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9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

<div class='caption-title'>A ribbon representation of the structure of Ctβ20–390 (dark green) Ctδ2–150 (orange) as viewed from the vesicle membrane. The α-helices within the α-solenoid of β-COP are numbered according to their position in the sequence. δ-COP consists of a longin domain and two downstream helices: helix a and b. Helix b projects away from the α-solenoid of β-COP. The schematic at the bottom of the panel shows the subunit domain composition. The subunit regions that are visualised in the x-structure are marked. The β-COP sequence numbering includes the N-terminal His-tag and thus is shifted by 15 residues relative to the annotated uniprot-Q9JIF7 Mus Musculus sequence. See also Figure 1—figure supplement 1 and Table 2.</div>
<div class='caption-title'>(A) In the crystal structure, β- and δ-COP share an extensive interface with a size of 1737 Å2. The longin domain, β–turn, and helix a of δ-COP are part of the interface with the inside of the β-COP α-solenoid, whereas helix b traverses the α-solenoid along the connection of helices α7 and α6 of β-COP and protrudes away from the solenoid (see also Figure 1). F133 in helix a is in close contact with α-solenoid residues Y114 and Y149. The side chain of δ-M136 anchors the short region between helix a and b onto β. Additionally, the side-chain of β-N112 forms a hydrogen bond to the main-chain oxygen of δ-M136. Other interactions are a salt bridge between δ-E141, which is located at the beginning of helix b, and side chain of β-K71, and a hydrogen bond between side chains of δ-S138 and β-E113. The side chains of I143, I146, and I147 in helix b are shown. These residues are essential for correct trafficking of HDEL motif containing cargo by COPI (Arakel et al., 2016). (B) In the crystal, δ-COP helix b (red) is involved in a contact with helix b of a symmetry-related molecule (grey). The crystal contact comprises an interface area of 371 Å2. We cannot assess whether the crystal contact influences the orientation of helix b, but superposition of corresponding parts of β- and δ-COP from the X-ray and cryo-ET structures (not shown) suggests that there is some flexibility in this region. (C) In the X-ray structure of γζ-COP with bound Arf1 (Yu et al., 2012), helices α2, α4, and α6 of γ-COP interact with Arf1. γζ-COP helix α4 shows standard α-helical geometry from residues T63 to T74 and ends in a 310 helical conformation (K75–Q78). In contrast, in our structure of βδ-COP, downstream of residue I61, β-COP helix α4 is shorter, and between residues I61 and F64 it forms a widened helix with a rare π-helix-like geometry. To highlight the quality of the structure in this region, electron density at contour level 4σ from the anomalous diffraction experiment with SeMet-labelled protein is shown for the two methionine residues M59 and M66. (D) The main chain oxygen of β-COP F64 in helix α4 forms a hydrogen bond with the amino group of the side chain of K37, which is located in the preceding helix α3. 2mFo-DFc electron density is shown at contour level 1σ. (E) The x-ray structure of Chaetomium thermophilum βδ-COP can be fit as a rigid body into the cryo-ET map of the Mus musculus COPI leaf at 9.2 Å resolution, indicating a high degree of structural conservation. β-COP is shown in dark green, δ-COP in orange.</div>
<div class='caption-title'>(A) CryoET reconstruction of the COPI asymmetric unit, the ‘leaf’ before local alignments. The density is colored according to the distance from the membrane - from red to blue. The displayed structure also contains part of Arf1-γ-ζ-COP from a neighboring leaf to show inter-leaf interactions. The density is displayed at 0.04 isosurface level in order to visualize the membrane. (B) CryoET reconstruction of the leaf at 9.2 Å resolution after local alignment. The membrane was masked out during local alignments and upon generation of the combined map. Note the definition of the α-helical densities in the structure. (C) A structural model of the COPI coat after flexible-fitting of structures and homology models into the cryoET structure. Note, that the C-terminal domain of α-COP, ε-COP, and the δ-COP MHD, are not visualized in the leaf structure, since they compose the inter-triad linkages. Color scheme: cryoET density - grey, Arf1 - pink, γ-COP – light green, β-COP - dark green, ζ-COP - yellow, δ-COP - orange, β’-COP - light blue, α-COP – dark blue. (D) The COPI triad. One asymmetric unit, the ‘leaf’, is outlined with an orange line. The part of the structure displayed in this figure A-C is outlined with the white line. The central Arf1 (γArf1) is marked with a blue asterisk, the peripheral Arf1 (βArf1) with a red asterisk. COPI subunits are displayed as molecular surfaces. See also Figure 2—figure supplement 1, Figure 2—figure supplement 2, and Video 1.</div>
Dodonova SO, Aderhold P, Kopp J, Ganeva I, Röhling S, Hagen WJH, Sinning I, Wieland F, Briggs JAG
OpenAccess logo Elife 6 (2017)
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Cited: 55 times
EuropePMC logo PMID: 28621666

