期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：18
页码：E2290-E2297
DOI：10.1073/pnas.1506409112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceIntracellular membrane trafficking relies on SNARE proteins from apposed membranes to form trans-complexes. Sec18 (N-ethylmaleimide-sensitive factor; NSF) and its cochaperone Sec17 (soluble NSF attachment protein; -SNAP) disassemble cis-SNARE complexes, liberating SNAREs for trans-complex assembly. We now describe an additional function of Sec17, its ability to trigger the fusion of trans-SNARE paired membranes. We propose a model in which Sec17 oligomerizes on trans-SNARE complexes, inserting apolar loops into the adjacent membranes. This precisely localized membrane interaction may disturb the lipid bilayer, lowering the energy barrier that prevents the two membranes from merging, and thereby facilitate fusion. Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; -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 ATP{gamma}S-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: Sec17L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17F21S,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 Sec17F21S,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.
