copi budding within the golgi stack - cshl...

COPI Budding within the Golgi Stack

Vincent Popoff, Frank Adolf, Britta Brugger, and Felix Wieland

Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany

Correspondence: [email protected]

The Golgi serves as a hub for intracellular membrane traffic in the eukaryotic cell. Transportwithin the early secretory pathway, that is within the Golgi and from the Golgi to theendoplasmic reticulum, is mediated by COPI-coated vesicles. The COPI coat shares struc-tural features with the clathrin coat, but differs in the mechanisms of cargo sorting andvesicle formation. The small GTPase Arf1 initiates coating on activation and recruits enbloc the stable heptameric protein complex coatomer that resembles the inner and theouter shells of clathrin-coated vesicles. Different binding sites exist in coatomer for mem-brane machinery and for the sorting of various classes of cargo proteins. During thebudding of a COPI vesicle, lipids are sorted to give a liquid-disordered phase composition.For the release of a COPI-coated vesicle, coatomer and Arf cooperate to mediate membraneseparation.

Eukaryotic cells are organized as a collectionof spatially separated internal organelles

embedded in the cytoplasm. Communicationbetween these internal compartments is medi-ated by trafficking events, some of which areaccomplished by vesicular transport. Lipidsand proteins are sorted at the membrane of adonor compartment and included into spheri-cal transport carriers with a typical diameterof 50–100 nm. After detachment, these carri-ers travel through the cytoplasm, and fusewith their target compartment to deliver theircontent. Trafficking can serve the targeted deliv-ery of newly synthesized proteins and lipids, theuptake of extracellular cargo, and regulatoryprocesses. The Golgi represents a cellular traf-ficking hub strategically positioned betweenthe endoplasmic reticulum (ER), the site of

synthesis of secretory and membrane proteins,and endocytic compartments. Its characteristicstack of cisternae combines the early secretorypathway receiving cargo from the ER via theintermediate compartment (IC, tubulo-vesi-cular structures between ER and Golgi) with theendosomal membrane system and the plasmamembrane. The process of vesicle budding ismainly controlled by vesicular coat proteins,protein complexes able to simultaneously selectcargo to be included in a transport carrier andto deform a membrane. Table 1 summarizesthe various coats identified to date, as well astheir sites of action. Although COPII contrib-utes to the import of newly synthesized proteinsto the cis-Golgi (Lee et al. 2004), and retromermediates input from the endosomes to thetrans-Golgi (Bonifacino and Hurley 2008), the

Editors: Graham Warren and James Rothman

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Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a005231

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Table 1. Vesicular coats identified to date, their subunit compositions, sites of action, and roles

Coat/

Adaptor Structure Site of action Role References

Clathrin Clathrin HeavyChainClathrin LightChain

TGN,endosomes,plasmamembrane

EndocytosisTGN endosomesortingEarly-late endosomesorting

Pearse 1976;McMahon and Mills2004

AP-1 gb1s1 m1 TGN, endosomes TGN-endosome sorting Pearse andRobinson 1984;McMahon andMills 2004

AP-2 ab2s2 m2 Plasmamembrane

Plasma membraneendocytosis

Pearse and Robinson1984; McMahon andMills 2004

AP-3 db3s3 m3 Endosomes Melanosome biogenesis Murphy et al. 1991;McMahon and Mills2004

AP-4 1b4s4 m4 TGN Basolateral sortingTGN-endosomesorting

Dell’Angelica et al.1999; McMahonand Mills 2004

GGA1-3 GGA1-3 TGN TGN-endosome/-lysosome sorting

Hirst et al. 2000;Takatsu et al. 2000;McMahon and Mills2004

COPI ab01/bgdz ER, Golgi,IntermediateCompartment

Sorting at the ER-Golgiinterface and withinthe GolgiEndosomal functions(see text)

Duden et al. 1991;Serafini et al. 1991b;Waters et al. 1991;Harrison-Lavoieet al. 1993; Stenbecket al. 1993 Bethuneet al. 2006

COPII Sec13,31/ Sec23,24 ER Protein export from theER

Barlowe et al. 1994;Hughes andStephens 2008

ESCRT Hrs-STAM1-2Vps23,28,37-MvB12Vps22,25,36Vps20,24,2-Snf7

Endosomes Multivesicular bodyformation (lysosomalpathway)Cytokinesis Autophagy

Katzmann et al. 2001;Hurley and Hanson2010

Retromer SNX1,2,5,6Vps26,29-35

Early endosome Endosome-TGN sorting Seaman et al. 1998;Bonifacino andHurley 2008

Exomer a Chs5,6-Bch1,2-Bud7 Golgi/endosome TGN-plasma membranesorting

Wang et al. 2006

BBsome BBS1,2,4,5,7,8,9,10 Primary cilium Plasmamembrane-ciliarymembrane sorting

Nachury et al. 2007;Nachury et al. 2010

Coats involved in trafficking steps from or to the Golgi are highlighted in bold.aExomer is restricted to yeast and fungi that expressed chitin.

V. Popoff et al.

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clathrin coat (McMahon and Mills 2004) andthe recently discovered exomer (Wang et al.2006) participate in the exit of molecules fromthe trans-Golgi network (TGN). Coatomer,the coat complex of COPI-coated vesicles, playsvarious roles at the interface between the ER andthe Golgi, as well as within the Golgi (Beck et al.2009b).

