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YEASTBOOK

CELL STRUCTURE & TRAFFICKING

Secretory Protein Biogenesis and Traffic in the EarlySecretory PathwayCharles K. Barlowe* and Elizabeth A. Miller†,1

*Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, and ySchool of Biological Sciences, Columbia University,New York, New York 10027

ABSTRACT The secretory pathway is responsible for the synthesis, folding, and delivery of a diverse array of cellular proteins. Secretoryprotein synthesis begins in the endoplasmic reticulum (ER), which is charged with the tasks of correctly integrating nascent proteins andensuring correct post-translational modification and folding. Once ready for forward traffic, proteins are captured into ER-derivedtransport vesicles that form through the action of the COPII coat. COPII-coated vesicles are delivered to the early Golgi via distincttethering and fusion machineries. Escaped ER residents and other cycling transport machinery components are returned to the ER viaCOPI-coated vesicles, which undergo similar tethering and fusion reactions. Ultimately, organelle structure, function, and cellhomeostasis are maintained by modulating protein and lipid flux through the early secretory pathway. In the last decade, structural andmechanistic studies have added greatly to the strong foundation of yeast genetics on which this field was built. Here we discuss the keyplayers that mediate secretory protein biogenesis and trafficking, highlighting recent advances that have deepened our understandingof the complexity of this conserved and essential process.

TABLE OF CONTENTS

Abstract 383

Introduction 384

Expanding Methodologies: From a Parts List to Mechanisms and Back to More Parts 384Classic screens lay the groundwork; in vitro reconstitution defines mechanism 384

Dynamics and organization revealed by live cell imaging 385

New technologies yield new players and define interplay between pathways 385

Secretory Protein Translocation and Biogenesis 386Polypeptide targeting and translocation 386

Maturation of secretory proteins in the ER: signal sequence processing 388

Maturation of secretory proteins in the ER: protein glycosylation 388

Maturation of secretory proteins in the ER: glycosylphosphatidylinositol anchor addition 389

Maturation of secretory proteins in the ER: disulfide bond formation 389

Glucosidase, mannosidase trimming, and protein folding 390

Control of ER homeostasis by the Unfolded Protein Response 391

Continued

Copyright © 2013 by the Genetics Society of Americadoi: 10.1534/genetics.112.142810Manuscript received June 14, 2012; accepted for publication September 25, 20121Corresponding author: School of Biological Sciences, Columbia University, 1212 Amsterdam Ave, MC2456, New York, NY 10027. Email: [email protected]

Genetics, Vol. 193, 383–410 February 2013 383

mailto:[email protected]

CONTENTS, continued

Transport From the ER: Sculpting and Populating a COPII Vesicle 391Structure and assembly of the COPII coat 392

Cargo capture: stochastic sampling vs. direct and indirect selection 393

Regulation of COPII function: GTPase modulation, coat modification 394

Higher-order organization of vesicle formation 395

Vesicle Delivery to the Golgi 395Vesicle tethering 395

SNARE protein-dependent membrane fusion 396

A concerted model for COPII vesicle tethering and fusion 397

Traffic Within the Golgi 397Transport through the Golgi complex 397

Lipid requirements for Golgi transport 398

The Return Journey: Retrograde Traffic viaCOPI Vesicles 398Composition and structure of the COPI coat 399

Cargo capture: sorting signals, cargo adaptors, and coat stimulators 400

Vesicle delivery: DSL-mediated tethering and SNARE-mediated fusion 401

Perspectives 401

LIKE all eukaryotes, yeast cells segregate various physio-logical functions into distinct subcellular compartments.

A key challenge is thus ensuring that appropriate proteinsare delivered to the correct subcellular destination, a processthat is driven by discrete sorting signals that reside in theproteins themselves. Perhaps the most prevalent type of sort-ing signal is that directing a protein to the secretory pathway,which handles the various proteins that are destined for theextracellular environment or retention in the internal endo-membrane system. Approximately one-third of the yeast pro-teome enters the secretory pathway. Protein secretion is notonly essential for cellular function but also provides thedriving force for cell growth via delivery of newly synthe-sized lipid and protein that permits cell expansion. Secretoryproteins enter this set of interconnected organelles at theendoplasmic reticulum (ER), which regulates protein trans-lation, protein translocation across the membrane, proteinfolding and post-translational modification, protein qualitycontrol, and forward traffic of suitable cargo molecules (bothlipid and protein). Once contained within the secretory path-way, proteins are ferried between compartments via trans-port vesicles that bud off from one donor compartment tofuse with a downstream acceptor compartment, therebymediating directional traffic of both lipid and protein. Theforward-moving, or anterograde pathway is balanced bya reverse, or retrograde pathway that returns escaped resi-dent proteins and maintains the homeostasis of individualorganelles. Early yeast screens pioneered the genetic dissec-tion of the eukaryotic secretory pathway and were rapidlyfollowed by biochemical approaches that permitted the mo-

lecular dissection of individual processes of protein biogen-esis and traffic. Here we discuss the methodologies thathave yielded great insight into the conserved processes thatdrive protein secretion in all eukaryotes and describe thefundamental processes that act to ensure efficient and ac-curate protein secretion. The reader is also referred to earliercomprehensive reviews on these topics (Kaiser et al. 1997;Lee et al. 2004) as we focus our coverage on more recentadvances.

Expanding Methodologies: From a Parts Listto Mechanisms and Back to More Parts

Classic screens lay the groundwork; in vitro reconstitutiondefines mechanism

There is no doubt that early seminal yeast genetics ap-proaches laid the foundation upon which our understand-ing of protein secretion is built. From the original Novickand Schekman screens that identified a host of secretion-defective (sec) mutants (Novick and Schekman 1979; Novicket al. 1980), to additional more targeted approaches fromthe Schekman (more secs; Deshaies and Schekman 1987;Wuestehube et al. 1996), Gallwitz (ypt; Gallwitz et al. 1983),Ferro-Novick (bet; Newman and Ferro-Novick 1987), Jones(pep; Jones 1977), Stevens (vps; Rothman et al. 1989), andEmr (vps; Bankaitis et al. 1986) labs that expanded the rep-ertoire of mutants with defects in secretory protein andmembrane biosynthesis, the field has been blessed with anabundance of reagents that permitted the characterizationof each branch of the secretory pathway (Schekman and

384 C. K. Barlowe and E. A. Miller

Novick 2004). Many of these processes are essential, con-served, and have direct relevance to issues of human health,yet yeast genetics approaches remain at the forefront indeciphering molecular mechanisms, unraveling cellular re-dundancy and complexity, and appreciating the cross-talkbetween different branches of the pathway. The strength ofyeast as a model system to probe this complexity lies in thecombination of facile genetics and robust biochemistry thatare afforded by this remarkable organism. Indeed, the fieldhas a long history of capitalizing on yeast mutants to informbiochemical reconstitution approaches that in turn informnew genetic screening approaches.

The most pertinent example of the strength of thisapproach is the mechanistic description of the COPII coatproteins that drive vesicle formation from the endoplasmicreticulum. Classic epistasis analyses of the Novick andSchekman sec mutants (Novick et al. 1980), placed the earlysec genes in order within the secretory pathway: sec12,sec13, sec16, and sec23 mutants blocked formation of trans-port vesicles and induced proliferation of the ER, whereassec17, sec18, and sec22 mutants blocked vesicle fusion andcaused accumulation of vesicles (Novick et al. 1981; Kaiserand Schekman 1990). The subsequent development ofin vitro assays relied in part on the use of these mutants inbiochemical complementation assays (Baker et al. 1988;Ruohola et al. 1988). Recapitulation of ER–Golgi traffic inpermeabilized yeast cells was perturbed in sec23 mutants,but could be restored by incubation with cytosol preparedfrom wild-type cells, placing Sec23 as a soluble factor re-quired for transport vesicle formation (Baker et al. 1988).Further refinement of these in vitro transport assays permit-ted the dissection of different transport stages (Rexach andSchekman 1991) and allowed the biochemical characteriza-tion of the COPII coat proteins (Barlowe et al. 1994) thatgenerate transport intermediates, and the membrane-boundand cytosolic factors required for tethering and fusion stepsthat consume vesicles at the Golgi membrane (Barlowe1997; Cao et al. 1998). Further mechanistic dissection camefrom even more refined reconstitution systems that permit-ted the identification of the minimal machinery required togenerate COPII vesicles from synthetic liposomes (Matsuokaet al. 1998a,b) and defined the dynamics of individualevents using real-time assays (Antonny et al. 2001).

Similar reconstitution of the COPI-mediated Golgi–ERretrograde pathway in yeast lagged somewhat behind, inpart due to equivalent biochemical experiments that wereunder way in mammalian cells (Balch et al. 1984; Waterset al. 1991). Furthermore, due to rapid perturbation in for-ward (ER–Golgi) traffic when the retrograde pathway isblocked, for some time there was confusion over the direc-tionality of COPI-mediated events (Gaynor and Emr 1997).Despite these difficulties, in vitro reconstitution of COPI-coated vesicle formation was ultimately achieved (Spangand Schekman 1998) and has been similarly dissectedin minimal systems using synthetic liposomes (Spang et al.1998).

In contrast to the genetics-informed biochemical ap-proaches described above, minimal reconstitution of themembrane fusion events that drive vesicle consumption tooka slightly different path. Armed with the knowledge thatfusion is driven by proteins known as SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein recep-tors) and with the full description of yeast SNAREs in handfrom computational analyses of the yeast genome, Rothmanand colleagues established liposome-based assays that dem-onstrated compartment specificity of different SNARE pairs(McNew et al. 2000). That this biochemical approach largelyrecapitulated known pathways, previously defined by ge-netic means, serves to highlight the success of mutually in-formed genetic and biochemical approaches to fully dissectthe molecular mechanisms of budding and fusion events.

Dynamics and organization revealed by live cell imaging

With budding and fusion machineries well described inminimal systems, it became apparent that there were stillpieces of the puzzle missing, including the roles of someessential proteins (e.g., Sec16; Espenshade et al. 1995) thatremained unexplained in terms of functionality. Further-more, some of the more pressing mechanistic questionscould not be answered by biochemical means. For example,the mode of protein and lipid traffic through the Golgiremained controversial: did COPI vesicles mediate forwardtraffic or did proteins proceed through the Golgi by a processof maturation of individual cisternae? These questions wereaddressed in part by the Glick and Nakano labs using high-resolution time-lapse imaging of living yeast cells (Losevet al. 2006; Matsuura-Tokita et al. 2006). Such experimentsdefined discrete sites of vesicle formation, known as transi-tional ER (tER) or ER exit sites (ERES), that are dynamic innature, can form de novo but also fuse with each other, andhave clear relationships with downstream Golgi elements(Bevis et al. 2002; Shindiapina and Barlowe 2010). Further-more, imaging of distinct Golgi elements lent support for thecisternal maturation model of protein secretion, althoughdirect imaging of cargo molecules remains to be fully dem-onstrated. Recent advances in superresolution imaging holdgreat promise in further understanding the nature of thesesubdomains and their relationships with distinct proteinmachineries and membrane compartments, although somelimitations will still apply, especially with respect to theproblem of detecting transient cargo molecules that arein flux through the system.

New technologies yield new players and define interplaybetween pathways

Since the yeast community entered the postgenomic world,a host of new tools has opened up many new approaches:the haploid deletion collection represents an accessiblelarge-scale analysis platform for novel screens (Tonget al. 2001), the GFP- (Huh et al. 2003) and TAP-tagged(Ghaemmaghami et al. 2003) fusion databases documentedthe localization and abundance of many gene products, and

Early Events in Protein Secretion 385

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microarray analyses of gene expression changes allow thedissection of cell-wide changes to a given perturbation(Travers et al. 2000). These new tools are being used withremarkable imagination, often capitalizing on the facile na-ture of yeast genetics, to define the interplay between relatedpathways in exciting ways. For example, microarray analysisof the changes in gene expression that occur upon inductionof ER stress via the unfolded protein response (UPR) iden-tified upregulation of machineries involved in ER-associateddegradation (ERAD), ultimately leading to the appreciationthat these discrete pathways are intimately coordinated tomanage the burden of protein within the ER (Travers et al.2000). A second example derives from the development ofsynthetic genetic array (SGA) technology, which allows therapid generation of haploid double mutant strains (Tonget al. 2001). Although the piecemeal application of this tech-nology was informative for individual genes, the broaderapplication to an entire pathway was revolutionary in termsof being able to define novel functions based on sharedgenetic fingerprints. The first so-called epistatic miniarrayprofile (E-MAP) made pairwise double mutations amongalmost 500 early secretory pathway components, quantify-ing the phenotypic cost of combined mutations (Schuldineret al. 2005). Analysis of the shared patterns of genetic inter-actions revealed (perhaps not surprisingly) that componentsin common pathways shared similar profiles, which allowedthe assignation of novel functions to previously uncharacter-ized and enigmatic proteins. An elaboration on the E-MAPapproach made elegant use of a fluorescent reporter systemto first assess the UPR state of individual strains in thegenomic deletion collection and then to probe how UPRactivation changes in double mutant backgrounds, yieldinga more subtle understanding of genetic interactions thangross life and death dichotomies, which usually form thebasis of synthetic interactions (Jonikas et al. 2009). With

further development of such reporters on cell status, thisarea of cross-talk between pathways will become moreand more integrated, allowing a detailed picture of cellu-lar physiology. However, as these new technologies yieldnew functional clues to previously uncharacterized genes,we need to continue to use and develop biochemical toolsthat allow true mechanistic insight. Again, the strength ofthe yeast system is the use of both genetic and biochemicaltools to mutually inform new discoveries.

