Figure 10.

Enhanced membrane fusion and priming kinetics via ERG6 induction. (A) Standard fusion reactions contained wild-type vacuoles from strains KTY1 and 2, or wild-type sterol-enriched vacuoles from strains KTY3 and 4. At various times aliquots were placed on ice to stop further fusion. Reactions were then assayed for alkaline phosphatase activity (top panel), and the rate of fusion was calculated as follows: ALP units_{(tn + 30 min)} - ALP units_(tn)/time (bottom panel). (B and C) Fusion reactions, supplemented with 5 μg/ml Sec18p, were analyzed for Sec17p release by coprecipitation with vacuoles. Immunoblots show the decrease of membrane-bound levels of Sec17p over time (0, 5, 10, 15, 30, and 60 min). Vacuoles were prepared from wild-type (B) and vrp1Δ (C) strains either with sterol enrichment via P_GAL1-ERG6 induction (▴) or without (□). (D) Sec17p release from vacuoles isolated from the erg6Δ strain (KTY15). Immunoblots from four experiments were analyzed by densitometry (see Materials and Methods), and values were normalized to maximum signals. Error bars, SE.