MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1 -ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly

The Journal of Biological Chemistry
Stuti SharmaStephan Wilkens

Abstract

Vacuolar H+-ATPases (V-ATPases; V1Vo-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 V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V1 However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full-length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysi...Continue Reading

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