The signaling lipid PI(3,5)P(2) stabilizes V(1)–V(o) sector interactions and activates the V-ATPase

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V(1) domain from the integral membrane V(o) domain. Multiple stresses induce changes in V(1)-V(o) assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)). V-ATPase activation through V(1)-V(o) assembly in response to salt stress is strongly dependent on PI(3,5)P(2) synthesis. Purified V(o) complexes preferentially bind to PI(3,5)P(2) on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P(2) levels in vivo recruits the N-terminal domain of V(o)-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V(1)-V(o) interaction, suggesting that interaction of Vph1p with PI(3,5)P(2)-containing membranes stabilizes V(1)-V(o) assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1∆ and vac14∆ mutants and suggest that human disease phenotypes associated with PI(3,5)P(2) loss may arise from compromised V-ATPase stability and regulation.