Role of the H subunit C-terminal domain in the assembly of the vacuolar H+-ATPase

Abstract The vacuolar H + -ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V 1 -ATPase and V o proton channel sectors, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor and recent structural analysis revealed that disassembled V 1 and V o are in different rotary states, a mismatch that is likely responsible for the inability to reconstitute holo V-ATPase from its functional sectors in vitro . Here, using the model organism S. cerevisiae , we show that a key impediment for binding of autoinhibited V 1 to V o is the conformation of the inhibitory C-terminus of subunit H (H CT ). Using biolayer interferometry and biochemical analysis, we show that selective disruption of H CT ’s binding site on V 1 allows in vitro assembly of a structurally and functionally coupled V-ATPase complex. The resultant mutant V-ATPase, however, does not disassemble as readily as the wild type enzyme, highlighting the importance of H CT ’s conformation in the mechanism of reversible disassembly. These findings pave the way for identifying molecules that allow for therapeutic modulation of aberrant V-ATPase activity in the disease state.