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

Svetlana O Dodonova,Felix Wieland,Iva Ganeva,Patrick Aderhold,Simone Röhling,John A G Briggs,Wim J H Hagen,Juergen Kopp,Irmgard Sinning

doi:10.7554/elife.26691

Svetlana O Dodonova, Felix Wieland + Show 7 more

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https://doi.org/10.7554/elife.26691

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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.

Highlights

Coated vesicles mediate transport between intracellular organelles
Crystal structures were available for the majority of the components of the Coat protein 1 (COPI) coat, but not for the bd-COP subcomplex
As a first step towards a complete structural model, we expressed and crystallized a complex containing b-COP19-391 and d-COP1-175 from C. thermophilum (Ct), from which we obtained a molecular model of bd-COP comprising residues 20–390 of b-COP (‘trunk region’) and residues 2– 150 of d-COP (Figure 1, Table 1)

Summary

Introduction

Coated vesicles mediate transport between intracellular organelles. Of a COPI coated vesicle is initiated by recruitment of the small GTPase Arf (ADP-ribosylation factor 1) to the membrane, where it is activated by a guanine exchange factor and transitions into a GTP-bound state in which its amphipathic N0 helix is inserted into the membrane. Arf1-GTP recruits the protein complex coatomer from the cytoplasm and anchors it at the membrane. Polymerization of coatomer at the membrane assembles the COPI coat, which recruits cargo molecules and membrane machinery to the assembly site (Harter and Wieland, 1998; Jackson et al, 2012). GTP hydrolysis by Arf, activated by ArfGAPs (Arf GTPase Activating Proteins), mediates uncoating of the vesicle which subsequently fuses with its target membrane

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