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FEBS Letters 581 (2007) 2076–2082

Minireview Mechanisms of COPII vesicle formation and protein sorting

Ken Satoa,*, Akihiko Nakanoa,b a Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, Hirosawa, Wako, Saitama 351-0198, Japan b Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received 29 November 2006; revised 23 January 2007; accepted 30 January 2007

Available online 14 February 2007

Edited by Thomas So¨llner

cations with the aid of a variety of cellular chaperones. Once Abstract The evolutionarily conserved coat protein complex II (COPII) generates transport vesicles that mediate protein trans- correctly folded and assembled, proteins are then segregated port from the endoplasmic reticulum (ER). COPII coat is respon- from ER resident proteins and exported from the ER in trans- sible for direct capture of cargo proteins and for the physical port vesicles, the so-called ‘‘COPII vesicles’’, to either the Golgi deformation of the ER membrane that drives the COPII vesicle complex or the ER–Golgi intermediate compartment (ERGIC) formation. In addition to coat proteins, recent data have indicated [3]. The ER membrane makes up more than half of the total that the Ras-like small GTPase Sar1 plays multiple roles, such as membrane in the cell, and genomic and proteomic analyses COPII coat recruitment, cargo sorting, and completion of the have estimated that approximately one-third of all yeast pro- final fission. In the present review, we summarize current know- teins are targeted to the ER for delivery to other organelles ledge of COPII-mediated vesicle formation from the ER, as well or the cell surface [4]. Because protein export by COPII vesicle as highlighting non-canonical roles of COPII components. from the ER is the default ER-to-Golgi route that has been pro- Ó 2007 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved. posed in yeast and mammals, the COPII vesicle accommodates an extraordinary variety of cargo proteins with different struc- Keywords: COPII, Sar1; Small GTPase; Endoplasmic tures, functions and ultimate destinations. reticulum; Vesicular transport A variety of reconstituted in vitro assays as well as high-res- olution structural information of key players are now available to study the molecular mechanisms of COPII vesicle formation and therefore COPII vesicle is one of the best-understood among various transport vesicle formations. 1. Introduction

The secretory pathway of the eukaryotic cell has evolved to 2. COPII coat recruitment and assembly transport newly synthesized proteins destined for the mem- brane compartments (Fig. 1). Transport from one subcom- In Saccharomyces cerevisiae, COPII vesicle formation ap- partment of this pathway to the next is thought to be pears to proceed throughout the ER. However, in most other mediated by vesicular carriers, which are formed by the regu- eukaryotes, COPII vesicle-mediated protein export is found at lated assembly of coat protein complexes (COPs) on donor specialized regions termed transitional ER (tER) or ER exit organelles [1]. The coat proteins form a shell around the form- sites (ERES) [5,6]. In these distinct zones of the ER, a set of ing vesicle, shaping it into a transport vesicle [2]. A common cytoplasmic proteins, collectively known as the COPII coat, feature of those vesicular carriers is that they employ small generates COPII vesicles through a sequence of events [7] GTPases to direct coat assembly at the donor membrane. Dis- (Fig. 2). Assembly of the COPII coat is initiated through acti- tinct sets of coat proteins and small GTPases are used at differ- vation of the small Ras-like GTPase Sar1 [8]. Similar to other ent steps in the secretory pathway. At each vesicle formation small GTPases, conversion of Sar1-GDP to Sar1-GTP is step, secretory cargo proteins must be selectively and efficiently mediated by guanine nucleotide exchange factor (GEF). sorted into transport vesicles, while the composition of the Sec12 is an ER-bound transmembrane GEF for Sar1 [9,10]. donor membrane must be maintained. The process of accurate Sec12 is strictly regulated to localize to the ER, and thereby cargo packaging into transport vesicles represents a fundamen- Sar1 activation is restricted to the ER [11]. The GDP-to- tal and largely unresolved problem in cell biology; how are GTP transition triggers the exposure of the N-terminal amphi- specific proteins captured into a forming transport vesicle in pathic a-helix element of Sar1 that inserts into the ER mem- a highly selective manner? How does cell determine which pro- brane [12,13]. This N-terminal helix insertion into the teins to exclude and retain? membrane is a prerequisite for GTP exchange and thus the Transport of proteins along the secretory pathway starts GTP loading to Sar1 proceeds only in the presence of a mem- from the endoplasmic reticulum (ER). Secretory proteins are brane surface. Membrane-bound Sar1-GTP recruits Sec23- translated at the rough ER and translocated into the ER, where Sec24 heterodimer by binding to the Sec23 portion, and they undergo folding, assembly and post-translational modifi- Sec23/24-Sar1 complex selects cargo to form a so-called ‘‘pre- budding complex’’ [14]. Subsequently, the prebudding complex *Corresponding author. Fax: +81 48 462 4679. recruits Sec13-Sec31 heterotetramer onto the prebudding com- E-mail address: [email protected] (K. Sato). plex, providing the outer layer of the coat [15]. Sec13/31 is

