title: Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18.
abstract: Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17(L291A,L292A) did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17(F21S,M22S), with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17(F21S,M22S), interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.
authors.affiliation: Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755; and michael.zick@dartmouth.edu bill.wickner@dartmouth.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Zick
authors.affiliation: Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755; and.
authors.forename: Amy
authors.initials: A
authors.lastname: Orr
authors.affiliation: Departments of Biochemistry and.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Schwartz
authors.affiliation: Departments of Biochemistry and Physiology and Biophysics, University of Washington, Seattle, WA 98195.
authors.forename: Alexey J
authors.initials: AJ
authors.lastname: Merz
authors.affiliation: Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755; and michael.zick@dartmouth.edu bill.wickner@dartmouth.edu.
authors.forename: William T
authors.initials: WT
authors.lastname: Wickner
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008561:Membrane Fusion; D009154:Mutation; D011485:Protein Binding; D011510:Proteolipids / Q000737:chemistry; D050600:SNARE Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D008055:Lipids; D008081:Liposomes; D011510:Proteolipids; C075137:SEC17 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; C020485:proteoliposomes; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae
keywords: NSF; SNAREs; membrane fusion; α-SNAP