Characterization of Ca2+/H+ exchange in the plasma membrane of Saccharomyces cerevisiae

Abstract

The characteristics of the Ca2+/H+ exchange were directly investigated in functionally inverted (inside-out) plasma membrane vesicles isolated from yeast using an aqueous two-phase partitioning method. Results showed that following the generation of an inside-acid pH gradient (fluorescence quenching), addition of Ca2+ caused movement of H+ out of the vesicles (fluorescence recovery). The Ca2+/H+ exchange displayed saturation kinetics with respect to extravesicular Ca2+ and ATP concentrations in the plasma membrane, and showed specificity for Ca2+. The protonophore FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone), abolished the fluorescence quenching and consequently inhibited Ca2+/H+ exchange in plasma membrane vesicles. Vanadate, which is known to inhibit the plasma membrane H+-ATPase, significantly decreased the Ca2+-dependent transport of H+ out of vesicles. When the electrical potential across the plasma membrane was dissipated with valinomycin and potassium, the rate of Ca2+/H+ exchange increased compared to that of the control without valinomycin, indicating that the stoichiometric ratio for this exchange is greater than 2H+:Ca2+. These data suggest that Ca2+ is transported out of yeast cells through a Ca2+/H+ exchange system that is driven by the proton-motive force generated by the plasma membrane H+-ATPase.