The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport

Vps45p loses membrane association upon inactivation of PP1. (A) Tlg1p-containing complexes were immunoprecipitated from cells harboring the glc7–10 mutation (NOzY23) grown at 25°C (25°C), or grown at 25°C and then incubated at 37°C for 10 min before cell lysate preparation (37°C). Immunoblot analysis was used to detect the amount of Tlg1p, Tlg2p, Vti1p, HA-tagged Snc2p, and Vps45p in the immunoprecipitated complexes. (B) wild-type (SF838–9D), sec18–1 (NOzY22), vps21D (SGY79), and glc7–10 (PAY704–1) cells were incubated at 37°C for 10 min before fractionation by differential centrifugation to yield a whole cell extract (WCE), a low-speed membrane pellet (P13), a high-speed membrane pellet (P100), and a soluble, cytosolic fraction (S100). The amount of Vps45p in each fraction was assessed using immunoblot analysis.