Regulation and Reversibility of Vacuolar H+-ATPase

Tomoyuki Hirata,Norihiro Nakamura,Hiroshi Omote,Yoh Wada,Masamitsu Futai

doi:10.1074/jbc.275.1.386

Tomoyuki Hirata, Norihiro Nakamura + Show 3 more

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https://doi.org/10.1074/jbc.275.1.386

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Abstract

Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally 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 (DeltamuH) 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 (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established 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 DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.

Highlights

Arabidopsis thaliana vacuolar H؉-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H؉-ATPase (VATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles
In this study we demonstrated that V-ATPase can synthesize ATP coupled with ⌬␮H generated by V-PPase and that proton translocation by the two pumps is not strictly regulated by ⌬pH
Expression of Arabidopsis V-PPase in Saccharomyces cerevisiae—We introduced a multicopy expression plasmid, pKT10N-myc-AVP3, carrying the cDNA (AVP3) for A. thaliana V

Summary

Introduction

Arabidopsis thaliana vacuolar H؉-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H؉-ATPase (VATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (⌬␮H) 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 (⌬pH). Chimeric vacuolar membranes showed ATP synthesis coupled with ⌬␮H established by V-PPase.

Results

Conclusion