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

The Journal of Biological Chemistry
Stuti SharmaStephan Wilkens

Abstract

The vacuolar H+-ATPase (V-ATPase; V1Vo-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 V1-ATPase and Vo 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 V1 and Vo, 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 V1 to Vo is the conformation of the inhibitory C-terminal domain of subunit H (HCT). Using biolayer interferometry and biochemical analyses of purified mutant V1-ATPase and Vo proton channel reconstituted into vacuolar lipid-containing nanodiscs, we further demonstrate that disruption of HCT's V1-binding site facilitates assembly of a functionally coupled and stable V1Vo-ATPase. Unlike WT, this mutant enzyme was resistant to MgATP hydrolysis-induced dissociation, further highlighting HCT's role in the mechanism of V-ATPase regulation. Our findings provide key insight into the molec...Continue Reading

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Citations

Feb 1, 2020·Trends in Biochemical Sciences·Thamiya Vasanthakumar, John L Rubinstein
Dec 17, 2020·American Journal of Physiology. Cell Physiology·Amity F EatonDennis Brown
Mar 21, 2021·Nature Communications·Rong WangXiaochun Li
Apr 26, 2021·The Journal of Biological Chemistry·Michael C JaskolkaPatricia M Kane
Jul 13, 2021·Frontiers in Cell and Developmental Biology·Michael C JaskolkaPatricia M Kane
Jul 17, 2021·The Journal of Biological Chemistry·Rebecca A OotStephan Wilkens

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