Figure 5.

Actin ligands inhibit vacuole fusion. (A) Inhibition of vacuole fusion by latrunculin B and jasplakinolide. Drugs were added to standard fusion reactions. (B) Standard vacuole fusion reactions were arrested while undocked by incubation with 64 μg/ml Ypt7 antibodies or when docked (but not fused) by incubation with 3.5 mM BAPTA for 35 or 45 min, respectively. The indicated second inhibitors were added after reversal of the initial blocks by the addition of either 30 μg/ml YPT7 peptide or 4 mM calcium, respectively. (C) Kinetic analysis of inhibition by latrunculin B. A large standard fusion reaction was incubated at 27°C. At various times, 30-μl aliquots were placed on ice (defining the fusion curve) or added to tubes containing anti-Sec18p antibodies (priming curve), anti-Ypt7p antibodies (docking curve), or latrunculin B (300 μM final concentration), and further incubated at 27°C for 90 min total.