Oxidative stress protein Oxr1 promotes V-ATPase holoenzyme disassembly in catalytic activity-independent manner

Abstract

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.