23p stimulates the GTP hydrolysis activity of Sar1p Yoshihisa et al 1993 by contributing an argi nine finger that pokes into the GTP binding site and aids catalysis Bi et al 2002 This activity of Sec23p as a GTPase activating protein GAP is augmented ap proximately tenfold by addition of Sec1 SpoSec81 p An onny et al 2001 A paradoxical implication of this mechanism is that COPII coat assembly should trigger disassemny by promoting GTP hydrolysis How can the COPII coat polymerize to cover a forming vesicle if the basic unit of the polymer is unstable A possible expla nation is that the kinetics of GTP hydrolysis might be slower than the kinetics of vesicle budding in which case there would be time for a vesicle to form before the coat fell apart Alternatively GTP hydrolysis might cause Sar1 p to be released from the coat while the other subunits remained assembled on the membrane The r thec I u ity in the absence of Sar1pGTP In addition the cyto solic domains of transmembrane cargo proteins could act as secondary membrane tethers or could modulate Ar9342 Site Figure 6 Surface Representation of the Crystal Structure of the PreBudding Complex The locations of the A B and Arg342 sites for binding different ER surface of the complex is represented by a curved line The bottom image is rotated by 120 along a longitudinal axis relative to the top image Adapted from Mossessova et al 2003 and Miller et al 2003 Images were generated using GRASP Nicholls et al 1991 Cell 158 Table 2 ER Export Signals Proteins Functions Signals Sys1p Golgi protein high copy suppressor of ypt6 mutants QLE Gap1p General amino acid permease Big VSVG Envelope glycoprotein of vesicular stomatitis virus YTQIE Kir21 lnwardly rectifying potassium channe ECYENE Kir11 lnwardly rectifying potassium channel EVETQ Prm8p Pheromoneregulated membrane protein E ERGICSG Mammalian type I 39 39 FD no uu e1 ul E gous to yeast Emp46p and Emp47p hp245 p24 family member putative ER export receptor homologous to yeast Erv25p and Emp24p E Erv46p ER vesicle transmembrane protein part of a complex with Erv4 Erv41p ER vesicle transmembrane protein part of a complex with Erv46 E Emp46p Type transmembrane lectin cycles between the ER and the Golgi homologous to Emp47p M E Emp47p Type transmembrane lectin cycles between the ER and the Golgi homologous to Emp46p E Erv25p p24 family member putative ER export receptor part of a complex with Emp24p E E Emp24p p24 family member ER export receptor for Gas1p part of a complex with Erv25p E E Sed5p Golgi tSNARE YNNSNPF LMLE Bet1 p ERGolgi vSNARE EASE GalT2 Golgi enzyme E GalNAcT Golgi enzyme E lnforrnation was obtained from Barlowe 2003 and Giraudo and Maccioni 2003 Underlining indicates known key residues the GAP activity of Sec28p Any of these alternative explanations would imply that Sar1pGTP is dispens able for the integrity of the central area of the coat and LL a 39Q 1 J J quotu Schekman 2001 Cargo Selection The majority of cargo proteins are actively concentrated in COPIIcoated buds and vesicles prior to export from the ER Balch et al 1994 Malkus et al 2002 Most transmembrane cargo proteins exit the ER by binding directly to COPII Kuehn et al 1998 Aridor et al 1998 Votsmeier and Gallwitz 2001 but sometransmembrane and most soluble cargo proteins bind indirectly to COPII through transmembrane export receptors I39 able 2 Ap penzeller et al 1999 Muniz et al 2000 Powers and Barlowe 2002 Export receptors leave the ER together with their ligands unload their cargo into the acceptor compartment and recycle back to the ER The sorting signals recognized by the COPII coat are found in f quot quot 39 3 proteins These signals are quite diverse I39 able 2 Bar lowe 2008 Some consist of diacidic motifs fitting the consensus DEJXDEJ where D is aspartate X is any 39 acid and E is glutamate Nishimura and Balch 1997 Votsmeier and Gallwitz 2001 whereas others are based on short hydrophobic motifs such as FF YYM FY LL or lL F is phenylalanine Y is tyrosine M is methionine L is leucine and l is isoleucine Kappeler et al 1997 Nakamura et al 1998 In addition a di basic RKXRK R is arginine and K is lysine motif has recently been shown to promote ER exit of Golgi glycosyltransferases Giraudo and Maccioni 2008 