FIGURE 7.

Inositol depletion inhibits V-ATPase activity and reduces H⁺ pumping in ino1Δ cells. ino1Δ cells were grown in SM I+ buffered to pH 5 overnight and then washed twice and resuspended in either SM I+ or SM I−, both buffered to pH 5, and incubated for 4 h at 30 °C. Vacuolar vesicles were isolated as described under “Experimental Procedures.” A, mean concanamycin A-sensitive ATPase activity (in μmol of ATP hydrolyzed per min/mg of protein) for three independent vacuole preparations is shown. Error bars represent S.E. B, proton pumping rate was assayed using the ACMA fluorescence-quenching assay as described under “Experimental Procedures.” Results represent the mean ± S.E. for three independent vacuole preparations. p values for significant differences based on unpaired two-tailed t test are indicated. C, immunoblots of vacuoles were probed for V₀ subunit Vph1 and V₁ subunits A–C as described under “Experimental Procedures.” 7 μg of vacuolar protein were loaded for detection of Vph1 and subunit C, and 5 μg were loaded for detection of the A and B subunits.