Secretory Protein Translocation and Biogenesis

Polypeptide targeting and translocation

The first step in biogenesis of most secretory proteins issignal sequence-directed translocation of the polypeptideinto the ER. Both cotranslational and post-translationalmechanisms operate in yeast to target diverse sets of solubleand integral membrane secretory proteins to the ER (Figure1). The cotranslational translocation process is initiatedwhen a hydrophobic signal sequence or transmembranesequence is translated and recognized by the signal-recognitionparticle (SRP) for targeting to the SRP receptor at ER trans-location sites (Figure 1a). In the case of post-translationaltranslocation, cytosolic chaperones play a critical role inbinding hydrophobic targeting signals to maintain the na-scent secretory protein in an unfolded or loosely folded trans-location competent state until delivery to the ER membrane(Figure 1b). Progress on identification and characterizationof the translocation machinery will be described in turnbelow as the start of a continuum of events in biogenesisof secretory proteins.

Genetic approaches in yeast uncovered key componentsin both the co- and post-translational translocation path-ways. Appending a signal sequence to the cytosolic enzyme

Figure 1 Membrane transloca-tion of secretory proteins. Threewell-characterized pathways oper-ate to deliver secretory proteinsto the ER for membrane trans-location. (A) The signal recogni-tion particle (SRP) recognizes ahydrophobic signal sequence ortransmembrane segment duringprotein translation followed bytargeting of the ribosome–nascentchain complex to the SRP receptorfor cotranslational membrane in-sertion. (B) Post-translational inser-tion of secretory proteins dependson cytosolic Hsp70 ATPases suchas Ssa1 to maintain the nascentsecretory protein in an unfolded

translocation competent state until delivery to the Sec63 complex formed by Sec62/Sec63/Sec71/Sec72. The Sec61 complex forms an aqueouschannel for both post- and cotranslational polypeptide translocation. Kar2, a luminal Hsp70 ATPase, facilitates directed movement and foldingof nascent polypeptides. (C) In GET-mediated insertion of C-terminal tail-anchored proteins, the Sgt2–Get4–Get5 complex targets nascentpolypeptides to Get3 for Get1/Get2 dependent translocation. Tail-anchored proteins are integrated into the membrane in Sec61-independentpathway.


encoded by HIS4 targets this enzyme to the ER where itcannot function and produces histidine auxotrophy. A ge-netic selection for mutants that are partially defective intranslocation of this signal peptide-bearing fusion protein,and therefore restore histidine prototrophy was used toidentify conditional mutations in three essential genes;SEC61, SEC62, and SEC63 (Deshaies and Schekman 1987;Rothblatt et al. 1989). Sequencing indicated that all threegenes encode integral membrane proteins, with the 53-kDaSec61 protein a central component that contained 10 trans-membrane segments and striking sequence identity with theEscherichia coli translocation protein SecY (Stirling et al.1992; Jungnickel et al. 1994). Similar genetic selectionapproaches using the HIS4 gene product fused to integralmembrane proteins identified SEC65, which encodes a com-ponent of the SRP (Stirling and Hewitt 1992; Stirling et al.1992) as well as mutations in SEC71 and SEC72 (Greenet al. 1992).

Concurrent with these genetic approaches, cell-freereconstitution assays that measured post-translationaltranslocation of radiolabeled pre-pro-a-factor into yeastmicrosomes were used to dissect molecular mechanisms inthis translocation pathway (Hansen et al. 1986; Rothblattand Meyer 1986). Fractionation of cytosolic components re-quired in the cell-free assay revealed that Hsp70 ATPasesstimulated post-translational translocation (Chirico et al.1988). Yeast express a partially redundant family of cyto-solic Hsp70s encoded by the SSA1–SSA4 genes that are col-lectively essential. An in vivo test for Hsp70 function intranslocation was demonstrated when conditional expres-sion of SSA1 in the background of the multiple ssaD strainresulted in accumulation of unprocessed secretory proteinsas Ssa1 was depleted (Deshaies et al. 1988). ATPase activityof Hsp70 family members is often stimulated by a corre-sponding Hsp40/DnaJ partner and in the case of poly-peptide translocation in yeast the YDJ1 gene encodesa farnsylated DnaJ homolog that functions in ER transloca-tion (Caplan et al. 1992). Ydj1 has been shown to directlyregulate Ssa1 activity in vitro (Cyr et al. 1992; Ziegelhofferet al. 1995) and structural studies indicate that Ydj1 binds tothree- to four-residue hydrophobic stretches in nonnativeproteins that are then presented to Hsp70 proteins such asSsa1 (Li et al. 2003; Fan et al. 2004). Finally, genetic experi-ments connect YDJ1 to translocation components in addi-tion to multiple other cellular pathways presumably due toaction on a subset of secretory proteins (Becker et al. 1996;Tong et al. 2004; Costanzo et al. 2010; Hoppins et al. 2011).

Several lines of experimental evidence indicate that themultispanning Sec61 forms an aqueous channel for polypep-tide translocation into the ER. Initial approaches probinga stalled translocation intermediate in vitro revealed thatdirect cross-links formed only between transiting segmentsof translocation substrate and Sec61 (Musch et al. 1992;Sanders et al. 1992; Mothes et al. 1994). Purification offunctional Sec61 complex revealed a heterotrimeric complexconsisting of Sec61 associated with two �10-kDa proteins

identified as Sss1 and Sbh1 (Panzner et al. 1995). For effi-cient post-translational translocation, the Sec61 complexassembles with another multimeric membrane complextermed the Sec63 complex, which consists of the geneticallyidentified components Sec63, Sec62, Sec71, and Sec72(Deshaies et al. 1991; Brodsky and Schekman 1993; Panzneret al. 1995). Purification of these complexes combined withproteoliposome reconstitution approaches have demon-strated that the seven polypeptides comprising the Sec61and Sec63 complexes plus the lumenal Hsp70 protein,Kar2, are sufficient for the post-translational mode oftranslocation (Panzner et al. 1995). Further biochemical dis-section of this minimally reconstituted system in addition tocrystal structures of the homologous archaeal SecY complex(Van den Berg et al. 2004) have provided molecular insightsinto the translocation mechanism (Rapoport 2007). Currentmodels for post-translational translocation suggest that thehydrophobic N-terminal signal sequence is recognized andbound initially by the Sec63 complex, which then transmitsinformation through conformational changes to the Sec61complex and to lumenally associated Kar2 (Figure 1b). Ina second step that is probably coordinated with opening ofthe translocation pore, the signal sequence is detected at aninterface between membrane lipids and specific transmem-brane segments in Sec61 where it binds near the cytosolicface of the channel (Plath et al. 1998). Opening of the porewould then permit a portion of the hydrophilic polypeptideto span the channel where association with lumenal Kar2would capture and drive directed movement in a ratchetingmechanism through cycles of ATP-dependent Kar2 binding(Neupert et al. 1990; Matlack et al. 1999). Well-documentedgenetic and biochemical interactions between Kar2 and thelumenal DnaJ domain in Sec63 are thought to coordinatedirected movement into the ER lumen (Feldheim et al.1992; Scidmore et al. 1993; Misselwitz et al. 1999). TheN-terminal signal sequence is thought to remain boundat the cytosolic face of the Sec61 complex as the nascentpolypeptide chain is threaded through the pore where, atsome stage, the signal sequence is cleaved by a translocon-associated signal peptidase for release into the lumen (Antoninet al. 2000).

Of course, a major pathway for delivery of nascentsecretory proteins to the ER employs the signal recognitionparticle in a co-translational translocation mechanism. Here,the ribosome–nascent chain–SRP complex is targeted toSec61 translocons through an initial interaction betweenSRP and the ER-localized SRP receptor (SR) encoded bySRP101 and SRP102 (Ogg et al. 1998). In an intricateGTP-dependent mechanism, paused SRP complexes boundto SR transfer ribosome–nascent chains to Sec61 tranloconsas polypeptide translation continues in a cotranslationaltranslocation mode (Wild et al. 2004). Genetic screens un-covered the Sec65 subunit of SRP, and purification of nativeSRP identified the other core subunits termed Srp14, Srp21,Srp54, Srp68, and Srp72 in addition to the RNA componentencoded by SCR1 (Hann and Walter 1991; Brown et al.











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1994). Somewhat surprisingly, deletion of the SRP compo-nents in yeast produced yeast cells that grow slowly butremain viable. These findings indicate that the SRP-dependentpathway is not essential, unlike the core translocation porecomponents, and indicates that other cytosolic machinerycan manage delivery of all essential secretory proteins tothe translocon. Although yeast cells can tolerate completeloss of the SRP pathway, it became clear that certain secre-tory proteins displayed a preference for the SRP-dependentroute, whereas others were efficiently translocated into theER in a post-translational mode (Hann et al. 1992; Stirlingand Hewitt 1992). In general, integral membrane proteinsand signal sequences of relatively high hydrophobicity pref-erentially engage the SRP-dependent pathway, whereas sol-uble and lower hydrophobicity signal sequences depend ona Sec63-mediated post-translational mode of translocation(Ng et al. 1996).

More recently a third post-translational translocationpathway to the ER has been characterized in yeast andother eukaryotes whereby short integral membrane proteinsand C-terminal tail-anchored proteins are integrated intothe membrane (Figure 1c) (Stefanovic and Hegde 2007;Schuldiner et al. 2008). For this class of proteins, transmem-brane segments are occluded by the ribosome until trans-lation is completed, thereby precluding SRP-dependenttargeting. Bioinformatic analyses suggest that up to 5%of predicted integral membrane proteins in eukaryoticgenomes may follow this SRP-independent route includingthe large class of SNARE proteins that drive intracellularmembrane fusion events and are anchored by C-terminalmembrane domains. Interestingly, this post-translational tar-geting pathway operates independently of the Sec61 andSec63 translocon complexes (Steel et al. 2002; Yabal et al.2003) and instead depends on recently defined soluble andmembrane-bound factors. Large-scale genetic interactionanalyses in yeast identified a clustered set of nonessentialgenes that produced Golgi-to-ER trafficking deficiencies thatwere named GET genes (Schuldiner et al. 2005). Get3shares high sequence identity with the transmembrane do-main recognition complex of 40 kDa (TRC40) that had beenidentified through biochemical strategies in mammaliancell-free assays as a major interaction partner for newly syn-thesized tail-anchored proteins (Stefanovic and Hegde2007; Favaloro et al. 2008). Subsequent synthetic geneticarray analyses and biochemical approaches in yeast (Jonikaset al. 2009; Battle et al. 2010; Chang et al. 2010; Chartronet al. 2010; Costanzo et al. 2010) have implicated five Getproteins (Get1–5) and Sgt2 in this process. Current modelsfor the GET targeting pathway in yeast suggest that a Sgt2–Get4–Get5 subcomplex loads tail-anchored substrates ontothe targeting factor Get3 (Figure 1c). The Get3-boundsubstrate then delivers these newly synthesized proteinsto an integral membrane Get1/Get2 complex. In an ATP-dependent process, Get3 in association with Get1/Get2then inserts the hydrophobic segment to span across theER membrane bilayer (Shao and Hegde 2011). Although

structural and biochemical studies are rapidly advancingour understanding of the GET-dependent targeting path-way, the mechanisms by which tail-anchored proteins areinserted into ER membrane bilayer remain to be defined.