Yet other signals consist of longer sequences folded deter minants or combinations of any of the above Table 2 The involvement of so many different signals in the same a I I elulel I ing sites on the same recognition protein or a family of recognition proteins Both of these solutions have evolved for COPII Genetic biochemical and structural analyses have demonstrated that most ER export sig nals bind to Sec24p Miller et al 2002 2008 Mosses sova et al 2008 Sec24p displays at least three distinct signal binding sites term 1 A B an Arg842 this latter site is named after an arginine residue that is criti cal for binding of Sec22p Miller et al 2008 Mosses sova et al 2008 Figure 6 The Sec24p paralogs Lst1 p Roberg et al 1999 and lss1p Kurihara et al 2000 may interact with export signals different from those recognized by Sec24p In addition Sar1p may partici pate in signal recognition either by direct binding to the signals or by allosteric modulation of Sec24p Springer and Schekman 1998 Aridor et al 1998 Giraudo and Maccioni 2008 In this regard it is interesting that muta tions in one of two human Sar1p homologs Sar1 b re sults in a specific defect in chylomicron export from the ER Jones et al 2008 This diversity of signals and recognition modes explainsthe ability of COPII to pack a e a wide variety of exported proteins Vesicle Budding How do the properties of the COPII proteins lead to vesicle formation A mixture of purified Sec28po Sec24p Sec18poSec81p and GTPlocked Sar1 p is suffi cient to generate coated vesicles from liposomes indi cating that these proteins are intrinsically capable of deforming the membrane and pinching off a vesicle Matsuoka et al 1 998 An important clue to the genesis of the curvature of COPIIcoated buds comes from the crystal structure of the prebudding complex which has a positively charged concave surface that likely apposes L quot 39 quot quot 439 39quot39 t al 2002 Figure 6 The contribution of the Sec18poSec81p subcomplex to m mbrane deformation is still not clear although this subcomplex might stabilize the curvature generated by the prebudding complex The final stage in vesicle formation is scission of the neck of the bud To date no proteins have been identified as being spe cifically involved in this process One possibility is that coat polymerization itself may drive membrane scission by closing the spherical COPII cage Review 159 Comparison with Other Coats It is now clear that other vesicle coats follow the basic COPII paradigm but with variations Kirchhausen 2000 Bonifacino and LippincottSchwartz 2003 For exam ple I b I U tion and membrane recruitment of Arf GTPases that are closely related to San p Donaldson et al 1992 Helms and Rothman 1992 But unlike Sar1 p which has an exclusive relationship with COPII Arf proteins have many effectors including COPI and other coats see below as well as lipidmodifying enzymes Nie et al 2003 Many different GEFs and GAPs activate and in active Arf respectively in an effector or compart mentspecific fashion Nie et al 2003 During COPI coat assembly ArfoGTP simultaneously recruits the mem braneproximal Se5 and the membranedistal 043 s sub complexes HaraKuge et al 1994 in apparent contrast to the stepwise assembly of COPII Like COPII COPI recognizes specific signals in the cytosolic domains of transmembrane cargo proteins although in this case the signals function to retrieve proteins from the ERGIC or the Golgi complex to the ER Cosson and Letourneur 1994 Bremser et al 1999 Clathrin coats are considerably more complex than COPII and COPI ArfoGTP andor specific phosphoinosi tides eg phosphatidylinositol 45bisphosphate and phosphatidylinositol 4phosphate recruit a variety of clathrin adaptors from the cytosol to membranes Bonifacino and LippincottSchwartz 2003 Wang et al 2003 Examples of adaptors are the heterotetrameric AP1 AP2 and AP3 complexes and the monomeric GGA Hrs Epsin 1 and ARH proteins specific combina tions of which form a heterogeneous membraneproxi mal layer onto which clathrin is subsequently deposited The adaptors also bind to transmembrane cargo pro teins by recognizing cytosolic sorting signals that con tain either critical tyrosine or dileucine residues or con jugated ubiquitin Bonifacino