The structure of the COPII transport-vesicle coat assembled on membranes

Proteins often need to move between different compartments within cells. To do this they are packaged into transport pods called vesicles. Many trafficked proteins are synthesized in an organelle called the endoplasmic reticulum, or ER; these proteins are transported away from the ER in ‘COPII’ vesicles, which are formed when the COPII proteins assemble on the ER membrane and force it to bulge outward. The bulge pinches off from the ER membrane, forming the vesicle, which can then move to, and fuse with, a different compartment in the cell.

The COPII proteins assemble in a particular order to form the vesicle—Sar1 inserts into the membrane of the ER; Sec23 and Sec24 form an inner coat and capture the proteins that the vesicle will transport; and Sec13 and Sec31 form an outer coat. Although the structures of the coat proteins are known, how they bind to each other—and to the ER membrane—to form vesicles of many shapes and sizes is less well understood. Now, Zanetti et al. show how the inner and outer coat proteins can interact flexibly to accommodate a variety of cargoes.

Zanetti et al. mixed purified Sar1 and COPII coat proteins with artificial membranes in vitro. As in cells, the proteins assembled a coat on the membranes, creating bulges and vesicles of different shapes. These coats were imaged using an electron microscope, and the images were analysed using computational image-analysis methods. In this way, Zanetti et al. produced a detailed 3D view of the assembled coat.

It was found that the inner and outer proteins each arranged to form lattice structures—like fishing nets—which showed flexibility and variability in the way the individual proteins interact, as well as imperfections in the arrangement. Both coats may help to reshape the membrane, and the inner-coat and outer-coat lattices were also found to move with respect to each other. These flexible properties could allow the coat to assemble on membranes with different shapes and curvatures, forming COPII vesicles with distinct sizes and shapes that can carry a range of cargoes.

In eukaryotic cells, newly synthesized proteins are transported from the endoplasmic reticulum (ER) to the Golgi apparatus through the action of the coat protein complex II (COPII). Assembly of COPII coat proteins on the membrane leads to generation of coated membrane vesicles carrying cargo molecules. Vesicle formation proceeds via sequential assembly of the coat components. It is initiated by the small GTPase, Sar1. Upon exchange of GDP for GTP (catalysed by Sec12), Sar1 exposes an N-terminal amphipathic helix that inserts into the outer ER membrane leaflet, promoting curvature (Lee et al., 2005). Sar1 recruits heterodimers of Sec23 and Sec24 to the membrane, thereby forming the inner layer of the COPII coat (Matsuoka et al., 1998). Sec23/24 is an adaptor complex: Sec24 binds transport cargo while Sec23 interacts with Sar1 and recruits Sec13/31 (Miller et al., 2002; Bi et al., 2007). Sec13/31 heterotetramers constitute the outer coat layer, thought to polymerise into cages that enclose the budding membrane (Fath et al., 2007; Stagg et al., 2008). GTP hydrolysis on Sar1, activated by Sec23 and further accelerated by Sec31, completes the cycle by promoting fission of the bud and coat depolymerization (Zanetti et al., 2012).

X-ray crystallography has been used to obtain structural models for all the coat subunits (Bi et al., 2002; Fath et al., 2007). Available structural data for the inner coat is limited to isolated subcomplexes. Progress has been made in understanding how the outer coat subunits assemble into a coat by using single-particle cryo-electron microscopy to derive models of Sec13/31 cages formed in vitro under high salt conditions in the absence of membrane (Stagg et al., 2006, 2008; Bhattacharya et al., 2012; Noble et al., 2012). A comparison of the vertices and edges in cages of different sizes (60–100 nm) has suggested geometrical rules governing outer coat assembly, and indicated regions of flexibility in Sec13/31 that permit envelopment of vesicles with sizes ranging from 60 to 120 nm (Fath et al., 2007; Stagg et al., 2008; Bhattacharya et al., 2012). Nevertheless, a higher degree of flexibility in COPII architecture is needed to explain the ability of COPII to mediate secretion of cargoes such as 300 nm pro-collagen fibres, which are much larger than the 60–100 nm in vitro assembled cages. There is increasing evidence that incorporation of pro-collagen into COPII coated vesicles is a highly regulated process, and that modifications in the outer coat proteins—such as ubiquitination—as well as in timing of GTP hydrolysis and coat recycling may be necessary for COPII ability to coat large vesicles (Saito et al., 2009; Jin et al., 2012; Kim et al., 2012; Venditti et al., 2012). COPII-dependent defects in collagen transport are linked to a genetic syndrome characterized by late-closing fontanels, sutural cataracts, facial dysmorphisms and skeletal defects: Cranio-lenticulo-sutural dysplasia (Boyadjiev et al., 2006).

