Journal
EMBO JOURNAL
Volume 20, Issue 15, Pages 4035-4040Publisher
OXFORD UNIV PRESS
DOI: 10.1093/emboj/20.15.4035
Keywords
ergosterol; membrane fusion; Sec17p; Sec18p; vacuoles
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In vitro homotypic fusion of yeast vacuoles occurs in three stages: priming, the Sec18 (NSF)-mediated changes that precede vacuole association; docking, the Ypt7 and SNARE-mediated pairing of vacuoles; and fusion, mediated by calmodulin/V0/t-SNARE interactions. Defects in catalysts of each stage result in fragmented (unfused) vacuoles. Strains with deletions in any of ERG genes 3-6, lacking normal ergosterol biosynthesis, have fragmented vacuoles. The ergosterol ligands filipin, nystatin and amphotericin B block the in vitro fusion of vacuoles from wild-type cells. Each of these inhibitors acts at the priming stage to inhibit Sec17p release from vacuoles. A reversible delay in Sec18p action prevents vacuoles from acquiring resistance to any of these three drugs, confirming that their action is on the normal fusion pathway. Ergosterol or cholesterol delivery to wild-type vacuoles stimulates their in vitro fusion, and the in vitro fusion of erg Delta vacuoles requires added sterol. The need for ergosterol for vacuole priming underscores the role of lipids in organizing the membrane elements of this complex reaction.
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