Maturation of secretory proteins in the ER: signalsequence processing

For the many secretory proteins that contain an N-terminalsignal sequence, the signal peptidase complex (SPC) removesthis domain by endoproteolytic cleavage at a specific cleav-age site during translocation through the Sec61 complex(Figure 2a). The SPC consists of four polypeptides termedSpc1, Spc2, Spc3, and Sec11 (Bohni et al. 1988; YaDeauet al. 1991). Spc3 and Sec11 are essential integral mem-brane proteins that are required for signal sequence cleav-age activity, with the Sec11 subunit containing the proteaseactive site (Fang et al. 1997; Meyer and Hartmann 1997).Based on structural comparisons with E. coli leader pepti-dase, the active site of SPC is thought to be located very nearthe lumenal surface of the ER membrane and presumablyclose to translocon exit sites. The Spc1 and Spc2 subunitsare not required for viability; however, at elevated temper-atures the corresponding deletion strains accumulate unpro-cessed precursors of secretory proteins in vivo (Fang et al.1996) and are required for full enzymatic activity of the SPCin vitro (Antonin et al. 2000). Interestingly, Spc2 is detectedin association with the Sbh1 subunit of the Sec61 complexand is thought to physically link the SPC and Sec61 complex(Antonin et al. 2000). Given that SEC11 was identified inthe original SEC mutant screen as required for ER-to-Golgitransport of secretory proteins, signal sequence cleavage isregarded as an essential step for maturation of secretoryproteins that contain N-terminal signal sequences.

Maturation of secretory proteins in the ER:protein glycosylation

In addition to signal sequence cleavage, attachment ofasparagine-linked oligosaccharide to nascent glycopro-teins occurs concomitantly with polypeptide translocationthrough the Sec61 pore (Figure 2b). The addition of coreoligosaccharides to consensus Asn-X-Ser/Thr sites in transit-ing polypeptides is catalyzed by the oligosaccharyltrans-ferase (OST) enzyme. OST is composed of eight integralmembrane polypeptides (Ost1, Ost2, Ost3 or Ost6, Ost4,Ost5, Wbp1, Swp1, and Stt3) and is also detected in com-plex with the Sec61 translocon (Kelleher and Gilmore2006). Indeed, for N-linked glycosylation sites that are nearsignal sequence cleavage sites, cleavage must occur beforeaddition of N-linked oligosaccharide, demonstrating the se-quential stages of polypeptide translocation, signal sequencecleavage, and N-linked glycosylation (Chen et al. 2001). TheStt3 subunit is critical for catalytic activity and in addition toStt3, most of the OST subunits are required for cell viability,indicating a critical role for N-linked glycosylation in matu-ration of secretory proteins. OST transfers a 14-residue oli-gosaccharide core en bloc to most (but not all) Asn-X-Ser/






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Thr sites in transiting polypeptides. The 14-residue oligosac-charide core is assembled on the lipid-linked carrier mole-cule dolichylpyrophosphate in a complex multistep pathway(Burda and Aebi 1999).

The precise role(s) for N-linked glycosylation of secretoryprotein is not fully understood because in many instancesmutation of single and multiple sites within a given proteinproduces only mild consequences. Hydrophilic N-linkedglycans influence thermodynamic stability and solubility ofproteins, and in the context of nascent secretory proteinsin the ER, the N-linked structure is also thought to be anintegral part of a system that assists in protein folding andquality control to manage misfolded glycoproteins (Schwarzand Aebi 2011). This quality control process will be exploredfurther after covering other folding and post-translationalmodification events in secretory protein maturation.

In addition to N-linked glycosylation, some secretoryproteins undergo O-linked glycosylation through attach-ment of mannose residues on Ser/Thr amino acids byprotein O-mannosyltransferases (Pmts). Saccharomyces cer-evisiae contains a family of seven integral membrane man-nosyltranferases (Pmt1–Pmt7) that covalently link mannoseresidues to Ser/Thr residues using dolichol phosphate man-nose as the mannosyl donor (Orlean 1990; Willer et al.2003). Both O-linked mannose residues and N-linked coreoligosaccharides added in the ER are extended in the Golgicomplex by the nine-membered KRE2/MNT1 family of man-nosyltranferases that use GDP-mannose in these polymeri-zation reactions (Lussier et al. 1997a,b). O-linked mannosylmodification of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wallintegrity as well as normal morphogenesis (Strahl-Bolsingeret al. 1999). The role of O-linked glycosylation in ER qualitycontrol processes remains unclear although investigatorshave reported influences of specific pmt mutations on turn-over rates of misfolded glycoproteins (Harty et al. 2001;Vashist et al. 2001; Hirayama et al. 2008; Goder and Melero2011) and the PMT genes are upregulated by activation ofthe UPR (Travers et al. 2000).

Maturation of secretory proteins in the ER:glycosylphosphatidylinositol anchor addition

Approximately 15% of proteins that enter the secretorypathway are post-translationally modified on their C termi-nus by addition of a lipid-anchored glycosylphosphatidyli-nositol (GPI) moiety. The synthesis and attachment of GPIanchors occur in the ER through a multistep pathway thatdepends on .20 gene products (Orlean and Menon 2007).GPI synthesis and attachment are essential processes inyeast and GPI anchored proteins on the cell surface arethought to play critical roles in cell wall structure and cellmorphology (Leidich et al. 1994; Pittet and Conzelmann2007). As with assembly of the N-linked core oligosaccha-ride, the GPI anchor is fully synthesized as a lipid anchoredprecursor and then transferred to target proteins en bloc bythe GPI transamidase complex (Fraering et al. 2001). TheGPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalentlinkage to what becomes the terminal amino acid (termed thev residue) and removal of the �30-amino-acid C-terminalGPI signal sequence (Udenfriend and Kodukula 1995). Bio-informatic approaches are now reasonably effective in pre-dicting GPI anchored proteins. These algorithms scan foropen reading frames that contain an N-terminal signal se-quence and a C terminus that consists of an v residuebracketed by �10 residues of moderate polarity plus a hy-drophobic stretch near the C terminus of sufficient lengthto span a membrane bilayer (Eisenhaber et al. 2004). GPIprecursor proteins that do not receive GPI-anchor additionand removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al. 1993; Doeringand Schekman 1996) and are probably retained through anER quality control mechanism.

Maturation of secretory proteins in the ER: disulfidebond formation

Most secretory proteins contain disulfide bonds that formwhen nascent polypeptides are translocated into the oxidiz-ing environment of the ER lumen. A family of protein-

Figure 2 Folding and matura-tion of secretory proteins. A se-ries of covalent modificationsand folding events accompanysecretory protein biogenesis inthe ER. (A) Signal peptidase com-plex consisting of Spc1/Spc2/Spc3/Sec11 cleaves hydrophobicsignal sequences during polypep-tide translocation. (B) Coincidentwith polypeptide translocationand signal sequence cleavage,N-linked core-oligosaccharide isattached to consensus N-X-S/Tsites within the transiting poly-

peptide by the multisubunit oligosaccharyl transferase complex. (C) In the oxidizing environment of the ER lumen, disulfide bond formation is reversiblycatalyzed by protein disulfide isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents. (D) Trimming of individual glucose and mannoseresidues from the attached core-oligosaccharide assists protein folding and quality control processes, which involve the calnexin family member Cne1.For terminally misfolded glycoproteins, sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation.


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disulfide isomerases that contain thioredoxin-like domainscatalyze the formation, reduction, and isomerization ofdisulfide bonds to facilitate correct protein folding in theER lumen (Figure 2c). In yeast, Pdi1 is an essential pro-tein disulfide isomerase that is required for formation ofcorrect disulfide bonds in secretory and cell surface proteins(Farquhar et al. 1991; Laboissiere et al. 1995). Pdi1 obtainsoxidizing equivalents for disulfide formation from the es-sential flavoenzyme Ero1, which is bound to the luminalface of the ER membrane (Sevier et al. 2007). Ero1 andPdi1 form the major pathway for protein disulfide bondformation by shuttling electrons between Ero1, Pdi1, andsubstrate proteins (Tu and Weissman 2002; Gross et al.2006). In reconstituted cell-free reactions, FAD-linked Ero1can use molecular oxygen as the electron acceptor to drivePdi1 and substrate protein oxidation. The electron acceptor(s)used by Ero1 in vivo remain to be fully characterized (Hatahetand Ruddock 2009).

In addition to Pdi1, yeast express four other nonessentialER-localized protein disulfide isomerase homologs, Mpd1,Mpd2, Eug1, and Eps1. Overexpression of Mpd1 or mutantforms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al. 2001; Norgaard and Winther 2001). Inaddition to oxidoreductase activity, Pdi1 can act as a molec-ular chaperone in protein folding even for proteins that lackdisulfide bonds (Wang and Tsou 1993; Cai et al. 1994).More recently, Pdi1 and other members of this family werereported to interact with components of the ER folding ma-chinery including calnexin (Cne1) and Kar2 (Kimura et al.2005) as well as the quality control mannosidase enzymeHtm1 (Gauss et al. 2011). Growing evidence indicates thatthis family of protein disulfide isomerases contains differentdomain architectures (Vitu et al. 2008) to dictate interac-tions with specific ER-chaperone proteins and thus shepherda broad range of client proteins into folded forms or into ER-associated degradation pathways (Figure 2d).

Glucosidase, mannosidase trimming, and protein folding

The initial 14-residue N-linked core oligosaccharide that isattached en bloc to nascent polypeptides is subsequentlyprocessed by glycosylhydrolases in a sequential and proteinconformation-dependent manner to assist protein foldingand quality control in the ER lumen (Helenius and Aebi2004). The Glc3Man9GlcNAc2 glycan, which comprises theN-linked core, is rapidly processed by glucosidase I (Gls1/Cwh41) and glucosidase II (Gls2/Rot2) enzymes to removethe three terminal glucose residues and generate Man9-GlcNAc2. Molecular chaperones collaborate in protein fold-ing during these glucose-trimming events and Rot1 alonehas been shown to possess a general chaperone activity(Takeuchi et al. 2008). In many cell types, a calnexin-dependent folding cycle operates to iteratively fold andmonitor polypeptide status through the coordinated activi-ties of glucosidase I, glucosidase II, UDP-glucose;glycopro-tein glucosyltransferase (UGGT), and calnexin (Cne1). Afterremoval of terminal glucose residues by the glucosidase

enzymes, UGGT can add back a terminal glucose to theglycan if the polypeptide is not fully folded to generate theGlc1Man9GlcNAc2 structure. This Glc1Man9GlcNAc2 form ofan unfolded protein binds to calnexin, which keeps the na-scent polypeptide in an iterative folding cycle. Once fullyfolded, UGGT does not act after glucosidase II and the na-scent protein exits the cycle (Helenius and Aebi 2004). Thiscalnexin cycle operates in many eukaryotes but it is cur-rently unclear how or if the cycle works in yeast since de-letion of Cne1, Gls1, Gls2, or Kre5 (potential UGGT-likeprotein) do not produce strong delays in biogenesis of se-cretory proteins but are known to produce defects in bio-synthesis of cell wall b-1,6-glucan (Shahinian and Bussey2000). Although a precise molecular understanding of thecalnexin cycle components in yeast folding remains to bedetermined, there are clear genetic (Takeuchi et al. 2006;Costanzo et al. 2010) and biochemical (Xu et al. 2004;Kimura et al. 2005) interactions that indicate a coordinatedrole for these factors in protein folding.

In addition to the glucose trimming of core oligosaccha-ride, two additional ER-localized mannosidase enzymestermed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d).Mns1 and Htm1 are related enzymes with distinct specific-ities. Mns1 removes the terminal mannosyl residue of the Bbranch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exportedfrom the ER (Jakob et al. 1998). Htm1 is thought to act afterMns1 on terminally misfolded proteins (or misfolded pro-teins that have lingered in the ER folding cycle for too long)to remove the outermost mannosyl residue from the Cbranch of the glycan to generate Man7GlcNAc2 (Clercet al. 2009). This form of the glycan is then recognized bythe ER lectin Yos9 and targets misfolded proteins for ER-associated degradation (Carvalho et al. 2006; Denic et al.2006). Although Mns1- and Htm1-deficient cells appear totransport folded secretory proteins at normal rates, bothdisplay significant delays in turnover of terminally misfoldedglycoproteins (Jakob et al. 1998, 2001), which serves tohighlight an important role for mannosidase activity in ERquality control.

