Dissection of GTPase-activating proteins reveals functional asymmetry in the COPI coat of budding yeast

Eric C Arakel,E-Ming Rau,Anne Spang,Blanche Schwappach,Alejandro F Estrada,Martina Huranova

doi:10.1242/jcs.232124

Abstract

ABSTRACTThe Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI.This article has an associated First Person interview with the first author of the paper.

Highlights

Coated vesicles transport proteins and lipids between compartments of the secretory pathway
We demonstrate that Glo3/ArfGAP2/3 triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat
Elucidation of the structure of the COPI coat on reconstituted vesicles (Dodonova et al, 2017; Dodonova et al, 2015) and insights into the molecular interactions of either Arf GTPase-activating protein (ArfGAP) with coatomer (Rawet et al, 2010; Schindler et al, 2009; Suckling et al, 2014; Watson et al, 2004) leads to the hypothesis that they exist in contrasting molecular environments (Fig. 1A), just like the two Arf1 GTPase molecules associated with the coat (Dodonova et al, 2017)

Summary

Introduction

Coated vesicles transport proteins and lipids between compartments of the secretory pathway. Coats are macromolecular assemblies that associate with membranes, selectively capture proteins and lipids, deform the underlying membrane to form vesicles, and help accurately target these vesicles to their physiological destinations. The COPI coat is formed by an obligate heptamer – termed coatomer – consisting of the α, β’, ε, β, γ, δ, and ζ subunits. The COPI coat mediates the retrograde trafficking of proteins and lipids from the Golgi to the ER and within intraGolgi compartments (Arakel et al, 2016; Beck et al, 2009; Pellett et al, 2013; Spang and Schekman, 1998). Several reports have implicated COPI in endosomal recycling and regulating lipid droplet homeostasis (Aniento et al, 1996; Beller et al, 2008; Xu et al, 2017)

Methods

Results

Conclusion