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-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 V-1, 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 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.

Automated summary

The vacuolar H+-ATPase (V-ATPase) is a proton pump responsible for acidifying organelles in cells. The RAVE complex plays a crucial role in the reassembly of V-ATPases through the rapid and reversible disassembly and reassembly processes. Native RAVE and RAVE-V1 complexes purified using an inducible overexpression system contain substoichiometric levels of subunit C, suggesting their importance in V-ATPase reassembly.