Folding of nascent polypeptides throughout transloca-tion and within the ER is also managed by Hsp70 ATPasesystems, which handle partially folded intermediates. Ingeneral, Hsp70 proteins hydrolyze ATP when binding toexposed hydrophobic stretches in unfolded polypeptidesto facilitate protein folding. The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70ATPase cycle governed by specific DnaJ-like proteins thatstimulate ATP hydrolysis and nucleotide exchange factors thatdrive ADP release (Hartl 1996; Bukau and Horwich 1998). Inyeast, the Hsp70 Kar2 plays a prominent role in ER folding inconcert with the related Hsp70 protein Lhs1 (Rose et al.1989; Baxter et al. 1996; Brodsky et al. 1999; Steel et al.2004). For Kar2, the known DnaJ-like stimulating factorsinclude Sec63, Scj1, and Jem1 (Schlenstedt et al. 1995;


















































Nishikawa and Endo 1997), whereas the GrpE family mem-ber Sil1 and, surprisingly, the unrelated ATPase Lhs1 serve asnucleotide exchange factors (Hale et al. 2010). Complexity inregulating the Kar2 ATPase cycle probably reflects the range ofunfolded substrates that Kar2 must handle in maintaining ERhomeostasis, and there are likely to be additional factors thatcouple Kar2 activity to other specific ER processes. As mentionedabove, Kar2 chaperone activity is tightly linked with the PDI,calnexin, and glycan trimming pathways (Figure 2d). Finally,Kar2 also plays a prominent role in ER-associated degradation(ERAD) pathways to dispose of terminally misfolded proteins(Nishikawa et al. 2001). Although our understanding of Kar2biochemical activity is advanced, the coordinated control ofKar2-dependent folding and modification cycles in the contextof an ER lumenal environment remains a challenging area.

ERAD of misfolded and unassembled proteins proceedsthrough a series of pathways that remove targeted proteinsfrom the ER for ubiquitin- and proteasome-dependent deg-radation in the cytoplasm. ERAD is thought to play a keyrole in ER homeostasis and cellular physiology. Since thesepathways divert misfolded secretory proteins from theirroutes of biogenesis, this important topic is beyond thescope of this current review and the reader is referred toexcellent recent reviews (Vembar and Brodsky 2008; Smithet al. 2011).

Control of ER homeostasis by the UnfoldedProtein Response

Much of the folding and biogenesis machinery in the ER isunder a global transcriptional control program referred toas the UPR. The yeast UPR is activated by an increase inthe level of unfolded proteins in the ER, which can beexperimentally induced by treatment with inhibitors ofER protein folding (e.g., tunicamycin, dithiothreitol) or byoverexpression of terminally misfolded proteins (Bernaleset al. 2006). Regulation of the UPR was initially examinedthrough identification of a 22-nucleotide segment in theKAR2 promoter region, termed the unfolded protein re-sponse element (UPRE), which was required for UPR ac-tivation of Kar2 expression. Fusion of this KAR2 promoterelement to a lacZ reporter provided an elegant screen forgene mutations that blunted UPR reporter expression (Coxet al. 1993; Mori et al. 1993). Genetic screening led to thediscovery that IRE1, HAC1, and RLG1 were required fora robust UPR under ER stress conditions (Cox and Walter1996; Sidrauski et al. 1996). Further studies revealed thatIRE1 encodes an ER transmembrane protein with cytosolickinase/ribonuclease domains and a lumenal sensor domainthat together are thought to serve as readout on unfoldedprotein levels. HAC1 encodes a basic leucine zipper tran-scription factor that binds to UPRE-containing segments ofDNA and induces their expression (Cox and Walter 1996).Surprisingly, RLG1 encodes a tRNA ligase that is required forthe nonconventional splicing of HAC1 pre-mRNA. Structuraland mechanistic dissection of these core components is nowadvanced. Current models indicate that the Ire1 lumenal

domain interacts with Kar2 and unfolded proteins to senseprotein folding status (Bertolotti et al. 2000; Pincus et al.2010; Gardner and Walter 2011). When unfolded proteinsaccumulate in the ER, Ire1 forms oligomers that activate thecytoplasmic kinase and ribonuclease domains. ActivatedIre1 ribonuclease then acts on HAC1 pre-mRNA to removea nonconventional intron and this splicing intermediate isthen ligated by the Rlg1 ligase to produce mature HAC1mRNA. Translation of HAC1 message produces Hac1 pro-tein, which is a potent transcriptional activator of UPR targetgenes (Bernales et al. 2006).

In addition to Kar2, the UPR was known to induce otherER folding components including Pdi1 and Eug1 (Cox et al.1993; Mori et al. 1993). To comprehensively assess the tran-scriptional profile of the yeast UPR, DNA microarray analysiswas powerfully applied to monitor mRNA levels under ERstress conditions (Travers et al. 2000). Comparing transcrip-tion profiles in wild-type, ire1D, and hac1D strains after UPRinduction revealed 381 genes that passed stringent criteriaas UPR targets. Not surprisingly, 10 genes involved in ERprotein folding were identified as UPR targets and includedJEM1, LHS1, SCJ1, and ERO1. In addition, dozens of genesinvolved in ER polypeptide translocation, protein glycosyla-tion, and ER-associated degradation were induced. Perhapsmore surprisingly, 19 genes involved in lipid and inositolmetabolism as well as 16 genes encoding proteins that func-tion in vesicle trafficking between the ER and Golgi wereupregulated by the UPR. These findings highlight a globalrole for the UPR in regulating ER homeostasis through bal-ancing ER lipid and protein biosynthetic rates. In the contextof cellular physiology, the UPR is now thought to serve a cen-tral role in sensing and integrating secretory pathway func-tion to finely tune ER capacity in response to cellulardemands (Walter and Ron 2011).

Transport From the ER: Sculpting and Populatinga COPII Vesicle

Once secretory proteins have completed their synthesis andmodification regimes, they become competent for forwardtraffic through the secretory pathway, a process mediatedby a series of transport vesicles that bud off from onecompartment, traverse the cytoplasm, and fuse with a down-stream organelle (Figure 3). ER-derived vesicles are createdby the COPII coat that, like other coat protein complexes, ischarged with the dual tasks of creating a spherical transportvesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes. Biochemicalcharacterization of this process, first from complex mi-crosomal membranes using purified COPII coat proteins(Barlowe et al. 1994), then in more reduced form from syn-thetic liposomes (Matsuoka et al. 1998b), and subsequentlyat the structural level through cryo-EM (Stagg et al. 2006)and X-ray crystallography (Bi et al. 2002; Fath et al. 2007),has been remarkably fruitful in defining the molecular basisof these events. What has emerged is an elegant mechanism







































whereby the minimal COPII machinery, composed of fiveproteins (Sar1, Sec23, Sec24, Sec13, and Sec31), sufficesto fulfill these multiple functions. However, recent insightsinto how this process is regulated suggest there is still muchto learn about coat dynamics in the cell, and the precisephysical basis for various steps, including membrane scissionduring vesicle release, vesicle uncoating, and the formationof large transport carriers capable of shuttling large cargoes.

Structure and assembly of the COPII coat

COPII coat assembly (Figure 3) is initiated by the local re-cruitment and activation of the small G protein, Sar1(Nakano and Muramatsu 1989; Barlowe et al. 1993) uponexchange of GDP for GTP, catalyzed by an ER membraneprotein, the guanine nucleotide exchange factor (GEF)Sec12 (Nakano et al. 1988; d’Enfert et al. 1991). GTP load-ing on Sar1 exposes an amphipathic a-helix that likelyinduces initial membrane curvature by locally expandingthe cytoplasmic leaflet relative to the lumenal leaflet (Leeet al. 2005). GTP-bound, membrane-associated Sar1 sub-sequently recruits the heterodimeric complex of Sec23and Sec24 (Matsuoka et al. 1998b). Sec23 is the GTPase-activating protein (GAP) for Sar1 (Yoshihisa et al. 1993),contributing a catalytic arginine residue analogous to GAPstimulation in many Ras-related G proteins (Bi et al. 2002).Sec24 provides the cargo-binding function of the coat, con-taining multiple independent domains that interact directlywith specific sorting signals on various cargo proteins (Milleret al. 2002, 2003; Mossessova et al. 2003). The Sar1/Sec23/Sec24 “prebudding” complex in turn recruits the hetero-tetrameric complex of Sec13 and Sec31 (Matsuoka et al.1998b). Sec31 also contributes to the GTPase activity ofthe coat by stimulating the GAP activity of Sec23 (Antonnyet al. 2001; Bi et al. 2007). Thus, the fully assembled coat is

composed of two distinct layers: the “inner” membraneproximal layer of Sar1/Sec23/Sec24 that intimately asso-ciates with lipid headgroups (Matsuoka et al. 2001) andcontributes cargo-binding function, and the “outer” mem-brane distal layer composed of Sec13/Sec31. Both layerscontribute to the catalytic cycle of Sar1 and endowingmaximal GTPase activity when the coat is fully assembled(Antonny et al. 2001).

Our mechanistic understanding of COPII coat action hasbeen significantly enhanced by the structural characteriza-tion of the different coat components. A structure of theSec23/Sec24 dimer showed a bow-tie shaped assembly witha concave face that is presumed to lie proximal to the mem-brane and is enriched in basic amino acids (Bi et al. 2002).These charged residues may facilitate association with theacidic phospholipid headgroups of the ER membrane. Sub-sequent structural, genetic, and biochemical analyses ofSec24 revealed multiple discrete sites of cargo interactiondispersed around the perimeter of the protein (Miller et al.2003; Mossessova et al. 2003). Structural analysis of theouter coat was facilitated by the observation that undersome conditions, the purified coat proteins can self-assembleinto “cages” of the approximate size of a COPII vesicle(Antonny et al. 2003). Further experiments using mamma-lian Sec13/Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPIIcage, which forms a lattice-like structure with geometry dis-tinct from that of the clathrin coat (Stagg et al. 2006). Het-erotetrameric Sec13/Sec31 complexes form straight rods,known as “edge” elements, four of which come together at“vertex” regions to drive cage assembly (Figure 3). Subse-quent crystal structures of Sec13 and a portion of Sec31revealed an unexpected domain arrangement within theedge element, whereby Sec31 forms both the dimerization

Figure 3 Coat assembly drivesvesicle formation. Both the COPII(left) and COPI (right) coats aredirected in their assembly bysmall GTPases of the Arf/Sar1family. In the COPII coat, Sar1is activated by its guanine nu-cleotide exchange factor (GEF),Sec12, which localizes to the ERmembrane. Activated Sar1–GTPrecruits the Sec23/Sec24 dimer,which corresponds to the “in-ner coat” layer and provides thecargo-binding function. A heter-otetramer of Sec13/Sec31 is sub-sequently recruited, forming the“outer coat” and polymerizinginto a lattice-like structure thatdrives membrane curvature. Inthe COPII cage formed by Sec13/Sec31, four molecules of Sec31

assemble head-to-head via b-propeller domains to form the “vertex” of the cage (inset). The COPI coat assembles upon activation of Arf1, which isdriven by either of the redundant GEFs, Gea1 or Gea2. Arf1 in turn recruits the inner coat complex of Sec21/Sec26/Ret2/Ret3, which has homologyto the clathrin AP-2 adaptor complex. The COPI outer coat is formed by Sec27/Ret1/Sec28, which assembles in a triskelion structure via interactionsof three b-propeller domains of Sec27 (inset).







































interface along the edge element and the vertex assemblyunit with Sec13 sandwiched between these structural ele-ments (Fath et al. 2007). However, the fragment of Sec31that fits well into the density of the cryo-EM structurerepresents only about half of the protein: an additionalproline-rich domain contains the GAP-stimulatory activityof Sec31. Again, the crystal structure of this region boundto Sar1/Sec23 has yielded great insight into the mecha-nism of GAP activity, whereby the active fragment of Sec31lies along the membrane-distal surface of Sec23/Sar1 andoptimizes the orientation of the catalytic histidine of Sar1(Bi et al. 2007).

