Figure 3.

Reversal of inhibitors of vacuole fusion (A, B). Inhibitors were added at time zero with standard fusion components. After 25 min at 27°C, the reversal agent was added. Reactions were incubated at 27°C for a total of 90 min and alkaline phosphatase activity was assayed. Inhibitor concentrations: 1.23 μM 3sQ (an equimolar mixture of MBP-Vti1, GST-Vam7, and His6-Vam3), anti-Sec18p IgG (378 nM), anti-Sec17 IgG (378 nM), anti-Vam3p IgG (353 nM), anti-Vps33p IgG (32 nM), U73122 (Calbiochem) dissolved in DMSO (80 μM), ENTH domain (29 μM), GST-PX domain of Vam7 (3.9 μM), C1b domain from PKC (Johnson et al, 2000; 41 μM), MED (Marcks effector domain; Wang J et al, 2001; synthesized by Keck Center, Yale; 10 μM), and anti-Ypt7 affinity-purified IgG (133 nM). The reversal agents were added at the following concentrations: chlorpromazine (CPZ, from Sigma) dissolved in 33% DMSO (74 μM), PIP₂ (di-palmitoyl C-16 D-myo-phosphatidylinositol 4,5-bisphosphate, from Echelon; 300 μM), his₆-Sec18p (259 nM for reversal of 3sQ, 346 nM for reversal of anti-Sec18p), and Ypt7 peptide (66.7 μg/ml). Vam7p (prepared from a C-terminal intein construct; a generous gift from A Merz, University of Seattle, WA) was used at 833 nM for reversal of C1b, anti-Sec18p, anti-Sec17p, U73122, or anti-Ypt7p and at 370 nM for reversal of anti-Vam7p or GST-PX domain of Vam7. The percent fusion is the inhibited value minus the ice control value divided by uninhibited fusion value minus the ice value, times 100%. The average uninhibited fusion was 5.02±0.21 U (Merz and Wickner, 2004). The s.e. was calculated from three or more experiments.