Effect of membrane voltage on the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

A novel system for generating large interior positive membrane potentials in proteoliposomes was used to examine the effects of membrane voltage on reconstituted plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The membrane potential-generating system was dependent upon the lipophilic electron carrier tetracyanoquinodimethane, located within the bilayer, to mediate electron flow from vesicle entrapped ascorbate to external K3Fe(CN)6. Membrane potential formation was followed by the potential-dependent probe oxonol V and was found to rapidly reach a steady-state which lasted at least 90 s. A membrane potential of approximately 254 mV was determined under optimal conditions and ATP hydrolysis by wild-type H(+)-ATPase was inhibited from 34 to 46% under these conditions. In contrast, membrane potential had little effect on pma1-105 mutant enzyme suggesting that it is defective in electrogenic proton translocation. Applied membrane voltage was also found to alter the sensitivity of wild-type enzyme to vanadate at concentrations less than 50 microM. These data suggest a coupling between the charge-transfer and ATP hydrolysis domains and establish a solid basis for future probing of the electrogenic properties of the yeast H(+)-ATPase.