The ability of Sec13/Sec31 to assemble into a sphericalstructure that matches closely the size of a COPII vesiclesuggests that the primary membrane bending force maycome from the scaffolding effect of this structure on theER membrane. Indeed, when the curvature-inducing amphi-pathic helix of Sar1 is replaced with an N-terminal histidinetag to drive recruitment to Ni-containing liposomes, subse-quent recruitment of Sec23/Sec24 and Sec13/Sec31 is suf-ficient to drive the generation of spherical buds that remainattached to the donor liposome (Lee et al. 2005). Thus anadditional function of the Sar1 helix is to drive vesicle scis-sion, a model supported by experiments that link GTPaseactivity to vesicle release in a manner analogous to thatproposed for dynamin (Pucadyil and Schmid 2009; Kunget al. 2012). Although the concave face of Sec23/Sec24may also contribute to membrane curvature, it has beensuggested that the relatively paltry dimer interface betweenthese two molecules is not robust enough to impart curva-ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006). Thus, although Sar1 andSec23/Sec24 may participate in membrane curvature, themajority of membrane bending force likely comes fromSec13/Sec31. Indeed, recent genetic and biochemicalexperiments support this model: Sec31 likely forms all thecontacts needed to make the COPII cage (Fath et al. 2007)with Sec13 providing structural rigidity to the cage edgeelement to overcome the membrane bending energy ofa cargo-rich membrane (Copic et al. 2012).

Cargo capture: stochastic sampling vs. directand indirect selection

The fundamental function of vesicles is to ensure directionaltraffic of protein cargoes, making cargo capture an in-tegral part of coat action. To some extent, cargo can enterinto vesicles in a nonspecific manner known as bulk flow,whereby stochastic sampling of the ER membrane andlumen occurs during vesicle formation, capturing localmolecules by chance. Although this mode of transport couldtraffic some abundant cargoes, the random nature of thisprocess cannot explain the efficiency with which some ERexport occurs. In particular, some cargoes are dramaticallyenriched in vesicles above their prevailing concentration inthe ER, suggesting a more efficient and selective packagingprocess. Although the concentrative mode of cargo selectionhas gained favor in the last decade, recent experimentsreevaluating the potential for bulk flow to explain forwardtraffic of some proteins warrants a more detailed analysis ofthe potential prevalence of this nonspecific pathway, espe-cially with respect to abundant, nonessential proteins wherethe efficiency of secretion may not be central to cellularviability (Thor et al. 2009).

Selective enrichment of cargo in transport vesicles viaspecific sorting signals is a common paradigm in intracellu-lar protein trafficking, first characterized in endocytosis.Deciphering a similar mode of transport for the entirespectrum of cargoes handled by the COPII coat, however,has been hindered by the absence of a single common signalused by the entire secretome. Instead, multiple signals seemto drive selective capture, meaning the COPII coat mustrecognize various signals employed by structurally diversecargoes. Such signals range from simple acidic peptides(Malkus et al. 2002) to folded epitopes (Mancias and Goldberg2007) and can act either by interacting directly with theCOPII coat or by binding to a cargo adaptor that links themto the coat indirectly (Figure 4) (Dancourt and Barlowe2010).

Genetic, biochemical, and structural data support Sec24as the cargo binding adaptor for the COPII coat, forming

Figure 4 Cargo selection can be direct or indirect. Selec-tive cargo capture during vesicle formation can occur viadirect interaction of cargo molecules with the COPI andCOPII coats. ER export signals (e.g., DxE, LxxLE, andYxxNPF) interact directly with Sec24 to facilitate captureinto COPII vesicles. Similarly, dilysine and diaromatic sig-nals mediate interaction with the COPI coat to direct ret-rograde traffic back to the ER. Soluble secretory proteinsmay be captured indirectly via specific cargo receptors thatserve to recognize the transport-competent cargo and linkit to the coat. Erv29 is the cargo receptor for many solublesecretory proteins. Soluble ER residents are returned backto the ER via a similar cargo receptor system, driven byErd2, which recognizes HDEL signals. Membrane proteinsmay also require cargo adaptor proteins such as Erv14 andRer1, although the basis for cargo recognition is not aswell defined.





























a relatively static platform that has multiple binding sites forinteraction with distinct sorting signals. The so-called A sitebinds the SNARE, Sed5, via a NPF motif (Mossessova et al.2003; Miller et al. 2005); the B site is most diverse, recog-nizing acidic sorting signals such as those found on theSNARE, Bet1, the Golgi membrane protein, Sys1, and un-known signals on additional cargoes (Miller et al. 2003;Mossessova et al. 2003); the C site binds a folded epitopeformed by the longin domain of the SNARE, Sec22 (Milleret al. 2003; Mancias and Goldberg 2007). The repertoire ofbinding sites is further expanded by the presence of addi-tional Sec24 isoforms, the nonessential Iss1 and Lst1 pro-teins (Roberg et al. 1999; Kurihara et al. 2000; Peng et al.2000). Sec24–cargo interactions are in general fairly lowaffinity (Mossessova et al. 2003), which is compatible withthe transient nature of the association of cargo with coat:proteins must bind during vesicle formation but must also bereleased prior to vesicle fusion to allow coat recycling andexposure of fusogenic domains. The possibility remains thatadditional layers of regulation impact coat dissociation fromcargo molecules after vesicle release: Sec23 is both ubiquiti-nated (Cohen et al. 2003) and phosphorylated (Lord et al.2011) and similar activity on Sec24 may promote uncou-pling of coat from cargo.

Some cargoes, by topology or preference, do not interactdirectly with Sec24 but instead use adaptor/receptor pro-teins to link them to the coat indirectly (Dancourt andBarlowe 2010). Some of these adaptors likely function ascanonical receptors, binding to their ligands in one compart-ment and simultaneously interacting with Sec24 to couplecargo with coat, then releasing their ligand in another com-partment, perhaps as the result of a change in ionic strengthor pH of the acceptor organelle (Figure 3). Although theirprecise mechanisms of ligand binding and release remain tobe fully explored, such receptors include Erv29, which medi-ates traffic of soluble secretory proteins like pro-a-factor andCPY (Belden and Barlowe 2001) and Emp46/Emp47, whichare homologous to the mammalian ERGIC-53 family of pro-teins that mediate traffic of coagulation factors (Sato andNakano 2002). Other receptors function to enrich vesicleswith membrane protein cargoes. The p24 proteins, Emp24,Erv25, Erp1, and Erp2, are required for efficient ER ex-port of GPI-anchored proteins, whose lumenal orientationprecludes direct coupling to the COPII coat (Belden andBarlowe 1996; Muniz et al. 2000; Belden 2001). Others, likeErv26 (Bue et al. 2006; Bue and Barlowe 2009) and Erv14(Powers and Barlowe 1998; Powers and Barlowe 2002;Herzig et al. 2012), mediate efficient export of transmem-brane proteins that have cytoplasmically oriented regionsbut either do not contain ER export signals or require addi-tional affinity or organization to achieve efficient capture.The requirement for receptors for such transmembrane car-goes remains unexplained, but may derive from the ancestralhistory of the cargoes whereby previously soluble proteinsbecame membrane anchored as a result of gene fusion events(Dancourt and Barlowe 2010). Alternatively, the receptor

proteins may provide additional functionality required forefficient ER egress, like a chaperoning function that wouldprotect the long transmembrane domains of plasma mem-brane proteins from the relatively thinner lipid bilayer char-acteristic of the ER (Sharpe et al. 2010). Indeed, some cargoproteins have specific chaperoning needs, with ER resi-dent proteins that are not themselves captured into COPIIvesicles likely functioning to promote assembly and foldingof polytopic membrane proteins. For example, the aminoacid permeases all depend on an ER resident, Shr3, for cor-rect folding and quaternary assembly, which is itself a pre-requisite for COPII capture (Ljungdahl et al. 1992; Kuehnet al. 1996; Gilstring et al. 1999; Kota et al. 2007).

Regulation of COPII function: GTPase modulation,coat modification

The GTPase activity of the coat is the primary mode ofregulation, known to govern initiation of coat assembly/disassembly through canonical GEF and GAP activities ofSec12 (d’Enfert et al. 1991) and Sec23 (Yoshihisa et al.1993) respectively, but also contributing to additional func-tions, like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al. 2005;Lee et al. 2005). Unlike other coat systems, the COPII coatuses a combinatorial GAP activity that is provided by com-ponents of the coat themselves, Sec23 (Yoshihisa et al.1993) and Sec31 (Antonny et al. 2001). The effect of thisautonomous GAP in minimal systems is that as soon as thecoat fully assembles, GTP is hydrolyzed and the coat is rap-idly released (Antonny et al. 2001), creating a paradox as tohow coat assembly might be sustained for a sufficient lengthof time to generate vesicles. One solution to this conundrumis that constant Sec12 GEF activity feeds new coat elementsinto a nascent bud (Futai et al. 2004; Sato and Nakano2005); coat release from the membrane might also bedelayed by the increased affinity afforded by cargo proteins(Sato and Nakano 2005). However, recent findings suggestthat a GAP inhibitory function, contributed by the peripheralER protein, Sec16, also modulates the activity of the coat(Kung et al. 2012; Yorimitsu and Sato 2012). Sec16 isa large, essential protein that associates with the cytoplas-mic face of the ER membrane at ERES (Espenshade et al.1995; Connerly et al. 2005). It interacts with all of the COPIIcoat proteins (Gimeno et al. 1996; Shaywitz et al. 1997) andis thus thought to scaffold and/or organize coat assembly atthese discrete domains (Supek et al. 2002; Shindiapina andBarlowe 2010). In addition to this recruitment function,a fragment of Sec16 dampens the GAP-stimulatory effectof Sec31, probably by preventing Sec31 recruitment toSar1/Sec23/Sec24 (Kung et al. 2012). The GAP-inhibitoryeffect of Sec16 was diminished in the context of a point muta-tion in Sec24 (Kung et al. 2012), raising the tantalizing possi-bility that cargo engagement by Sec24 could trigger interactionwith Sec16 to inhibit the full GTPase activity of the coat in sucha manner that a vesicle is initiated around a cargo-bound com-plex of Sar1/Sec23/Sec24/Sec16 (Springer et al. 1999).














































Another poorly explored aspect of COPII regulation ispost-translational modification of the coat. Sec23 is a targetfor ubiquitination and is seemingly rescued from degrada-tion by the action of the ubiqutin protease complex, Bre5/Ubp3 (Cohen et al. 2003). Whether this activity only con-trols expression levels of the protein or contributes moresubtly to regulate protein–protein interactions remains tobe tested. Furthermore, the potential ubiquitination of otherCOPII coat components also warrants investigation: recentexperiments in mammalian cells identified Sec31 as a targetfor a specific monoubiquitination event that is important forER export of collagen fibers (Jin et al. 2012). Whether yeastSec31 is similarly modified by the equivalent E3 ubiquitinligases, and how such a modification might influence coataction, perhaps by contributing to the structural integrityof the coat to drive membrane bending around rigid car-goes, remains to be tested. Like ubiquitination, the role ofcoat phosphorylation is only starting to be explored. It haslong been known that Sec31 is a phosphoprotein and thatdephosphorylation specifically impacted vesicle release(Salama et al. 1997). However, despite the many sites ofSec31 phosphorylation being revealed by high throughputphosphoproteomics, the precise function of these modifi-cations remains unclear. In contrast, progress has recentlybeen made in understanding phosphorylation of Sec23and how this event probably influences the directionalityof vesicle traffic by controlling sequential interactions withdifferent Sec23 partners (Lord et al. 2011). It is tempting tospeculate that similar phosphorylation of Sec24 might alsoregulate coat displacement from cargo molecules to furtherpromote coat release and expose the fusogenic SNARE pro-teins, that would otherwise be occluded by their interactionwith the coat. Indeed, at least partial uncoating of COPIIvesicles is required for fusion to ensue since when GTP hy-drolysis is prevented, vesicles fail to fuse (Barlowe et al.1994). Whether additional protein–protein interactions orpost-translational modifications contribute to coat sheddingremains to be seen.

Higher-order organization of vesicle formation

Although the minimal COPII coat can drive vesicle forma-tion from naked liposomes (Matsuoka et al. 1998b), thisprocess in vivo is likely tightly regulated to enable both ef-ficient vesicle production and adaptability to suit the secre-tory burden of the cell (Farhan et al. 2008). In part, thisregulation occurs at the level of the subdivision of the ERinto discrete ERES from which vesicles form. These smalldomains are marked by both the COPII coat proteins them-selves and accessory proteins such as Sec16, and, in somecells, Sec12 (Rossanese et al. 1999; Connerly et al. 2005;Watson et al. 2006). ERES are located throughout the ER,with a seemingly random distribution that may in fact cor-respond to regions of high local curvature induced by the ERmembrane proteins, Rtn1, Rtn2, and Yop1 (Okamoto et al.2012). In related yeasts, these sites are dynamic, with theability to form de novo, fuse, and divide (Bevis et al. 2002).

