MgATP hydrolysis destabilizes the interaction between subunit H and yeast V-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

Vacuolar H-ATPases (V-ATPases; VV-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V-ATPase and V proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V and V H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V However, how H fulfills these two functions is poorly understood. To characterize the H-V interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (H) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit (), and V lacking subunit H (VΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that H binds EG with moderate affinity, that full-length H binds weakly, and that both H and H bind VΔH with high affinity. We also found that only one molecule of H binds VΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in VΔH. Moreover, MgATP hydrolysis-driven conformational changes in V destabilized the interaction of H or H with VΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V points to H's role in the mechanism of reversible disassembly.