and Traub 2003 Indeed the tyrosinebased signal present in the cytosolic do main of the lowdensity lipoprotein receptor was the first cytosolic sorting signal to be identified a finding that was reported in another landmark paper by Mike Brown and Joe Goldstein Davis et al 1986 This partic uldl 39 39 lU interact 39 He et al 2002 Mishra et al 2002 Clathrin and clathrin adaptor complexes can polymerize into spherical cage like structures Kirchhausen and Harrison 1981 as can COPII Antonny et al 2003 indicating that these pro teins have an intrinsic ability to sculpt buds and vesicles from membranes Thus the clathrinadaptor complexes appear to perform the same basicfunctions as the COPII 4 J mation However the clathrin vesicle cycle involves ad ditional classes of proteins that do not seem to operate during COPII vesicle formation Clathrin vesicle assem bly is regulated by an ensemble of kinases phospha tases and other accessory proteins Lafer 2002 In addition clathrin vesicle scission depends on accessory factors such as dynamins Sever 2002 Finally clathrin vesicle uncoating is mediated by the cytosolic chaper ones Hsc70 and auxilin Rothman and Schmid 1986 Ungewickell et al 1995 Why does clathrin utilize all of this extra machinery Part of the answer may lie in the participation of clathrin in multiple postGolgi sorting events each of which requires a specific set of adaptors and regulators Role of SNARE Proteins in Vesicle Fusion After a vesicle sheds its coat it must be targeted to the appropriate acceptor compartment The final step in a vesicle s existence is fusion with the acceptor mem brane Remarkably the targeting and fusion reactions both rely on the same class of proteins which were identified in a biochemical tour de force Discovery of the SNAREs An early contribution of the cellfree intra Golgi transport assay Figure 4 was the identification of an Nethylmalei mideSensitive Factor NSF which could exist in cyto solic or membrane bound forms Glick and Rothman 1987 Electron microscopy by Lelio Orci demonstrated that when NSF was inactivated uncoated vesicles accu mulated on Golgi membranes implying that NSF is re quired for membrane fusion Malhotra et al 1988 By treating Golgi membranes with Nethylmaleimide the intra Golgi transport reaction was converted into a spe cific assay that allowed for the purification of NSF Block etal 1988 Cloning of the corresponding gene revealed that NSF was the mammalian ortholog of yeast Sec1 8p which had been implicated in ERtoGolgi transport Wil son et al 1989 Eakle et al 1988 It soon became apparent that NSF acts in a wide range of membrane fusion steps in the secretory and endocytic pathways Beckers et al 1989 Diaz et al 1989 Despite the obvious importance of NSF its role in membrane fusion was initially unclear NSF forms a hex americ ring Whiteheart et al 2001 and is a founding member of the AAA protein family ATPases associated with diverse cellular activities a group of enzymes that catalyze the structural remodeling of protein complexes Lupas and Martin 2002 A crucial step toward under standing NSF function came from identifying a partner protein called DASNAP soluble NSF association pro tein which binds NSF to membranes Clary et al 1990 048 NAP turned out to be the mammalian ortholog of yeast Sec17p Griff et al 1992 At this point it was evident that NSF and DASNAP formed a complex with additional unidentified membrane proteins Using NSF DASNAP as an affinity reagent to fractionate a brain ly sate Thomas Sollner and colleagues identified a set of three membraneassociated SNAP Receptors or SNAREs Sollner et al 1993 These same membrane proteins had previously been implicated in linking syn aptic vesicles to the plasma membrane WalchSoli mena etal1993 One of the proteins known as VAMP or synaptobrevin was known to be associated with syn aptic vesicles whereas the other two proteins syntaxin and SNAP25 no relation to otSNAP had been local ized to the presynaptic plasma membrane From today s perspective it may seem obvious that synaptic vesicle exocytosis is mechanistically related to other vesicular transport steps but until 1993 most researchers as sumed that these processes were distinct The discov ery of the link between NSF DASNAP