Although the precise mechanisms that regulate the steadystate distribution and size of these domains remain unclear,activity of both Sec12 and Sec16 seems to play a role(Connerly et al. 2005), as does the lipid composition ofthe ER (Shindiapina and Barlowe 2010). In mammaliancells, misfolded proteins that are incompetent for forwardtraffic are excluded from ERES (Mezzacasa and Helenius2002) and this also seems to be true for some proteinsin yeast, most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled, whichare not concentrated at ERES but instead remain dispersedwithin the bulk ER (Castillon et al. 2009).

Vesicle Delivery to the Golgi

After release of COPII vesicles from ER membranes, tetheringand fusion machineries guide ER-derived vesicles to Golgiacceptor membranes through the action of over a dozengene products (Figure 5). Although ER–Golgi transportcan be separated into biochemically distinct stages usingcell-free assays, evidence suggests that these events maybe organized in a manner that couples the budding andfusion stages. In general, budded vesicles become tetheredto Golgi membranes through the action of the Ypt1 GTPaseand tethering proteins Uso1 and the transport protein par-ticle I (TRAPPI) complex. Membrane fusion between vesicleand Golgi acceptor membranes is then catalyzed throughassembly of SNARE protein complexes from the apposedmembrane compartments. How the budding, tethering,and fusion events are coordinated in cells remains an openquestion, although genetic, biochemical, and structuralstudies have advanced our understanding of underlyingmolecular mechanisms in vesicle tethering and membranefusion described below.

Vesicle tethering

Initial cell free transport assays, coupled with genetic ap-proaches, placed ER–Golgi transport requirements intodistinct vesicle budding and vesicle consumption/fusionstages (Kaiser and Schekman 1990; Rexach and Schekman1991). Ypt1, identified as a founding member of the Rabfamily of GTPases, was implicated in the vesicle targetingstage in the ER–Golgi transport pathway (Schmitt et al.1988; Segev et al. 1988; Baker et al. 1990). In reconstitutedvesicle fusion reactions, Ypt1 was found to act in concertwith the extended coil-coiled domain protein Uso1 to tetherCOPII vesicles to Golgi acceptor membranes (Nakajima et al.1991; Barlowe 1997). In these assays, freely diffusible COPIIvesicles could be tethered to and sedimented with washedGolgi acceptor membranes upon addition of purified Uso1.Interestingly, the Uso1- and Ypt1-dependent tethering stagedoes not appear to require the downstream SNARE proteinfusion machinery (Sapperstein et al. 1996; Cao et al. 1998).

In addition to the extended structure of Uso1, which ispredicted to span a distance of .180 nm (Yamakawa et al.1996), the multisubunit TRAPPI complex is required for




























COPII-dependent transport to Golgi acceptor membranes(Rossi et al. 1995; Sacher et al. 1998). In vitro assaysrevealed that TRAPPI can also function to physically linkCOPII vesicles to Golgi membranes (Sacher et al. 2001).Structural analyses show that TRAPPI is a 170-kDa particleconsisting of six subunits (Bet3, Bet5, Trs20, Trs23, Trs31,and Trs33) that assemble into a flat bilobed arrangementwith dimensions of �18 nm · 6 nm · 5 nm (Kim et al.2006). Bet3 can bind directly to Sec23, and with TRAPPIperipherally bound to membranes, this activity is thoughtto link partially coated COPII vesicles to Golgi acceptormembranes (Cai et al. 2007). In a recent study, the Golgi-associated Hrr25 kinase was reported to phosphorylateSec23/Sec24 and regulate interactions between Sec23 andTRAPPI to control directionality of anterograde transport (Lordet al. 2011). Moreover, TRAPPI functions as a GEF for Ypt1in a manner that is thought to generate activated Ypt1 onthe surface of Golgi acceptor membranes and/or COPIIvesicles (Jones et al. 2000; Wang et al. 2000; Lord et al.2011). A subassembly of TRAPPI consisting of Bet3, Bet5,Trs23, and Trs31 binds Ypt1p and catalyzes nucleotide ex-change by stabilizing an open form of this GTPase (Cai et al.2008). TRAPPI does not appear to interact directly withUso1, although Ypt1 activation could serve to coordinatethe long-distance tethering mediated by Uso1 with a closerTRAPPI-dependent tethering event. The precise orientationof TRAPPI on Golgi and vesicle membranes is not known,but current models suggest that this multisubunit complexlinks COPII vesicles to the cis-Golgi surface and serves as acentral hub in coordinating vesicle tethering with SNARE-mediated membrane fusion.

Genetic and biochemical evidence indicate that othercoiled-coil domain proteins also act in COPII vesicle tether-ing and/or organization of the early Golgi compartment inyeast. The GRASP65 homolog Grh1 is anchored to cis-Golgi

membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termedBug1 (Behnia et al. 2007). Grh1 and Bug1 are not essential,but deletion of either protein reduces COPII vesicle tether-ing and transport levels in cell-free assays and the grh1Dand bug1D mutants display negative genetic interactionswith thermosensitive ypt1 and uso1 mutants (Behnia et al.2007). These findings suggest a redundant network ofcoiled-coil proteins that act in tethering vesicles and orga-nizing the cis-Golgi compartment. Indeed, additional coiled-coil proteins including Rud3 and Coy1 localize to cis-Golgimembranes and are implicated in organization of the cis-Golgi and interface with COPII vesicles (VanRheenen et al.1999; Gillingham et al. 2002, 2004). Although some doubledeletion analyses have been performed with these genes,multiple deletions may be required to severely impact thisredundant network.

SNARE protein-dependent membrane fusion

Fusion of tethered COPII vesicles with cis-Golgi membranesdepends on a set of membrane-bound SNARE proteins. Sev-eral lines of evidence indicate that the SNARE proteinsSed5, Bos1, Bet1, and Sec22 catalyze this membrane fusionevent in yeast (Newman et al. 1990; Hardwick and Pelham1992; Sogaard et al. 1994; Cao and Barlowe 2000). TheSNARE protein family is defined by a conserved �70-amino-acid heptad repeat sequence, termed the SNARE mo-tif, which is typically adjacent to a C-terminal tail-anchoredmembrane segment (Rothman 1994; Fasshauer et al. 1998).Cognate sets of SNARE proteins form stable complexesthrough assembly of their SNARE motifs into parallel four-helix coiled-coil structures (Hanson et al. 1997; Sutton et al.1998). The close apposition of membranes that follows as-sembly of SNARE complexes in trans is thought to drivemembrane bilayer fusion (Weber et al. 1998). Structural

Figure 5 Vesicle tethering and fu-sion. Anterograde delivery of COPII-coated vesicles is mediated by avariety of tethering and fusion com-plexes. The TRAPP complex binds toSec23 on the surface of a COPII ves-icle and mediates local activation ofthe Rab family member, Ypt1. Ypt–GTP recruits downstream effectorssuch as the long coiled-coil tether,Uso1. AGolgi-localized kinase, Hrr25,phosphorylates Sec23 and displa-ces TRAPP, perhaps contributing tocoat shedding. Removal of the coatexposes the fusogenic SNARE pro-teins, which assemble to drivemembranemixing. In the retrogradepathway, COPI-coated vesicles em-ploy the DSL1 complex, composedof Dsl1/Sec39/Tip20, to recognizethe incoming vesicle and coordinatecoat release and SNARE pairing.






































studies of the four-helix bundle reveal that the central or“zero layer” consists of ionic residues such that three of theSNARE proteins contribute a glutamine residue, and arethus termed Q-SNARES, whereas the fourth helix containsan arginine residue, and is known as the R-SNARE (Fasshaueret al. 1998; Sutton et al. 1998). Further refinement of theQ-SNARE proteins based on sequence conservation iden-tifies each as a member of the Qa, Qb, or Qc subfamily(Kloepper et al. 2007). SNARE-dependent membrane fusionis though to proceed through a conserved mechanism inwhich three Q-SNARES (Qa, Qb, and Qc) and one R-SNAREzipper together from the N-terminal side of the SNARE motiftoward the membrane (Sudhof and Rothman 2009). Inthe case of COPII vesicle fusion with Golgi membranes,Sed5 serves as the Qa-SNARE, Bos1 the Qb-SNARE, Bet1the Qc-SNARE, and Sec22 the R-SNARE. Furthermore, thisSNARE set is sufficient to catalyze membrane fusion whenreconstituted into synthetic proteoliposomes (Parlati et al.2000).

In addition to Sed5, Bos1, Bet1, and Sec22, other regu-latory factors are required to control fusion specificity andgovern SNARE complex assembly/disassembly. Members ofthe Sec1/Munc18-1 (SM) family of SNARE-binding proteinsregulate distinct SNARE-dependent fusion events (Sudhofand Rothman 2009). The SM family member Sly1 is re-quired for fusion of COPII vesicles with Golgi membranein yeast (Ossig et al. 1991; Cao et al. 1998). SLY1 was ini-tially identified as a suppressor of loss of YPT1 functionwhen the gain-of-function SLY1-20 allele was isolated ina selection for mutations that permit growth in the absenceof YPT1 (Dascher et al. 1991). Sly1 binds directly to Sed5and increases the fidelity of SNARE complex assembly be-tween Sed5, Bos1, Bet1, and Sec22 compared to noncognateSNARE complexes (Peng and Gallwitz 2002). Crystallo-graphic studies of Sly1 reveal a three-domain arch-shapedarchitecture that binds a 45-amino-acid N-terminal domainof Sed5 as observed for other SM protein interactions withQa-SNAREs (Bracher and Weissenhorn 2002). Workingmodels for Sly1 and SM protein function in general arebased on multiple binding modes wherein Sly1 initiallybound to the N terminus of Sed5 would subsequently bindto other cognate SNARE proteins to regulate assembly andultimately to act as a clamp in stabilizing a trans-SNAREcomplex (Furgason et al. 2009; Sudhof and Rothman 2009).

After SNARE-mediated membrane fusion is complete,stable four-helix bundles of cis-SNARE complexes are nowpresent on the acceptor membrane compartment. To recycleassembled Sed5–Bos1–Bet1–Sec22 complexes for use in ad-ditional rounds of membrane fusion, the general fusion fac-tors Sec17 and Sec18 catalyze SNARE complex disassembly(Sogaard et al. 1994; Bonifacino and Glick 2004). Sec18belongs to the AAA family of ATPase chaperones and usesthe energy of ATP hydrolysis to separate stable cis-SNAREcomplexes. Sec17 is thought to recruit Sec18 to SNARE pro-tein complexes and couples ATPase dependent disassemblyof cis-SNARE complexes (Bonifacino and Glick 2004). How

Sec17/Sec18-mediated disassembly is coordinated withcoat-dependent capture of SNARE proteins into vesiclesand Sly1-dependent assembly of trans-SNARE complexesduring fusion remain open questions.

A concerted model for COPII vesicle tethering and fusion

Although distinct stages in vesicle tethering and fusion canbe defined through biochemical and genetic analyses, theseare likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5). The multi-subunit TRAPPI may serve as an organizational hub on cis-Golgi membranes or vesicles to coordinate vesicle tetheringand fusion events. TRAPPI interactions with the COPIIsubunit Sec23, with the Ypt1 GTPase and potentially withSNARE proteins (Jang et al. 2002; Kim et al. 2006) couldlink tethering and fusion stages. TRAPPI-activated Ypt1could recruit Uso1 to Golgi membranes and as COPIIvesicles emerge from the ER, Uso1 could forge a long-distance link between newly formed vesicles and acceptormembranes. With tethered vesicles aligned to fusion sites,TRAPPI interactions with vesicle-associated Sec23 and GolgiSNARE machinery would then position vesicles in closerproximity to acceptor membranes. TRAPPI-bound vesiclescould transmit signals to the SNARE machinery by directcontact or perhaps through generation of elevated levels ofactivated Ypt1. The result of such a signal may be to disas-semble cis-SNARE complexes or to generate a Sly1–Sed5conformation that promotes assembly of fusogeneic SNAREcomplexes. Assembly of trans-SNARE complexes would thenpresumably lead to rapid hemifusion followed by bilayerfusion and compartment mixing.