ROLES OF THE COPI COAT

Role in the Early Secretory Pathway

Coatomer is a stable protein complex of sevensubunits. According to immunogold electronmicroscopy (EM) studies, membrane associ-ated coatomer is mainly localized to the Golgiapparatus (Duden et al. 1991; Serafini et al.1991b; Griffiths et al. 1995), IC (Griffiths et al.1995), coated vesicles surrounding the Golgi(COPI vesicles) (Duden et al. 1991; Serafiniet al. 1991b; Griffiths et al. 1995), and the ER(Orci et al. 1994). These locations highlight arole of coatomer in transport at the ER-Golgiinterface. COPI vesicles have been shown toparticipate in the retrieval of proteins from theGolgi back to the ER (Cosson and Letourneur1994; Letourneur et al. 1994). In contrast, arole of these carriers in anterograde ER-to-Golgitransport, is still a matter of debate. Newly syn-thesized proteins are first exported from the ERto the IC by COPII vesicles (Aridor et al. 1995;Scales et al. 1997). Inhibition of COPI transportby a dominant negative mutant of Arf1, mi-croinjection of anti-ß-COP antibodies, or BFAresults in an impairment of subsequent IC-to-Golgi trafficking, which can be interpretedeither as a direct effect, or as a secondary conse-quence of the default of coatomer-mediatedrecycling of membrane components of theCOPII system (for a review see Pelham 1994),and/or as a fault in IC maturation (Aridoret al. 1995; Scales et al. 1997; Shima et al. 1999).

The prominent presence of COPI vesiclesaround the cisternae of the Golgi suggests arole of these carriers in intra-Golgi trafficking(Duden et al. 1991; Serafini et al. 1991b).Although these carriers have been known formore than 20 years, their roles are far from being

completely established, and several models existwith regard to their functions (see other chap-ters of this book). According to the vesicularmodel, the Golgi is static, and anterogradeCOPI vesicles deliver newly synthesized proteinsand lipids to successive cisternae. In the matura-tion and progression model, cisternae form atthe cis-Golgi and progress along the Golgi tofinally disassemble at the trans-Golgi. Hereretrograde transport of Golgi resident proteinsby COPI vesicles would ensure the maturationof the cisternae along the cis-trans axis of theGolgi. Observation of both anterograde andretrograde cargoes within COPI vesicles (Orciet al. 1997) gave rise to the percolating model,a combination of the two mechanisms above(Orci et al. 2000). COPI vesicles would mediatebidirectional transport between two adjacentcisternae by a random walk. Anterograde trans-port is simply driven by protein biosynthesis,entry of biosynthetic cargos at the cis-Golgiand their exit at the trans-Golgi, generating aflow across the Golgi.

Lipids droplets, another kind of structureoriginating at the ER-Golgi interface, also re-quire coatomer activity for proper functioning(Guo et al. 2008). By mediating the delivery ofenzymes to this compartment, the COPI systemmight help control the homeostasis of lipids inthe cell (Beller et al. 2008; Soni et al. 2009),although carriers with cargo indicative of thispathway have not yet been described.

Role in Mitosis and Golgi Positioning

In addition to its role in interphase, coatomerhas an active role during mitosis. COPI activity,concomitantly with repression of COPII vesicleformation (Farmaki et al. 1999), strongly con-tributes to the fragmentation of the Golgi ap-paratus into vesicles (Misteli and Warren 1994;Tang et al. 2008). In addition, recruitment ofcoatomer by a nucleoporin may induce thebreakdown of the nuclear envelope (Liu et al.2003).

An interaction of coatomer with cdc42 (Wuet al. 2000) and dynein was attributed to posi-tioning of the Golgi (Chen et al. 2005; Hehnlyet al. 2010).

Early Secretory Pathway

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Role in the Endocytic Pathway?

The early secretory and the endocytic pathwaysare similar and mirror each other as newly syn-thesized protein from the ER and proteins ofthe plasma membrane follow a similar traffick-ing scheme. Both protein populations are firsttransferred to a sorting station, the Golgi appa-ratus or the early endosomes, before beingeither recycled back to their starting compart-ment (ER or plasma membrane), or furthertransported along the late secretory pathwayor to the late endosomal or lysosomal compart-ments. Pools of coatomer have also been identi-fied at endosomal membranes (Whitney et al.1995; Aniento et al. 1996; Gu and Gruenberg2000). In this context it is of note that coatomerhas been implicated to take part in the matura-tion of early endosomes (Whitney et al. 1995;Aniento et al. 1996; Daro et al. 1997; Gu et al.1997; Gu and Gruenberg 2000; Gabriely et al.2007), and/or recycling toward the plasmamembrane (Daro et al. 1997; Razi et al. 2009).Moreover, coatomer may participate in thematuration of specialized endosomes such asphagosomes (Botelho et al. 2000; Beron et al.2001; Hackam et al. 2001), autophagosomes(Razi et al. 2009), and peroxisomes (Lay et al.2006). It is, however, not known if in theseendosomal functions coatomer directly servesas a coat, or if these processes directly dependon the service of COPI-coated vesicles. Further-more, recruitment of coatomer to endosomes,in contrast to Golgi binding, was reported tobe pH-sensitive (Aniento et al. 1996), and torequire only a subset of coatomer subunits(Whitney et al. 1995; Aniento et al. 1996), sug-gesting differences in mechanisms underlyingthe function of the complex in the endocyticand in the early secretory pathways.

How Can COPI Mediate VariousTrafficking Steps?

How coatomer could serve different traffickingroutes does not seem trivial to explain at firstsight, specifically because only one form of thecomplex was known to begin with. The com-plex is organized in two subcomplexes: a trimercomposed ofa-COP,b01- COP, and 1-COP, and

a tetramer of b-COP, g-COP, d-COP, and z-COP (Lowe and Kreis 1995; Fiedler et al.1996a; Pavel et al. 1998). More recently, thetwo coatomer subunits g-COP and z- COPwere found to exist in two isoforms, called g1,g2, and z1, z2, (Futatsumori et al. 2000). Eachisoform is, like all other subunits, present incoatomer as one copy, resulting in four possibledifferent heptameric protein complexes (Weg-mann et al. 2004). These coatomer isoformslocalize differently within the Golgi apparatusof mammalian cells (Moelleken et al. 2007),suggesting different sites of budding for eachof them. COPI shares structural aspects andmechanistic properties with the well-character-ized clathrin coat (see Fig. 1). Clathrin heavyand light chains polymerize into a cage consti-tuting the outer layer of the coat. An innerlayer composed of adaptor proteins takes therole of a link between the outer layer and themembrane. Various adaptors allow recruitmentof the clathrin system to different organellemembranes, and thus selection of differentsets of cargo for inclusion into coated vesicles(Robinson 2004).

