Defining steps in RAVE-catalyzed V-ATPase assembly using purified RAVE and V-ATPase subcomplexes

The vacuolar H⁺-ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid and reversible disassembly of the peripheral V₁ domain from the integral membrane V_o domain, accompanied by release of the V₁ C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H⁺-ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE-V₁ complexes. Both purified RAVE and RAVE-V₁ contain substoichiometric levels of subunit C. RAVE-V₁ binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE-V₁ interacts with the N-terminal domain of V_o subunit Vph1 in vitro. RAVE-V₁ complexes, like isolated V₁, have no MgATPase activity, suggesting that RAVE cannot reverse V₁ inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V₁ carrying a mutant subunit H incapable of inhibition with V_o complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.