and SNAREs revo lutionized the analysis of both intracellular transport and synaptic transmission and brought these two fields to gether in a spectacular collision Cell 160 Properties of the SNAREs The product of this collision was the SNARE hypothesis which proposed that each type of transport vesicle car ries a speci c vSNARE that binds to a cognate tSNARE on the target membrane Rothman 1994 This idea fits with the observations that cells contain families of proteins related to the synaptic SNAREs and that various SNAREs localize to different intracellular compartments Bennett and Scheller 1998 Weimbs et al 1998 Chen and Scheller 2001 Most SNAREs are Cterminally anchored transmembrane proteins with their functional Nterminal domains facing the cytosol Each of 39 39 r motif of 60 70 amino acids that can participate in coiledcoil formation Bock et al 2001 An exception is SNAP25 which contains two SNARE motifs and binds to the membrane via covalently linked palmitate groups attached to the central part of the protein Struc tural and biochemical studies showed that the SNARE complex generated by the pairing of a cognate v and tSNARE is a very stable fourhelix bundle with one oi helix contributed by the monomeric vSNARE and the other three oi helices contributed by the oligomeric tSNARE Fasshauer et al 1997 Sutton et al 1998 Figure 7A The tS NARE usually consists of three sepa I I I although in u l r Arquot plex two of the SNARE motifs are supplied by SNAP 25 All of the SNARE complexes in the cell appear to fit this general pattern in which the four SNARE motifs are contributed by a protein related to synaptobrevin a protein related to syntaxin a protein or protein domain related to the Nterminal part of SNAP25 and a protein or protein domain related to the Cterminal part of SNAP 25 Misura et al 2002 In some cases the distinction between vesicles and target membranes is not meaningful for example dur ing the homotypic fusion of organelles but the general classification scheme of vSNAREs one othelix and scheme uses the terminology R or QSNAREs re flecting the presence of an arginine or a glutamine re spectively at a characteristic position within the SNARE motif Fasshauer et al 1998 In each SNARE complex three glutamines and one arginine form a central ionic la erinw u I f wh Ii bundle Sutton et al 1998 Although the two classifica tion schemes are based on different principles there is a rough correspondence of RSNAREs with vSNAREs and of QSNAREs with tSNAREs A major insight from structural analysis of the SNARE complex was that v and tSNAREs pair in a parallel fashion Hanson et al 1997 Lin and Scheller 1997 Sutton et al 1998 Therefore v and tS NAREs in sepa rate membranes can pair to form a transSNARE com plex or v and tS NAREs in the same membrane can pair to form a cisSNARE complex A transSNARE complex persists throughout the fusion reaction to become a cis SNARE complex in the fused membrane Figure 7B DASNAP then binds along the edge of the SNARE com plex Rice and Brunger 1999 and recruits NSF ATP hydrolysis by NSF dissociates the cisSNARE complex Mayer et al 1996 possibly by exerting rotational force to untwist the fourhelix bundle May et al 1999 Yu et al 1999 Thus NSF and DASNAP do not participate directly in the fusion reaction but instead act to recycle the SNAREs for another round of complex formation What Exactly Do SNAREs Do SNAREs seem to perform two major functions One function is to promote fusion itself In all transport reac tions that have been examined the formation of trans SNARE complexes is essential for fusion Assembly of the four 39 39 3 0 supply 3 needed to bring apposing membranes close enough to fuse Hanson et al 1997 Weber et al 1998 Chen and Scheller 2001 This model is appealing because a transSNARE complex also known as a SNAREpin has r 39 segments in two apposing membrane bilayers and is therefore structurally analogous to the activated form of viral fusion proteins Jahn et al 2003 Support for the idea that SNAREs act as fusogens came from reconstitution experiments showing that purified recombinant SNAREs can promote the fusion of lipo somes provided that v and tSNAREs are in different liposomes Weber et al 1998 In an elegant extension of this