0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.01.091 K. Sato, A. Nakano / FEBS Letters 581 (2007) 2076–2082 2077

Lysosome/Vacuole likely to cross-link the prebudding complexes and drive mem- brane deformation to form COPII vesicles (60–70 nm in diam- ER eter). Additional factors are known to contribute to the COPII Golgi assembly. Several studies indicate the roles for Sec16 and Sed4 in COPII vesicle formation. Sec16 is a large peripheral protein that associates with the ER membrane [16]. Although COPII vesicle formation can be minimally reconstituted using Sec23/24, Sec13/31, and GTP-locked Sar1 with non-hydrolyz- Endosome able GTP analog [17], Sec16 is shown to be an essential gene in S. cerevisiae [16]. In vitro COPII vesicle budding reaction per- formed with ER-enriched microsomes stripped of their Sec16 reveals that vesicle formation is significantly reduced. More- Plasma membrane over, a temperature-sensitive mutation in Sec16 causes tER fragmentation in Pichia pastoris and seems to be required for the integrity of tER sites. Sec16 contains domains that make direct contact with the COPII coat components and may act COPII coats as a scaffold for assembly of the coat [18,19]. Considerably COPII vesicle more work is required to fully understand the role of Sec16. Another related molecule isolated as a dosage-dependent sup- pressor of temperature-sensitive SEC16 mutations is Sed4. Sed4 is an integral membrane protein located at the ER mem- brane, and deletion of the SED4 gene from wild-type cells re- tards transport from the ER to the Golgi [20]. Although the cytoplasmic domain of Sed4 shares significant homology with Sar1p (small GTPase) that of Sec12, no GEF activity has been found [21]. This cyto- plasmic domain interacts directly with Sec16 at the ER mem- brane [20] and these factors are likely to function together, Fig. 1. Intracellular vesicular traffic and transport vesicles. Schematic though their roles are less understood. representation of organelles and protein transport routes in the secretory pathways. Transport vesicles (50–100 nm in diameter) are generated by the actions of both coat proteins and small GTPases. The 3. Cargo selection and ER export signals vesicle formation is initiated by the recruitment of cytoplasmic coat proteins to the surface of the donor membrane, which then induce deformation of the membrane into a coated vesicle. The COPII coat is Whereas some proteins are shown to exit the ER via a non- known to mediate export from the ER to either the ER–Golgi specific process known as ‘‘bulk-flow’’, this avenue seems to be intermediate compartment (ERGIC) or the Golgi complex. remarkably inefficient and contributes in a relatively minor

