Sec18 ATP hydrolysis selectively releases Sec17 from <i>trans</i>-SNARE complexes, inhibiting membrane fusion

Intracellular membrane fusion is catalyzed by SNAREs, Rabs, SM proteins, tethers, Sec18/NSF, and Sec17/SNAP. Fusion has two engines, completion of SNARE zippering (without needing Sec17/Sec18) and Sec17/Sec18 (which need SNAREs but not energy from their complete zippering). We have reconstituted membrane fusion with purified vacuolar proteins to address three questions: 1) whether Sec18 ATP hydrolysis affects fusion, 2) whether Sec17 and Sec18 only promote fusion with mutant SNAREs or also work with wild-type SNAREs, and 3) whether Sec17 and Sec18 can separately promote fusion. We find that 1) Sec18 ATP hydrolysis blocks fusion at limiting Sec17 levels by releasing Sec17 without concomitant <i>trans</i>-SNARE complex disassembly. At higher (physiological) Sec17 levels, or without ATP hydrolysis, fusion prevails over Sec17 release. 2) With entirely wild-type SNAREs and with unimpeded SNARE zippering, Sec17, Sec18, and either ATP or a nonhydrolyzable ATP analogue stimulate fusion. 3) Sec17 and Sec18 work together but can act independently. Fusion blocked by impaired zippering can be restored by concentrated Sec17 alone (but not by Sec18), while fusion inhibited by stiff fatty acyl chains is partially restored by Sec18 alone (but not by Sec17). Optimal fusion needs zippering, Sec17, and Sec18.