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 molecular events underlying regulation of V-ATPase activity by reversible disassembly.
Highlights
The vacuolar H؉-ATPase (V-ATPase; V1Vo-ATPase) is an ATP-dependent proton pump that acidifies subcellular compartments in all eukaryotic organisms
We show that replacement of endogenous H in yeast V1 with Hchim (V1Hchim) [16] permits binding to vacuolar lipid containing nanodiscs (VoND), and formation of a coupled holo V-ATPase (V1HchimVoND) with catalytic properties similar to the ones of the recently characterized WT V1VoND [29]
Using a biolayer interferometry (BLI) setup similar to the one we recently employed to analyze binding of H to V1⌬H [30], we found that V1⌬C binds C with a Kd of ϳ0.7 nM (Fig. S4), indicating that one of the EG heterodimers bound to V1 is in a conformation that is more favorable for C binding compared with the isolated heterodimer
Summary
Purification and characterization of native lipid nanodisc reconstituted Vo (VoND) and V1 mutants. The experiment demonstrated that binding of V1Hchim and C to VoND resulted in the formation of a coupled V1HchimVoND complex and that the reconstitution under these conditions was complete in ϳ2 h, with a final specific activity of 7.2 Ϯ 1.09 units/mg, similar to what is reported for purified WT V1VoND (6.9 Ϯ 0.6 units/mg) [29] (Fig. 3B, inset, pink and blue bars, respectively). A more detailed analysis indicated a good match between averages obtained by reference free alignment and classification of a small data set of V1HchimVoND and corresponding projections of a cryoEM map of yeast V1Vo [23] (Fig. 4C) Taken together, these data show that reconstitution of V1Hchim with VoND and C result in a stable and coupled holo V-ATPase that is structurally similar to the WT enzyme
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