Figure 5.

Vacuolar acidification. (A) Sensitivity of the acidification of wild-type and mutant vacuoles to CA, CCCP, and Ca²⁺. Vacuoles (20 μg) from BJ3505 wild-type or pmc1Δvcx1Δ strains were incubated at 27°C for 30 min in the presence (lanes 2–7) or absence of ATP (lane 1). CA (250 μM), CCCP (50 μM), apyrase (40 units/ml), or Ca²⁺ (5 mM) were added where indicated. Quinacrine (7) was added at 200 μM to all samples just before the start of the incubation. At the end of the incubation, vacuoles were reisolated (3 min, 8,000 × g, 4°C), resuspended in 900 μl of 0.4% Triton X-100 in water, and assayed for OD₄₃₀. (B) Partial reversal of CA-mediated block in acidification by Ca²⁺ and CaM. The experiment was performed as described in A. Ca²⁺ (1.5 mM) and CaM (10 μM) were added where indicated.