The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V 1 -ATPase and V o proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted V o and mutant V 1 . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V 1 to V o provides sufficient free energy to overcome V o autoinhibition. Moreover, the structures suggest that the unequal spacing of V o 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V 1 and V o motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V 1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V 1 -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.

The vacuolar H ؉-ATPase (V-ATPase; V 1 V o-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 V 1-ATPase and V o 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 V 1 and V o , 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 V 1 to V o is the conformation of the inhibitory C-terminal domain of subunit H (H CT). Using biolayer interferometry and biochemical analyses of purified mutant V 1-ATPase and V o proton channel reconstituted into vacuolar lipid-containing nanodiscs, we further demonstrate that disruption of H CT 's V 1-binding site facilitates assembly of a functionally coupled and stable V 1 V o-ATPase. Unlike WT, this mutant enzyme was resistant to MgATP hydrolysis-induced dissociation, further highlighting H CT '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. The vacuolar H ϩ-ATPase (V-ATPase, V 1 V o-ATPase) 3 is an ATP-dependent proton pump found on the endomembrane system of all eukaryotic organisms. This multisubunit nanomotor acidifies subcellular compartments and, in certain specialized tissues, the extracellular space. V-ATPase is essential for vital cellular processes such as pH homeostasis, protein

Edited by Henrik DohlmanThe vacuolar H + -ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid and reversible disassembly of the peripheral V 1 domain from the integral membrane V o domain, accompanied by release of the V 1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H + -ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE-V 1 complexes. Both purified RAVE and RAVE-V 1 contain substoichiometric levels of subunit C. RAVE-V 1 binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE-V 1 interacts with the N-terminal domain of V o subunit Vph1 in vitro. RAVE-V 1 complexes, like isolated V 1 , have no MgATPase activity, suggesting that RAVE cannot reverse V 1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V 1 carrying a mutant subunit H incapable of inhibition with V o complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.

Vacuolar ATPases (V-ATPases, V 1 V o -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V 1 -ATPase disengages from V o proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex. Recently, an alternative pathway of holoenzyme disassembly was discovered that involves binding of Oxidation Resistance 1 (Oxr1p), a poorly characterized protein implicated in oxidative stress response. Unlike conventional reversible disassembly, which depends on enzyme activity, Oxr1p induced dissociation can occur in absence of ATP. Yeast Oxr1p belongs to the family of TLDc domain containing proteins that are conserved from yeast to mammals, and have been implicated in V-ATPase function in a variety of tissues. This brief perspective summarizes what we know about the molecular mechanisms governing both reversible (ATP dependent) and Oxr1p driven (ATP independent) V-ATPase dissociation into autoinhibited V 1 and V o subcomplexes.

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