Vesicle Coats: Structure, Function, And

Home , AP2 adaptor complex, Clathrin, Coatomer, Copi, COPII, SEC13

Review

Vesicle coats: structure, function, and

general principles of assembly

1 2 2 1,3

Marco Faini , Rainer Beck , Felix T. Wieland , and John A.G. Briggs

Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

Heidelberg University Biochemistry Center, Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany

Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

The transport of proteins and lipids between distinct The three best-characterized types of vesicular carrier

cellular compartments is conducted by coated vesicles. involved in intracellular trafﬁcking are distinguished by

These vesicles are formed by the self-assembly of coat their different coat proteins and their different trafﬁcking

proteins on a membrane, leading to collection of the routes. Clathrin-coated vesicles (CCVs) act in the late

vesicle cargo and membrane bending to form a bud. secretory pathway and in the endocytic pathway,

Scission at the bud neck releases the vesicle. X-ray COPII-coated vesicles export proteins from the endoplas-

crystallography and electron microscopy (EM) have re- mic reticulum (ER), and COPI-coated vesicles shuttle

cently generated models of isolated coat components within the Golgi organelle and from the Golgi back to

and assembled coats. Here, we review these data to the ER. Despite having different compartment speciﬁci-

present a structural overview of the three main coats: ties and different structural components, the mechanisms

clathrin, COPII, and COPI. The three coats have similar of their formation follow similar rules. The time and place

function, common ancestry, and structural similarities, at which vesicle formation occurs are most often regulated

but exhibit fundamental differences in structure and by small GTP-binding proteins. In these cases, vesicle

assembly. We describe the implications of structural formation is initiated by activation of a small GTPase,

similarities and differences for understanding the func- stimulated by speciﬁc guanine exchange factors. The

tion, assembly principles, and evolution of vesicle coats. small GTPase exposes an N-terminal amphipathic helix

that anchors the protein to the outer leaﬂet of the mem-

Transport vesicle formation brane, then recruits coat protein complexes that further

Eukaryotic cells segregate functions in membrane-delim- interact with cytosolic cargo-recognition sequences [2–4].

ited compartments. These intracellular compartments are In endocytosis, a small GTPase is not required for initia-

not static: they exchange proteins and lipids continuously tion; instead, the AP2 adaptor complex is recruited to the

in a directional and regulated manner [1]. The exchange of membrane by phosphatidylinositol phosphates (PIPs) [5].

material (cargoes) between compartments is mostly con- Coat protein complexes have a common organization: they

ducted by coated transport vesicles that bud from one can be functionally divided into adaptor and cage com-

membrane and fuse with another. Transport vesicles are plexes. In the case of clathrin or COPII, the adaptor

hence essential for maintaining organelle identity and complexes (including AP1–5, AP180, and the Golgi-local-

lipid homeostasis and for the secretion of proteins. izing, g-adaptin ear containing, ARF-binding (GGA) pro-

The formation of transport vesicles is mediated by teins for clathrin, and Sec23-24, for COPII) are ﬁrst

cytosolic coat proteins. These proteins can bind each other recruited to the membrane, followed by the cage com-

as well as the membrane of a compartment and can inter- plexes that polymerize to form the protein lattice or mesh-

act with cargoes. To form a transport vesicle, the coat work that constitutes the ‘cage’ of a coated vesicle. In the

proteins must collect cargo, must induce membrane bend- case of the COPI coat, the adaptor and cage complexes are

ing to form a coated bud, must coordinate membrane associated as a single heptameric complex, which is

scission to release a vesicle, and must then disassemble recruited to the membrane en bloc [6]. Assembly of the

to allow fusion of the vesicle with the target membrane. protein coat, in some cases with the assistance of other

The molecular mechanisms underlying these processes cellular machineries such as the actin cytoskeleton, leads

are, despite extensive research, still not fully understood. to concentration of the vesicle cargo and membrane cur-

Recent advances using structural biology approaches in- vature to form a bud. Additional activities, either present

cluding X-ray crystallography, cryo-EM, and cryoelectron within the coat proteins or mediated through the GTPase

tomography (cryo-ET) have given new structural insights [7,8], then induce scission at the neck of the bud, releasing

into the protein complexes involved (Box 1). Combining the vesicle from the donor membrane (Box 2). Lastly, the

structural and biochemical approaches is advancing our coat depolymerizes under the effect of GTP hydrolysis

understanding of the dynamic and complex mode of assem- mediated by GTPase-activating proteins (GAPs) [9] or

bly and disassembly of coated transport carriers. by GAP activity within the coat protein complex. Alterna-

tively, clathrin coats are destabilized by ATP hydrolysis of

Corresponding authors: Wieland, F.T. ([email protected]);

HSC70 [10]. Depolymerization uncoats the vesicle, mak-

Briggs, J.A.G. ([email protected]).