Traffic Within the Golgi

Transport through the Golgi complex

Newly synthesized secretory proteins arrive at the cis-Golgiin COPII vesicles and after membrane fusion progressthrough the Golgi complex. Secretory cargo may receiveouter-chain carbohydrate modifications and proteolytic pro-cessing in a sequential manner as cargo advances throughdistinct Golgi compartments. For glycoproteins, the N-linkedcore carbohydrate is extended by addition of a-1,6-mannoseresidues in the cis-Golgi and by addition of a-1,2- anda-1,3-mannose residues in the medial compartment. Kex2-dependent proteolytic processing of certain secretory cargooccurs in the trans-Golgi compartment. Each of these eventscan be resolved by blocking membrane fusion through in-activation of the thermosensitive sec18-1 allele (Graham andEmr 1991; Brigance et al. 2000). In support of this sequen-tial organization, distinct Golgi compartments can be visu-alized through fluorescence microscopy or immuno-EMby monitoring components of the glycosylation and pro-cessing machinery (Franzusoff et al. 1991; Preuss et al. 1992;Wooding and Pelham 1998; Rossanese et al. 1999). However,genetic and morphological approaches have not uncovered


















































a vesicle-mediated anterograde transport pathway throughdistinct compartments of the yeast Golgi complex. Instead,a model of cisternal maturation, in which Golgi cisternae arethe anterograde carriers of secretory cargo, is most consis-tent with a range of experimental observations (Bonifacinoand Glick 2004). In the cisternal maturation model, Golgicisterna containing nascent secretory cargo are formed atthe cis-face of the Golgi and mature into a medial and thentrans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrogradevesicles to preceding cisternae. Indeed, the dispersed orga-nization of Golgi compartments in S. cerevisiae are resolv-able by fluorescence microscopy and provided a powerfultest of the maturation model through live cell imaging ofcis- and trans-Golgi proteins labeled with different fluores-cent tags. In such a dual labeled strain, a cis-compartmentshould be observed to change color to a trans-compartmentover the time period required for secretory cargo to transitthe Golgi complex. Strikingly, two independent researchgroups using time resolved high resolution microscopy docu-mented individual cisterna transitioning from early to latecompartments in accord with the cisternal maturationmodel (Losev et al. 2006; Matsuura-Tokita et al. 2006).

In addition to retrograde transport from cis-Golgi to ER(discussed below), the COPI coat is thought to mediate ret-rograde transport within the Golgi complex to retrieve recy-cling Golgi machinery to earlier compartments as Golgicisternae mature (Bonifacino and Glick 2004). In currentworking models, anterograde-directed COPI vesicles are tar-geted to preceding Golgi compartments by the conservedoligomeric Golgi (COG) complex, a large multisubunit teth-ering complex identified through a combination of geneticand biochemical approaches (Miller and Ungar 2012). COGconsists of eight subunits and belongs to the larger CATCHR(complex associated with tethering containing helical rods)family of tethering factors that includes the exocyst andGARP complexes (Yu and Hughson 2010). In intra-Golgiretrograde transport, the COG complex appears to operateas a tethering and fusion hub with multiple interactions thatlink COG to the g-COPI subunit, to Ypt1 and to Golgi SNAREproteins (Suvorova et al. 2002). More specifically, fusionof retrograde-directed COPI vesicles with cis-Golgi mem-branes is thought to depend on COG complex interactionswith a distinct SNARE complex consisting of Sed5 (Qa),Gos1 (Qb), Sft1 (Qc), and Ykt6 or Sec22 as the R-SNARE(Shestakova et al. 2007). Mutations in COG complex subu-nits disrupt Golgi transport and glycosylation of secretorycargo, fully consistent with this model. However, at thisstage there are no cell-free assays to measure COG-dependentfusion of COPI vesicles to fully dissect underlying molecularmechanisms (Miller and Ungar 2012).

Lipid requirements for Golgi transport

While the protein machinery underlying Golgi transport hasreceived much attention, the role of specific lipid biosyn-thetic and transfer pathways in Golgi trafficking remain

relatively understudied. One of the first connections fora lipid requirement in transport through the Golgi complexwas the identification and characterization of Sec14 as anessential phosphatidylinositol/phosphatidylcholine (PI/PC)transfer protein in yeast (Novick et al. 1981; Bankaitiset al. 1989; Cleves et al. 1991). The trafficking blocks asso-ciated with Sec14 deficiencies lead to an accumulation ofGolgi membranes and Golgi forms of secretory cargo. Sec14probably does not play a major role in transporting bulkphospholipids but rather is thought to function in regulatingphospholipid homeostasis through presentation of PIs tomodifying activities such as the PI4 kinases (Schaaf et al.2008). Interestingly, PI4P levels in the Golgi complex alsoplay a critical role in Golgi structure and function as dem-onstrated by mutations in the essential PI4 kinase Pik1,which block transport through the Golgi (Walch-Solimenaand Novick 1999; Audhya et al. 2000). More recently, a di-rect requirement for PI4P levels on Golgi organization hasbeen documented through characterization of the Golgi-localized PI4P binding protein encoded by VPS74 (Schmitzet al. 2008; Tu et al. 2008). Loss of Vps74 function resultsin mislocalization of Golgi mannosyltransferases from earlyGolgi compartments to the vacuole. Vps74 appears to bindto cytoplasmic sorting signals contained on Golgi residentenzymes and to the COPI coat in addition to PI4P in sortingGolgi-localized proteins into retrograde-directed vesicles. Inthis manner, PI4P levels and Vps74 may function togetherin dynamic recycling of Golgi modification enzymes as cis-terna containing nascent secretory cargo mature in accordwith Golgi maturation models. Indeed, the polarized dis-tribution of PI4P across the Golgi with increasing concen-trations from cis- to trans-compartments appears to playseveral important roles in organization and transport throughthe Golgi complex (Graham and Burd 2011).

The Return Journey: Retrograde Traffic viaCOPI Vesicles

Although it remains to this day somewhat controversial as tothe precise function (and thus direction) of COPI-mediatedvesicular traffic within the Golgi (Emr et al. 2009), the roleof these vesicles in retrograde Golgi–ER transport is wellestablished. This is despite the original confusion in the fieldas to the directionality of COPI-mediated traffic: yeast COPImutants generally have anterograde trafficking defects thatprobably stem from indirect effects of blocking retrogradetransport rather than impacting forward traffic directly(Gaynor and Emr 1997). Although one COPI component,Sec21, was identified in the original sec mutant screen(Novick et al. 1980), advances in understanding this step ofthe secretory pathway largely lagged behind and was informedby the biochemical advances made in mammalian systems(Serafini et al. 1991). Once Sec21 was cloned and realizedto be an ortholog of the mammalian coatomer complex(Hosobuchi et al. 1992), biochemical analyses allowed theidentification of all equivalent yeast subunits, which were


















in turn also subsequently identified in a variety of geneticscreens as additional sec/ret/cop mutants (Duden et al.1994; Cosson et al. 1996). The major advances in dissectingthe mechanisms of retrograde traffic have continued to beled by biochemical approaches (Spang et al. 1998; Spangand Schekman 1998), with many recent high resolutionstructures of the relevant coat (Lee and Goldberg 2010;Faini et al. 2012; Yu et al. 2012) and tether proteins (Renet al. 2009; Tripathi et al. 2009). Given the strong homologybetween the mammalian and yeast proteins, it seems likelythat the global structure of the yeast COPI coat is broadlysimilar to that of mammals (Yip and Walz 2011). Indeed,current approaches make good use of yeast genetics ap-proaches to test functional relevance of the structural data,yielding insight into areas including cargo selection (Michelsenet al. 2007), directionality of vesicle delivery (Kamena andSpang 2004), and coat/tether influences on vesicle fusion(Zink et al. 2009).

Composition and structure of the COPI coat

Originally characterized from mammalian cells as a singlecoat protomer, or coatomer (Waters et al. 1991), the COPIcoat is composed of seven subunits: a-, b-, b9-, g-, d-, e-, andz-COP that correspond to the yeast proteins Cop1/Sec33/Ret1, Sec26, Sec27, Sec21, Ret2, Sec28, and Ret3, respec-tively. Although found as a large cytosolic complex, it is nowappreciated that, like the COPII coat, COPI comprises twoseparable layers: an inner layer that functions in cargo bind-ing composed of g-, d-, z-, and b-COP and an outer layerformed by a-, b9-, and e-COP (Figure 3). Furthermore, sig-nificant sequence homology was apparent between the innerCOPI coat and the adaptor subunits of the clathrin coatsystem. Indeed, a recent structural analysis of the g/z sub-complex of the inner COPI coat shows clear homology withthe a/s subunits of the AP2 clathrin adaptor, with Arf1bound at a site that corresponds spatially to the PI(4,5)P2binding site on AP2 (Yu et al. 2012). Although the structureof the b/d subcomplex remains to be determined, homologymodeling suggests that it adopts a conformation very similarto the b2–AP2 subunit, and biochemical analyses suggestthat a second Arf1 molecule can bind to the PI(4,5)P2 bind-ing site on b2–AP2 (Yu et al. 2012). Unlike the inner coat,which is most similar to the clathrin coat adaptors, the outerCOPI coat shows homology with both clathrin and COPIIcoats, with b-propeller and a-solenoid domains formingthe building blocks of the putative cage. Structural analysisof stable fragments of the a-/b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat isintermediate between that of the COPII and clathrin coats:the individual b-barrel and a-solenoid structures mostclosely resemble the Sec13/Sec31 structure of the COPIIcage but they assemble in a clathrin-like triskelion (Leeand Goldberg 2010). It remains unclear exactly how theinner and outer layers come together, either in solutionprior to assembly on the membrane or during vesicle forma-tion, although purified yeast coatomer examined by single

particle electron microscopy suggests a somewhat flexibleconfiguration that would need to stabilize during poly-merization or oligomerization on the surface of the mem-brane (Yip and Walz 2011). This concept of structuralflexibility for the COPI coat is supported by recent EM anal-ysis of COPI vesicles budded from synthetic liposomes,which showed striking structural diversity of coat arrange-ment on the surface of the budded vesicles (Faini et al.2012). Although all the crystallographic, and much of thebiochemical analysis of the COPI coat has employed mam-malian proteins, the yeast orthologs are highly likely toadopt similar conformations. Indeed, the known structuresare consistent with the nonessential nature of Sec28; itsortholog, e-COP, is a helical structure that interacts witha-COPI but likely does not form part of the cage (Hsia andHoelz 2010; Lee and Goldberg 2010), probably renderingit dispensable in vivo despite some destabilization of Cop1(a-COP) in the sec28 mutant (Duden et al. 1998).

Like the COPII coat, COPI assembly on the membrane isinitiated by a small GTPase, Arf1, which in addition to the N-terminal amphipathic a-helix also contains a myristoylgroup that facilitates membrane anchorage (Antonny et al.1997a). GDP–GTP exchange on Arf1 and its paralogs makesuse of a common structural motif, the Sec7 domain, namedfor the late Golgi GEF that is the target of the fungal me-tabolite, Brefeldin A (Sata et al. 1998, 1999). In Golgi–ERretrograde traffic, two redundant GEFs, Gea1 and Gea2,each with a Sec7 domain, likely initiate coat assembly bytriggering local recruitment of Arf1 (Peyroche et al. 1996;Spang et al. 2001). Unlike the COPII system, the GAP activ-ity for the COPI coat is not an integral part of the coat itself,but is instead contributed by a separate protein, known (notsurprisingly) as ArfGAP1 in mammalian cells. In yeast Arf–GAP activity derives from two distinct proteins, Gcs1 andGlo3, with partially overlapping roles (Poon et al. 1996,1999). Mammalian ArfGAP1 employs a lipid-packing sensordomain to regulate its activity according to membrane cur-vature, becoming active on highly curved membranes, likelyafter vesicle formation has completed or at least progressedenough as to permit Arf release without destabilizing thecoat (Bigay et al. 2003, 2005). Yeast Gcs1 also showeda binding preference for conical lipids, suggesting a similarmechanism could regulate GTPase activity of the yeast COPIcoat (Antonny et al. 1997b). However, curvature-responsiveactivity may not be the only mode of regulation of the COPIGTPase cycle. Coatomer itself also seems to influence Arf-GAP activity (Goldberg 1999), although the mechanismremains to be fully defined (Luo and Randazzo 2008). Fur-thermore, the ability of some sorting signals on cargo pro-teins to inhibit the coatomer-stimulated GAP activity directlylinks coat recruitment to cargo selection (Springer et al.1999; Goldberg 2000), an appealing model whereby thecoat stably associates with the membrane only when boundto cargo proteins (Springer et al. 1999). Further complicat-ing the problem, is evidence that implicate ArfGAP proteinsas positive regulators of the COPI coat rather than negative




























regulators: overexpression of any of the four yeast ArfGAPssuppressed the lethality of an arf1 mutant (Zhang et al.1998, 2003). Further yeast experiments also support anactive role for Gcs1 and Glo3 in cargo selection, actingon SNARE proteins prior to incorporation into vesicles topromote Arf1 and coatomer interaction (Rein et al. 2002;Schindler and Spang 2007; Schindler et al. 2009). Clearly,the precise role of the GAP in the COPI system remainsto be fully understood, complicated by conflicting resultsfrom different labs and/or systems and may in fact be mul-tifaceted by serving both positive and negative roles at dif-ferent stages during the vesicle formation process (Spanget al. 2010).

