Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion.
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 ergDelta 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.
Kato
M
M
Department of Biochemistry, Dartmouth Medical School, Vail Building, Hanover, NH 03755-3844, USA.
Wickner
W
W
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004875:Ergosterol / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D008561:Membrane Fusion / Q000502:physiology*; D008780:Methyltransferases / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000502:physiology; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; C022673:delta 24-sterol reductase; C039306:sterol delta-5 desaturase; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; D004875:Ergosterol