There are currently no structural data on the COPII coat assembled in its functional state on a membrane, leaving important mechanistic questions unanswered. How do the inner and outer coat components arrange to form a membrane coat? How do they interact with the membrane? How do they interact with each other? Are the existing models for the outer COPII coat cages representative of the complete coat assembled on a membrane? What is the structural basis for COPII ability to transport large or elongated cargoes such as pro-collagen? Here we address these questions. We provide evidence for coordinated assembly of the two layers of the coat on a lipid membrane and suggest how this interplay may effect shape changes essential to the capture of large and unusually-shaped secretory cargo complexes and particles.

To answer the questions above, it is necessary to understand the structural arrangement of the complete coat in its membrane bound form. To this end we have studied a reconstituted COPII assembly reaction (Bacia et al., 2011). We incubated giant unilamellar vesicles (GUVs) with yeast COPII proteins (Sec12, Sar1, Sec23/24, Sec13/31) in the presence of a non-hydrolysable GTP analogue GMP-PNP. We imaged the reaction mixture by cryo-electron tomography, observing that coated membranes of a variety of shapes and sizes were generated. These included round vesicles, either independent or still attached to the donor membrane, as well as curved or extended tubes, reminiscent of those detected budding from ER exit sites in vivo (Mironov et al., 2003; Fromme et al., 2007; Venditti et al., 2012; Figure 1). All of these structures were surrounded by two protein layers indicating that the COPII coat can assemble on membranes with a range of different curvatures. The outer coat appeared in sections as points or lines (Figure 1A), and in grazing slices as a regular arrangement of X-shaped features arrayed to form a rhomboidal lattice (a lozenge pattern) (Figure 1B,C). Other geometries, such as triangles and pentagons, were observed on vesicles and tube ends (Figure 1D). These geometries are similar to those formed when purified Sec13/31 proteins assemble in vitro in the absence of a membrane (Stagg et al., 2006, 2008). The inner coat layer also appeared as a regular array (Figure 1B), implying that Sar1 and Sec23/24 can also assemble to form a lattice.

Figure 1

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The structure of the outer coat

To understand the architecture of the Sec13/31 outer coat we applied reference free, contrast-transfer function (CTF) corrected, subtomogram averaging to solve the 3D structure of the vertex, and of the connecting rods to resolutions of ∼40 Å (Figure 2A–B, Figure 2—figure supplement 1, and ‘Materials and methods’). The vertex structure was a twofold symmetric X-shape, similar to that seen in in vitro assembled Sec13/31 cages (Stagg et al., 2006, 2008) (Figure 2D, Figure 2—figure supplement 2). The connecting rods are consistent in shape and size with Sec13/31 heterotetramers, and are bent in the middle by approximately 15°. This same bend is seen in the solved X-ray crystal structure, which can be fitted into the densities as a rigid body (Figure 2B,C). In contrast, there is a 45° bend in the rods of in vitro-assembled protein cages (Stagg et al., 2008; Figure 2E,F). These data indicate that the central hinge between Sec31 molecules can adapt to assemble coats of different curvatures.

