Figure 6. Vacuoles treated with the Pah1p inhibitor propranolol have a decreased level of Sec18p bound to cis-SNARE complexes.

Vacuoles were harvested from BJ3505 yeast expressing CBP-Vam3p and assayed for Sec18p binding to cis-SNARE complexes. Fusion reactions were incubated at 27°C for the indicated times in the presence of propranolol or ATPγS or fusion buffer. At the indicated times, vacuoles were isolated by centrifugation and solubilized. Calmodulin agarose was used to pull down CBP-Vam3p and attached proteins were resolved by SDS-PAGE and imaged by immunoblotting. Fusion reactions were treated with PS buffer, 2 mM propranolol (A) or 1 mM ATPγS (B). (C) Liposome binding and effect of the nucleotide-binding state of Sec18p was tested using liposomes containing 10% PA. His₆-Sec18p (2 µg) was incubated with PA liposomes in binding buffer alone or in the presence of 1 mM Mg²⁺, 1 mM EDTA, or 1 mM ATPγS. Densitometry values for Sec18p pull down were normalized against the corresponding pull down Vam7p value. Graph shows the normalized average ratios (n=3). * P<0.05; ** P<0.001; *** P<0.0001.