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© The Rockefeller University Press, 0021-9525/1998//61 $5.00
The Journal of Cell Biology, Volume 140, Number 1, , 1998 61-69

Article

A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion



Christian Ungermann*, Benjamin J. Nichols{ddagger}, Hugh R.B. Pelham{ddagger}, and William Wickner*

* Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844; and {ddagger} Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [{alpha}-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, {alpha}-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.

Abbreviations used in this paper: NSF, N-ethylmaleimide–sensitive fusion protein; SNAP, soluble NSF attachment proteins; SNARE, SNAP receptor; t, target membrane; v, vesicle membrane.

The work was supported by grants from the Medical Research Council to H. Pelham and B. Nichols; the National Institute of General Medical Sciences to W. Wickner and the Deutsche Forschungsgemeinschaft to C. Ungermann.

Address all correspondence to W. Wickner, Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844. Tel.: (603) 650-1701. Fax: (603) 650-1353. E-mail: Wickner.William{at}Dartmouth.edu


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