work Rothman and colleagues recently demon strated that the fusion of natural biological membranes can be driven by SNAREs in the absence of accessory proteins Hu et al 2003 Cells were engineered to pro duce flipped SNAREs that faced the outside of the cell rather than the cytoplasm When cells containing a flipped vSNARE were mixed with cells containing the cognate flipped tSNARE efficient fusion occurred The combined data leave little doubt that SNAREs form the conserved essential core of the fusion machinery than in vivo fusion reactions Weber et al 1998 im plying that additional components cooperate with SNAREs to tickle the membranes and accelerate fu sion The best documented example is the yeast vacuo lar ATPase V0 subunit which has been reported to act downstream of the SNAREs in vacuolar fusion Bayer et al 2003 Under some conditions fusion can apparently proceed even if the transSNARE complex has already dissociated Szule and Coorssen 2003 The meaning of these observations is still being debated but they suggest that assembly of a transSNARE complex is not always temporally coupled to membrane fusion This point may be particularly relevant for the reversible kissandrun fusion that occurs during regulated exo cytosis Palfrey and Artalejo 2003 Despite these com plexities it is likely that in all of the transport steps in the same function of overcoming the energy barrier to fusion The second major function of SNAREs is to help en sure the specificity of membrane fusion Different v tSNARE complexes form at different steps of intracellu lar transport Surprisingly purified SNAREs can pair pro miscuously in vitro But when purified SNAREs were tested in the liposome fusion assay the formation of productive transSNARE complexes was almost ex clusively restricted to physiologically relevant v and tSNARE combinations McNew et al 2000 As a result the biophysical fusion assay actually has predictive power for identifying SNARE complexes that form in vivo Parlati et al 2002 SNAREs cannot however be the only specificity de 39 39 a39 QMADI Review 161 Vesicle vSNARE Synaplohrevln lSNAR E Syntaxin SNAP25 Figure 7 Structure and Function of SNAREs A Crystal structure of a synaptic trans SNARE complex drawn after Sutton et al 1998 The structures of the two membrane anchors and of the peptide that links the two SNAP25 ol helices are hypothetical B The SNARE cycle A transSNARE com plex assembles when a monomeric vSNARE on the vesicle binds to an oligomeric tSNARE on the target membrane forming a stable fourhelix bundle that promotes fusion The result is a cisSNARE complex in the fused membrane olSNAP binds to this complex and recruits NSF which hydrolyzes ATP to dissociate the complex Unpaired vSNAREs can then be packaged again into vesicles The depictions of the SNARE complex and olSNAP are from Sutton et al 1998 and Rice and Brunger 1999 respectively A complete crystal structure of NSF is not yet available but the protein is known to form a double Target B CE SN F39E barreled hexameric ring that binds to the end gsSNARE amp e of the SNARE complex Lupas and Martin tSNARE mp ex i gt A SNARE Vesicle Complex Fusion V39SNAHE Formatlcm 39 W Veslcle Binding of Buddlng NSF llSNAP m NSF ocSNAP N vSNARE H ATPDriven Recycling SNARE g 4 Complex Dissociation recycles and is therefore present in both anterograde and retrograde vesicles Figure 7B Additional specific ity is provided by tethering proteins that link the appos ing membranes prior to SNARE complex formation These tethering proteins come in several flavors Whyte and Munro 2002 The heteromeric quatrefoil tethers are exemplified by the exocyst which links secretory carriers to the plasma membrane Guo et al 1999 Six of the original set mutants defined different subunits of the exocyst Schekman and Novick 2004 Related quatrefoil tethers function in Golgi traffic For example the COG complex is believed to mediate the tethering of COPI vesicles to Golgi cisternae and was identified by several approaches Whyte and Munro 2002 including biochemical purification using the cellfree intra Golgi transport assay Ungar et al 2002 A different type of tether is EEA1 a long coiledcoil protein that promotes the homotypicfusion of early endosomes Christoforidis