Keywords: coated vesicles; clathrin; COPII; COPI; structure; assembly. ing it competent for fusion with its target membrane.

Review Trends in Cell Biology June 2013, Vol. 23, No. 6

Box 1. Methods in structural biology of coats

Coat proteins are complex machineries that represent a challenge for can often be sorted from one another and analyzed separately. The

structural biology. They have a broad range of sizes (from 50 to 600 resulting electron density reveals the shape of the protein and may

kDa) and they are often flexible, because they have to interact with reveal structural differences between conformations. When combined

multiple cargo proteins and bend membranes. To understand them, it with atomic models of individual subunits, pseudoatomic models can

is necessary to use a combination of diverse structural techniques be built to identify protein-interaction interfaces.

that span different sizes and resolutions. Cryo-electron tomography is a related technique whereby a unique

X-ray crystallography is a structural technique whereby a crystal, object is imaged from several directions by rotating it within the

formed from purified protein, is irradiated with X-rays and the electron microscope. These views are reconstructed as a 3D density

resulting diffraction pattern is interpreted to obtain an atomic model. map. It has been used to show the structure of unique assembled coated

Proteins can be cocrystallized with binding peptides or other proteins vesicles and to assess their heterogeneity. The resolution is limited to

to identify sites of interaction. The main limitation of the technique is about 40 A and is not the same in all directions (anisotropic resolution).

the requirement for the formation of a protein crystal. This involves Subtomogram averaging is an emerging structural technique based

selection of suitable protein constructs that will usually only include on local averaging of volumes extracted from electron tomograms.

stable, less flexible parts of a complex. For example, where cryo-ET has been applied to coated vesicles,

In single particle electron microscopy, thousands of noisy images subtomogram averaging can subsequently be used to identify and

of copies of the same biological object are combined in a 3D electron- average the many copies of the basic building block of the coat

density model. The sample is either stained with a heavy metal salt or contained within the tomogram. In this way, higher-resolution

frozen in vitreous ice, preserving all of its physiological conforma- structural data can be obtained, typically at a resolution of between

˚ ˚

tions. The technique is applicable to purified complexes, with an 20 A and 40 A, and the resolution is the same in all directions. The

effective minimum size limitation of about 150 kDa. The resolution positions at which the many copies of the structure were identified

attainable is limited by the conformational flexibility and size of the can be mapped in 3D in the original position in the tomogram to

˚ ˚

sample, but is typically between 4 A and 30 A. Where the sample observe, for example, the arrangement of the building blocks within

contains more than one conformation of the protein complex, these the assembled coat.

Here, we will describe and compare the structural Structural biology can help us to answer questions such

biology of the coats at multiple levels: their component as: ‘how is cargo identiﬁed and distinguished?’; ‘how does

proteins and protein domains, their cytosolic complexes coat polymerization form a curved protein shell?’; ‘how can

and subcomplexes, and their assembled cage-like forms. the same proteins form different-sized vesicles?’; and ‘how

In all cases, the structural biology has profound implica- is formation of a coated vesicle initiated and regulated?’.

tions for understanding function and mechanism. For other recent reviews of related topics, the reader is

Box 2. A function of coats in membrane scission involving GTPases?

Various models have been advanced to explain the function of that the vesicles were not released by a biochemical mechanism but

dynamin in endocytosis [60–63]. Repeated cycles of GTP loading and rather by mechanical shearing during the experimental preparation,

hydrolysis trigger conformational changes in a helical dynamin [64,70,71]. Nevertheless, we are convinced by the multiple reports

lattice on the bud neck. This leads to narrowing of the helix and showing that small GTPase-dependent COPI and COPII vesicles form

constriction of the bud neck [64]. Recent work suggests that the and are released without hydrolysis of GTP from native membranes

change in curvature at the boundary of the dynamin lattice causes an [33,68,72–74] or chemically defined liposomes [56,57,75]. Free vesicles

increase in the local elastic energy of the membrane, reducing the are observed by cryo-EM of incubations of liposomes with coat

scission energy barrier [65]. Scission therefore occurs at the edge of proteins and GTPgS under conditions where a ‘fission-arrest’ point

the dynamin helix. Another recent report on scission in clathrin- mutant of Arf1 yields no free vesicles but a series of buds that are

mediated endocytosis implicates epsin as a major contributor to continuous with the donor membrane [7]. A similar fission arrest was

scission [66]; shallow insertion of the amphipathic helix of epsin into observed in vitro for COPII when a Sar1p variant was used, lacking its

the membrane induced scission. Downregulation of epsin isoforms N-terminal amphipathic helix [76]. This suggests it is unlikely that GTP

led to fission arrest of clathrin-coated structures in mammalian cells, hydrolysis-independent scission is an experimental artifact. When

whereas slight overexpression of epsin could palliate a CCV scission directly compared in semi-intact cells, COPI and COPII vesicles are

defect on downregulation of dynamin. When dynamin disassembly formed (and released without any further manipulation) with GTP,

was blocked, epsin did not support formation of CCVs [66]. These GTPgS or GMPPNP or using constitutively activated small GTPases

findings hint that dynamin may play a regulatory role where its (our observation).

