Functional reconstitution of vacuolar H-ATPase from V proton channel and mutant V-ATPase provides insight into the mechanism of reversible disassembly.

The vacuolar H-ATPase (V-ATPase; VV-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-ATPase and V 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 and V, a mismatch that is likely responsible for their inability to reconstitute into holo V-ATPase Here, using the model organism , we show that a key impediment for binding of V to V is the conformation of the inhibitory C-terminal domain of subunit H (H). Using biolayer interferometry and biochemical analyses of purified mutant V-ATPase and V proton channel reconstituted into vacuolar lipid-containing nanodiscs, we further demonstrate that disruption of H's V-binding site facilitates assembly of a functionally coupled and stable VV-ATPase. Unlike WT, this mutant enzyme was resistant to MgATP hydrolysis-induced dissociation, further highlighting H'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.