Salt-washed vacuoles require Sec18p, LMA1, CoA, and CaM for optimal fusion. Vacuoles (11) were incubated for 100 min at 27°C with ATP and Sec18p (0.33 μg/ml), LMA1 (0.33 μg/ml), CoA (20 μM), and CaM (7.5 μM) as indicated. Alkaline phosphatase activity was measured (27).