disassembly is required to allow shallow insertion of the epsin A role in regulating vesicle formation has recently been discussed

amphipathic helix contributing to scission. for arfaptin-1. Depending on arfaptin-1’s phosphorylation state, it

Like epsin, small GTPases tubulate liposomes in vitro using can sequester Arf1-GTP and antagonize the formation of insulin

amphipathic helices that are inserted shallowly into the outer leaflet granules in vivo or COPI vesicles in vitro [77].

of membranes, inducing bilayer curvature. In vivo, shallow inser- These findings suggest to us that, in all three systems – clathrin,

tion into a membrane does not necessarily cause curvature in the COPI, and COPII – scission may be mediated by the insertion of

absence of additional proteins, such as vesicular coat proteins, but amphipathic helices into the membrane by epsin, Arf1, or Sar1. This

can be a prerequisite for curvature. We define the contribution of mechanism would be independent of GTP hydrolysis (e.g., epsin is

shallow insertion of amphipathic helices into membranes as not a GTPase). There are many possible models of how such

‘potentiating membrane curvature’. In the COPII system, Sar1p insertion could drive scission. We have suggested [7] that insertion

was shown in vitro to potentiate membrane curvature [8], depend- of the amphipathic helices creates a high-energy state at the neck of

ing on its N-terminal amphipathic helix. In the COPI system, Arf1 forming buds in zones of growing negative curvature. The helices

with its myristoylated N-terminal amphipathic helix potentiates would be prevented from moving out of this zone by their

membrane curvature, strictly depending on dimerization of the interactions with the coat proteins, so the high-energy state would

small GTPase [67]. be relaxed by separation of the vesicular membrane from the donor

COPI- and COPII-coated vesicles were originally accumulated and membrane. Regulation of this process would then be mediated by

purified using non-hydrolyzable analogs of GTP [33,68,69]. This preventing access of the scissase to the neck of the bud (by dynamin)

independence of GTP hydrolysis was questioned with the suggestion or by sequestering the activated scissase (by arfaptin).

280

Review Trends in Cell Biology June 2013, Vol. 23, No. 6

directed to [9,11]. In this review, we do not deal with that interact with clathrin and with accessory proteins

accessory proteins like tethers, fusion factors, or regulators, [14,15]. Adaptor complexes are therefore able to link the

for which we refer the reader to recent reviews [12,13]. membrane, membrane-embedded proteins, and the cage

components.

Structures of coat components Clathrin heavy and light chains are the main structural

Vast efforts have been invested in unraveling the struc- components of the cage complex. The heavy chain com-

tures of protein components of the three archetypal coats. prises a long polypeptide composed of an N-terminal b-

X-ray crystallography (Box 1) has provided atomic models propeller domain followed by a stretch of a helices that

of coat fragments and also identiﬁed protein–protein inter- assemble into a long a-solenoid. Early rotary shadowing

actions involved in coat polymerization [14–16] and cargo and EM experiments revealed that clathrin heavy chains

binding [3,17]. In some cases, X-ray crystallography has form bent rods that trimerize at their C terminus (the hub

been combined with single-particle EM (Box 1) to derive domain) to form a triskelion (Figure 1a) [27]. Each leg of

the overall shape of coat subcomplexes [18,19]. the triskelion is about 50 nm long. In the past decade,

various parts of the molecule have been crystallized. First,

Clathrin coat components the N-terminal b-propeller was shown to contain binding

The coat of CCVs comprises two distinct complexes: an sites for adaptor proteins within its blades [16,28]. Then,

adaptor complex and a cage complex (Figure 1). There are the long a-solenoid – part of the hub domain – revealed the

multiple different adaptor complexes associated with dif- structural basis for interaction between clathrin heavy

ferent intracellular membranes, varying from the large chains and clathrin light chains [29]. Another crystal

heterotetrameric adaptors AP1 to AP5 to largely disor- structure revealed the clathrin heavy chain trimerization

dered proteins such as the monomeric Golgi-localizing, g- domain (Table 1) [30]. Overall, atomic structures describe

adaptin ear containing, ARF-binding (GGA) proteins [20] the structure of clathrin heavy and light chains, their

and AP180 [21]. Adaptors are recruited to the membrane interactions to form triskelia, and their interactions with

by GTPases or, in the case of AP2, by the membrane adaptor proteins. They reveal a complex whose structure

phospholipid PIP2 [5]. Among the best-characterized adap- directly tells the story of its function: the long, extended

tors are AP1 and AP2; AP1 mediates trafﬁcking between arms of the triskelion are ideal building material for a

the trans-Golgi network (TGN) and endosomes [22,23], protein cage.

