Reversible redox control of plant vacuolar H+-ATPase activity is related to disulfide bridge formation in subunit E as well as subunit A

The plant vacuolar proton pump can be subjected to reversible redox regulat ion in vitro. The redox-ependent activity change involves disulfide bridge formation not only in Vatp A, as reported for bovine V-ATPase, but also in the stalk subunit Vatp E. Microsomal membranes isolated from barley leaves were analysed for their activity of bafilomycin-sensitive ATP hydrolysis an d proton pumping using quinacrine fluorescence quenching in vesicle prepara tions. ATP hydrolysis and proton pumping activity were inhibited by H2O2. H 2O2-deactivated ATPase was reactivated by cysteine and glutathione. The glu tathione concentration needed for half maximal reactivation was 1 mmol l(-1 ). The activity loss was accompanied by shifts in electrophoretic mobility of Vatp A and E which were reversed upon reductive reactivation. The redox- dependent shift was also seen with recombinant Vatp E, and was absent follo wing site-directed mutagenesis of either of the two cys residues conserved throughout all plant Vatp E sequences. V-ATPase was also inhibited by oxidi zed thioredoxin. These results support the hypothesis that tuning of vacuol ar ATPase activity can be mediated by redox control depending on the metabo lic requirements.