title: Distinct role of subcomplexes of the COPI coat in the regulation of ArfGAP2 activity.
abstract: COPI vesicles serve for transport of proteins and membrane lipids in the early secretory pathway. Their coat protein (coatomer) is a heptameric complex that is recruited to the Golgi by the small GTPase Arf1. Although recruited en bloc, coatomer can be viewed as a stable assembly of an adaptin-like tetrameric subcomplex (CM4) and a trimeric 'cage' subcomplex (CM3). Following recruitment, coatomer stimulates ArfGAP-dependent GTP hydrolysis on Arf1. Here, we employed recombinant coatomer subcomplexes to study the role of coatomer components in the regulation of ArfGAP2, an ArfGAP whose activity is strictly coatomer-dependent. Within CM4, we define a novel hydrophobic pocket for ArfGAP2 interaction on the appendage domain of γ₁-COP. The CM4 subcomplex (but not CM3) is recruited to membranes through Arf1 and can subsequently recruit ArfGAP2. Neither CM3 nor CM4 in itself is effective in stimulating ArfGAP2 activity, but stimulation is regained when both subcomplexes are present. Our findings point to a distinct role of each of the two coatomer subcomplexes in the regulation of ArfGAP2-dependent GTP hydrolysis on Arf1, where the CM4 subcomplex functions in GAP recruitment, while, similarly to the COPII system, the cage-like CM3 subcomplex stimulates the catalytic reaction.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Irit
authors.initials: I
authors.lastname: Pevzner
authors.forename: Jeroen
authors.initials: J
authors.lastname: Strating
authors.forename: Lena
authors.initials: L
authors.lastname: Lifshitz
authors.forename: Anna
authors.initials: A
authors.lastname: Parnis
authors.forename: Fabian
authors.initials: F
authors.lastname: Glaser
authors.forename: Alexia
authors.initials: A
authors.lastname: Herrmann
authors.forename: Britta
authors.initials: B
authors.lastname: Brügger
authors.forename: Felix
authors.initials: F
authors.lastname: Wieland
authors.forename: Dan
authors.initials: D
authors.lastname: Cassel
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D020755:Coat Protein Complex I / Q000737:chemistry*; D019295:Computational Biology / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D020755:Coat Protein Complex I; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D020727:ADP-Ribosylation Factors; C525860:ARFGAP2 protein, human