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The published EM reconstructions of in vitro assembled COPII outer coat protein cages reveal that the Sec31 β-propeller domains at the ends of two Sec13/31 rods (referred to in the literature as the plus ends) contact each other directly at the center of the vertex, whereas the ends of the other two rods (the minus ends) contact the plus ends at the side and are more distant from the center of the vertex. The geometrical relationship of the ‘+’ and ‘−’ rod ends at the vertex can be described by two angles: alpha (clockwise between + and − ends, which is 60° in the cages), and beta (clockwise between − and + ends which is variable at least between 90° and 108° in the cages) (Figure 2A,B, Figure 2—figure supplement 2, Figure 3—figure supplement 1) (Stagg et al., 2008). Within the in vitro assembled cages each Sec13/31 rod makes a ‘+’ contact at one end and a ‘−’ contact at the other end. This arrangement allows assembly of a coat that curves in two directions, appropriate for coating spherical membranes. (Figure 3C, Figure 3—figure supplement 1). In our reconstructions, the arrangement of Sec13/31 rods we observed at the rounded tips of tubes and on spherical membranes (Figure 1D) was similar to that seen in in vitro assembled cages, consistent with the presence of such ‘+/−’ rods (Figure 3C). When we analysed the distribution of vertices (Figure 3A) and rods (Figure 3B) on the tubular membranes we found that they assembled regions of rhomboidal lattice. A rhomboidal lattice could be built in two ways: each vertex could be rotated by approximately 90° in the plane of the membrane relative to the adjacent vertices, or each vertex could have the same orientation (see schematic in Figure 3—figure supplement 2C). The final structure of the vertex on the tubes clearly shows the expected twofold features of the vertex previously described by Stagg et al. (Noble et al., 2012), indicating that the majority of vertices have the same orientation (‘Materials and methods’). In this arrangement of vertices the rods oriented in one direction (the right handed rods, purple in Figures 2C and 3B) each make two ‘+’ contacts (+/+), while the left-handed rods (green in Figures 2C and 3B) each make two ‘−’ contacts (−/−). The alpha angle is 79.7° ± 5.9° and is oriented around the tube circumference while the beta angle is 95.7° ± 5.8° and is oriented along the tube axis (Figure 3—figure supplements 2 and 3). Together these data imply that three properties contribute to outer coat adaptability: (i) variability of both alpha and beta angles at the vertices, (ii) flexibility of the central rod hinge, and (iii) the absence of any inherent asymmetry in the Sec13/31 rods, allowing them to make ‘+’ contacts at both ends, ‘−’ contacts at both ends, or a ‘+’ contact at one end and a ‘−’ contact at the other. Together this versatility allows coating of not only spherical, but also of tubular membranes and therefore accommodation of large elongated cargoes such as pro-collagen.

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Proteins able to form both spherical and tubular structures are found in other biological systems, most notably virus capsids. For example, the elongated heads of T-even phages, as well as the capsid cores of retroviruses, are assembled as closed structures built from hexamers and pentamers of the component protein and can have regions with spherical and with tubular curvatures (Baschong et al., 1988; Ganser et al., 1999). This structural flexibility can be understood within the framework of quasi-equivalence: while subunits are found in symmetrically different positions with different local curvatures, the contacts between the subunits are only subtly different (Caspar and Klug, 1962). In some virus capsids, subtle changes in the contacts are supplemented by structural switches that mediate the change from hexameric to pentameric assembly (Johnson and Speir, 1997). It seems likely that flexibility in the outer COPII cage geometry is primarily mediated by subtly varying contacts at multiple hinge positions in the rod and vertex (Noble et al., 2012), but we cannot rule out the presence of structural switches.

The structure of the inner coat

Our images showed that the inner coat can also form a regular lattice, suggesting it may not only function to link cargo and membrane to the outer coat, but also play a structural role in determining vesicle shape. We applied reference-free, CTF corrected, subtomogram averaging to solve the structure of the repeating inner coat unit with a resolution of approximately 23 Å (Figure 4A, Figure 4—figure supplement 1; ‘Materials and methods’ and Faini et al., 2012). The bow-tie-shaped repeating unit interacts tightly with the outer membrane leaflet. The units interact with each other to form an approximately helical array on the tube surface (Figure 4B). In many instances we see step changes in tube diameter, associated with displacement of a few subunits from the lattice (Figure 4B).

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We docked the crystal structures of Sec23/Sar1 (PDB 2QTV), and of Sec23/24 (PDB 1M2V) (Bi et al., 2002) into the subtomogram averaging electron density map. Both fit unambiguously into one bow-tie shaped subunit as rigid bodies, in each case placing Sec23 in the same position (‘Materials and methods’; Figure 5A). Analysis of the fitted models reveals the novel network of interactions that mediate lattice formation and membrane binding. Two interfaces between the long edges of the Sar1/Sec23/24 heterotrimer mediate formation of lattice rows. The larger involves Sar1, the gelsolin-like and the C-terminal domain of Sec23 from one heterotrimer interacting with the Zn-finger and part of the beta barrel domains of Sec23 in the adjacent heterotrimer. The smaller interface is between the Zn-finger domain of Sec24 in one heterotrimer and the gelsolin-like and C-terminal domains of Sec24 in the neighbouring heterotrimer (Figure 5B). These contacts are characterised by extended surfaces with opposite charges, a property that is conserved (Figure 5—figure supplement 1). The rows contact each other where the beta barrel domain of Sec23 approaches the Sec24 N-terminus. In the Sec23/24 crystal structure (PDB ID 1M2V), the Sec24 N-terminal helix 61-73 forms a crystallographic contact with the beta-barrel domain of Sec23 in the neighbouring asymmetric unit (Bi et al., 2002). When placed in this position, the 61–73 helix fills an unoccupied region of electron density in our structure, suggesting this contact is present in the assembled coat (Figure 5A,B, and ‘Materials and methods’).

