Article
- The EMBO Journal (2008) 27, 2031 - 2042
- doi:10.1038/emboj.2008.139
Published online: 24 July 2008
Subject Category:
Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones
Joji Mima1, Christopher M Hickey1, Hao Xu1, Youngsoo Jun1,a and William Wickner1
- Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA
Correspondence to:
William Wickner, Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Room 425 Remsen, Hanover, NH 03755-3844, USA. Tel.: +1 603 650 1701; Fax: +1 603 650 1353; E-mail: Bill.Wickner@dartmouth.edu
aPresent address: Department of Biological Sciences, Seoul National University, Seoul 151-747, South Korea.
Received 6 May 2008; Accepted 27 June 2008
Abstract
The homotypic fusion of yeast vacuoles, each with 3Q- and 1R-SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q-SNAREs on one and the R-SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion-competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.
Keywords:
- membrane fusion,
- reconstitution,
- SNARE chaperones,
- regulatory lipids
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