X-ray crystallographic analyses of partialstructures revealed similarities between the coat-omer and the clathrin coat. On one hand, thethree-dimensional structures of coatomer sub-units g- COP and z-COP show a striking resem-blance to bAP2 (Hoffman et al. 2003; Watsonet al. 2004) and sAP2 (Yu et al. 2009), two sub-units of the tetrameric clathrin adaptor proteinsadaptin (AP2). The overall structure of the tetra-meric coatomer subcomplex b/d/g/z-COP istherefore likely to be similar to the AP complexes.Likewise, the a/b0/1-COP-trimer presents aspatial organization similar to the clathrin sub-units (Hsia and Hoelz 2010; Lee and Goldberg2010). Thus, it is tempting to speculate that,like the various adaptins, different coatomerisoforms provide ways to modulate the cargorepertoire of the COPI system, leading to distinctpools of coated vesicles involved in differentpathways. In agreement with this hypothesis,different subpopulations of COPI vesicles withdifferent cargo compositions can be observedin the living cell (Orci et al. 1997) and in vitro(Lanoix et al. 2001; Malsam et al. 2005).

V. Popoff et al.




α

αβ′-COP

γ-COP appendage

AP2 σ2

β2 appendage

Coatomer

ε

β

δγ

β′

ζα

μβ

σ

Clathrin

Clathrin

ζCOP

Figure 1. The heptameric complex coatomer is compared with the clathrin/adaptor complex AP2. The stablecomplex coatomer can be dissociated in vitro into subcomplexes: a trimer (ab01) and a tetramer (bgdz). Struc-tural similarities exist between the trimeric coatomer subcomplex (Lee and Goldberg 2010) and the triskelion ofclathrin heavy and light chains (Xing et al. 2010). Within the tetrameric subcomplex of coatomer structural sim-ilarities are reported for z-COP and AP2s2 (Yu et al. 2009), and for the g-COP and the b2 appendage domains(Watson et al. 2004). Thus, the trimeric COPI-subcomplex is thought to resemble the clathrin part, and the tet-rameric COPI-subcomplex the adaptor part of a clathrin-coated vesicle.

Box. Lipids and COPI Vesicles

Molecular mechanisms that underlie the sorting of proteins have been elucidated to quite some detailin the recent years. Much less is known, however, of how a living cell can maintain the identities ofits various organelles with respect to their unique lipid composition, although lipidomes of a fewmembrane carriers have become available recently (Brugger et al. 2000; Takamori et al. 2006;Klemm et al. 2009). Lipidomic analysis of COPI vesicles revealed a depletion of cholesterol andsphingomyelin (SM), two lipids characteristic of the liquid-ordered (Lo) phase, when comparedwith the donor Golgi membrane (Brugger et al. 2000). In vitro analysis of vesicle formation fromgiant unilamellar vesicles supports the idea that COPI budding occurs exclusively from liquid-disordered (Ld) phases (Manneville et al. 2008). In accordance, ER membranes contain less choles-terol and SM than Golgi membranes. Combined with retrograde transport by COPI vesicles from theGolgi to the ER, selective transport of Ld lipids would allow maintaining lipid homeostasis within theearly secretory pathway. In addition, selective sorting of Ld lipids by the COPI coat could induce aphase separation within the forming bud. The line tension thus created between Ld and Lo phasewould favor fission of the formed vesicle (for a review, see Pinot et al. 2010).

Additionally, an active role of lipids in COPI vesicle formation has been proposed. Lipids likephosphatidic acid (PA) (Yang et al. 2008), diacylglycerol (DAG) (Fernandez-Ulibarri et al. 2007;Asp et al. 2009), and phosphatidyinositol (PI) (Simon et al. 1998; Carvou et al. 2010) can affect mem-brane curvature and thus have the potential of facilitating budding and/or scission of vesicles.However, a direct involvement in budding/fission of lipids is experimentally not easy to access,





MOLECULAR MECHANISM OF COPIVESICLE FORMATION

The generation of a fusion-competent COPItransport vesicle can be conceptually subdi-vided into partially interdependent steps: coatrecruitment, uptake of cargo, budding, mem-brane separation (scission), and uncoating(see Fig. 2). Whereas the minimal require-ments for coat formation, budding, fission,and uncoating have been defined by reconstitu-tion experiments using chemically defined lip-ids and purified protein components (Spanget al. 1998; Bremser et al. 1999; Reinhard et al.

1999), the mechanisms underlying cargo up-take are still not completely understood.

Coat Recruitment

The small Ras-like GTPase ADP-ribosylationfactor 1 (Arf1) plays a central role in the forma-tion of COPI-coated vesicles at the Golgi appa-ratus. However, action of Arf1 is not limited tothis organelle and can function at various intra-cellular compartments, involving different setsof effectors (Nie et al. 2003; Donaldson et al.2005; Volpicelli-Daley et al. 2005). It is thusvery likely that additional factors are required

and data existing so far are indirect observations based on inhibition of lipid modifying enzymes.Furthermore, none of these enzymes seems to be required for vesicle formation in in vitro reconsti-tution experiments. More investigation is thus needed to clarify whether local regulation of mem-brane lipid composition is a basis for fine-tuning of the formation of a COPI vesicle in the living cell.

UncoatingArfGAP2/3 (?)

GBF1

cytosol

Golgi lumen

D

α β′β

δ

ε γζ

D

T T T T T TT

ArfGAP1 (?)

Budding

Coatpolymerization

Cargop23/p24dimer

CoatomerArf1-GDPD

T Arf1-GTP

Scission(Arf1)2

Pi

Figure 2. Individual steps in the formation of a COPI vesicle. (Scheme adapted from Beck et al. [2009b] andreprinted, with permission, from Elsevier # 2009.) For details see text.

