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.
Mesh Terms:
Alleles, Antibodies, Carrier Proteins, Chromatography, Affinity, Fungal Proteins, Intracellular Membranes, Membrane Fusion, Membrane Proteins, N-Ethylmaleimide-Sensitive Proteins, Nerve Tissue Proteins, Nuclear Proteins, Phenotype, Precipitin Tests, Protein Binding, Qa-SNARE Proteins, Qb-SNARE Proteins, R-SNARE Proteins, Receptors, Cytoplasmic and Nuclear, SNARE Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins, Synaptosomal-Associated Protein 25, Temperature, Vacuoles, Vesicular Transport Proteins
J. Cell Biol.
Date: Jun. 28, 1999
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