et al 1999 Similar coiledcoil tethers called golgins are present in the Golgi Barr and Short 2003 These various tethers assemble with the aid of Rab family GTPases known as th proteins in yeast to promote the initial association of two membranes Segev 2001 Jahn et al 2003 Multiple Rab proteins operate at differ ent steps of transport Rabs tethers and SNAREs col laborate to ensure that membranes fuse at the correct time and place Thus like many biological processes membrane fusion employs sequential partially redun dant mechanisms to achieve high fidelity Accessory and Regulatory Proteins Not surprisingly a plethora of accessory components and regulatory reactions modulate the action of SNAREs Gerst 2003 Table 3 This modulation is important to prevent inappropriate events of SNARE complex forma tion For example after two membranes fuse and the sisSNARE complex is dissociated by NSFOASNAP the SNAREs need to be kept inactive until the next round of fusion Cytosolic factors such as GATE16 and LMA1 bind the individual v and tSNAREs and help to keep them separate Elazar et al 2003 In some cases SNARE complex formation is regulated by phosphoryla tion of SNAREs or of interacting components Gerst 2003 Key regulatory elements for SNARE complex as sembly are present in the SNAREs themselves many of Cell 162 Table 3 Selected Protein Families lmplicated in Vesicle Targeting and Fusion5 Family Names or Representative Family Members Functions VAMPSynaptobrevin Monomeric vSNARE contributing a single RSNARE helix Syntaxin tSNARE subunit contributing one QSNARE helix SNAP25 39 39 39 39 as in SNAP25 or in two separate polypeptides NSFSec18p ATPase that promotes dissociation of clsSNARE complexes olSNAPSec17p Sec1P Munc18 plex assembl GATE16 LMA1 Va tnrntam analogous to GATE16 Synaptotagmins Vacuolar ATPase V0 subunit Quatrefoil tethers EEA1 Golgins Rabth GTPases with cytoskeletal motors and m Links NSFSec18p to SNAREs Bind to syntaxin family proteins and perform diverse essential functions regulating SNARE com Small ubiquitinrelated mamamlian protein that binds and shields unpaired SNAREs Putative Ca sensors for regulated exocytosis Promotes a late step of vacuolar fusion in yeast Heteromeric tethering factors that act at various transport steps Long coiledcoil tethering factor involved in early endosome fusion Coiledcoil proteins that mediate vesicle tethering and cisternal stacking in the Golgi apparatus vesicle tethering interaction of vesicles embrane subdomain formation are given by Whyte and Munro 2002 1 luv u re which contain extensions upstream of the SNARE motif Misura et al 2002 Dietrich et al 2008 For example syntaxins have an Nterminal threehelix bundle which family have an Nterminal longin domain that may have a similar autoinhibitory function Dietrich et al 2008 In some cases transSNARE complex assembly seems to be arrested at an intermediate stage with accessory proteins preventing the complete zipping up of the fourhelix bundle until a fusion signal is received Chen and Scheller 2001 The best candidate for such an accessory protein is the putative Ca sensor synapto t L L mm agmln I aptic vesicle fusion in response to Ca influx Jahn et al 2003 An intriguing group of SNAREinteracting proteins is the SM family whose founding members are yeast Sec1p the product of thefirst gene identified by Novick and Schekman 1979 and neuronal Munc1 8 The SM proteins can be viewed as comparable in importance to the SNAREs because each membrane fusion step requires a specific SM protein l39oonen and Verhage 2008 Gallwitz and Jahn 2008 However the function of SM proteins is still enigmatic These proteins bind to syntaxins but the mode of binding is not conserved and various SM proteins either stimulate or inhibit SNARE complex assembly Thus much remains to be learned about the regulatory aspects of SNAREdependent membrane fusion Intracellular Targeting of SNAREs A typical SNARE is a transmembrane protein with an Nterminal cytosolic domain and a single membrane spanning sequence near the C terminus Such tail anchored proteins insert into the ER membrane post translationally and reach their final destinations by traversing the secretory pathway Borgese et al 2008 Little is known about how