V. Popoff et al.




to provide a spatio-temporal regulation ofcoatomer recruitment at specific sites suitablefor COPI vesicle biogenesis. Like most of thesmall GTPases, Arf1 cycles between a GDP-loaded inactive cytosolic state and an active,membrane anchored, GTP-loaded state (Ran-dazzo et al. 1993). This molecular switch ispositively regulated by guanine nucleotide ex-change factors (GEFs), which catalyze exchangeof GDP with GTP. On activation of an intrinsiclow GTPase activity by Arf GTPase activatingproteins (ArfGAPs), Arf1 is converted into itsinactive GDP form and released from themembrane.

Specific recruitment of the small GTPase tothe membrane is a prerequisite for its function.In agreement with its role in COPI vesicle for-mation, Arf1 contains Golgi localization signalsencoded in its sequence. Arf1-GDP binds to adimeric complex of members of the p24 family,a group of type I transmembrane proteins. Invitro cross-linking experiments reveal a specificinteraction between a carboxy-terminal site ofArf1 with a dimer of p23 or p24 (Gommelet al. 2001). Foerster resonance energy transfer(FRET) experiments confirmed the existenceof this interaction in the living cell (Majoulet al. 2001). In addition, at the cis-Golgi, Arf1is recruited to the membrane via membrin, aSNARE protein. This interaction is mediatedby a MXXE motif within the GTPase. Mutationof this motif impaired recruitment of Arf1 tothe cis-Golgi without affecting its binding tothe trans-Golgi (Honda et al. 2005). Thus, othersignals within Arf1 must mediate targeting ofthe GTPase to the trans-Golgi.

Arf1 is activated on membranes by large Arf-GEFs that are members of the Sec7 super family.Based on sequence homologies, mammalianArfGEFs can be classified into the followinggroups: (1) Golgi Brefeldin A (BFA)-resistancefactor 1/BFA-inhibited GEF (GBF/BIG), (2)Arf nucleotide binding site opener (ARNO)/cytohesins, (3) exchange factor for Arf6 (EFA6),(4) BFA-resistant Arf GEF (BRAG), and (5)F-box only protein 8 (FBX8) (Casanova 2007).

GBF1 and the BIGs, as well as their yeasthomologs (Gea1/2 and Sec7p, respectively)activate Arf1 at the Golgi membrane (Claude

et al. 1999; Spang et al. 2001; Kawamoto et al.2002; Zhao et al. 2002), whereas the otherArfGEFs act at post-Golgi compartments. Themammalian Golgi GEFs are not evenly distrib-uted across the stack of cisternae. BIG1 andBIG2 are present at the trans-Golgi, the TGN,and the recycling endosomes (Mansour et al.1999; Shinotsuka et al. 2002; Shin et al. 2004).Their mode of recruitment to the membraneis not well understood. GBF1 localizes to ICand Golgi, acting as a direct mediator of retro-grade transport between these compartmentsand the ER (Kawamoto et al. 2002; Garcia-Mataet al. 2003; Zhao et al. 2006). This localization isdirectly dependent on an interaction betweenthe GEF and Rab1b (Monetta et al. 2007).Recruitment of GBF1 also requires PI4P, and itwas proposed that Rab1 contributes to GBF1recruitment by locally activating phosphati-dylinositol 4-kinase (PI4KIIIalpha) (Dumar-esq-Doiron et al. 2010).

Once both Arf1 and its exchange factor arerecruited to the membrane, a Sec7 domainwithin the ArfGEF triggers the activation ofthe small GTPase. A critical feature of this cata-lytic domain is its “glutamic finger,” a conservedglutamate residue exposed at the tip of a hy-drophilic loop between helices 6 and 7 (Beraud-Dufour et al. 1998). Nucleotide exchange ismediated by electrostatic competition of thisglutamate side chain with the nucleotide’s b–phosphate. On exchange of GDP to GTP, aconformational change within Arf1 leads toexposure of its amino-terminal amphipathicand myristoylated helix, which in turn causesinsertion of this helix into the lipid bilayerand thereby secures membrane anchorage ofArf1 (Franco et al. 1996; Antonny et al. 1997).This model was challenged by recent structuraldata. Nuclear magnetic resonance spectrometryof full-length myristoylated Arf1 embeddedinto bicelles showed an unexpected localiza-tion of the myristic acid residue. Instead ofbeing inserted within the bilayer parallel to thephospholipid-acyl chains, the myristic fattyacyl chain appears on top of the bicelles, per-pendicular to the phospholipids (Liu et al.2010). Whether this orientation is because ofthe physicochemical nature of the bicelles, or





reflects Arf1’s positioning on physiologicalmembranes, is presently not known.

Recruitement of coatomer to Golgi mem-branes is tightly correlated to Arf1 activation(Donaldson et al. 1991; Serafini et al. 1991a;Palmer et al. 1993). The complex is recruited tothe Golgi membrane en bloc (Hara-Kuge et al.1994), in contrast to clathrin/AP and COPIIthat are composed of two successively recruitedlayers. Coatomer forms multiple interfaces withArf1-GTP: site-directed photolabeling studieshighlighted specific contacts between theGTPase and the subunits b0-COP, b-COP, d-COP, as well as the trunk domain of g-COP(Zhao et al. 1997, 1999; Sun et al. 2007). Yeasttwo hybrid analysis further points to an interac-tion of Arf1 with 1-COP (Eugster et al. 2000).Three to four Arf1 molecules bind to one coat-omer complex (Serafini et al. 1991a; Beck et al.2009a). Thus, interaction of coatomer with Arfprovides multiple protein–protein interfaces.Binding of g-COP, via its trunk and appendagedomains, to dimers of p24 transmembrane pro-tein family members (COPI transmembranemachinery proteins, see later) further stabilizescoatomer on the Golgi membrane (Harter andWieland 1998; Bethune et al. 2006). Membersof the p24 family carry variants of a dilysinemotif in combination with a double F motif,FFXX(K/R)(K/R)Xn (n � 2). Binding of coat-omer to these signals depends more stronglyon the phenylalanine than on the dilysineresidues (Fiedler et al. 1996b; Sohn et al. 1996).These machinery signatures are recognizedexclusively by g-COP (Bethune et al. 2006).p24 proteins are present in COPI vesicles inamounts stoichiometric to coatomer (Sohnet al. 1996). In a yeast strain defective for vesiclefusion, a p24 knockout reduced the numberof COPI-coated vesicles (Stamnes et al. 1995).These data suggested that type I transmembraneproteins represent membrane receptors for coat-omer and are actively required for the formationof COPI vesicles (Stamnes et al. 1995; Sohn et al.1996). Surprisingly, however, in yeast the dele-tion of all p24 proteins showed only a reductionin the rate of transport of some cargo proteins(Springer et al. 2000). More recently, additionalbiochemical evidence was reported that in yeast

