Membrane fusion reactions limited by defective SNARE zippering or stiff lipid fatty acyl composition have distinct requirements for Sec17, Sec18, and adenine nucleotide

Abstract

Intracellular membrane fusion is catalyzed by SNAREs, Rab GTPases, SM proteins, tethers, Sec18/NSF and Sec17/SNAP. Membrane fusion has been reconstituted with purified vacuolar proteins and lipids to address 3 salient questions: whether ATP hydrolysis by Sec18 affects its promotion of fusion, whether fusion promotion by Sec17 and Sec18 is only seen with mutant SNAREs or can also be seen with wild-type SNAREs, and whether Sec17 and Sec18 only promote fusion when they work together or whether they can each work separately. Fusion is driven by two engines, completion of SNARE zippering (which does not need Sec17/Sec18) and Sec17/Sec18-mediated fusion (needing SNAREs but not the energy from their complete zippering). Sec17 is required to rescue fusion that is blocked by incomplete zippering, though optimal rescue also needs the ATPase Sec18. ATP is an essential Sec18 ligand, but at limiting Sec17 levels Sec18 ATP hydrolysis also drives release of Sec17 without concomitant trans-SNARE complex disassembly. At higher (physiological) Sec17 levels, or without ATP hydrolysis, fusion prevails over Sec17 release. Stiff 16:0, 18:1 fatty acyl chain lipids provide an alternative route to suppressing fusion, with entirely wild-type SNAREs and without impediment to zippering. In this case, Sec17 and Sec18 restore comparable fusion with either ATP or a nonhydrolyzable analog. 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). With distinct fusion impediments, Sec18 and Sec17 have both shared roles and independent roles in promoting fusion. SignificanceSec17/SNAP and Sec18/NSF catalyze cis-SNARE complex disassembly through ATP hydrolysis, but also drive fusion itself without ATP hydrolysis. Fusion can be inhibited by either "stiff" fatty acyl chains while allowing full zippering of entirely wild-type SNAREs or by incomplete SNARE zippering. In either case, Sec17 and Sec18 restore full fusion, but with clear differences. With defective zippering, high levels of Sec17 can restore fusion without Sec18. With stiff fatty acyl chains, Sec18 alone can restore fusion but Sec17 alone will not. In both cases, Sec17 and Sec18 are more efficient together. SNARE-dependent membrane fusion thus relies on two energy sources, complete SNARE zippering and interactions with Sec17 and Sec18.