Regulation and reversibility of vacuolar H+-ATPase

Arabidopsis thaliana vacuolar H+-translocating pyrophosphatase (V-PPase) wa s expressed functionally in yeast vacuoles with endogenous vacuolar H+-ATPa se (V-ATPase), and the regulation and reversibility of V-ATPase were studie d using these vacuoles. Analysis of electrochemical proton gradient (Delta mu H) formation with ATP and pyrophosphate indicated that the proton transp ort by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (Delta pH). On the other hand, vacuolar membranes may have a regu latory mechanism for maintaining a constant membrane potential (Delta Psi). Chimeric vacuolar membranes showed ATP synthesis coupled with Delta mu H e stablished by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K-m values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates Delta pH but not Delta Psi, suggest ing that Delta pH is essential for ATP synthesis.