FIGURE 8.

VPA results in an inositol-reversible loss of H⁺ pumping in vacuolar vesicles from wild-type cells. WT cells were grown overnight, with or without 100 μm inositol and 0.6 mm VPA in SM buffered to pH 5 with 50 mm potassium phosphate and 50 mm potassium succinate. Vacuolar vesicles were isolated as described under “Experimental Procedures.” A, concanamycin A-sensitive ATPase activity was measured in vacuolar vesicles isolated from cells grown overnight in the presence or absence of inositol and VPA as indicated. The mean activity (in μmol of ATP/min/mg protein) of four independent preparations from cells grown in I+ or I− medium without VPA and three independent preparations from VPA-treated cells is shown, and error bars indicate S.E. There were no significant differences between the V-ATPase activities between the four different conditions. B, initial rate of proton pumping was measured in the same preparations of vacuolar vesicles and the mean ± S.E. × 10⁴ is shown. Three independent preparations of vacuolar vesicles from cells grown in the presence of VPA and the absence of inositol showed no proton pumping. p values for significant differences based on unpaired two-tailed t test are indicated.