whereas AP2 localizes at the plasma membrane and med-

iates endocytosis. Structures of AP1 and AP2 trunk COPII coat components

domains have been solved at atomic resolution [3,24,25], The COPII coat, like the clathrin coat, assembles sequen-

showing that they share a similar architecture: two large, tially. A membrane-bound activated Sar1 GTPase recruits

L-shaped subunits (a and b2, using the nomenclature of the adaptor complex Sec23-24, a heterodimer of about 200

the AP2 complex) assemble to form a pseudo-twofold sad- kDa [31]. Negative-staining single-particle EM showed

dle. The a subunit surrounds a small (s2) subunit and the that the heterodimer has a bow tie shape, with a rigid

b2 subunit interacts with the C terminus of the medium interaction between monomers [32]. The atomic model of a

subunit (m2) (Figure 1a). In the ﬁrst atomic model for the Sec23-24-Sar1 heterotrimer (assembled from crystal struc-

AP2 adaptor, the N terminus of the a2 subunit was bound tures of a Sec23-24 and of a Sec23-Sar1 complex) revealed

to an analog of PIP2 [24] and an additional PIP2-binding that, despite low sequence identity, Sec23 and Sec24 share

site was found on the m2 subunit, suggesting that the the same fold (Figure 1b) [17]. One side of the complex is

adaptor is competent for membrane binding at multiple positively charged and curved and may mediate binding to

positions. Interestingly, the previously mapped YxxF car- a curved membrane. The interaction between Sar1 and

go motif-binding site on m2 [26] was occluded in the struc- Sec23-24 positions this curved surface and the amphipath-

ture by the b2 subunit, rendering it inaccessible. For this ic helix of Sar1 such that they could both interact with the

reason, despite being able to interact with the membrane, membrane. Sec23 has a Sar1-GAP activity and the struc-

this conformation of the adaptor could not interact with ture revealed that it stimulates GTP hydrolysis by insert-

cargo proteins, leading to speculation that a conformation- ing an arginine ﬁnger into the GTPase. The Sec23-24

al change would be required for function. Several years complex has been solved bound to several different cargoes,

later, the structure of the adaptor AP2 in a different revealing that cargo-binding sites are distributed over the

conformation was described (Figure 1a, inset) [3]. A dra- complex, mostly around the edge of the membrane-binding

matic conformational difference had moved the C terminus surface.

of m2, exposing two endocytic motif-binding sites (YxxF The cage complex of the COPII coat is made of Sec13 and

and ExxxLL motifs) and four PIP2-binding sites. All of Sec31, which associate to form a dimer of heterodimers

these binding sites are coplanar, deﬁning the side of the [33]. EM of the full-length Sec13-31 complex showed a

complex that binds to the membrane. Together, these two 30 nm long linear association of four or ﬁve ﬂexibly at-

structures suggest that the complex ﬁrst interacts with the tached globular domains with the central domain acting as

membrane in a closed state through lipid binding and a a hinge [32,34]. The atomic structure of the heterotetramer

subsequent conformational change then exposes the endo- revealed that Sec31 has an N-terminal b-propeller and an

cytic motif-binding sites. extended a-solenoid, between which is found the b-propel-

The large adaptor components have C-terminal append- ler of Sec13 (Figure 1b) [35]. Tight dimerization mediated

age domains linked to the trunk of the adaptor by ﬂexible by the a-solenoid leads to formation of the long, rod-like

linkers. Structures of appendage domains describe regions structure. As in the clathrin triskelion, the a-solenoids are

281 2 6 of of

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2013,

TRENDS in Cell Biology chain

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Review Subunits in the the complexes shows (PIP2) as 282 Figure

