Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complexon isolated vacuoles, are essential for homotypic fusion

Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the V-SNARE Nyv1 p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional c omponents of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a compl ex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that i s complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all togeth er in the same cis multi-SNARE complex. After priming, which disassembles t he cis-SNARE complex, antibodies to any of the five SNARE proteins still in hibit the fusion assay until the docking stage is completed, suggesting tha t each SNARE plays a role in docking. Furthermore, vti1 temperature-sensiti ve alleles cause a synthetic fusion-defective phenotype in our reaction. Ou r data show that vacuole-vacuole fusion requires a cis-SNARE complex of fiv e SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Y kt6p.