Molecular Interactions and Cellular Itinerary of the Yeast RAVE (Regulator of the H+-ATPase of Vacuolar and Endosomal Membranes) Complex

Anne M Smardon,Negin Dehdar Nasab,Maureen Tarsio,Theodore T Diakov,Patricia M Kane

doi:10.1074/jbc.m115.667634

Anne M Smardon, Negin Dehdar Nasab + Show 3 more

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https://doi.org/10.1074/jbc.m115.667634

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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.

Highlights

The RAVE complex is required for glucose-sensitive V-ATPase assembly, but its mechanism is unknown
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
RAVE/V-ATPase Binding Sites Suggest a Role for RAVE in Orienting V1 Peripheral Stalks, V1C and Vo Vph1NT in VATPase Assembly—Consistent with RAVE functioning as a V-ATPase assembly factor, RAVE binds to all three components of the V-ATPase that are dissociated during glucose deprivation: the V1 subcomplex via the E and G subunits [10], the V1 C subunit [18], and Vo subunit Vph1 [13]

Summary

Background

The RAVE complex is required for glucose-sensitive V-ATPase assembly, but its mechanism is unknown. 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). Under conditions of V-ATPase disassembly, RAVE binds to V1 sectors in cytosolic fractions via the E and/or G subunits of V1, but this interaction is not intrinsically glucose-sensitive [10, 11]. Given the high level of sequence conservation among all eukaryotic V-ATPases and the evidence that RAVE and rabconnectin complexes both regulate V-ATPase activity at the level of V1-Vo assembly, we hypothesized that functionally critical V-ATPase subunit binding sites might be located in the conserved regions of Rav. We demonstrate that the RAVE complex shows glucose-dependent association with the vacuolar membrane in vivo, consistent with its role in glucose-dependent V-ATPase assembly

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