Fig. 2. COPII vesicle formation and the selective uptake of cargo proteins. The COPII vesicle formation is initiated by GDP–GTP exchange on Sar1 catalyzed by the transmembrane guanine nucleotide exchange factor Sec12. Activated Sar1-GTP binds to the ER membrane and recruits the Sec23/24 subcomplex. The cytoplasmically exposed signal of transmembrane cargo is captured by direct contact with Sec24, forming the ‘‘prebudding complex’’. It is currently not clear whether the membrane-bound Sar1-GTP associates with cargo before the recruitment of Sec23/24 or lateral diffusion of Sar1-GTP-Sec23/24 complex captures cargo. These prebudding complexes are clustered by the Sec13/31 subcomplex, generating COPII- coated vesicles. 2078 K. Sato, A. Nakano / FEBS Letters 581 (2007) 2076–2082 way to protein secretion [22]. The most of membrane and sol- an adaptor that links transmembrane protein, Axl2, to the CO- uble cargo proteins are enriched in COPII vesicles at concen- PII vesicle coat [38]. Vma21 mediates integral membrane com- trations À3- to 50-fold higher than the bulk flow markers, ponent (Vo) of the vacuolar H+-ATPase packaging into COPII and it is now quite evident that most secretory proteins are ac- vesicles [39]. Emp47, which accompanies type-I membrane tively sorted into COPII vesicles. This selective capture is basi- protein Emp46 out of the ER [40]. It is not clear why such cally driven by export signals within the amino acid sequence transmembrane secretory proteins depend on adaptor/receptor of each cargo protein contained on their cytoplasmically protein for ER export, though these proteins could in principle exposed regions [23]. Export signals of most transmembrane interact directly to the COPII coat subunit with an exposed cargo proteins are thought to interact directly with the COPII cytoplasmic domain. A possible explanation for the require- coat subunits, but some transmembrane and most soluble car- ment of adaptor/receptor protein is that the cytoplasmic region go proteins bind indirectly to COPII through transmembrane of such transmembrane cargo might interact with other factors cargo adaptors/receptors, offering a mechanistic explanation to generate a functional complex or contains a sorting signal for their specific capture into COPII vesicles. destined for downstream compartments. In those cases, addi- tion of the ER export signal may be incompatible with those 3.1. Transmembrane cargo functions, and an integral membrane adaptor/receptor may Several classes of ER export signals have been identified on be required for linkage to cytoplasmic COPII coat subunits. transmembrane cargo proteins in various organisms. For exam- ple, vesicular stomatitis virus glycoprotein (VSV-G), mammalian 3.4. Oligomeric assembly Kir2.1 potassium channel protein, and the yeast Sys1p and Many trafficking routes generally accommodate a smaller Gap1p possess cytoplasmically exposed di-acidic motifs subset of proteins, and employ a simple, well-defined signal. ((D/E)x(D/E), with x representing any amino acid residue) that In the ER, however, there is an enormous diversity in the cargo are required for efficient transport from the ER [24–26].Di- captured by the COPII coat and many exported proteins re- hydrophobic motifs (FF, YY, LL, or FY) comprising a simple quire multiple signals to be packaged into COPII vesicles, such combination of two adjacent hydrophobic residues at or near as a combination of any of the above motifs or homo- or hete- the C-terminus of a protein define a second class of ER export rooligomeric association. Well-known examples include the signals that are well-studied in p24 family proteins and the VSV-G, potassium channels, ERGIC-53, the Emp46/47 com- Erv41/46 complex [27,28]. In addition to the di-hydrophobic mo- plex, and the Erv41/46 complex [28,40–42]. The ER contains tifs, the tyrosine-containing motif is found in the membrane- a certain amount of newly synthesized unfolded or unassem- proximal region of the C-terminus of the ERGIC-53/Emp47 bled cargos, which should be segregated from secretory pro- family proteins [29,30]. Recent structural and biochemical char- teins to be exported. It is tempting to speculate that the acterization of cytoplasmic domains of several yeast SNARE COPII coat may not recognize a signal unless it is displayed proteins has identified additional export signals, YxxxNPF and in a specific assembly that is achieved only through proper LxxME motifs on Sed5 and LxxLE motif on Bet1 [31]. oligomerization, ensuring efficient incorporation of fully assembled cargos into COPII vesicles. 3.2. Soluble cargo On the other hand, selective export of soluble luminal cargo 3.5. Cargo recognition by the Sec24 would require specific transmembrane cargo receptors to medi- Such a diverse array of cargo proteins containing a variety of ate the interaction of cargo in the ER lumen with COPII sub- different sorting signals is specifically recognized by the COPII units in the cytoplasm. The ERGIC-53, a mannose-selective coat. A combination of structural, biochemical, and genetic and calcium-dependent transmembrane lectin, appears essen- approaches have revealed that these divergent signals are tial for the transport of a subset of glycoproteins, including recognized by distinct cargo binding sites on the Sec24 subunit blood coagulation factors, cathepsin C, and cathepsin Z of the Sec23/24 complex [31,43]. These studies indicate the [32–34]. Yeast Erv29 has been shown to be required for presence of at least three independent signal-binding sites on efficient packaging of the glycosylated a factor pheromone pre- the membrane-proximal surface of Sec24, termed ‘‘A-site’’, cursor (gpaf) into COPII vesicles and for efficient secretion of ‘‘B-site’’, and ‘‘C-site’’. The ‘‘A-site’’ recognizes a YxxxNPF carboxypeptidase Y (CPY) [35]. The pro region of gpaf has an motif on the SNARE, Sed5, the ‘‘C-site’’ binds to unidentified Erv29 recognition motif containing hydrophobic residues, motif on the SNARE, Sec22, whereas the ‘‘B-site’’ interacts termed I-L-V motif [36]. According to database searches, other with multiple motifs: (D/E)x(D/E), and Lxx(L/M)E found on soluble secretory proteins seem to bear such hydrophobic res- the SNARE, Bet1 and Sed5. Moreover, the existence of idues in similar positions, but much less are known about ER various subtypes of Sec24 expands the cargo multiplicity cap- export signal on soluble secretory cargo. tured by COPII coat. Several distinct Sec24 family members, Iss1p and Lst1p in yeast, are able to pair with Sec23 and bind 3.3. Cargo-specific adaptor protein to export signals different from those recognized by Sec24 Similar to soluble cargo proteins, GPI-anchored proteins ex- [44–46]. For instance, COPII vesicles generated in vitro with pose no amino acid residues to the cytoplasm, suggesting a Lst1 in place of Sec24 showed that a specific subset of cargo requirement for a transmembrane adaptor/receptor. Yeast proteins was absent from these vesicles, suggesting distinct car- p24 protein, Emp24, is likely to function as the adaptor/recep- go specificities for the different isoforms of Sec24. Moreover, a tor for efficient ER exit of the GPI-anchored luminal cargo number of cargo proteins are unaffected in those uptakes into Gas1 [37]. On the other hand, some transmembrane cargo pro- vesicles generated in the presence of the mutant forms of teins with cytoplasmically exposed region(s) are known to de- Sec24. This observation suggests that additional cargo recogni- pend on adaptor/receptor for ER export. Yeast Erv14 serves as tion sites will map to Sec24, and also to either Sec23 or Sar1. K. Sato, A. Nakano / FEBS Letters 581 (2007) 2076–2082 2079

In fact, recent studies have suggested that Sar1 also plays a role prebudding complex polymerization. How kinetically stable in cargo recognition by direct binding to the export signals prebudding complexes are maintained in the presence of GAP [47]. during prebudding complex polymerization? Recent in vitro study with liposomes have shown that the Sec23/24 is main- tained on the membrane by the presence of the GEF Sec12, 4. Sar1-GTP hydrolysis and cargo selection which counteracts the GTPase-stimulating activity of the coat by continually recharging Sar1 with GTP [51]. Furthermore, re- In vitro reconstitution experiments using fractionated ER cent kinetic data from our laboratory with cargo-reconstituted membranes or synthetic liposomes have defined components proteoliposomes have demonstrated that the Sec23/24 can re- required for generation of COPII vesicles; these are Sec23/24, main transiently associated with properly assembled cargo even Sec13/31, and GTP-locked Sar1 with a non-hydrolyzable after Sar1 GTP hydrolysis, while the interaction between Sec23/ GTP analog [7,17]. Therefore, the GTP hydrolysis by Sar1 is 24 and unassembled cargo or phospholipid on membranes is dispensable for coat polymerization and subsequent COPII disrupted immediately upon Sar1-GTP hydrolysis [52]. In the vesicle formation. Instead, COPII vesicles shed their coats be- presence of Sec12, stable binding of Sec23/24 with assembled fore fusion with cis-Golgi membrane and this uncoating reac- cargo molecules has been observed due to continuous reactiva- tion is thought to be achieved by the Sar1 GTP hydrolysis tion of Sar1 to its GTP-bound form before Sec23/24 release. In [48]. GTP hydrolysis by Sar1 is mediated by the activity of contrast, continuous Sec23/24 binding and release occurs on GTPase activating protein (GAP), which accelerates GTP unassembled cargo and phospholipids accompanied by Sar1 hydrolysis and reverses the Sec23/24-Sar1 interaction. A para- GTPase cycles. Thus, the Sec23/24 stability is biased toward doxical implication of this mechanism is that the Sec23 subunit the prebudding complex containing properly assembled cargo of Sec23/24 complex is the GAP for Sar1 [49], and the GAP molecules during Sar1 GTPase cycles. In other words, Sar1 activity is further stimulated by recruitment of Sec13/31 [50]. selectively promotes exclusion of unassembled cargo molecules Therefore, assembly of the prebudding complex triggers disas- from forming COPII vesicles by virtue of its GTP hydrolysis sembly, which compromises the capture of cargo molecules and [53] (Fig. 3). This explains why Sec23/24 in prebudding com-

Fig. 3. A model for GTPase-driven proof-reading by Sar1. The assembled cargo has a high affinity for the Sec23/24 because of the combined export signals. This cargo-Sec23/24 association persists during the nucleotide exchange reaction of Sar1 catalyzed by Sec12 (A). In contrast, the Sar1 GTP hydrolysis dissociates the weak association between the coat and unassembled cargo before polymerizing into COPII coats (B). Thus, the prebudding complex stabilities are biased towards the complex including assembled cargo, ensuring that the fully assembled cargo is preferentially incorporated into COPII vesicles. 2080 K. Sato, A. Nakano / FEBS Letters 581 (2007) 2076–2082 plexes can remain associated with cargo proteins during Sec13/ the ER. Subsequently, procollagen-I exits the ER in structures 31 mediated clustering, and how cargo molecules can be con- containing Sec24, implicating that COPII proteins play a key centrated into COPII vesicles. role in the ER export of procollagen. One issue of interest is whether the COPII vesicle is sufficiently flexible to accommo- date a large cargo molecule. This may be achieved through 5. COPII vesicle budding and pinch-off the flexibility of COPII coat geometry. A recent cryo-elec- tron-microscopy study has claimed that Sec13/31 complex Sar1 serves not only as a spatial landmark that recruits CO- forms a cuboctahedral cage whose faces are squares or trian- PII coat components but also as a mediator of membrane gles [57]. With adequate combinations of squares and triangles deformation and vesicle fission. Recent results have shown that a closed cage is formed. Consequently, it is possible that mam- Sar1 alone can deform liposomes into long, narrow tubules malian COPII coats may be able to shape vesicles of much lar- about 26 nm in diameter [54]. This was achieved only with acti- ger size by coordinating the relative numbers of triangles and vated Sar1-GTP, suggesting an involvement of the N-terminal squares. However, despite the theoretical ability to form a amphipathic a-helix of Sar1. The capacity of the Sar1 to de- large vesicle carrier, no such COPII-associated large vesicles form liposomes into tubules is thought to correlate with its are ever observed in vivo [61]. Thus, although COPII compo- ability to generate vesicles. Insertion of the N-terminal helix nents are no doubt necessary for large cargo transport into the outer leaflet of the ER membrane would act to dis- in vivo, it remains unclear whether COPII coat cages the mem- place the lipid head-groups, and this asymmetric expansion brane encircling large macromolecular complexes or instead could promote curvature toward the cytoplasmic region by serves a more indirect role in transport carrier biogenesis. the bilayer couple mechanism [55,56]. Sar1-GTP then stimu- lates the binding of an inner shell of Sec23/24 to form prebud- ding complex. It has been shown in crystallization studies that 7. Involvement of COPII components in quality control the Sec23/24 complex has a concave surface that matches the and autophagy size of a 60-nm vesicle and is thus thought to facilitate mem- brane bending [13]. Such prebudding complexes only give a The ER has quality control machinery that discriminates be- random distribution of local membrane deformations because tween correctly folded proteins and misfolded ones, ensuring they have no ability to interact with each other. Sec13/31 alone that only properly folded and, in some cases, glycosylated or is shown to self-assemble in solution into COPII cage-like assembled into multiprotein complexes are transported out of structures [57], and hence the spherical shape of the COPII the ER [62]. The misfolded proteins are retrotranslocated from coat seems to be generated by prebudding complex clustering the ER to the cytoplasm and subjected to ER-associated by Sec13/31. Importantly, although Sec23/24 and Sec13/31 degradation (ERAD) by the ubiquitin-proteasome system. complexes can form budded vesicles, the buds rarely closed The requirement for the COPII machinery for the ERAD of sev- off to generate free vesicles when Sar1 is anchored in the nick- eral misfolded proteins has been suggested. Several soluble el-conjugated lipids containing liposomes by a polyhistidine (luminal) ERAD substrates are known to require ER-to-Golgi tag in place of N-terminal helix [54]. So, the neck of a COPII transport and retrieval back to the ER for degradation, but coated bud is not likely to break spontaneously and the N-ter- the relevance of such cycling between the ER and Golgi has minal helix of Sar1 may have an active role in membrane fis- not been explored [63,64]. Yeast Rer1p protein is known as a re- sion. Other experiments suggest that fission is more efficient trieval receptor for ER-resident transmembrane proteins in the when Sar1 hydrolyses GTP [58], suggesting that the Sar1 Golgi. For its role in ER quality control, there is evidence that GTP hydrolysis may play some roles in vesicle fission. Further Rer1p is responsible for the ER retrieval of unassembled iron analyses are needed to determine how Sar1 N-terminal helix transporter subunits from the Golgi [65]. However, most mem- initiates and completes the fission of a COPII vesicle. brane ERAD substrates are retained in the ER and sequestered into specialized ER subdomains before degradation [66–68]. 6. Transport of large cargo Strikingly, recent study has implicated that this sequestration and degradation of the membrane ERAD substrate are im- As visualized by electron microscopy, in vitro generated CO- paired by mutations in sec12, sec13, and sec23 strains, but not PII vesicles are approximately 60–70 nm in diameter [7]. How- by the mutation in sec18 (mutant in transport vesicle fusion) ever, some specialized mammalian cells secrete large biological [69]. These results suggest that the sequestration and degrada- complexes from the ER such as a 400 nm chylomicron or a tion of membrane ERAD substrates require the COPII compo- 300 nm procollagen-I bundle. Such large cargo assemblies nent but are independent of ER-to-Golgi traffic. Although there would clearly be too large for transport within conventional remains possibility that a defect in ER-to-Golgi transport leads COPII vesicles. Recent work has shown that in vitro produced to an overall perturbation of the ER, COPII components are pre-chylomicron transport vesicles (PCTVs) contain both likely to mediate sorting of misfolded substrates into subcom- COPII subunits and common cargo molecules [59]. However, partments of the ER from which they are subsequently de- COPII components are seen to be dispensable for generation graded. of PCTVs. Instead, COPII is necessary for creation of PCTVs In addition to ERAD system, studies on autophagic path- that can subsequently fuse with Golgi membranes, suggesting ways have demonstrated the contribution of the COPII com- that some factor necessary for fusion with the Golgi apparatus ponent in autophagy [70,71]. Autophagosome formation is does not included in the absence of COPII components. It is completely blocked when some early Sec proteins such as also implicated that COPII proteins are involved in the ER ex- Sec12, Sec24, Sec23, and Sec16, are defective. The blockage port of procollagen-I [60]. Cells transfected with a dominant- of autophagy in these mutants was not dependent on mem- negative mutant of Sar1 displayed procollagen-I retention in brane flow from the ER to the Golgi, because autophagosome K. Sato, A. 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