the p24 complex participates in retrograde trans-port from Golgi to ER, by promoting the forma-tion of COPI vesicles (Aguilera-Romero et al.2008). p24 members are indispensible in mam-mals: knockout of p23 is lethal at the earliest pos-sible time point in the development of a mouseembryo (Denzel et al. 2000).

Specific binding of the g-COP trunk do-main to dimers of p23 and p24 (not p25, p26,or p27), results in a conformational change ing-COP (Reinhard et al. 1999; Bethune et al.2006) that is transmitted to the a-COP subunit(Langer et al. 2008). This spatial rearrangementcauses aggregation of the complex and is likelyto initiate coatomer polymerization (Reinhardet al. 1999), providing the energy to bend themembrane and sculpting a COPI-coated bud(R Beck et al., unpubl.).

A machinery component of COPI vesicleswith another dilysine motif is the seven-helixtransmembrane protein KDEL receptor thatfunctions as a transmembrane adaptor. Itscytosolic tail interacts with coatomer via aKKXSXXX signal, active only when its serineresidue is phosphorylated (Cabrera et al.2003), whereas its luminal part interacts withsoluble proteins that harbor a C-terminalKDEL-sequence (Lewis and Pelham 1992). Asa result, KDEL-proteins are included intoCOPI vesicles and retrieved to the ER (Pelham1991; Majoul et al. 1998), where they dissociatefrom the KDEL receptor, probably because of adifference of pH between Golgi and ER (Wilsonet al. 1993). The free KDEL-receptor is thencycled back to the Golgi.

Identification of a subpopulation of COPIvesicles, which lacks p24 proteins (Malsamet al. 2005), raises the question of whether thesecarriers contain transmembrane machinerycomponents, other than p24 family proteins,that are crucial for their biogenesis.

Cargo Sorting/Sorting Motifs

Proteins are directed into COPI vesicles byvarious mechanisms based on direct or indirectbinding to the coat.

Membrane proteins to be included intoCOPI vesicles can also be recognized directly

V. Popoff et al.




by coatomer through sorting motifs present intheir sequence. The first signals characterizedwere dilysine motifs present at the extreme car-boxyl terminus of membrane proteins (Nilssonet al. 1989). They bind directly to coatomer atsites different to the above machinery proteinsand induce the retrieval from the Golgi to theER of the host protein (Cosson and Letourneur1994; Letourneur et al. 1994). Various subsetsof dilysine motifs exist that are recognized dif-ferentially by the coatomer complex (Schroder-Kohne et al. 1998). In contrast to the p24 familiycarboxy-terminal signatures, KKXX motifsinteract with the WD40 domain of a-COP,and KXKXX binds to a similar domain withinb0-COP (Eugster et al. 2004). Thus structuraldifferences constitute the molecular basis forcoatomer to discriminate machinery compo-nents that are recycled from cargo proteinsthat are transported and delivered unidirection-ally. The affinity between coatomer and suchcargo sorting signals depends on the nature ofthe X amino acids following the lysine residues(Zerangue et al. 2001), thus giving rise to differ-ences in efficiency of retrieval to the ER.

Together with a modulation of the efficiencyof ER exit, these tuned affinities may provide amechanism to control the steady-state locali-zation of membrane proteins at the ER-Golgiinterface (Zerangue et al. 2001).

Furthermore, Arginine-based motifs thatconform to the consensus sequence (F/C/R)RXR (where F/C is an aromatic or bulkyhydrophobic residue) (Zerangue et al. 1999)are recognized by coatomer subunits b- andd-COP (Michelsen et al. 2007). In contrast todilysine motifs, they are not restricted to car-boxyl termini and can have a more flexiblepositioning within the cytosolic tail of thehost protein (Shikano and Li 2003). These sig-nals can control the maturation of membraneprotein complexes (Michelsen et al. 2005). Aslong as a complex is not fully assembled, sucharginine motifs present in its subunits areexposed, inducing retrograde transport to theGolgi. On complete assembly of a complex,the signals become masked, allowing export ofthe complex to the cell surface (Zerangue et al.1999). Several mechanisms for masking of the

signals have been proposed, including stericmasking by a partner subunit (Michelsenet al. 2005), inactivation of the signal by phos-phorylation of nearby residues (Scott et al.2001), or competition for motif binding ofcoatomer with 14-3-3 proteins (O’Kelly et al.2002; Yuan et al. 2003), or PDZ-domain pro-teins (Standley et al. 2000).

Additional sorting motifs are based on aro-matic residues. A “dL” motif confers bindingto d-COP and retrieval to the ER (Cossonet al. 1998), and a FXXXFXXXFXXLL motif inthe Dopamin1 receptor mediates interactionwith g-COP, which is necessary for physiologi-cal trafficking of the receptor (Bermak et al.2002). However, not all the membrane proteinstransported within COPI vesicles carry coat-omer-interacting motifs. Notably, this is thecase of glycosylation enzymes with their tailslacking known sorting signal. Recent studiesin yeast implicated Vps74 as an essential factorfor the packaging of glycosylation enzymesinto transport carriers (Schmitz et al. 2008; Tuet al. 2008). By binding simultaneously tocoatomer and the cytosolic tails of glycosyl-transferases, Vps74 works as a coat-cargo adap-tor (Tu et al. 2008). Although further studieswill be needed to unravel similar mechanismsin mammalian cells, Vps74 suggests the exis-tence of a set of similar cargo adaptors in highereukaryotes.