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Our structure reveals that the concave surface of the inner coat heterotrimer is oriented towards the membrane and the Sec31 binding site towards the coat exterior (Figure 5A,C), as previously predicted (Bi et al., 2002). We identify two primary membrane interacting regions. One is formed by a small part of Sec23 together with Sar1, orienting Sar1 to direct its N-terminal amphipathic helix into the outer leaflet. The other is a large basic surface in Sec24, including the Sec24 Zn finger, beta barrel and alpha helical domains (Figure 5B). While the Sec24 Zn finger domain binds the membrane, the Sec23 Zn finger domain does not, instead it faces the neighbouring heterotrimer, suggesting that the role of the Zn fingers in Sec23 and Sec24 has diverged. Consistent with this difference in function, the Sec24 Zn finger, essential for cell survival (Peng et al., 1999), contains a conserved basic patch that is absent in Sec23. We speculate that, analogous to the basic Zn fingers in FYVE domains (Gaullier et al., 2000), this represents a phospholipid-binding site. The trunk domains of Sec23 and Sec24 do not contact the membrane but are suspended above it, leaving space that would allow binding to more highly curved vesicles (Figure 5B).

Inner coat assembly into lattices must remain compatible with the well-characterised cargo binding activity of Sec24. To test this, we modelled the X-ray structures of Sec24 co-crystallised with cargo-derived peptides (Mossessova et al., 2003; Mancias and Goldberg, 2007, 2008), or the cytosolic domain of a globular cargo Sec22 (Mancias and Goldberg, 2007) in our map. None of the characterised cargo-binding sites overlap with the protein–protein and protein–membrane interfaces we identified in the lattice: all remain accessible to cargo in the assembled inner coat (Figure 5C,D). Strikingly, comparison of the co-crystal structure of Sec23/24/Sec22 with our density map reveals that the space between heterotrimers in the assembled inner coat lattice forms a pocket into which Sec22 would bind (Figure 5C).

We cannot rule out that the large-scale order seen in the inner coat lattice is promoted by carrying out assembly in vitro in the absence of GTP hydrolysis. Three factors suggest, however, that the interactions we observe between heterotrimers are relevant in vivo and are not simply an artefact of in vitro assembly. Firstly, they result from a productive assembly reaction that bends the GUV membrane into a variety of shapes. Secondly, the interfaces have conserved electrostatic properties. Thirdly, the interactions that mediate inner coat lattice assembly are distinct from those that mediate membrane binding, outer coat binding, and cargo binding and can accommodate Sec22 in the correct position: they are fully compatible with available biochemical and structural data.

The relationship between inner and outer coats, and implications for assembly

The low resolution that can be obtained in cryo-EM reconstructions of Sec23/24 bound to in vitro assembled Sec13/31 cages, and the necessity of implying symmetry which may not be appropriate for both coat layers, has prevented a clear understanding of the structural relationship between the inner and outer coats (Stagg et al., 2008; Bhattacharya et al., 2012). Our structure of the outer coat does not reveal density corresponding to an ordered inner coat, and vice versa, indicating that the relative positions of the two coat layers are not fixed. Nevertheless, the two layers are approximately aligned both in spacing and orientation. The ‘rows’ in the inner coat are aligned to the left-handed rods of the outer coat, with four inner coat heterotrimers spanning one outer coat rod. The inner coat ‘columns’ are aligned to the right-handed outer coat rods, with two inner coat heterotrimers spanning one outer coat rod (Figure 3A,B, Figure 3—figure supplement 3). This relationship suggests that the unstructured C-terminal region of Sec31 (Fath et al., 2007; Noble et al., 2012), which connects the inner and outer coats, constrains the coat layers but does not fix their absolute positions, much like multiple anchor cables on a ship. This flexibility in the relative positions of the two layers would permit adaptation of the coat to variable curvatures. Gaps and dislocations in both inner and outer coat lattices may further contribute to curvature variability.

