The RAVE complex (regulator of the H+-ATPase of vacuolar and endosomal mem-branes) is required for biosynthetic assembly and glucose-stimulated reassembly of the yeast vacuolar H+-ATPase (V-ATPase). Yeast RAVE contains three subunits, Rav1, Rav2 and Skp1. Rav1 is the largest subunit, and it binds Rav2 and Skp1 of RAVE, the E, G, and C subunits of the V-ATPase peripheral V1 sector and Vph1 of the membrane Vo sector. We identified Rav1 regions required for interaction with its binding partners through deletion analysis, co-immunoprecipitation, two-hybrid assay, and pull-down assays with expressed proteins. We find that Skp1 binding requires sequences near the C-terminus of Rav1, V1 subunits E and C bind to a cons... More
The RAVE complex (regulator of the H+-ATPase of vacuolar and endosomal mem-branes) is required for biosynthetic assembly and glucose-stimulated reassembly of the yeast vacuolar H+-ATPase (V-ATPase). Yeast RAVE contains three subunits, Rav1, Rav2 and Skp1. Rav1 is the largest subunit, and it binds Rav2 and Skp1 of RAVE, the E, G, and C subunits of the V-ATPase peripheral V1 sector and Vph1 of the membrane Vo sector. We identified Rav1 regions required for interaction with its binding partners through deletion analysis, co-immunoprecipitation, two-hybrid assay, and pull-down assays with expressed proteins. We find that Skp1 binding requires sequences near the C-terminus of Rav1, V1 subunits E and C bind to a conserved region in the C-terminal half of Rav1, and the cytosolic domain of Vph1 binds near the junction of the Rav1 N- and C-terminal halves. In contrast, Rav2 binds to the N-terminal domain of Rav1, which can be modeled as a double β-propeller. Only the V1 C subunit binds to both Rav1 and Rav2. Using GFP-tagged RAVE subunits in vivo, we demonstrate glucose-dependent association of RAVE with the vacuolar membrane, consistent with its role in glucose-dependent V-ATPase assembly. It is known that V1 subunit C localizes to the V1-Vo interface in assembled V-ATPase complexes and is important in regulated disassembly of V-ATPases. We propose that RAVE cycles between cytosol and vacuolar membrane in a glucose-dependent manner, positioning V1 and V0 subcomplexes and orienting the V1 C subunit to promote assembly.
