Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion.
Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p.
Ungermann
C
C
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844, USA.
von Mollard
G F
GF
Jensen
O N
ON
Margolis
N
N
Stevens
T H
TH
Wickner
W
W
D000483:Alleles; D000906:Antibodies / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D005656:Fungal Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010641:Phenotype; D011233:Precipitin Tests; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050683:R-SNARE Proteins; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; D013696:Temperature; D014617:Vacuoles / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D033921:Vesicular Transport Proteins
D000906:Antibodies; D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050683:R-SNARE Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; C107706:VAM3 protein, S cerevisiae; C106736:VTI1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C107127:YKT6 protein, S cerevisiae; D052066:N-Ethylmaleimide-Sensitive Proteins