Inclusion of Cargo into COPI Vesicles

Two modes of incorporation of membraneproteins into COPI vesicles are observed. Pro-teins directly involved in the budding processare incorporated into vesicles in a GTP-inde-pendent manner and are further referred to asmachinery components (Nickel et al. 1998;Malsam et al. 1999). Cargo proteins, on theother hand, require GTP hydrolysis for theiruptake into the transport carrier. Indeed, inthe presence of GTPgS, a poorly hydrolysableform of GTP, or Arf1-Q71L (an Arf1 variantlocked in its GTP-loaded state), COPI vesiclescan still form, but appear devoid of anterogradeand retrograde cargo (Nickel et al. 1998; Lanoixet al. 1999; Malsam et al. 1999; Pepperkok et al.





2000). In contrast, uptake of p23, p24, or KDELreceptor is still effective under these conditions(Nickel et al. 1998). As previously described,activation of Arf1 is a prerequisite for therecruitment of coatomer to the membrane. Asa corollary, hydrolysis of Arf1-GTP intoArf1-GDP leads to membrane uncoating (seelater). This seems in contradiction with a roleof GTPase activity in cargo sorting. In in vitroexperiments with the COPII coat, primingcomplexes weakly bound to unassembled cargoare instantaneously released, whereas thoseinteracting more strongly to oligomerized cargoremain transiently associated to the membrane,increasing their probability to be incorporatedinto a final COPII vesicle (Sato and Nakano2005). This characteristic may provide themolecular basis of a GTPase-driven kineticproofreading mechanism (Sato and Nakano2007; Tabata et al. 2009). Kinetic analysis of theCOPI system performed in living cells revealedthat Arf1 dissociates faster from membranesthan coatomer (Presley et al. 2002), suggestingthat coatomer is not immediately released afterArf1 inactivation but stays metastably associatedto the membrane for an additional period oftime. This difference is likely because of theadditional binding of coatomer to membraneproteins (e.g., p24 family proteins), and to lateralinteractions of the polymerized complexeswithin the coat network. It is open at presentwhether the different dissociation kinetics ofArf and coatomer would reflect a kinetic proof-reading mechanism, a model that was suggestedby Weiss and Nilsson (2003).

A different, but not mutually exclusive,mechanism has been proposed for specific en-richment of cargo in a nascent COPI vesiclebased on the curvature sensitivity of ArfGAP1(Liu et al. 2005). Preferential binding of theprotein to positively curved membranes (Bigayet al. 2003) increases local ArfGAP1 activity.As a result, GTP hydrolysis, and thus coatrelease, from the positive curvature of a formingbud would be increased. This GTPase activitywould lead to a flux of coatomer from therim, where Arf and coatomer are recruited tothe center of a growing bud, thereby mediatingcargo concentration (Liu et al. 2005). Several

machinery components can locally modulateGTP hydrolysis. Coatomer itself can stimulateArfGAP1 (Goldberg 1999; Szafer et al. 2001),whereas the cytosolic tails of p23 and p24 slowdown the hydrolysis of Arf1 (Goldberg 2000;Lanoix et al. 2001). Interaction between aKDEL receptor and ArfGAP1 may recruit anadditional activating protein to the mem-brane, and thus increase local GTPase activity(Aoe et al. 1997, 1998). This would give rise toanother, less characterized, level of regulation.

Budding and Scission

More than 20 years after the discovery of COPI-coated vesicles (Orci et al. 1986; Malhotra et al.1989), there is still an ongoing discussion aboutthe structural components of this class of ve-sicular carrier (Beck et al. 2009b; Hsu andYang 2009; East and Kahn 2010). The two cyto-solic components essential for COPI vesicle for-mation in vitro are Arf1 and the heptameric coatcomplex coatomer (Serafini et al. 1991a; Orciet al. 1993).

In a chemically defined in vitro system,using synthetic liposomes with a nonphysiolog-ical lipid composition, Arf1-GTP and coatomeralone were sufficient to induce vesicle formation(Spang et al. 1998). Nevertheless, in the pres-ence of a cytoplasmic domain of a p24 familyprotein, vesicle formation is stimulated, andindependent of a wide range of lipid com-positions (Bremser et al. 1999). As describedabove, the p24 proteins were initially found tobe a major component of COPI-coated vesicles(Stamnes et al. 1995; Sohn et al. 1996) and serveas membrane machinery for these carriers.

More recently a membrane surface activitywas observed of the activated, GTP-loadedform of Arf1 on synthetic membranes in theabsence of coatomer (Beck et al. 2008; Krausset al. 2008; Lundmark et al. 2008). This activityresults in tubulation of giant unilamelar vesi-cles (GUVs), or of membrane sheets tetheredto glass surfaces, and strictly depends on adimerization of the small GTPase. This dimeri-zation is essential in the living cell, as a yeaststrain devoid of Arfs (Takeuchi et al. 2002) can-not be rescued with an Arf point mutant unable

V. Popoff et al.




to dimerize (Beck et al. 2008). The surface activ-ity of Arf1 is a prerequisite for COPI vesicleformation (Beck et al. 2008). Thus, during for-mation of a COPI bud, two different activitieshave the potential to deform a flat donor mem-brane into a curved bud. These are the polymer-ization of coatomer on its recruitment and/orthe dimerization-dependent membrane surfaceactivity of activated Arf.

