RAVE Is Essential for the Efficient Assembly of the C Subunit with the Vacuolar H+-ATPase

The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V1 and V0 sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V1 peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V1 complexes, suggesting that RAVE may play a role in docking of the V1 peripheral stalk to the V0 complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V1 subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V1 and V0 sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V1/V0 complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V1 peripheral stalk subunit interactions with V0 during V-ATPase assembly.