title: Molecular Interactions and Cellular Itinerary of the Yeast RAVE (Regulator of the H+-ATPase of Vacuolar and Endosomal Membranes) Complex.
abstract: The RAVE complex (regulator of the H(+)-ATPase of vacuolar and endosomal membranes) 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 pulldown 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Smardon
authors.affiliation: From the Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.
authors.forename: Negin Dehdar
authors.initials: ND
authors.lastname: Nasab
authors.affiliation: From the Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.
authors.forename: Maureen
authors.initials: M
authors.lastname: Tarsio
authors.affiliation: From the Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.
authors.forename: Theodore T
authors.initials: TT
authors.lastname: Diakov
authors.affiliation: From the Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210 kanepm@upstate.edu.
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Kane
mesh_terms: D001665:Binding Sites; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism*; D011992:Endosomes / Q000201:enzymology*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases
keywords: cell signaling; glucose; protein-protein interaction; vacuolar ATPase; vacuolar acidification