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© The Rockefeller University Press, 0021-9525/1999//1435 $5.00
The Journal of Cell Biology, Volume 145, Number 7, , 1999 1435-1442

Regular Articles

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{ddagger}, Ole N. Jensen§, Nathan Margolis*, Tom H. Stevens||, and William Wickner*

* Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844; {ddagger} 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 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.

Key Words: SNAREs • membrane fusion • yeast vacuoles • NSF • {alpha}-SNAP

Abbreviations used in this paper: MALDI, matrix-assisted laser desorption/ionization; ts, temperature-sensitive.

C. Ungermann's present address is Biochemie Zentrum Heidelberg (BZH), Universität Heidelberg, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany.


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