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

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 bd-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.

AP‐3 vesicle uncoating occurs after HOPS‐dependent vacuole tethering

“…Recent work on COPI revealed a close interplay of two ArfGAPs, Glo3, and Gcs1 in uncoating, one of which is controlled in activity by phosphorylation (Arakel et al, 2019). COPI binds two Arf1 proteins in different molecular environments (Bykov et al, 2017;Dodonova et al, 2017), as does AP-1 (Shen et al, 2015), suggesting that one Arf GAP protein may destabilize the coat partially. Age2mediated Arf1 hydrolysis and final uncoating may thus occur only very late, possibly after tethering of AP-3 vesicles at the vacuole and engagement of AP-3 localized SNAREs with the vacuolar HOPS complex, which has several SNARE-binding sites Lü rick et al, 2015;Orr et al, 2017).…”