Functional reconstitution of vacuolar H+-ATPase from Vo proton channel and mutant V1-ATPase provides insight into the mechanism of reversible disassembly

The vacuolar H + -ATPase (V-ATPase; V 1 V o -ATPase) is an ATP-dependent proton pump that acidifies subcellular compartments in all eukaryotic organisms. V-ATPase activity is regulated by reversible disassembly into autoinhibited V 1 -ATPase and V o proton channel subcomplexes, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor, and recent structural analyses have revealed different rotary states for disassembled V 1 and V o , a mismatch that is likely responsible for their inability to reconstitute into holo V-ATPase in vitro . Here, using the model organism Saccharomyces cerevisiae , we show that a key impediment for binding of V 1 to V o is the conformation of the inhibitory C-terminal domain of subunit H (H CT ). Using biolayer interferometry and biochemical analyses of purified mutant V 1 -ATPase and V o proton channel reconstituted into vacuolar lipid-containing nanodiscs, we further demonstrate that disruption of H CT 's V 1 -binding site facilitates assembly of a functionally coupled and stable V 1 V o -ATPase. Unlike WT, this mutant enzyme was resistant to MgATP hydrolysis-induced dissociation, further highlighting H CT 's role in the mechanism of V-ATPase regulation. Our findings provide key insight into the molecular events underlying regulation of V-ATPase activity by reversible disassembly.