SNAREs are targeted to spe cific organelles For the few SNAREs that have been examined targeting determinants are present in the transmembrane sequence the cytosolic domain or both Joglekar et al 2008 An important mechanism for SNARE localization is interaction with vesicle coats For example SNAREs involved in ERtoGolgi transport must be packaged into COPII vesicles during ER export and then into COPI vesicles during retrieval from the Golgi Springer and Schekman 1998 Rein et al 2002 Hfll Plll quot nated the process by which three S cerevisiae SNAREs involved in ERtoGolgi transport Sed5p Be p and Sec22p interact with the COPquot coat Miller et al 2008 Mossessova et al 2008 These SNAREs bind to distinct sites on the Sec24p subunit a YNNSNPF N is aspara gine S is serine and P is proline signal from Sed5p binds to the A site a LXXLME signal from Sed5p and Bet1p binds to the B site as does a diacidic signal from the Golgi protein Sys1 p and an unidentified deter minant on Sec22p binds to a site that includes Arg842 Miller et al 2008 Mossessova et al 2008 Figure 6 Sec24p apparently cannot bind an assembled SNARE complex but instead selects for the uncomplexed fuso genic forms of the SNAREs Mossessova et al 2008 Thus vesicle budding is mechanistically integrated with vesicle fusion Perspectives Over the past 80 years we have progressed from the L am way to the present molecular understanding of vesicular transport The experimental approaches introduced by Novick et al 1980 and Balch et al 1984 were crucial to this endeavor and remain among the most powerful tools available to probe the workings of the protein traf ficking machinery The advent of genomics and proteo mIc quot 39 39 39 3v rr methods and the development of fluorescent livecell imaging technologies have further contributed to mak Review 163 ing protein trafficking one of the most vibrant areas of modern cell biology In the near future we can expect progress toward a better understanding of the structure assembly regula tion and function of vesicle coats It is remarkable that to date the only coats with wellestablished functions are COPII export from the ER and plasma membrane clathrin coats endocytosis The exact roles of other coats including COPI and TGNendosomal clathrin coats are less clear Moreover a look at Figure 2 reveals that the arrows representing single transport steps far outnumber the known coats ls each of these steps mediated by a different coat If so there must be many coats yet to be discovered A more likely alternative is that some transport steps occur by mechanisms other than coatmediated budding For example transport from the TGN to the plasma membrane is not known to be concentrative or to involve conventional coats phoinositides regulate the membrane recruitment of clathrin coats and of other trafficking components De Matteis et al 2002 Wang et al 2003 while other lipids eg glycosphingolipids and cholesterol may help to 2003 How are local variations in lipid composition es tablished and maintained How do lipid modification and localization regulate protein trafficking What lipid rearrangements occur during membrane fission and fu sion The fundamental nature of these questions illus trates that lipids are a new research frontier inally we can expect to learn more about the trans port vesicles themselves When and how do they lose their coats How do they interact with the cytoskeleton Do all transport vesicles have a uniformly small size and spherical shape Regarding this last question the best 39 Iran pn 39 39 are indeed small spherical coated vesicles But emerging evidence from mammalian cells points to an alternative form of trans port by large pleiomorphic intermediates Bonifacino and LippincottSchwartz 2003 Awelldocumented ex mple of such a ransport intermediate is the COPI containing vesicular tubular carriers that move from ER exit sites to the Golgi complex Aridor et al 1995 Presley et a 1997 Mironov et al 2003 There are undoubtedly similarities between the transport mechanisms em ployed by large pleiomorphic intermediates and those employed by small vesicles but there are probably sig nificant quotquot quot 39 i m imi on the vesicular 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