Fig 5.

ATPase activity of the V656A mutant. ATPase was measured as described in the experimental procedures using 16 μg of PM vesicle protein from double-copy strains (see reference 23). Reactions with a final volume of 100 μl were initiated by addition of ATP and incubated at 35°C for 8 min before being terminated with 2.5% (final volume) SDS. The nonspecific activity (<5%) present in the Δpdr5 strain is subtracted prior to calculating the activity. (A) Activity as a function of ATP concentration. Data represent the averages of at least two independent vesicle preparations per strain, and 3 independent experiments were performed. (B) Activity in the presence of clotrimazole (3.1 to 50 μM). Standard ATPase conditions were used. PM vesicles were incubated at room temperature for 5 min in the presence of clo before initiation of the reactions with the addition of ATP to 3 mM. ■, WT; ▲, V656A mutant. n = 3.