Review Trends in Cell Biology June 2013, Vol. 23, No. 6

Table 1. References and database codes for relevant coat protein structures

Description Refs Database code

Clathrin coat

CHC, terminal domain ter Haar, 1998 [16] 1BPO

CHC, proximal segment Ybe, 1999 [29] 1B89

CHC trimerization domain and CLC Wilbur, 2010 [30] 3LVH

CHC and CLC, D6 basket cage Fotin, 2004 [18] EMD-5119

CHC and b arrestin peptide Kang, 2009 [28] 3GD1

AP2 trunk, closed state Collins, 2002 [24] 2VGL

AP2 trunk, unlatched state Kelly, 2008 [78] 2JKR

AP2 trunk open state, cargo bound Jackson, 2010 [3] 2XA7

AP2, a2 appendage Brett, 2002 [14] 1KY7

AP2, b2 appendage Edeling, 2006 [15] 2G30

AP1, trunk closed state Heldwein, 2004 [25] 1W63

COPII coat

Sec13-31 Fath, 2007 [35] 2PM6, 2PM9

Sec13-31, cuboctahedron cage Stagg, 2006 [19] EMD-1232

Sec13-31-Sec23-24, icosidodecahedron cage Stagg, 2008 [11] EMD-1511

Sec13-31-Sec23 cuboctahedron cage Bhattacharya, 2012 [55] EMD-5408

Sec23-Sar1 Bi, 2002 [17] 1M2O

Sec23-24 Bi, 2002 [17] 1M2V

Sec23-24 with SNARE peptides Mossessova, 2003 [79] 1PCX, 1PD0, 1PD1

Sec23-24 with cargo peptides Mancias, 2008 [4] 3EFO, 3EGD, 3EG9, 3EGX

Sec23-Sar1 with active fragment of Sec31 Bi, 2007 [36] 2QTV

COPI coat

b -a-COP Lee, 2010 [41] 3MKQ

a-e-COP Hsia, 2010 [43] 3MV2

g-COP appendage Watson, 2004 [80] 1R4X

g-z-COP-Arf1 Yu, 2012 [38] 3TJZ

Coatomer Yip, 2011 [44] EMD-5269

COPI triad and triad connections Faini, 2012 [57] EMD-2084 to EMD-2087

used to assemble an extended structure, appropriate for mutagenesis. Together, these data suggest that the COPI

building a cage. The cage complex interacts with the Sec23- adaptor subcomplex interacts with two molecules of Arf1,

24 complex via a disordered proline-rich domain of Sec31. conﬁrming earlier data that showed interactions between b-

This interaction further activates the Sar1-GAP activity of and g-COPs and Arf1 [39,40]. Based on these data and on

Sec23 by correctly orienting Sec23 residues that stimulate structural homology with AP2, a model for the orientation of

GTP hydrolysis, providing a ‘two-gear’ Sar1 activity: ﬁrst the COPI adaptor on the membrane was proposed [38].

gear when Sec23 binds and second gear when Sec31 binds The cage complex of coatomer comprises the large a and

[36]. b -COP subunits and the medium subunit e-COP. An

atomic model for a fragment of the COPI cage subcomplex

COPI coat components [41] shows that the N terminus of b -COP contains two b-

The COPI coat comprises coatomer, a 600-kDa cytosolic propellers followed by a curved a-solenoid. The a-solenoid

complex made of seven subunits (a- to z-COP). Unlike the intertwines with a symmetrical solenoid from the central

clathrin and COPII coats, coatomer is recruited en bloc as a region of a-COP in a manner similar to the Sec31 dimer-

single complex to the membrane [6]. Nevertheless, two ization interface (Figure 1b). a-COP is predicted to have

subcomplexes can be distinguished based on homology to the same domain organization as b -COP. This suggests

the other coats: the adaptor subcomplex (g-b-d-z-COPs), that a and b -COP together form an extended b-propeller-

homologous to the AP adaptors [37], and a cage-like sub- a-solenoid-a-solenoid-b-propeller structure, as in the

complex (a-b -e-COPs), which shares domain architecture COPII cage complex. The structures of the b-propellers

with the COPII cage comprising Sec13-31 (see below) of b -COP have also been solved bound to a KxKxx motif

(Figure 1c). [42]. This motif targets transmembrane proteins to assem-

X-ray crystallography has recently revealed the structure bling vesicles. The structure reveals how the motif is

of g-z-COP interacting with Arf1-GTP [38]. The ﬁrst 300 speciﬁcally recognized by electrostatically mediated con-

residues of g-COP adopt a curved a-solenoid, creating a tacts between b -COP and both the C terminus of the

binding pocket for z-COP. The association is structurally peptide and the lysine in position 3.

similar to the homologous subunits in the AP2 complex (a Interestingly, the b -a-COP complex [41] was found to

and s2). The g-z-COP-Arf1 structure revealed the residues assemble in the crystal as a triskelion where the trimeriza-

in g-COP responsible for Arf1 binding and equivalent resi- tion is mediated by b-propellers of b -COP (Figure 1c). This

dues could be identiﬁed based on homology in b-COP. A arrangement makes it tempting to speculate that the tri-

role for these residues in Arf1 binding was conﬁrmed by skelion is a key building block of the COPI coat. The smallest

