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 V(1) domain from the integral membrane V(o) domain, accompanied by release of the V(1) 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–V(1) complexes. Both purified RAVE and RAVE–V(1) contain substoichiometric levels of subunit C. RAVE–V(1) binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE–V(1) interacts with the N-terminal domain of V(o) subunit Vph1 in vitro. RAVE–V(1) complexes, like isolated V(1), have no MgATPase activity, suggesting that RAVE cannot reverse V(1) inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V(1) 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.