Cryo electron microscopy and tomographyof liposomes incubated with Arf wt, GTP andcoatomer revealed production of regular freeCOPI vesicles, as expected from earlier work(Reinhard et al. 2003). With the nondimerizingmutant of Arf, however, hardly any free vesiclesare formed. Instead, the liposomes are trans-formed into flower-like structures that representmultiple buds linked by a continuous bilayer,indicating a block in scission. It is of note thatneither mechanical shearing nor high salt con-ditions are needed to create a free COPI-coatedvesicle (R Beck et al., unpubl.). In the mechanis-tically similar COPII system, a membrane sur-face activity of Sar1 was revealed earlier (Bielliet al. 2005; Lee et al. 2005). Here, the fissionstep in the formation of COPII-coated vesi-cles from liposomes was dependent on the N-terminal amphipathic helix of the small GTPase(Lee et al. 2005). Most recently, a regulated scaf-fold assembly by Sar1 was observed (Long et al.2010), opening a possibility that Sar1 assemblyon membranes controls constriction of the budneck similar to the fission mechanism proposedfor dynamin in the clathrin system (Bashkirovet al. 2008; Pucadyil and Schmid 2008). How-ever, the dynamin mechanism requires hy-drolysis of GTP for the scission process, and invarious reports the formation of COPII vesicleswas described in the presence of nonhydolyz-able analogs of GTP (Barlowe et al. 1994; Okaand Nakano 1994). Likewise, Arf1-dependentformation of COPI vesicles is independent ofGTP hydrolysis (Malhotra et al. 1989). Takentogether, this indicates that the small GTPasesArf1 and Sar1 not only mediate coat assemblybut also promote the fission step to release anascent vesicle in a mode other than that pro-posed for dynamin. What mechanism canexplain these two functions of a small GTPase?

A Model for Membrane Separation Basedon Cooperation of Coatomer with Arf1

Within the growing coat, Arf interacts with themembrane via its myristoylated amphipathichelix, and with its covering layer of coatomervia several defined interfaces (Zhao et al. 1997,1999; Sun et al. 2007). Two Arf1 dimers arebound to one coatomer complex (Serafiniet al. 1991b; Beck et al. 2008) (R Beck et al.,unpubl.). In the positive curvature of the grow-ing bud, Arf ’s interaction with the membrane isenergetically favorable. As the bud becomesmore complete, a neck forms at the site of Arfand coatomer recruitment, with increasinglynegative curvature, which is highly unfavorablefor Arf binding. This energy strain could berelaxed by diffusion of the small GTPase fromthe negatively curved zone. If the stability ofthe complex of the dimerized Arfs with thecovering network of coatomer is strong enough,however, to prevent Arf from escaping, thelocal high-energy state can only be relaxedby fusion of the adjacent membranes in theneck, causing membrane separation. The needfor Arf to dimerize is simply explained bycomparing the stability of a complex of coat-omer formed with either two dimers of Arf(Arf wt) or with four monomers (nondimeriz-ing mutant). With Arf wt the complex’s stabilityis higher because of the energy released by thetwo Arf dimerization interfaces (R Beck et al.,unpubl.).

Additional Proteins Implicated inCOPI Fission

A variety of proteins have been shown to be in-volved in COPI vesicle formation. Among those,Brefeldin-A ADP-ribosylated Substrate (BARS)(Yang et al. 2005), endophilin (Yang et al. 2006),and ArfGAP1 (Yang et al. 2002) are thought toplay a role in membrane scission and/or act as acoat component. Some of these functions havebeen challenged by more recent investigations(Gallop et al. 2005; Beck et al. 2009b).

Whereas the basic mechanisms of the coremachinery as deduced from experiments inreconstituted systems seem quite clear, themechanisms underlying the contributions of





such additional components that might playimportant roles in the living cell represent chal-lenges for future research.

Uncoating

To allow fusion with its target membrane, aCOPI-coated vesicle must be uncoated. In aprevailing model, this process is coupled to hy-drolysis of GTP by Arf1 (Tanigawa et al. 1993),triggered by ArfGAPs (Cukierman et al. 1995).The members of this protein family are definedby the presence of a catalytically active domainbearing a zinc finger motif, followed by aninvariant arginine residue (CX2CX16CX2CX4R)(Cukierman et al. 1995).

Three mammalian ArfGAPs (ArfGAPs1, 2/3) are implicated in the COPI system as knockdown of all three ArfGAPs increased the levelof membrane bound Arf1 in the living cell.As an additional phenotype, the cycling proteinERGIC-53, the Golgi tethering protein GM130,and coatomer accumulated in the ER-Golgiintermediate compartment. Golgi-to-ER retro-grade transport was blocked as consequence ofthe triple knock down, a phenotype similar toa b-COP knock down (Saitoh et al. 2009). Yeasthomologs, Gcs1 for ArfGAP1, and Glo3 forArfGAPs 2 and 3, function as an essential pairfor retrograde transport from the Golgi tothe ER (Poon et al. 1999). These data suggestthat both types of ArfGAPs in mammals (Arf-GAP1 and ArfGAP2/3) and in yeast (Gcs1and Glo3) are interchangeable.

It came as a surprise when ArfGAP1 activityturned out to be regulated by membrane curva-ture (Bigay et al. 2003), with increasing enzymeactivity correlated to decreasing diameter of aliposome (i.e., increasingly steeper curvature).This sensitivity is based on the presence of amotif termed ALPS (for ArfGAP1 Lipid Pack-ing Sensor) that on membrane contact formsan amphipathic helix that inserts bulky hydro-phobic side chains between the loosely packedlipid head-groups of the outer leaflet of a posi-tively curved membrane (Bigay et al. 2005).

More recently comparison of ArfGAP1 withthe related ArfGAP2/3 revealed functional dif-ferences of these auxiliary proteins. ArfGAPs 2

and 3 lack an ALPS motif and hence do notshow sensitivity to membrane curvature (Wei-mer et al. 2008). In contrast, the activities ofArfGAPs2 and 3 depend on coatomer (Weimeret al. 2008; Kliouchnikov et al. 2009). ArfGAP2and 3 were shown to directly interact with coat-omer (Frigerio et al. 2007; Kliouchnikov et al.2009), and can also be detected on purifiedCOPI vesicles derived from liposomes or nativeGolgi membranes (Frigerio et al. 2007; Weimeret al. 2008).

