Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones.
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.
Mima
Joji
J
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.
Hickey
Christopher M
CM
Xu
Hao
H
Jun
Youngsoo
Y
Wickner
William
W
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D005800:Genes, Fungal; D008055:Lipids / Q000737:chemistry / Q000502:physiology*; D008081:Liposomes; D008561:Membrane Fusion / Q000502:physiology*; D018832:Molecular Chaperones / Q000737:chemistry / Q000502:physiology*; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D011485:Protein Binding; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000737:chemistry / Q000502:physiology; D050766:Qb-SNARE Proteins / Q000737:chemistry / Q000502:physiology; D050767:Qc-SNARE Proteins / Q000737:chemistry / Q000502:physiology; D050683:R-SNARE Proteins / Q000737:chemistry / Q000502:physiology; D050600:SNARE Proteins / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000737:chemistry / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*
D008055:Lipids; D008081:Liposomes; D018832:Molecular Chaperones; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C075137:SEC17 protein, S cerevisiae; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae