Traffic 2010; 11: 303–310 © 2010 John Wiley & Sons A/S

doi:10.1111/j.1600-0854.2009.01026.x

Review

New Insights into the Structural Mechanismsof the COPII Coat

Christopher Russell and Scott M. Stagg∗

Institute for Molecular Biophysics, Department ofChemistry and Biochemistry, Florida State University,Tallahassee, FL 32306, USA*Corresponding author: Scott M. Stagg,sstagg@fsu.edu

In eukaryotes, coat protein complex II (COPII) proteins

are involved in transporting cargo proteins from the

endoplasmic reticulum (ER) to the Golgi apparatus. The

COPII proteins, Sar1, Sec23/24, and Sec13/31 polymerize

into a coat that gathers cargo proteins into a coated

vesicle. Structures have been recently solved of individual

COPII proteins, COPII proteins in complex with cargo,

and higher-order COPII coat assemblies. In this review,

we will summarize the latest developments in COPII

structure and discuss how these structures shed light on

the functional mechanisms of the COPII coat.

Key words: coated vesicle, COPII, Sar1, Sec13/31,

Sec23/24, secretory pathway, structure

Received 30 September 2009, revised and accepted for

publication 3 December 2009, uncorrected manuscript

published online 7 December 2009, published online 8

January 2010

A distinctive feature of eukaryotic cells is the presence ofan endomembrane system enclosing various intracellularcompartments. Transport between these compartmentsis mediated by coat protein complexes such as COPI,COPII, and clathrin (1). Coat protein complex II (COPII) isinvolved in transporting proteins from the endoplasmicreticulum (ER) to the Golgi apparatus. The basicfunctional units of the COPII coat are the proteins Sar1,Sec23/24, and Sec13/31 (2). X-ray crystal structures ofeach of these proteins have been solved, and cryogenicelectron microscopy (EM) structures have been solved ofassembled COPII coats and cages. Here we will describestructural features of the COPII coat protein complexand discuss how these characteristics give rise to theirfunctional mechanisms.

The Structure of Sar1

Sar1 is involved in regulating the formation of COPII vesi-cles at the ER. Sar1 is a GTPase in the Ras superfamily of

GTPases and, as such, functions as molecular switch con-trolled by the exchange of GDP and GTP. Sar1 is cytosolicin its GDP form and is recruited to the ER by the exchangeof GDP for GTP. The GTP exchange factor (GEF) for Sar1 isSec12, which is a membrane protein that is localized to theER. Thus, the localization and activity of Sec12 promotesthe localization of Sar1 to the ER. Upon exchanging GDP toGTP, Sar1 exposes an amphipathic α-helix which becomesburied in the ER membrane. Sar1•GTP then recruitsSec23/24 which in turn recruits Sec13/31, and these to-gether promote the formation of a COPII-coated vesicle.

The structure of Sar1 has been characterized by X-raycrystallography (3–5) (Figure 1, Table 1). This revealedSar1 to have four distinct domains: a guanine nucleotidebinding pocket, switch I, switch II, and N-terminal amphi-pathic α-helix each of which play a specific role in Sar1function (Figure 1). The core of the protein is com-prised of six β sheets (five parallel [β6,5,4,1,3] andone antiparallel [β2]) that form the guanine nucleotide-binding pocket (the G domain) typical of the Ras super-family with a characteristic highly conserved nucleotide-binding motif GxxxxGKT39 (5). The G domain is essentialfor Sar1 function; mutation of Thr39 to Asn creates aGDP restricted dominant negative mutant by interferingwith the association to its guanine nucleotide exchangefactor, Sec12 (6). The switch I domain mediates theinteractions between the guanine nucleotide and Mg2+.Switch I encompasses residues 44–57 which are locatedclose to the guanine nucleotide (Figure 1, red). Mg2+ iscoordinated by the β phosphate of GDP and the hydroxylof a conserved Thr54 residue in switch I (3). Switch II(residues 76–89) plays an important role in the hydrolysisof GTP (Figure 1, blue). In the structure of Sar1 boundto GppNHp, Mg2+ is coordinated to the γ phosphateposition and interacts with the backbone of Gly76 (4). IfHis79 of the switch II region is mutated, so that it losesits ablility to H-bond with Asp34, Asp34 swings awayfrom His79 and Sar1 loses the ability to hydrolyze GTPthus inhibiting the disassembly of the COPII complex (5).The N-terminal amphipathic α-helix (α1′) is essential foranchoring Sar1 to the lipid bilayer (5,7,8). Sar1 in the GDPform keeps the α1′ helix retracted into a pocket formedby the β2–β3 hairpin linker region (Figure 1, orange). Inthe GDP bound conformation, a conserved Asp upstreamfrom switch II mimics the charges of the γ phosphate inGTP. Exchange of GDP for GTP causes a two residue shiftthat pulls switches I and II up to the active conformation(Figure 1B). This causes a 7 A shift of the linker (Figure 1)eliminating the binding pocket for α1′ and causing it to

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Figure 1: The structure of Sar1. A) Structure of Sar1 bound toGDP. The N-terminal α-helix is shown in green, switch I is shownin red, switch II is shown in blue, and the mobile linker regionis shown in orange. The atoms of the nucleotide are shown assticks. B) Structure of Sar1 bound to GppNHp. A conformationalchange can be observed where switches I and II interact with thenucleotide and induce a shift in the linker region.

shift into a conformation that exposes the hydrophobicregion which facilitates binding to the ER membrane (9).

In addition to tethering Sar1 to the ER membrane, α1′may be involved in membrane deformation and vesiclefission (10). Upon membrane binding, the hydrophilicface of the Sar1 amphipathic helix lies close to thephospholipids and the hydrophobic face buries in themembrane interior disrupting the lipid interactions andcausing a deformation of the membrane. Addition ofexcess Sar1 to semi-permeabilized cells induces theformation of tubules from the ER, and likewise, additionof Sar1 to liposomes in vitro without Sec23/24 orSec13/31 causes the liposomes to be extruded into longtubules (10–12). Mutation of the hydrophobic residues ofthe α1′ were shown to reduce the tubulation activity (10).In addition, it was also observed that these mutations leadto a reduction in the formation of free vesicles in an invitro budding assay, despite the fact that Sec23/24 andSec13/31 were recruited to the membrane (10). Giventhese data, it has been proposed that Sar1 may havea role in both the membrane deformation and vesiclefission (10,13). In the early stages of vesicle formationSar1 recruits Sec23/24 and together these may create alocal region of curvature that is later organized into a moreglobal curvature by Sec13/31 (10,13,14). After a vesicle isformed but has not yet budded from the ER, Sar1 maybe involved in the fission of the vesicle from the donormembrane, and this role would be analogous to dynaminin the clathrin-coated vesicle system (15).

The Structure of Sec23/24

The Sec23/24 heterodimer is responsible for bindingcargo and serving as the Sar1 GTPase activating protein

(GAP) (16). As such, it plays a central role in the orga-nization of the COPII coat. The importance of thisheterodimer is evident in the fact that human diseasescranio-lenticulo-sutural dysplasia, and congenital dys-erythropoietic anemia type II result from mutations inSec23 (17,18). Sec23/24 is recruited to the ER membraneby Sar1. Crystal structures of the Sec23/24 heterodimerrevealed it to have a slightly curved surface thatpresumably facilitates interaction with and curvature of theER membrane. The heterodimer of Sec23/24 has a generalbow tie shape as revealed first by Lederkremer et al. (19).The dimer is 150 A long and 100 A wide at the widest pointnarrowing down to 30 A wide at the ‘knot’, where Sec23and Sec24 come together. It is 40 A tall (away from themembrane) except for a protrusion on each protein thatextend up to 60 A. The Sec23/24 dimer has a concaveinner face that closely matches the arc of the 60 nmcages, likely favoring deformation of the membrane (4).Moreover, binding Sar1•GTP causes a subtle rotationaround trunk domains increasing the curvature ofSec23/24 by 1.5◦ (4). The concave inner face (membranefacing) is generally positively charged enforcing therequirement for acidic phospholipid for Sec23/24-Sar1binding (20), while the outside is generally more acidic. Asingle Sec23/24 complex would cover ∼8200 A 2 roughly0.7% of the surface area of a 60 nm vesicle.

Table 1: COPII structures described in this review

PDB ID/Protein complexes EMDB ID References

Sar1Sar1H79G • GDP 2FA9 1Sar1 • GDP, low Mg2+ 2FMX 1Sec23 • Sar1 • GppNHp 1M2O 2Sar1 • GDP 1F6B 3

Sec23/24Sec23/24 1M2V 2Sec31(fragment) • Sec23 • Sar1 •

GppNHp 2QTV 4Sec23 • Sar1 • GppNHp 1M2O 2Sec23a/Sec24d • syntaxin 5 IxM

exit code peptide 3EFO 5Sec23a/Sec24d • membrin IxM

exit code peptide 3EG9 5Sec24 • Bet1 LxxLE exit code

peptide 1PCX 6Sec24 • Sed5 YNNSNPF exit code

peptide 1PD0 6Sec24 • Sys1 DxE exit code peptide 1PD1 6Sec23/24 • Sec22 2NUT 7

Sec13/31Sec31 (residues 370-763) • Sec13 2PM6 8Sec31 (residues 1-411) • Sec13 2PM9 8

COPII coat and cageSec13/31 cuboctahedron 1232 9Sec13/31 • Sec23/24icosidodecahedron

1511 10

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Figure 2: The structure of

Sec23/24. A) Superposition ofSec23and Sec24 shows the highdegree of homology betweenthese proteins. B) Domains ofSec23and Sec24. The trunk(green), Zn-finger (orange), β-barrel (red), helical (yellow), andgelsolin-like (blue) domains arehighlighted. C) and D) Structure ofSec23/24 in complex with Sec31(red), Sar1 (gold), Sec22 (yellow),and peptides containing the DxE(purple), LxxL/ME (purple), IxM(blue), and YNNSNPF (orange)exit codes. In both (C) and(D) Sec23 is represented in blueand Sec24 is green and (D) showsthe membrane proximal surfaceof Sec23/24.

The individual structures of Sec23 and Sec24 are very sim-ilar even though they share only 14% sequence identity(Figure 2A). Both Sec23 and Sec24 fold into five distinctdomains: β barrel, zinc finger, α-helical region, trunkdomain, and carboxyl-terminal domain (4) (Figure 2B). Theβ barrel, 180 residues long, lies roughly parallel to themembrane. The zinc finger is 55 residues and is situatednext to the β barrel. The Zn cluster is situated towardsthe membrane contributing to the basic inner surfaceof the protein. The trunk domain is located between β1and β19 of the β barrel, and presumably lies along thelipid membrane. The trunk domain plays an importantrole in the binding of Sar1 and is involved in the inter-action of Sec23 with Sec24. The two proteins dimerizethrough contacts between β14 and the β14–β15 loop oftheir respective trunk domains. Furthermore, Phe385 andPro387 form van der Waals interactions with conservedresidues (181–183) of Sec23. This presumably allowsmultiple isoforms of Sec24 to form dimers with Sec23.

The GTPase activity of Sar1 is activated by the carboxyl-terminal gelsolin-like domain of Sec23. Sec23 identifiesSar1•GTP through interactions between switches I andII and the linker region of Sar1 and the helical andtrunk domains of Sec23 (Figure 2C,D). Sec23 activatesthe Sar1 GTPase by inserting an arginine side chain intothe active site of Sar1 neutralizing negative charges of thephosphate; this mechanism is referred to as an argininefinger and is a feature of many Ras family proteins (21).In addition to the arginine finger, the active fragment ofSec23 inserts Trp922 and Asn923 with the indole ring ofTrp922 oriented close to parallel with the imidazole ringof His77 of Sar1. His77 bonds to a nucleophilic watermolecule and is solvent accessible on one side of theHis77 indole ring (4).

The lifetime of GTP bound to Sar1 is around 30 secondswhen Sar1 is bound to Sec23, and the hydrolysis isaccelerated by an additional order of magnitude by thebinding of Sec13/31 (22). A crystal structure of Sec23 and

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Figure 3: The structure of

Sec13/31. A) Surface rep-resentation of the Sec13/31heterotetramer. Sec13 is shownin orange and light blue, andSec31 is shown in red and blue.B) WD40 domains of Sec13and Sec31. Sec31 donates theseventh blade to the 6-bladedβ-propeller of the Sec13 WD40domain. C) The α-solenoid motifsof two Sec31s dimerize by foldingback on each other to form acontinuous α-solenoid bundle.

Sar1 together with a fragment of Sec31 (Figure 2C,D)show that this enhancement is because of conformationalchanges in Sec23 and Sar1 induced by the binding ofSec31 (23). In the Sec23 Sar1•GTP complex Gln720 ofSec23 is oriented away from the active site. Upon bindingof Sec31, the Gln720 swings towards the active site andforms hydrogen bonds with Trp922 and Asn923. Thesenew bonds improve the orientation with His77 of Sar1 andshield the solvent accessible side of the H77 imidazolering. This indicates that Sec31 plays a structural role inimproving the rate of hydrolysis and does not provide anycatalytic activity itself (23).

Structures of Sec23/24 in Complex withCargo

Sec24 functions as the primary cargo adaptor elementof the COPII coat. Initial work on yeast Sec24 identifiedthree separate binding sites (A, B, and C) that controlledwhich cargo was selected (24,25). The presence ofmultiple binding sites allows for diversity in the numberof exit codes that Sec24 can bind, and multipleSec24 isoforms expand the range and specificity ofSec24/cargo interactions. Here we will discuss thestructural interactions that facilitate the binding of cargoby Sec24. In order to simplify the naming, for the purposesof this review, we will simply refer to the binding sites onSec24 by the exit code sequences they recognize.

One of the first exit codes to be determined is thesequence DxE in the vesicular stomatitus virus Gprotein (26). The DxE exit code is also found in the SNAREprotein Sys 1. Crystallographic analysis of a Sys 1 peptidecontaining the DxE sequence has localized the binding siteon Sec24 to a groove on the membrane proximal surfaceof Sec23/24 (24) (Figure 2D, purple ribbon). In mammals,the DxE sequence is recognized by Sec24 isoforms aand b which are 75% identical in sequence, respectively.

The same binding site that binds the DxE sequence alsorecognizes the sequence LxxL/ME (24) (Figure 2D, purpleribbon). This binding pocket is modified in Sec24 isoformsc and d, where Leu582 is substituted to aspartic acid whichprevents binding of the DxE and LxxL/ME sequence.

An exit code that is found in the SNAREs membrin andsyntaxin is IxM. This sequence is recognized by the Sec24c and d isoforms, where a binding pocket is formed on thedistal side of Sec24 in the helical domain (27) (Figure 2D,blue ribbon). This binding site is obstructed in Sec24a andSec24b due to a short β strand on the N-terminus thatruns across the pocket and inhibits the binding of the IxMsequence.

The binding site for the SNARE Sec22 is unusual becauseit does not seem to recognize a specific sequence butrather a conformational epitope (28) (Figure 3C,D, yellow).Sec22 has ‘open’ and ‘closed’ conformations and onlybinds its cognate partner SNARES in its open conforma-tion and only binds to Sec23/24 when it is closed. Thisindicates that COPII will only transport Sec22 when it isnot interacting with other SNAREs, and this could serve asa timing device for SNARE transport. Sec22 binds specif-ically to Sec24a and Sec24b isoforms but is excludedfrom Sec24c and Sec24d. Furthermore the binding sitefor Sec22 is shared between Sec23 and Sec24. To datethis is the only COPII cargo identified to require binding toboth Sec23 and Sec24.

A site recognizing the sequence YNNSNPF has been char-acterized in yeast but that site appears to be uniqueto yeast SNARE Sed5 (24) (Figure 2D, orange ribbon). Themammalian homolog to Sed5, syntaxin 5, does not containthe YNNSNPF sequence and instead appears to bind tothe IxM site on Sec24c and Sec24d (27). In yeast Sed5, theYNNSNPF sequence is only exposed in the open confor-mation for this SNARE, and this conformation is promotedby the formation of the tSNARE complex (i.e. when it is

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bound to Bos1 and Sec22). It is unclear however, how thisrelates to the Sed5 homolog syntaxin 5 in mammals sincethe YNNSNPF sequence is only found in Sed5.

The Structure of Sec13/31

Two complementary structures were recently solved thatelucidated the structure of the outermost layer of theCOPII coat: what we refer to as the COPII cage. Acrystallographic study determined the atomic structuresof Sec13 and Sec31 and showed how they are organizedinto a heterotetramer, and a cryoEM study showed thatSec13/31 can self-assemble into a cage with the potentialto form a variety of structures that can accommodatecargos of widely varying shapes and sizes (14,29,30).Together these studies revealed the structural rules thatdictate the formation of the COPII cage.

Sec13 and Sec31 have long been known to associate witheach other (31), and evidence supported the idea that theyform a heterotetramer with two copies each of Sec13and Sec31 (19). Fath and colleagues (29) determined twoseparate structures that when combined revealed thecomplete structure of the Sec13/31 heterotetramer. Thefirst structure consisted of the N-terminal domain of Sec31bound together with Sec13. The other structure consistedof Sec13 bound to the C-terminal sequence of Sec31.They used Sec13 as a common alignment region andput the two structures together to form the completeSec13/31 heterotetramer (Figure 3A).

The Sec13/31 structure is composed primarily of WD40domains and α-solenoid motifs. WD40 domains consist ofa repeating β-propeller motif with the complete domaintypically being formed of seven β-propellers (Figure 3B).Sec13 is composed almost entirely of a WD40 domain,and Sec31 has a WD40 domain at its N-terminus with therest of the protein being comprised primarily of α-solenoidrepeats and a small unresolved proline-rich region. TheWD40 domain of Sec13 is unusual in that it contains onlysix blades instead of the usual seven. Remarkably, Sec31interacts with Sec13 by donating a seventh β-propellerblade into the WD40 domain of Sec13 (Figure 3B). Anotheroverlapping motif is observed in the Sec13/31 structurein the way that Sec31 forms a dimer with another Sec31.They form an interlocking and overlapping fold, wherethe C-termini of adjacent Sec31s in the heterotetramerloop back over each other, such that their adjacent α-solenoid motifs pack against each other and form acontinuous α-solenoid bundle via interprotein contacts(Figure 3C). Altogether, the protein/protein interactionsin the Sec13/31 heterotetramer are characterized byoverlapping and interlocking folds. One is tempted tospeculate that the degree of overlap may be important forstabilizing the heterotetramer in the cell so that the COPIIcoat can withstand the strains that may be encounteredin the cell during vesiculation.

The Structure of the COPII Cage

Sec13/31 self-assembles into a cage-like structure invitro, and these structures were solved by cryoEM andsingle particle reconstruction. This revealed the cage tobe a 600 A diameter cuboctahedron (30) (Figure 4A). Thediameter of the in vitro assembled cages is quite similarto that of COPII-coated vesicles which range from 500 to900 A (20). The cage structure exhibited a unique designfor cage complexes with four edges combining to formthe vertices of the cages as opposed to three edgeslike clathrin. The heterotetramer architecture of Sec13/31could be observed in the edges of the COPII cagestructure; the edge structure has two roughly sphericaldomains at one end, and these are related to two otherspherical domains by a 180◦ rotation around a continuouscurving region of density connecting the two (30). In theCOPII cage, the Sec13/31 heterotetramer is positionedoff-center with respect to the cuboctahedral vertices;that is, one end of the heterotetramer is closer to thevertex than the other end giving rise to a polarity in theedges. The end that is closer to the vertex is referredto as the plus end and the other end is the minusend (Figure 4B). The cages appear to be held togetherby relatively small-area protein–protein interactions atthe vertices, and these interactions contrast greatly withthe extensive overlapping arms of clathrin triskelia in theclathrin cage (32). The vertices in the COPII cage are heldtogether by interactions between adjacent Sec13 andSec31 WD40 domains (29,30) (Figure 4B). In contrast,clathrin cages exhibit extensively overlapping triskeliawhere every individual triskelion leg interacts with threedifferent cage edges and vertices (32,33).

The specific interactions mediating the formation of theCOPII cage were revealed by combining the crystalstructure of Sec13/31 with the EM structure of the COPIIcage. The Sec13/31 edge element, as it was crystallized,did not fit into the EM density. The EM density has a 135◦

bend in the middle of the edge while the crystal structureis much closer to straight with a bend angle of 165◦.A curve could be modeled into the crystal structure, andafter doing this, it fits very well into the contours of the EMstructure (14,29). Analysis of the crystal structure fit insidethe EM density showed that the vertices of the COPII cageare formed by interactions between adjacent Sec13 andSec31 WD40 domains. Four putative contact interfaceshave been identified that occur between WD40 domainsto facilitate the formation of the cage. Contact I (cI) occursbetween the WD40 domains of two adjacent Sec31 plusends. Contacts II and III (cII and cIII) occur between WD40domains of adjacent Sec31s at one plus end and oneminus end, and contact IV (cIV) occurs between WD40s ofSec13 at the plus end and the minus end of Sec31 (14,29)(Figure 4B). Thus it appears that interactions betweenWD40 domains facilitate the assembly of the COPII cage.

A larger COPII complex was observed when COPII cageswere assembled together with Sec23/24 (14) (Figure 4C).

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Figure 4: The structure of the COPII cage and coat. A) The structure of the Sec13/31 COPII cage from cryoEM. B) WD40domains mediate the formation of the vertices of the COPII cage. The vertices are formed through the interactions of four Sec13/31heterotetramers. One end of a given Sec13/31 heterotetramer is closer to its vertex than the other end, and it is called a ‘+’ end,while the other end is a ‘-’ end. Two angles are identified that dictate the COPII cage geometry; α occurs between a plus end and aminus end in a clockwise fashion, and β occurs between a minus end and a plus end in a clockwise fashion. C) The structure of theSec13/31•Sec23/24 COPII coat from cryoEM. Both (A) and (C) are colored by the magnitude of the EM density gradient. Regions withthe highest density gradient are colored red, while the lowest gradient are blue. D) and E) show the change in the β angle between thecuboctahedral COPII cage and the icosidodecahedral COPII coat. In (D)•β is 90◦, and this dictates the formation of the 600 A diametercuboctahedron. In (E)•β is 108◦, and this dictates the formation of the 1000 A diameter icosidodecahedron.

These structures were solved by cryoEM and singleparticle reconstruction, and this revealed a 1000 A diame-ter two-layered COPII assembly. Sec13/31 was observedto form the outer layer with Sec23/24 decorating theSec13/31 cage beneath the vertices. The outer cagestructure showed many similarities to the cuboctahedralCOPII cage assembled from Sec13/31 alone; the verticesare formed from the intersection of four edges, and theSec13/31 heterotetramers are off-center with respect tothe vertices. Even though these features are the same,the cage is quite a bit larger in diameter (1000 A versus600 A in the cuboctahedron structure) and forms with aicosidodecahedral geometry (Figure 4C). The cage accom-modates this larger size by changing one of the two anglesbetween edges at the vertices. The angles at the vertices

are identified as α and β, where α is the angle betweena plus end and a minus end in a clockwise manner andβ is the angle between a minus end and a plus end ina clockwise manner (Figure 4B,D,E ). α appears to befixed at 90◦, so this means that cages of different sizesare generated by varying the β angle. A demonstrationof this is seen in the transition from the cuboctahedralcage to the icosidodecahedral cage. The 600 A diametercuboctahedral cage has β angles of 90◦ and β expandsto 108◦ in the 1000 A diameter icosidodecahedral cage.It is not known what dictates the formation of particularβ angles, but Sec23/24 is located right beneath the cagevertices (Figure 4C,E ). It is speculated that Sec23/24 maybe involved in influencing the β angle in response to cargoof different shapes and sizes (14).

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The positioning of Sec23/24 with respect to Sec13/31also has relevance for the activation of the Sar1 GTPase.In the icosidodecahedral COPII coat structure, a clusterof four Sec23/24 dimers lies right beneath the Sec13/31vertices. The resolution of the EM structure was too lowto unambiguously determine the positioning of Sec23/24with respect to Sec13/31. Nonetheless, the approximatelocations of Sec23/24 indicate that it takes on two differentorientations relative to Sec13/31. One of the Sec23/24s inthe asymmetric unit of the cluster is much closer to theproline-rich region of Sec31 that increases the Sar1 GAPactivity than the other Sec23/24. This positioning suggeststhat only one of the two Sec23/24s in the asymmetricunit interacts with Sec31 and the other interacts withits neighboring Sec23/24. If this is the case, only one ofthe Sec23/24 dimers will have its GAP activity stimulatedby Sec31, and thus one Sar1 will have a short residenttime and the other will be longer lived. This idea dovetailsnicely with a study that showed that in the absenceof Sec13/31, Sar1 GTP hydrolysis precedes completeSec23/24 dissociation from membrane and cargo (34). Theauthors of this study suggested that multiple rounds ofSar1 GTP hydrolysis may serve to ‘proofread’ cargo beforeincorporation into a vesicle (34,35). In the model whereSar1 can have two residence times on a coated vesicle,the Sar1 molecules with a short resident time wouldpresumable fill the proofreading role. Further experimentsincluding higher resolution cryoEM reconstructions andreconstructions in the presence of Sar1 are required totest this model.

Transport of Large Cargoes

The variability of β angles has relevance to the transport oflarge or unusually shaped cargos such as procollagen andchylomicron particles. Procollagen is 3000 A in length (36)while chylomicron particles are greater than 4000 A indiameter both of which are too large to fit in the 1000 ACOPII cage (37). Indeed, early evidence suggested thatchylomicron particles and procollagen were transported bysome other means than COPII vesicles (37,38), but morerecent genetic and biochemical evidence support a rolefor COPII in transporting these unusual cargos. Mutationsin Sar1B have been shown to cause chylomicron retentiondisease (39,40), which is characterized by buildup ofchylomicron particles in the ER of enterocytes. Thefailure of chylomicrons to exit the ER because of aSar1 mutation strongly implicates COPII in their secretion.COPII also appears to be involved in the secretion ofcollagen. Depletion of Sec13/31 leads to the inhibitionof collagen secretion in fibroblasts and causes skeletalabnormalities in zebrafish (41). These abnormalities arephenotypically similar to developmental defects observedin cranio-lenticulo-sutural dysplasia, a human disease thatderives from a F382L substitution in Sec23A (17,42).Finally Saito et al. (43) have recently identified that theintegral membrane protein TANGO1 serves as an ERlumenal receptor that is involved in loading procollagen

into COPII-coated vesicles. These data support a role forthe COPII coat in the secretion of large cargos, but thequestion still remains if COPII is taking on a structural role,or if it simply serves to organize ER membrane domainsthat produce some other carrier besides a typical COPII-coated vesicle (38). If the COPII cage is to accommodatethese large procollagen or chylomicron particles, whichare bigger than the 1000 A diameter COPII coat structure,then the coat must be able to expand to accommodatethem. In other words, the β angle must be able to take onangles larger than 108◦. If β can accommodate angles upto 120◦, then any number of structures could be imaginedfrom planar polygonal sheets to helical tubes. The fact thatβ can take on different angles suggests that the COPII coathas the capacity to accommodate large cargo, but suchstructures have not yet been observed in vitro.

Conclusions and Future Perspectives

The last decade has seen much progress toward astructural understanding of the mechanisms employed bythe COPII coat, but we have only scratched the surface forthese complex and dynamic assemblies. Many questionsstill remain such as what are the molecular mechanismsby which the coat assembles and disassembles? Howis vesicle fission catalyzed? What is the structure of acomplete COPII-coated vesicle in complex with cargo?Answering these questions will require a combination oftraditional biochemistry and crystallography with cuttingedge biophysical and structural techniques.

