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 μM. 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.