283

Review Trends in Cell Biology June 2013, Vol. 23, No. 6

subunit of the cage subcomplex, e-COP, has a tetratricopep- the clathrin or the COPI coat adaptors, suggesting an

tide-repeat globular fold. The subunit interlocks with a b independent origin (Figure 1b). The cage complexes do

loop in the C terminus of a-COP and also forms a rod-shaped not share detectable sequence homology, but all three sys-

structure [41,43]. Understanding how these different com- tems contain a similar arrangement of protein domains: one

ponents can assemble to form a cage requires structural or two N-terminal b-propellers are followed by an elongated

study of the assembled cage (see below). a-solenoid [48]. b-propellers (comprising WD40 repeats)

A single particle reconstruction from cryo-negative-stain- (Figure 1) are often involved in mediating interactions with

ing EM (Box 1) showed that the cytosolic form of the com- protein motifs, making them appropriate for cargo recruit-

plete heptameric coatomer complex, including adaptor and ment [16,49]. a-Solenoids, due to their local ﬂexibility, can

cage subcomplexes, has the shape of a globular mass sur- arrange in straight rods (as in Sec31) or in bent conforma-

mounted by an extended domain [44]. The globular mass tions (clathrin and b -COP), making them ideal building

was assigned by antibody labeling and atomic ﬁtting to the blocks for cages. The association of a-solenoids and b-pro-

adaptor subcomplex, whereas the extended domain was pellers is also found in components of the nuclear pore

assigned to the outer cage subcomplex. The structural het- complex that interact with curved regions of the nuclear

erogeneity exhibited by even highly puriﬁed preparations of membrane, hinting at a common evolutionary origin of pores

coatomer does not allow higher-resolution reconstruction and coats [50]. Similarity between pores and coats is also

[44]. The cytosolic form of coatomer undergoes conforma- seen in the arrangement of the helices in the a-solenoid

tional change on assembling into a coat [45,46] and may at domain. A similar arrangement (called the ancestral coat-

this stage become more rigid in structure. omer element 1 domain) is seen in both the Nup85-Seh1

complex of the nuclear pore and in the Sec31 structure [51].

Structural homology between coats and evolutionary Despite the observed similarities in domain organization,

relationship the three cage complexes interact and polymerize to form

The three coat systems perform similar tasks on different remarkably different coat architectures, suggesting that

membranes, but how are they evolutionarily related? It is substantial divergence has occurred [48].

widely accepted that the tetrameric adaptors of clathrin

share distant sequence homology and structural similarity Structure of vesicles

with the adaptor subcomplex of COPI (b-, g-, d-, and z-COPs) Clathrin cages

[38,47]. Sequence similarities between g- and b-COPs and The ﬁrst high-resolution glimpse of how coat proteins

between z- and d-COPs suggest that the adaptor subcomplex assemble into a coated vesicle came from cryo-EM recon-

may originate from the duplication of a protodimer of a large struction of a clathrin cage assembled in vitro from

and a small subunit [37]. Conversely, the COPII adaptor puriﬁed cage components (Figure 2 and Box 1). The struc-

Sec23-24 has no sequence or structural homology to either ture of a hexagonal clathrin barrel with D6 symmetry could

CCV COPII COPI

TRENDS in Cell Biology

Figure 2. Assembly principles of clathrin, COPII, and COPI coats. Structures of protein cages from clathrin (EMD-5119), COPII (EMD-1232), and a COPI-coated vesicle. The

boxes show the vertices of the lattices where the position and orientation of repetitive units are symbolized by arrows. Red arrows indicate the repetitive units meeting in

the center of the box; blue arrows indicate repetitive units meeting at other vertices. In clathrin and COPII, all repetitive units meet with a fixed symmetry: threefold for

clathrin and twofold for COPII. Each unit makes the same number of interactions with other units. In COPI-coated vesicles, repetitive units adopt different conformations and

interact with different numbers of repetitive units. The purple triangles, symbolizing COPI triads (see text), meet with either two or three other triangles at each of their

corners. Modified from [57].

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CCV COPII COPI

90° 105°

TRENDS in Cell Biology

Figure 3. How coat proteins assemble vesicles of different sizes. Flexibility in the interactions between coat components (top) allows vesicle coats to form structures of

different sizes (bottom). In clathrin-coated vesicles (CCVs) (left), the interaction angle between the triskelia legs can be subtly modulated to change the curvature of the coat.