Acknowledgment

This work was supported by AHA grant #0835300N.

References

1. Bonifacino JS, Glick BS. The mechanisms of vesicle budding andfusion. Cell 2004;116:153–166.

2. Barlowe C, Orci L, Yeung T, Hosobuchi M, Hamamoto S, Salama N,Rexach M.F., Ravazzola M., Amherdt M., Schekman R. COPII: amembrane coat formed by Sec proteins that drive vesicle buddingfrom the endoplasmic reticulum. Cell 1994;77:895–907.

3. Rao Y, Bian C, Yuan C, Li Y, Chen L, Ye X, et al. An open conformationof switch I revealed by Sar1-GDP crystal structure at low Mg2+.Biochem Biophys Res Commun 2006;348:908–915.

4. Bi X, Corpina RA, Goldberg J. Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat. Nature 2002;419:271–277.

5. Huang M, Weissman JT, Beraud-Dufour S, Luan P, Wang C, Chen W,et al. Crystal structure of Sar1-GDP at 1.7 A resolution and the role ofthe NH2 terminus in ER export. J Cell Biol 2001;155:937–948.

6. Weissman JT, Plutner H, Balch WE. The mammalian guaninenucleotide exchange factor mSec12 is essential for activation ofthe Sar1 GTPase directing endoplasmic reticulum export. Traffic2001;2:465–475.

7. Antonny B, Beraud-Dufour S, Chardin P, Chabre M. N-Terminalhydrophobic residues of the G-protein ADP-ribosylation factor-1 insertinto membrane phospholipids upon GDP to GTP exchange. Biochem-istry 1997;36:4675–4684.

8. Goldberg J. Structural basis for activation of ARF GTPase: mecha-nisms of guanine nucleotide exchange and GTP-myristoyl switching.Cell 1998;95:237–248.

Traffic 2010; 11: 303–310 309

Russell and Stagg

9. Pasqualato S, Renault L, Cherfils J. Arf, Arl, Arp and Sar proteins: afamily of GTP-binding proteins with a structural device for ‘front-back’communication. EMBO Rep 2002;3:1035–1041.

10. Lee MC, Orci L, Hamamoto S, Futai E, Ravazzola M, Schekman R.Sar1P N-terminal helix initiates membrane curvature and completesthe fission of a COPII vesicle. Cell 2005;122:605–617.

11. Aridor M, Fish KN, Bannykh S, Weissman J, Roberts TH, Lippincott-Schwartz J, Balch WE. The Sar1 GTPase coordinates biosyntheticcargo selection with endoplasmic reticulum export site assembly. JCell Biol 2001;152:213–229.

12. Bielli A, Haney CJ, Gabreski G, Watkins SC, Bannykh SI, Aridor M.Regulation of Sar1 NH2 terminus by GTP binding and hydrolysispromotes membrane deformation to control COPII vesicle fission. JCell Biol 2005;171:919–924.

13. Lee MC, Miller EA. Molecular mechanisms of COPII vesicle forma-tion. Semin Cell Dev Biol 2007;18:424–434.

14. Stagg SM, LaPointe P, Razvi A, Gurkan C, Potter CS, Carragher B,Balch WE. Structural basis for cargo regulation of COPII coatassembly. Cell 2008;134:474–484.

15. Pucadyil TJ, Schmid SL. Conserved functions of membrane activeGTPases in coated vesicle formation. Science 2009;325:1217–1220.

16. Yoshihisa T, Barlowe C, Schekman R. Requirement for a GTPase-activating protein in vesicle budding from the endoplasmic reticulum.Science 1993;259:1466–1468.

17. Boyadjiev SA, Fromme JC, Ben J, Chong SS, Nauta C, Hur DJ, et al.Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutationleading to abnormal endoplasmic-reticulum-to-golgi trafficking. NatGenet 2006;38:1192–1197.

18. Schwarz K, Iolascon A, Verissimo F, Trede NS, Horsley W, Chen W,et al. Mutations affecting the secretory COPII coat componentSEC23B cause congenital dyserythropoietic anemia type II. Nat Genet2009;41:936–940.

19. Lederkremer GZ, Cheng Y, Petre BM, Vogan E, Springer S, Schek-man R, et al. Structure of the Sec23p/24p and Sec13p/31p complexesof COPII. Proc Natl Acad Sci USA 2001;98:10704–10709.

20. Matsuoka K, Orci L, Amherdt M, Bednarek SY, Hamamoto S, Schek-man R, Yeung T. COPII-coated vesicle formation reconstituted withpurified coat proteins and chemically defined liposomes. Cell1998;93:263–275.

