Interaction between the yeast RAVE complex and Vph1-containing V_o sectors is a central glucose-sensitive interaction...

The yeast vacuolar H⁺-ATPase (V-ATPase) of budding yeast (Saccharomyces cerevisiae) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low-glucose conditions by releasing peripheral V₁ subcomplexes from membrane-bound V_o subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H⁺-ATPase of vacuoles and endosomes (RAVE) complex, which binds to cytosolic V₁ subcomplexes and assists reassembly with integral membrane V_o complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V₁ Glucose-dependent RAVE localization to the vacuolar membrane required only intact V_o complexes containing the Vph1 subunit, suggesting that the RAVE-V_o interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1 in vivo and in vitro Mutations in this region resulted in the temperature- and pH-dependent growth phenotype characteristic of ravΔ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE's role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V₁.