Abstract

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

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  1. Assembly of COPI and COPII Vesicular Coat Proteins on Membranes. Béthune J, Wieland FT. Annu Rev Biophys 47 63-83 (2018)
  2. Formation of COPI-coated vesicles at a glance. Arakel EC, Schwappach B. J Cell Sci 131 jcs209890 (2018)
  3. COPII-mediated trafficking at the ER/ERGIC interface. Peotter J, Kasberg W, Pustova I, Audhya A. Traffic 20 491-503 (2019)
  4. COPII-dependent ER export in animal cells: adaptation and control for diverse cargo. McCaughey J, Stephens DJ. Histochem Cell Biol 150 119-131 (2018)
  5. Current data processing strategies for cryo-electron tomography and subtomogram averaging. Pyle E, Zanetti G. Biochem J 478 1827-1845 (2021)
  6. Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies. Ognjenović J, Grisshammer R, Subramaniam S. Annu Rev Biomed Eng 21 395-415 (2019)
  7. In situ structure determination by subtomogram averaging. Castaño-Díez D, Zanetti G. Curr Opin Struct Biol 58 68-75 (2019)
  8. Conformational regulation of AP1 and AP2 clathrin adaptor complexes. Beacham GM, Partlow EA, Hollopeter G. Traffic 20 741-751 (2019)
  9. Fine details in complex environments: the power of cryo-electron tomography. Hutchings J, Zanetti G. Biochem Soc Trans 46 807-816 (2018)
  10. Vesicle trafficking and vesicle fusion: mechanisms, biological functions, and their implications for potential disease therapy. Cui L, Li H, Xi Y, Hu Q, Liu H, Fan J, Xiang Y, Zhang X, Shui W, Lai Y. Mol Biomed 3 29 (2022)
  11. Bringing Structure to Cell Biology with Cryo-Electron Tomography. Young LN, Villa E. Annu Rev Biophys 52 573-595 (2023)
  12. Coat flexibility in the secretory pathway: a role in transport of bulky cargoes. Hutchings J, Zanetti G. Curr Opin Cell Biol 59 104-111 (2019)
  13. Alphavirus-Induced Membrane Rearrangements during Replication, Assembly, and Budding. Elmasri Z, Nasal BL, Jose J. Pathogens 10 984 (2021)
  14. Find your coat: Using correlative light and electron microscopy to study intracellular protein coats. Sochacki KA, Taraska JW. Curr Opin Cell Biol 71 21-28 (2021)
  15. Attachment, Entry, and Intracellular Trafficking of Classical Swine Fever Virus. Guo X, Zhang M, Liu X, Zhang Y, Wang C, Guo Y. Viruses 15 1870 (2023)
  16. Crowd-Sourcing of Membrane Fission: How crowding of non-specialized membrane-bound proteins contributes to cellular membrane fission. Manni MM, Derganc J, Čopič A. Bioessays 39 (2017)

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