21. Wennerberg K, Rossman KL, Der CJ. The Ras superfamily at a glance.J Cell Sci 2005;118:843–846.

22. Antonny B, Madden D, Hamamoto S, Orci L, Schekman R. Dynamicsof the COPII coat with GTP and stable analogues. Nat Cell Biol2001;3:531–537.

23. Bi X, Mancias JD, Goldberg J. Insights into COPII coat nucleationfrom the structure of Sec23.Sar1 complexed with the active fragmentof Sec31. Dev Cell 2007;13:635–645.

24. Mossessova E, Bickford LC, Goldberg J. SNARE selectivity of theCOPII coat. Cell 2003;114:483–495.

25. Miller EA, Beilharz TH, Malkus PN, Lee MC, Hamamoto S, Orci L,Schekman R. Multiple cargo binding sites on the COPII subunitSec24p ensure capture of diverse membrane proteins into transportvesicles. Cell 2003;114:497–509.

26. Nishimura N, Bannykh S, Slabough S, Matteson J, Altschuler Y,Hahn K, Balch WE. A di-acidic (DXE) code directs concentration of

cargo during export from the endoplasmic reticulum. J Biol Chem1999;274:15937–15946.

27. Mancias JD, Goldberg J. Structural basis of cargo membrane proteindiscrimination by the human COPII coat machinery. EMBO J2008;27:2918–2928.

28. Mancias JD, Goldberg J. The transport signal on Sec22 for packaginginto COPII-coated vesicles is a conformational epitope. Mol Cell2007;26:403–414.

29. Fath S, Mancias JD, Bi X, Goldberg J. Structure and organization ofcoat proteins in the COPII cage. Cell 2007;129:1325–1336.

30. Stagg SM, Gurkan C, Fowler DM, LaPointe P, Foss TR, Potter CS,et al. Structure of the Sec13/31 COPII coat cage. Nature2006;439:234–238.

31. Salama NR, Yeung T, Schekman RW. The Sec13p complex andreconstitution of vesicle budding from the ER with purified cytosolicproteins. EMBO J 1993;12:4073–4082.

32. Fotin A, Cheng Y, Sliz P, Grigorieff N, Harrison SC, Kirchhausen T,Walz T. Molecular model for a complete clathrin lattice from electroncryomicroscopy. Nature 2004;432:573–579.

33. Stagg SM, Lapointe P, Balch WE. Structural design of cage andcoat scaffolds that direct membrane traffic. Curr Opin Struct Biol2007;17:221–228.

34. Sato K, Nakano A. Dissection of COPII subunit-cargo assembly anddisassembly kinetics during Sar1p-GTP hydrolysis. Nat Struct Mol Biol2005;12:167–174.

35. Tabata KV, Sato K, Ide T, Nishizaka T, Nakano A, Noji H. Visualizationof cargo concentration by COPII minimal machinery in a planar lipidmembrane. EMBO J 2009;28:3279–289.

36. Bachinger HP, Doege KJ, Petschek JP, Fessler LI, Fessler JH. Struc-tural implications from an electronmicroscopic comparison of pro-collagen V with procollagen I, pC-collagen I, procollagen IV, and aDrosophila procollagen. J Biol Chem 1982;257:14590–14592.

37. Siddiqi SA, Gorelick FS, Mahan JT, Mansbach CM II. COPII proteinsare required for golgi fusion but not for endoplasmic reticulumbudding of the pre-chylomicron transport vesicle. J Cell Sci 2003;116:415–427.

38. Fromme JC, Schekman R. COPII-coated vesicles: flexible enough forlarge cargo? Curr Opin Cell Biol 2005;17:345–352.

39. Jones B, Jones EL, Bonney SA, Patel HN, Mensenkamp AR,Eichenbaum-Voline S, et al. Mutations in a Sar1 GTPase of COPIIvesicles are associated with lipid absorption disorders. Nat Genet2003;34:29–31.

40. Shoulders CC, Stephens DJ, Jones B. The intracellular transport ofchylomicrons requires the small GTPase, Sar1b. Curr Opin Lipidol2004;15:191–197.

41. Townley AK, Feng Y, Schmidt K, Carter DA, Porter R, Verkade P,Stephens DJ. Efficient coupling of Sec23-Sec24 to Sec13-Sec31drives COPII-dependent collagen secretion and is essential for normalcraniofacial development. J Cell Sci 2008;121:3025–3034.

42. Fromme JC, Ravazzola M, Hamamoto S, Al-Balwi M, Eyaid W, Boy-adjiev SA, et al. The genetic basis of a craniofacial disease providesinsight into COPII coat assembly. Dev Cell 2007;13:623–634.

43. Saito K, Chen M, Bard F, Chen S, Zhou H, Woodley D, et al. TANGO1facilitates cargo loading at endoplasmic reticulum exit sites. Cell2009;136:891–902.

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