The spherical tube ends have an outer coat arrangement similar to that previously observed in in vitro cages, in which adjacent vertices are rotated relative to one another. A helical array of the inner coat heterotrimer cannot be present on the spherically-curved tips. Instead, we consider it likely that on the tube tips, the inner coat lattice is arranged in smaller patches rotated relative to one another, each maintaining the same relationship to the overlying outer coat vertex. Gaps between or within lattice patches could accommodate cargoes with large membrane-proximal domains. A comparison of our results with previous hypotheses for the relationship between inner and outer coats (Stagg et al., 2008; Bhattacharya et al., 2012) is in Figure 6.

Figure 6

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The structural data we have presented shows that both inner and outer COPII layers have sufficient structural versatility to coat both spherical and tubular membranes, and thereby accommodate important large cargoes such as pro-collagen. We found that both coats can assemble regular lattices with constrained relative positions. This relationship would permit the inner coat to influence the arrangement of the outer coat and therefore to play a critical structural role in determining vesicle shape. Our data hint at the possibility that the formation of larger arrays of the inner coat and the formation of associated rhomboidal outer coat lattices, could favour the formation of larger tubular carriers able to accommodate cargoes such as pro-collagen. It has previously been proposed that proteins such as TANGO1 and Sedlin (Saito et al., 2009; Jin et al., 2012; Kim et al., 2012; Venditti et al., 2012) act in concert to stabilize the inner coat, modulating Sar1 GTPase activity and delaying both outer coat recruitment and scission. We suggest that stabilization of the inner coat could promote growth of larger carriers not only by delaying scission, but also by promoting tubular membrane morphology (Figure 7).

Figure 7

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1. 1. Zanetti G
   2. Prinz S
   3. Daum S
   4. Meister A
   5. Schekman R
   6. Bacia K

   et al. (2013) The structure of the COPII coat assembled on membranes: Sar1/Sec23/24 inner coat

   Publicly available at the Electron Microscopy Data Bank (http://www.ebi.ac.uk/pdbe/emdb/).

   http://www.ebi.ac.uk/pdbe/entry/EMD-2428
2. 1. Zanetti G
   2. Prinz S
   3. Daum S
   4. Meister A
   5. Schekman R
   6. Bacia K

   et al. (2013) The structure of the COPII coat assembled on membranes: Sec13/31 outer coat vertex

   Publicly available at the Electron Microscopy Data Bank (http://www.ebi.ac.uk/pdbe/emdb/).

   http://www.ebi.ac.uk/pdbe/entry/EMD-2429
3. 1. Zanetti G
   2. Prinz S
   3. Daum S
   4. Meister A
   5. Schekman R
   6. Bacia K

   et al. (2013) The structure of the COPII coat assembled on membranes: Sec13/31 outer coat left-handed rod

   Publicly available at the Electron Microscopy Data Bank (http://www.ebi.ac.uk/pdbe/emdb/).

   http://www.ebi.ac.uk/pdbe/entry/EMD-2430
4. 1. Zanetti G
   2. Prinz S
   3. Daum S
   4. Meister A
   5. Schekman R
   6. Bacia K

   et al. (2013) The structure of the COPII coat assembled on membranes: Sec13/31 outer coat right-handed rod

   Publicly available at the Electron Microscopy Data Bank (http://www.ebi.ac.uk/pdbe/emdb/).

   http://www.ebi.ac.uk/pdbe/entry/EMD-2431
5. 1. Zanetti G
   2. Prinz S
   3. Daum S
   4. Meister A
   5. Schekman R
   6. Bacia K

   et al. (2013) The structure of the COPII coat assembled on membranes: representative tomogram

   Publicly available at the Electron Microscopy Data Bank (http://www.ebi.ac.uk/pdbe/emdb/).

   http://www.ebi.ac.uk/pdbe/entry/EMD-2432
6. 1. Zanetti G
   2. Prinz S
   3. Daum S
   4. Meister A
   5. Schekman R
   6. Bacia K

   et al. (2013) The structure of the COPII coat assembled on membranes: fit of Sar1/Sec23/24 into EMD-2428

