A VACUOLAR V-T-SNARE COMPLEX, THE PREDOMINANT FORM IN-VIVO AND ON ISOLATED VACUOLES, IS DISASSEMBLED AND ACTIVATED FOR DOCKING AND FUSION

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide-se nsitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [alpha-SNAP]), and typical vesicle (v) and target membrane (t) SNAP re ceptors (SNAREs). We now report that vacuolar v- and t-SNAREs are main ly found with Sec17p as v-t-SNARE complexes in vivo and on purified va cuoles rather than only transiently forming such complexes during dock ing, 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 p riming, as the v-SNARE regulates the rate of Sec17p release and, in tu rn, Sec17p-dependent SNARE complex disassembly is required for indepen dent function of the two SNAREs. Sec17p physically and functionally in teracts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but n ot the v-SNARE, block Sec17p release. (b) Sec17p is associated with th e t-SNARE in the absence of V-SNARE, but is not bound to the v-SNARE w ithout 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 disassem ble the v-t-SNARE complex, prime the t-SNARE, and release the Sec17p t o allow SNARE participation in docking and fusion. These studies sugge st 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 deter gent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.