FIGURE 6.

Functional characterization of the K180G/G212K double mutant in comparison with wild-type. A, β-subunit content and OG-stimulated ATPase activity of everted vesicles isolated from the wild-type and mutant strains. The strains were grown in the semi-defined medium supplemented with 50 mm glucose at pH 10.5. The values for the mutants are given as % of wild-type with the wild-type set at 100%. Gray bars, OG ATPase; black bars, β-subunit content. B, growth of wild-type and double mutant strains as a function of pH and carbon source. Top graph, glucose; bottom graph, malate; gray bars, pH 7.5; black bars, pH 10.5. C, ATP synthesis by ADP + P_i-loaded RSO membrane vesicles at pH 7.5 and 10.5. The vesicles were prepared from cells grown in the semi-defined medium supplemented with 50 mm glucose at pH 10.5. Gray bars, pH 7.5; black bars, pH 10.5. D, the native ATPase activity with and without 100 μm DCCD was assayed as described under “Experimental Procedures.” Gray bars, no DCCD; black bars, plus DCCD. E, determination of the cytoplasmic pH of cells grown on 50 mm malate at pH 8.5 and subsequently subjected to a shift in the external pH from 8.5 to 10.5. See “Experimental Procedures” for details. The results shown in all panels are the mean results from duplicate determinations from 2–4 independent experiments for panels A–C and 6 independent experiments for panel D. The error bars show ±S.D.