Fig. 8. The molecular requirements for the late-stage GTPase activity and Ca²⁺ are functionally linked. (A) Reactions blocked for fusion by either the calcium chelator BAPTA or GTPγS are still sensitive to the other inhibitor. Fusion reactions (120 µl) were incubated at 27°C for 45 min in the presence of 2 mM Mg-GTPγS (middle panel) or 2.5 mM BAPTA (bottom panel) to allow docking without fusion. One set of reactions was not pre-treated (top panel). Vacuoles were re-isolated and aliquots added to tubes containing reaction buffer on ice or reaction buffer with either no inhibitor, anti-Vam3p antibodies, Gdi1p + Gyp7p, 2 mM GTPγS, 50 µM MAS7 or 2.5 mM BAPTA. Reactions were supplemented with LMA1 and calmodulin (5 µg/ml and 150 µg/ml, respectively) and incubated (27°C, 90 min) before assaying for alkaline phosphatase activity. (B) Non-hydrolyzable GTP analogs and G-protein ligands inhibit the release of Ca²⁺ during vacuole fusion reactions. Fusion reactions (700 µl) were incubated at 27°C in the presence of Gdi1p, 2 mM Mg-GppNHp or no inhibitor. A control reaction did not contain an energy source. At the indicated times, 100 µl samples were removed, centrifuged at 9 000 g for 5 min, and 90 µl of the supernatant were assayed for Ca²⁺ (see Material and methods).