Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).
In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles. We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p. Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro. Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion. We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion. Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p.
Haas
A
A
Department of Biochemistry, Dartmouth Medical School, NH 03775-3844, USA.
Wickner
W
W
D000214:Acyl Coenzyme A / Q000378:metabolism; D000251:Adenosine Triphosphatases; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005656:Fungal Proteins / Q000276:immunology / Q000378:metabolism*; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D014617:Vacuoles / Q000502:physiology*; D033921:Vesicular Transport Proteins
D000214:Acyl Coenzyme A; D000906:Antibodies; D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C075137:SEC17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; D003065:Coenzyme A