Abstract:
Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V₁ region that hydrolyzes ATP and a membrane-embedded V_O region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V₁ and V_O complexes becoming autoinhibited on disassembly and subunit C subsequently detaching from V₁. In yeast, assembly of the V₁ and V_O regions is mediated by the regulator of the ATPase of vacuoles and endosomes (RAVE) complex through an unknown mechanism. We used cryogenic-electron microscopy of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V₁ complex and the V₁ complex lacking subunit C. On separation, V₁ undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with V_O. Loss of subunit C allows V₁ to match the rotational state of V_O, suggesting how RAVE could reassemble V₁ and V_O by recruiting subunit C.