   Publicly available at the Protein Data Bank (http://www.rcsb.org/pdb/).

   http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BZI
7. 1. Zanetti G
   2. Prinz S
   3. Daum S
   4. Meister A
   5. Schekman R
   6. Bacia K

   et al. (2013) The structure of the COPII coat assembled on membranes: fit of Sec13/31 into EMD-2430

   Publicly available at the Protein Data Bank (http://www.rcsb.org/pdb/).

   http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BZJ
8. 1. Zanetti G
   2. Prinz S
   3. Daum S
   4. Meister A
   5. Schekman R
   6. Bacia K

   et al. (2013) The structure of the COPII coat assembled on membranes: fit of Sec13/31 into EMD-2431

   Publicly available at the Protein Data Bank (http://www.rcsb.org/pdb/).

   http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BZK

1. 1. Amat F
   2. Moussavi F
   3. Comolli LR
   4. Elidan G
   5. Downing KH
   6. Horowitz M

   (2008) Markov random field based automatic image alignment for electron tomography

   J Struc Biol 161:260–275.

   https://doi.org/10.1016/j.jsb.2007.07.007

   • Google Scholar
2. 1. Bacia K
   2. Futai E
   3. Prinz S
   4. Meister A
   5. Daum S
   6. Glatte D

   et al. (2011) Multibudded tubules formed by COPII on artificial liposomes

   Sci Rep 1:17.

   https://doi.org/10.1038/srep00017

   • Google Scholar
3. 1. Baschong W
   2. Aebi U
   3. Baschong-Prescianotto C
   4. Dubochet J
   5. Landmann L
   6. Kellenberger E

   et al. (1988) Head structure of bacteriophages T2 and T4

   J Ultrastruct Mol Struct Res 99:189–202.

   https://doi.org/10.1016/0889-1605(88)90063-8

   • Google Scholar
4. 1. Bhattacharya N
   2. O‘Donnell J
   3. Stagg SM

   (2012) The structure of the Sec13/31 COPII cage bound to Sec23

   J Mol Biol 420:324–334.

   https://doi.org/10.1016/j.jmb.2012.04.024

   • Google Scholar
5. 1. Bi X
   2. Corpina RA
   3. Goldberg J

   (2002) Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat

   Nature 419:271–277.

   https://doi.org/10.1038/nature01040

   • Google Scholar
6. 1. Bi X
   2. Mancias JD
   3. Goldberg J

   (2007) Insights into COPII coat nucleation from the structure of Sec23.Sar1 complexed with the active fragment of Sec31

   Dev Cell 13:635–645.

   https://doi.org/10.1016/j.devcel.2007.10.006

   • Google Scholar
7. 1. Boyadjiev SA
   2. Fromme JC
   3. Ben J
   4. Chong SS
   5. Nauta C
   6. Hur DJ

   et al. (2006) Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulum-to-Golgi trafficking

   Nat Genet 38:1192–1197.

   https://doi.org/10.1038/ng1876

   • Google Scholar
8. 1. Caspar DL
   2. Klug A

   (1962) Physical principles in the construction of regular viruses

   Cold Spring Harb Symp Quant Biol 27:1–24.

   https://doi.org/10.1101/SQB.1962.027.001.005

   • Google Scholar
9. 1. Castano-Diez D
   2. Kudryashev M
   3. Arheit M
   4. Stahlberg H

   (2012) Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments

   J Struct Biol 178:139–151.

   https://doi.org/10.1016/j.jsb.2011.12.017

   • Google Scholar
10. 1. Faini M
    2. Prinz S
    3. Beck R
    4. Schorb M
    5. Riches JD
    6. Bacia K

    et al. (2012) The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions

    Science 336:1451–1454.

    https://doi.org/10.1126/science.1221443

    • Google Scholar
11. 1. Fath S
    2. Mancias JD
    3. Bi X
    4. Goldberg J

    (2007) Structure and organization of coat proteins in the COPII cage

    Cell 129:1325–1336.

    https://doi.org/10.1016/j.cell.2007.05.036

    • Google Scholar
12. 1. Förster F
    2. Medalia O
    3. Zauberman N
    4. Baumeister W
    5. Fass D

    (2005) Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography

    Proc Natl Acad Sci USA 102:4729–4734.