In the living cell ArfGAP2/3 and ArfGAP1show fundamental differences. ArfGAP2/3 fol-lowed the dynamics of membrane associationof coatomer more closely than does ArfGAP1.Furthermore, ArfGAP2/3 knock down causedunstacking of the Golgi and cisternal shorten-ing, similar to conditions in which vesicleuncoating was blocked (Kartberg et al. 2010).Taken together, ArfGAP1, independent ofcoatomer, is likely to drive the cycle of activa-tion and inactivation of Arf1 explained above,whereas the coatomer-dependent ArfGAPs2/3are candidate-uncoating, GTPase-activatingproteins.

A possible contribution to uncoating oftethering proteins emerges from studies inyeast, which requires the Dsl1 complex, consist-ing of Dsl1p, Dsl3p, and Tip20 (Andag et al.2001; Tripathi et al. 2009) for Golgi to ER trans-port. Dsl1p was shown to interact directly withthe subunits a and d of the COPI complex(Reilly et al. 2001; Andag and Schmitt 2003;Zink et al. 2009). The observations that COPI-coated vesicles accumulate in Dsl1p-depletedcells led to the suggestion that this multi proteincomplex has, with its role in tethering, a func-tion in uncoating (Zink et al. 2009). A moredetailed explanation of tethers within the earlysecretory pathway as well as their functions isgiven by Malsam and Sollner (2011).

OPEN QUESTIONS

With the X-ray structures available for clathrinand COPII vesicle coat proteins and partialstructures of the COPI coat, it becomes clearthat common protein modules are used to buildthe various coats. This structural information

V. Popoff et al.




relates to crystallized coat protein components,however, the structures of the proteins withinthe coats they form are still elusive. How dothe structures of coat proteins in solution andwithin the network of polymerized complexeson the coated vesicle compare? This questionis of particular interest for COPI vesicles,because in this system coatomer is recruited enbloc, and undergoes a conformational changeafter its contact with p24 family proteins.Cryo electron microscopic tomography mayallow extracting such structural informationfrom coated liposomes or vesicles. Insightsderived from a combination of X-ray crystallog-raphy and electron microscopy analysis of coatsand cocrystals of coat components will thenhelp us understand basic open questions aboutthe molecular mechanism of energy-drivencargo uptake into a vesicle. More biochemicalapproaches will be needed to elucidate the dif-ferent roles isoforms of coatomer take in differ-ent locations within the Golgi. To this end,coated vesicles need to be prepared with indi-vidual coatomer isoforms and their proteomescompared. In this context, it is also of interestto know whether the coatomer-dependentArfGAP proteins 2 and 3 have preferences tobinding individual coat complexes. Likewise,comparison of binding of the ArfGAPs to thesoluble and the polymerized states of the coatprotein will allow attribution of functions tothe various ArfGAPs in uncoating of COPIvesicles.

What happens to coatomer during uncoat-ing? Does hydrolysis of GTP by Arf1 leave a net-work of polymerized coatomer that falls off inpieces, and is the conformation of the complexreversed to its soluble form afterward by energydependent chaperons, similar to the clathrinsystem (Schmid and Rothman 1985), or iseach individual coatomer complex reversed inconformation during the uncoating reaction?Investigation of such questions may reveal novelproteins or roles of chaperons and will helpfinally establish the molecular mechanisms ofthe process of uncoating.

The small GTPase Arf1 takes part not onlyin the formation of the COPI coat, but is alsoa component of all types of clathrin-coated

vesicles that bud from the endomembrane sys-tem but not the plasma membrane. What isthe mechanism of membrane scission of thesedynamin-independent vesicles? Does Arf1 playa role as a dimer in their scission reaction, sim-ilar to the COPI system?

We have learned about structures and func-tions of core components and individual ba-sic molecular mechanisms of COPI buddingin the Golgi, mainly from reconstituted systemsin vitro. Although this is a prerequisite for ourfuture integrated understanding, we need tolearn a lot more about the roles of additional,only partly known, players as they operate inthe context of a living cell.

Thus, investigating the biosynthesis ofcoated vesicular carriers will be a highly attrac-tive challenge in the future for biochemists,structural biologists, and cell biologists.

ACKNOWLEDGMENTS

We apologize to those colleagues whose work wecould not cite because of the limited space. Wethank Thomas Soellner and Patricia McCabefor critical reading the manuscript, and the Ger-man Research Council for supporting our work(SFB 638, projects A10 and A16, and GRK1188). V.P. is supported by a FEBS long-termfellowship.

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August 15, 20112011; doi: 10.1101/cshperspect.a005231 originally published onlineCold Spring Harb Perspect Biol

Vincent Popoff, Frank Adolf, Britta Brügger and Felix Wieland COPI Budding within the Golgi Stack

Subject Collection The Golgi

Structure of Golgi Transport ProteinsDaniel Kümmel and Karin M. Reinisch Identify

Golgi and Related Vesicle Proteomics: Simplify to

NilssonJoan Gannon, John J.M. Bergeron and Tommy

Golgi BiogenesisYanzhuang Wang and Joachim Seemann

Organization of SNAREs within the Golgi StackJörg Malsam and Thomas H. Söllner

DiseasesGolgi Glycosylation and Human Inherited

Hudson H. Freeze and Bobby G. Ng

Golgi during DevelopmentWeimin Zhong

Models for Golgi Traffic: A Critical AssessmentBenjamin S. Glick and Alberto Luini Golgi Complex

-Face of thecisEntry and Exit Mechanisms at the

Andrés Lorente-Rodríguez and Charles BarloweArchitecture of the Mammalian Golgi

Judith KlumpermanCOPI Budding within the Golgi Stack

Vincent Popoff, Frank Adolf, Britta Brügger, et al.Evolution and Diversity of the Golgi

Mary J. Klute, Paul Melançon and Joel B. DacksMechanisms of Protein Retention in the Golgi

David K. Banfield

Glycans Are Universal to Living CellsGlycosylation Machinery: Why Cell Surface Evolutionary Forces Shaping the Golgi

Ajit Varki

ApparatusThe Golgin Coiled-Coil Proteins of the Golgi

Sean Munro

Golgi PositioningSmita Yadav and Adam D. Linstedt

Signaling at the GolgiPeter Mayinger

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