Regulation and reversibility of vacuolar H⁺-ATPase

Arabidopsis thaliana vacuolar H⁺-translocating pyrophosphatase (V- PPase) was expressed funtionally in yeast vacuoles with endogenous vacuolar H⁺-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (ΔμH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (ΔpH. On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (ΔΨ). Chimeric vacuolar membranes showed ATP synthesis coupled with Δμh established by V-PPase. The ATP synthesis was sensitive to bafilomycin A₁ 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 ΔpH but not ΔΨ, suggesting that ΔpH is essential for ATP synthesis.