    https://doi.org/10.1073/pnas.0409178102

    • Google Scholar
13. 1. Fromme JC
    2. Ravazzola M
    3. Hamamoto S
    4. Al-Balwi M
    5. Eyaid W
    6. Boyadjiev SA

    et al. (2007) The genetic basis of a craniofacial disease provides insight into COPII coat assembly

    Dev Cell 13:623–634.

    https://doi.org/10.1016/j.devcel.2007.10.005

    • Google Scholar
14. 1. Ganser BK
    2. Li S
    3. Klishko VY
    4. Finch JT
    5. Sundquist WI

    (1999) Assembly and analysis of conical models for the HIV-1 core

    Science 283:80–83.

    https://doi.org/10.1126/science.283.5398.80

    • Google Scholar
15. 1. Gaullier JM
    2. Ronning E
    3. Gillooly DJ
    4. Stenmark H

    (2000) Interaction of the EEA1 FYVE finger with phosphatidylinositol 3-phosphate and early endosomes. Role of conserved residues

    J Biol Chem 275:24595–24600.

    https://doi.org/10.1074/jbc.M906554199

    • Google Scholar
16. 1. Jin L
    2. Pahuja KB
    3. Wickliffe KE
    4. Gorur A
    5. Baumgartel C
    6. Schekman R

    et al. (2012) Ubiquitin-dependent regulation of COPII coat size and function

    Nature 482:495–500.

    https://doi.org/10.1038/nature10822

    • Google Scholar
17. 1. Johnson JE
    2. Speir JA

    (1997) Quasi-equivalent viruses: a paradigm for protein assemblies

    J Mol Biol 269:665–675.

    https://doi.org/10.1006/jmbi.1997.1068

    • Google Scholar
18. 1. Kim SD
    2. Pahuja KB
    3. Ravazzola M
    4. Yoon J
    5. Boyadjiev SA
    6. Hammamoto S

    et al. (2012) The SEC23-SEC31 interface plays critical role for export of procollagen from the endoplasmic reticulum

    J Biol Chem 287:10134–10144.

    https://doi.org/10.1074/jbc.M111.283382

    • Google Scholar
19. 1. Kremer JR
    2. Mastronarde DN
    3. Mcintosh JR

    (1996) Computer visualization of three-dimensional image data using IMOD

    J Struct Biol 116:71–76.

    https://doi.org/10.1006/jsbi.1996.0013

    • Google Scholar
20. 1. Lee MC
    2. Orci L
    3. Hamamoto S
    4. Futai E
    5. Ravazzola M
    6. Schekman R

    (2005) Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle

    Cell 122:605–617.

    https://doi.org/10.1016/j.cell.2005.07.025

    • Google Scholar
21. 1. Mancias JD
    2. Goldberg J

    (2007) The transport signal on Sec22 for packaging into COPII-coated vesicles is a conformational epitope

    Mol Cell 26:403–414.

    https://doi.org/10.1016/j.molcel.2007.03.017

    • Google Scholar
22. 1. Mancias JD
    2. Goldberg J

    (2008) Structural basis of cargo membrane protein discrimination by the human COPII coat machinery

    EMBO J 27:2918–2928.

    https://doi.org/10.1038/emboj.2008.208

    • Google Scholar
23. 1. Matsuoka K
    2. Orci L
    3. Amherdt M
    4. Bednarek SY
    5. Hamamoto S
    6. Schekman R

    et al. (1998) COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes

    Cell 93:263–275.

    https://doi.org/10.1016/S0092-8674(00)81577-9

    • Google Scholar
24. 1. Miller E
    2. Antonny B
    3. Hamamoto S
    4. Schekman R

    (2002) Cargo selection into COPII vesicles is driven by the Sec24p subunit

    Embo J 21:6105–6113.

    https://doi.org/10.1093/emboj/cdf605

    • Google Scholar
25. 1. Mironov AA
    2. Mironov AA Jr
    3. Beznoussenko GV
    4. Trucco A
    5. Lupetti P

    et al. (2003) ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains

    Dev Cell 5:583–594.

    https://doi.org/10.1016/S1534-5807(03)00294-6

    • Google Scholar
26. 1. Mossessova E
    2. Bickford LC
    3. Goldberg J

    (2003) SNARE selectivity of the COPII coat

    Cell 114:483–495.