If the interaction angle between the clathrin legs is small (left), the clathrin lattice is more curved and the cages are smaller. If the angle is wider (a difference of 88), the

clathrin lattice is flatter and the cages are larger. For COPII (center), one of the angles (b) with which the Sec13-31 rods meet at the coat vertex can change [10]. If b is 908, the

protein cage has square faces forming a small cuboctahedron. If the angle is 1058, the cage has pentagonal faces forming a larger icosidodecahedron. In COPI (right),

positions where two triads (purple triangles) meet have higher local curvatures than positions where three triads meet. Local curvature is illustrated by yellow lines drawn

on slices through the vesicle surface. The ratio between twofold and threefold interactions therefore determines the size of the vesicles. Vesicles with more trivalent

interactions are larger than those with fewer trivalent interactions. Modified from [11,18,57].

accommodate the atomic structures of clathrin, leading to a Sec13-31 [35], showing that four Sec13-31 rods interact at

model for the assembly of clathrin triskelia [18]. Each each vertex. The vertices have twofold symmetry (Figure 2)

triskelion represents the vertex of a cage, with the legs and the in vitro assembled cage has the overall shape of a

of the triskelia intertwining to link the vertices together. cuboctahedron [19]. The interactions at the vertices of the

The interactions between the legs are mediated by a- cage are primarily mediated by b-propellers of Sec31 from

solenoids, similarly to elements of COPI and COPII coats. opposing edges (Figure 2). In a higher-resolution structure

The N-terminal b-propellers point inward toward the [19], a small interface between the b-propellers of Sec13

membrane and interact with adaptors [16] and cargo and Sec31 also appears to contribute to interactions at the

molecules [52]. The interaction at the triskelion vertex is vertex. A second single-particle EM structure showed that,

mediated by long a-helices that trimerize forming a tripod when incubated with Sec23-24, the cage complexes assem-

before onset of cage formation [30]. Threefold vertices and bled as a larger cage of the same polyhedral family, an

twofold edge interactions [53] create the cage, a lattice of icosidodecahedron (Figure 3) [11]. Both polyhedral struc-

hexagons and pentagons. tures have the same edge shape and assemble using simi-

To arrange six triskelia around a hexagonal hole or ﬁve lar twofold symmetric vertices. The different size is

triskelia around a pentagonal hole requires subtly different determined by a change in the angle with which the

angles between the legs of the triskelia. This is achieved by vertices meet (Figure 3) [11]. The rotation of edges is

changes in the distal ends of the triskelia arms, which form accommodated by a slight change in the interaction be-

sharper bends around pentagonal holes than around hexag- tween the b-propellers of adjacent Sec31 molecules. When

onal holes. Different arrangements of pentagons and hexa- Sec 13-31 cages are formed in the presence of only the

gons lead to cages of different sizes (Figure 3). In vitro, the Sec23 adaptor, the vertices adopt conformations charac-

cages generally have point-group symmetry, but there can teristic of both cuboctahedral and icosidodecahedral cages

be some deviation from this arrangement in puriﬁed CCVs, within the same cage. This leads to the assembly of cages

within which an irregular arrangement of hexagons and that do not have perfect point-group symmetry, expanding

pentagons, and indeed occasional heptagons, can be found the cage size distribution [55]. As for clathrin, in all of these

[54]. In all cases, however, the valence of interaction is structures the valence of interaction is conserved in cages

conserved – each clathrin molecule makes the same number of different size – each heterotetramer has the same num-

of interactions, three legs always meet at each threefold ber of interaction partners. Different-sized cages are

triskelion vertex, and the legs intertwine between vertices formed by small rotations around existing interfaces

in a twofold symmetrical manner. changing the angles between rods (Figure 3).

Due to the low resolution obtained in EM structures of

COPII cages cages assembled in the presence of the adaptor subunits

COPII cages assemble from rod-shaped Sec13-31 hetero- Sec23 and Sec24 (43 A), and due to the similarity between

tetramers. As for clathrin, the application of single-particle the Sec23 and Sec24 structures, the relative arrangements

EM methods (Box 1) to analyze cages assembled in vitro of the coat and adaptor complexes cannot be distinguished

allowed visualization of their 3D structure (Figure 2). This clearly [11,55]. It appears that there is more than one

structure could then be ﬁtted with the atomic structure of binding site on the cage subunits for the adaptor subunits.