Cargo capture: sorting signals, cargo adaptors,and coat stimulators

Like other vesicle trafficking events, retrieval of ER residentproteins via COPI vesicles employs sorting signals, mostnotably the canonical retrieval motifs, HDEL for solublelumenal cargoes and K(X)KXX for membrane proteins(Figure 4). Soluble proteins bind to a retrieval receptor,Erd2 (Semenza et al. 1990), which couples them to the COPIcoat to facilitate retrograde traffic. The COPI coat can dis-criminate between similar but distinct motifs, including thecanonical K(X)KXX, which must be located at the C terminusof the cargo and membrane-proximal to ensure efficientretrieval, R-based motifs that only function when spacedsome distance from the membrane surface, and other basicmotifs that remain to be fully dissected (Cosson et al.1998; Shikano and Li 2003). Yeast two-hybrid experi-ments and subsequent mutagenesis analyses suggest thatthe R-based motif binds at the interface between the b- andd-COP subunits (Sec26 and Ret2, respectively), in a mannerthat is distinct from KKXX binding to the coat (Michelsenet al. 2007). The site of KKXX recognition remains some-what unclear. Multiple lines of evidence support a role forthe a-/b9-/e-COP complex in KKXX binding (Cosson andLetourneur 1994; Letourneur et al. 1994; Fiedler et al. 1996),whereas direct cross-linking studies implicate the g-COPsubunit in KKXX binding (Harter et al. 1996; Harter andWieland 1998).

In addition to retrieval motifs based on basic residues,diaromatic retrieval signals have also been identified, per-haps best characterized for the p24 family of proteins, albeitlargely using the mammalian family members (Stratingand Martens 2009). This class of signal likely binds tothe inner COPI coat via the g-COP subunit, causing a con-formational change that may open up the cargo adaptorplatform to become receptive to additional cargo clients(Béthune et al. 2006; Strating and Martens 2009). Yet an-other mode of cargo binding is represented by the SNAREproteins that drive membrane fusion. Unlike SNARE inter-action with the COPII coat, direct binding of SNARE sortingsignals with COPI components has not been observed. In-stead, SNARE incorporation into COPI vesicles dependson the activity of the Arf–GAP, Glo3, although the precise

function of Glo3 in promoting a SNARE configuration thatis favorable for vesicle capture remains to be fully dissected(Rein et al. 2002).

As with the COPII coat, capture of cargo proteins intoretrograde COPI vesicles sometimes requires the action ofcargo adaptors. The first of these described was the HDELreceptor, Erd2, described above, where the lumenal domainlikely provides ligand-binding function (Scheel and Pelham1998), with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al.1993). Another well-described cargo adaptor is the mem-brane protein Rer1 (Nishikawa and Nakano 1993; Satoet al. 1995), which is important for the efficient retrieval,and thus steady-state ER localization, of some ER residentproteins, including the COPII GEF, Sec12, and the translo-con components, Sec63 and Sec71 (Sato et al. 1997). Thereason these proteins would require an escort back to the ERrather than employing their own retrieval motifs is unclear,but Rer1 seems to bind these clients within their transmem-brane domains, via polar residues embedded within the hy-drophobic environment (Sato et al. 1996, 2001). Sec12 andSec71 appear to use different sites on Rer1 to facilitate ret-rograde traffic, since mutation of the Sec12-binding site hadno effect on Sec71 retrieval, suggesting that Rer1 formsa multivalent cargo receptor that has the capacity to bindmultiple cargo clients simultaneously (Sato et al. 2003).

Yet another important player in COPI vesicle formationis the class of proteins that seem to serve as coat nucleators,increasing or stabilizing the recruitment of the COPI coaton the Golgi to stimulate retrograde traffic. Although themechanistic details remain to be fully understood, twoclasses of protein seem to stimulate retrograde traffic bymodulating the ability of the COPI coat to form vesicles. Thefirst description of this function was for a membrane protein,Mst27, which suppresses the lethality of a sec21-1 mutantwhen overexpressed (Sandmann et al. 2003). Mst27 and itsrelated binding partner, Mst28, both bind to yeast coatomervia KKXX motifs and this function is required for the sec21-1suppression. Although the endogenous function of Mst27/Mst28 is unclear, the ability of these cargo proteins to stim-ulate vesicle production was one of the first concrete piecesof evidence that cargo abundance can directly influencevesicle formation. More recently, a similar role has beenpostulated for the abundant class of p24 proteins; geneticinteractions between EMP24 and various COPI components,including SEC21 and the Arf–GAP, GLO3, are suggestiveof a functional relationship, and membranes isolated fromemp24D cells are diminished in their ability to form COPIvesicles in vitro (Aguilera-Romero et al. 2008). Since someof the mammalian p24 proteins showed a capacity to mod-ulate the GTPase activity of the COPI coat (Goldberg 2000),it is tempting to link these observations: by slowing theGTPase activity of Arf1, the COPI coat might be stabilizedon the membrane, prolonging the cargo-engagement stepand perhaps stimulating coat oligomerization to enhancevesicle production.



































Vesicle delivery: DSL-mediated tethering and SNARE-mediated fusion

Like other vesicle trafficking steps, the final stages ofdelivery of COPI vesicles employ a long-distance tether tobring the vesicle into proximity of the acceptor membraneand SNARE proteins to drive membrane fusion (Spang2012). The ER-localized tethering complex, the Dsl1 com-plex, performs the tethering function, recognizing COPIvesicles via their intact coat, and also participates in thefusion event by proofreading the SNARE pairing that occursprior to fusion (Figure 5). Originally identified as a mutantthat was dependent on the presence of the dominant sly1-20allele, dsl1 mutants showed accumulation of vesicles atrestrictive temperature and were suppressed by overex-pression of SEC21, although they also showed ER–Golgitransport defects, making a precise function difficult to dis-cern (VanRheenen et al. 2001). Dsl1 forms a complex withDsl3/Sec39 and Tip20 to form the Dsl1 complex, anothermember of the CATCHR family of tethering complexes notedfor their extended helical rod structures (Lees et al. 2010).Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgito the ER: tip20 and dsl1 mutants showed genetic interac-tions with a variety of ER–Golgi SNAREs (Sweet and Pelham1993; Andag et al. 2001; Kraynack et al. 2005), tip20mutantsshowed defects in fusion of COPI vesicles (Kamena and Spang2004), the Dsl1 complex was localized to the ER (Kraynacket al. 2005), and Dsl1 interacts directly with multiple compo-nents of the COPI coat (Andag and Schmitt 2003).

Recent structural analyses have generated an appealingmechanistic model by which the extended Dsl1 complexperforms three functions by virtue of its ability to interactwith both the COPI coat and the fusogenic SNAREs (Renet al. 2009; Tripathi et al. 2009; Zink et al. 2009). A com-posite crystal structure suggests that a long stalk, formedlargely by Sec39, extends away from the ER membrane,with Dsl1 located at the membrane-distal end to “catch”incoming COPI vesicles via an unstructured loop that wouldinteract directly with the coat via an a-helical structureformed by a- and e-COPI (Ren et al. 2009; Hsia and Hoelz2010). Sec39 itself binds to the N-terminal domain of the ERresident SNARE, Use1, via a region that likely lies proximalto the membrane (Tripathi et al. 2009), and Tip20 containsa second SNARE-binding site, interacting with the N-terminaldomain of Sec20 (Ren et al. 2009). In addition to bind-ing individual SNAREs, the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion:maintaining individual SNAREs in an unpaired, receptivestate, and scaffolding assembly of the fusogenic SNAREcomplex to promote fusion (Kraynack et al. 2005; Renet al. 2009). An additional role in vesicle uncoating is sug-gested by the tendency of vesicles to accumulate en masseunder conditions of Dsl1 depletion (Zink et al. 2009): COPIshedding might be assisted by a Dsl1–COPI interaction thatwould prevent repolymerization of disassembled coat sub-

units, or could be driven by conformational changes in theDsl1 complex that would capitalize on the ability of Dsl1 tointeract with both the outer a-/e-COPI domain and a secondsite on the inner d-COP subunit to prize the coat from themembrane (Ren et al. 2009; Zink et al. 2009). Indeed, neg-ative stain EM images of the Dsl1 complex suggest a varietyof possible configurations, although the mechanistic impactof the different conformations with respect to coat andSNARE binding remain to be tested (Ren et al. 2009).Clearly, the Dsl1 complex is a multifunctional tether thatmay serve as a useful paradigm for other vesicle “tethering”systems that may contribute to multiple layers of vesicleuncoating, docking, and fusion in addition to their canonicallong-distance vesicle trapping function.

Perspectives

Having moved from the “parts list” generated by numerousgenetic screens to molecular mechanisms defined by in vitroassays, where is the field currently heading? Emerging ques-tions currently center on how the varied processes that driveprotein secretion are coordinated and regulated, both at themolecular level and at the higher-order organizational level.The biosynthesis of secretory proteins can be thought of asa series of simple events (translation/translocation, post-translational modification, chaperone binding, forwardtransport) but are these events more closely entwined thanwe currently appreciate? How are protein quality controldecisions made: are they a simple outcome of a tug of warbetween the ER-associated degradation machinery and theforward transport machinery? Adding a dominant ER exportsignal to a misfolded protein could drive forward traffic(Kincaid and Cooper 2007), but the converse experimentof blocking ERAD of a different misfolded substrate didnot lead to its secretion (Pagant et al. 2007). Understandingthe interplay between the folding, degradation, and exportmachineries will be key in appreciating the intricate regula-tion of secretory protein production and how the differentmachineries might be coregulated to cope with the changingsecretory burden of the cell under different environmentalconditions.

Additional questions stem from our relatively poor un-derstanding of how the early secretory pathway is organizedand how this organization is maintained. Although it is clearthat ER exit sites form discrete subdomains of the ER(Rossanese et al. 1999; Shindiapina and Barlowe 2010),what is the functional significance of this organization? Isthe segregation of cargo molecules into different ER exitsites (Muniz et al. 2001) driven by active processes, or doesit reflect the passive influence of specific lipid and proteinrequirements for subsets of cargo molecules? Similarly, do allsecretory cargo proteins follow the same route through theGolgi, or are specific itineraries devised for distinct cargoesthat might also be driven by specific lipid microenvironmentsand/or post-translational modification needs? Larger-scalequestions also remain: How is the cis-Golgi founded, through






























homotypic fusion of COPII vesicles, by heterotypic fusion ofCOPII and COPI vesicles, or by templating from an existingcis-Golgi fragment that expands through delivery of COPIIand COPI vesicles? Electron tomography of yeast cells showdistinct transport vesicles and Golgi cisternae but no apparentintermediates (West et al. 2011). How are vesicles targeted tothe correct destination: Is there a role for the cytoskeleton invesicle delivery, and how do COPI vesicles that bud from theGolgi find the proper acceptor compartment? Indeed, arethere multiple types of COPI vesicles that drive differenttransport events between different Golgi cisternae and dotubular elements play a role in lipid and protein traffic asthey appear to do in mammalian cells? Finally, how are theprotein and lipid needs of the cell sensed and maintained toensure efficient protein secretion, which lies at the heart ofcell growth to permit cell division, and how are the rates ofanterograde and retrograde traffic balanced to maintain thecorrect morphology and distribution of the various secretoryorganelles? As in the past, the facile genetics and accessiblebiochemistry of the yeast system still hold promise in answer-ing these questions, with the development of new tools serv-ing to strengthen the field and provide new avenues forfurther exploration.

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Communicating editor: T. Davis


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