    https://doi.org/10.1016/S0092-8674(03)00608-1

    • Google Scholar
27. 1. Noble AJ
    2. Zhang Q
    3. O’Donnell J
    4. Hariri H
    5. Bhattacharya N
    6. Marshall AG

    et al. (2012) A pseudoatomic model of the COPII cage obtained from cryo-electron microscopy and mass spectrometry

    Nat Struct Mol Biol 20:167–173.

    https://doi.org/10.1038/nsmb.2467

    • Google Scholar
28. 1. Peng R
    2. Grabowski R
    3. De Antoni A
    4. Gallwitz D

    (1999) Specific interaction of the yeast cis-Golgi syntaxin Sed5p and the coat protein complex II component Sec24p of endoplasmic reticulum-derived transport vesicles

    Proc Natl Acad Sci USA 96:3751–3756.

    https://doi.org/10.1073/pnas.96.7.3751

    • Google Scholar
29. 1. Pettersen EF
    2. Goddard TD
    3. Huang CC
    4. Couch GS
    5. Greenblatt DM
    6. Meng EC

    et al. (2004) UCSF Chimera–a visualization system for exploratory research and analysis

    J Comput Chem 25:1605–1612.

    https://doi.org/10.1002/jcc.20084

    • Google Scholar
30. 1. Saito K
    2. Chen M
    3. Bard F
    4. Chen S
    5. Zhou H
    6. Woodley D

    et al. (2009) TANGO1 facilitates cargo loading at endoplasmic reticulum exit sites

    Cell 136:891–902.

    https://doi.org/10.1016/j.cell.2008.12.025

    • Google Scholar
31. 1. Stagg SM
    2. Gurkan C
    3. Fowler DM
    4. Lapointe P
    5. Foss TR
    6. Potter CS

    et al. (2006) Structure of the Sec13/31 COPII coat cage

    Nature 439:234–238.

    https://doi.org/10.1038/nature04339

    • Google Scholar
32. 1. Stagg SM
    2. Lapointe P
    3. Razvi A
    4. Gurkan C
    5. Potter CS
    6. Carragher B

    et al. (2008) Structural basis for cargo regulation of COPII coat assembly

    Cell 134:474–484.

    https://doi.org/10.1016/j.cell.2008.06.024

    • Google Scholar
33. 1. Venditti R
    2. Scanu T
    3. Santoro M
    4. Di Tullio G
    5. Spaar A
    6. Gaibisso R

    et al. (2012) Sedlin controls the ER export of procollagen by regulating the Sar1 cycle

    Science 337:1668–1672.

    https://doi.org/10.1126/science.1224947

    • Google Scholar
34. 1. Zanetti G
    2. Pahuja KB
    3. Studer S
    4. Shim S
    5. Schekman R

    (2012) COPII and the regulation of protein sorting in mammals

    Nat Cell Biol 14:20–28.

    https://doi.org/10.1038/ncb2390

    • Google Scholar
35. 1. Zanetti G
    2. Riches JD
    3. Fuller SD
    4. Briggs JA

    (2009) Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging

    J Struct Biol 168:305–312.

    https://doi.org/10.1016/j.jsb.2009.08.002

    • Google Scholar

Author details

1. Giulia Zanetti

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Present address

   Department of Biological Sciences, Institute of Structural and Molecular Biology, London, United Kingdom

   Contribution

   GZ, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Simone Prinz

   Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

   Contribution

   SP, Performed and optimized in vitro reconstitution, Acquisition of data

   Competing interests

   No competing interests declared.

3. Sebastian Daum

   HALOmem, Martin Luther University of Halle-Wittenberg, Halle, Germany

   Contribution

   SD, Performed and optimized protein expression and purification

   Competing interests

   No competing interests declared.

4. Annette Meister

   HALOmem, Martin Luther University of Halle-Wittenberg, Halle, Germany

   Contribution

   AM, Performed and optimized in vitro reconstitution

   Competing interests

   No competing interests declared.

5. Randy Schekman

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States

   Contribution

   RS, Conception and design

   Competing interests

   RS: Editor-in-Chief, eLife.

6. Kirsten Bacia

   HALOmem, Martin Luther University of Halle-Wittenberg, Halle, Germany

   Contribution

   KB, Conception and design

   Competing interests

   No competing interests declared.

7. John AG Briggs

   Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

   Contribution

   JAGB, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   john.briggs@embl.de

   Competing interests

   No competing interests declared.

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