285

Review Trends in Cell Biology June 2013, Vol. 23, No. 6

COPI vesicles to represent a budding scar and raises the possibility that

In contrast to CCV and COPII, the cage components of complete polymerization of the coat is not required for

COPI have not yet been assembled in vitro. Instead, we budding.

have recently described the structure of the complete COPI

coat assembled on membrane vesicles. To do this, COPI- Concluding remarks

coated vesicles were reconstituted from deﬁned proteins Different assembly principles are found in the three types

and liposomes [56] and imaged by cryo-ET [57] (Box 1 and of coated vesicle. In clathrin and COPII cages, the same

Figure 2). Such vesicles adopt a continuous size distribu- building blocks interact by making the same local contacts

tion (between 60 and 100 nm in diameter, including the with the same interaction valence. Changes in size of the

coat), suggesting that they need not have point-group cage are accommodated by local ﬂexibility of the triskelion

symmetry. Because the variability precludes averaging leg (clathrin) or by the interaction angle of different rods

of different vesicles, local 3D averaging was performed. (COPII). Clathrin cages puriﬁed from brain and COPII

3D volumes containing a fraction of the vesicle coat were cages assembled with the adaptor Sec23 can deviate from

extracted from tomograms and aligned to each other to point-group symmetry to form closed cages with a wide

identify the repeated unit of the coat (this method, ‘sub- size distribution. In COPI, triangular faces come together

tomogram averaging’, is described in Box 1). The average, in patterns of three or two; hence the same proteins can

with improved signal and resolution, revealed the symmet- make dimeric or trimeric interactions. Different patterns

ric basic unit of the coat, a triangularly shaped COPI triad. have different curvatures and smaller vesicles are

In a triad, three coatomer complexes are connected enriched for more curved patterns. Because membrane

through a thin central density platform. The triad interacts curvature is locally induced here, global symmetry is not

with the membrane bilayer below the coatomer complex enforced and a large range of vesicle arrangements and

and below the platform. Attempts to ﬁt the structure of sizes can be formed.

AP2 trunk, homologous to the coatomer adaptor complex, The structural determination of coat components and

into the cryo-ET density map failed, suggesting either that their arrangement on a vesicle is an ongoing challenge.

the coatomer adaptor has a different structure than AP2 or Crystal and EM structures have been proposed for most of

that its conformation on assembled vesicles is different the cytosolic components of clathrin and COPII coats.

from the one adopted by AP2 in crystals. Rigid body ﬁtting Atomic models of some coatomer subunits and a low-reso-

of the b -a-COP triskelion in the COPI triad suggest that lution EM structure of the holo complex were recently

either the triskelion does not represent the structural form described [38,41,43,44]. However, it remains unknown

of the coat or that the triskelion arms are differently how the subunits interact to form a coat lattice and how

oriented in the assembled structure [57]. A conﬁdent as- coats come together in different conformations during

sessment of the position of the coat components in the assembly. Numerous tasks lie ahead. High-resolution

assembled coat will therefore have to await higher-resolu- structures of the cytosolic states of coat complexes could

tion EM structures of the coat or more complete crystallo- reveal the mechanistic differences between coats; for ex-

graphic structures of coat subcomplexes. ample, the functional reasons for the en bloc recruitment of

Triads were found on the surface of vesicles in four the COPI coat as opposed to the sequential recruitment

characteristic patterns: three triads can meet with either that is characteristic of clathrin and COPII. In vitro bud-

their corners or their edges, or triads can meet with two ding reactions have been described both for the clathrin

corners, either singly or in a paired manner (purple trian- and the COPII system [58,59]. Applying cryo-ET and sub-

gles in Figures 2 and 3). This arrangement implies that in tomogram averaging techniques to these systems would

contrast to COPII and clathrin, the components of the reveal to what extent the assembly principles so far de-

COPI coat can have a variable valence of interaction; this scribed for membrane-free cages also apply to membrane-

means that coatomer, depending on its position on the coat derived vesicles. A high-resolution structure of the vesicu-

lattice, can form either homotrimeric or dimeric interac- lar COPI coat compared with a high-resolution structure of

tions. The change in interaction valences is made possible the cytosolic form would show the molecular basis for the

by substantial conformational changes. Depending on its conformational changes induced by cargo binding that lead

position within the lattice, the coatomer complex can adopt to coat assembly. Such structures would allow molecular

seven different conformations. The differences in confor- comparison of coat geometries and reveal the phylogenetic

mation are mostly concentrated at sites of interaction relationships between the main membrane-bending ma-

between triads [57]. chineries. Such structural analyses will need to be com-

In each of the triad patterns, the angle between the plemented by biochemical and functional studies of speciﬁc

adjacent triads is different, allowing the pattern to impose interactions of coat protein subunits.

or adapt to a speciﬁc local curvature on the underlying

membrane bilayer and therefore to coat vesicles of differ- Acknowledgments

The writing of this review was supported by a grant from the Deutsche

ent sizes. As a consequence, the more highly curved two-

Forschungsgemeinschaft within SFB638 (A16) to J.A.G.B. and F.T.W.

corner connections are enriched in smaller vesicles,

whereas the total number of three-corner connections

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