Three v-SNAREs and Two t-SNAREs, Present in a Pentameric cis-SNARE Complex on Isolated Vacuoles, Are Essential for Homotypic Fusion

doi:10.1083/jcb.145.7.1435

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J. Cell Biol., Volume 145, Number 7, June 28, 1999 1435-1442

Three v-SNAREs and Two t-SNAREs, Present in a Pentameric cis-SNARE Complex on Isolated Vacuoles, Are Essential for Homotypic Fusion

Christian Ungermann,^* Gabriele Fischer von Mollard, Ole N. Jensen,^§ Nathan Margolis,^* Tom H. Stevens, and William Wickner^*

^* Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844; Georg-August-Universität Göttingen, Biochemie II, 37073 Göttingen, Germany; ^§ Protein Research Group, Department of Molecular Biology, Odense University, 5230 Odense M, Denmark; and University of Oregon, Institute of Molecular Biology, Eugene, Oregon 97405

Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex onisolated organelles. We now identify the v-SNAREs Vti1p and Ykt6pby mass spectrometry as additional components of the immunoisolatedvacuolar SNARE complex. Immunodepletion of detergent extractswith anti-Vti1p removes all the Ykt6p that is in a complex withVam3p, immunodepletion with anti-Ykt6p removes all the Vti1p thatis complexed with Vam3p, and immunodepletion with anti-Nyv1p removesall the Ykt6p in complex with other SNAREs, demonstrating thatthey are all together in the same cis multi-SNARE complex. Afterpriming, which disassembles the cis-SNARE complex, antibodiesto any of the five SNARE proteins still inhibit the fusion assayuntil the docking stage is completed, suggesting that each SNAREplays a role in docking. Furthermore, vti1 temperature-sensitivealleles cause a synthetic fusion-defective phenotype in our reaction.Our data show that vacuole-vacuole fusion requires a cis-SNAREcomplex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREsNyv1p, Vti1p, andYkt6p.

Key words: SNAREs; membrane fusion; yeast vacuoles; NSF; $alpha$ -SNAP

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