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PMID: 18650938 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones.

The EMBO journal ·Vol. 27 ·No. 15 ·2008-08-06 ·Pages 2031-42

Mima J, Hickey CM, Xu H, Jun Y, Wickner W

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.

MeSH Terms
Adenosine Triphosphatases/chemistry,metabolism Genes, Fungal Lipids/chemistry,physiology Liposomes Membrane Fusion/physiology Molecular Chaperones/chemistry,physiology Phosphatidylcholines/chemistry Phosphatidylserines/chemistry Protein Binding Protein Transport Qa-SNARE Proteins/chemistry,physiology Qb-SNARE Proteins/chemistry,physiology Qc-SNARE Proteins/chemistry,physiology R-SNARE Proteins/chemistry,physiology SNARE Proteins/physiology Saccharomyces cerevisiae/metabolism Saccharomyces cerevisiae Proteins/chemistry,metabolism Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/chemistry,metabolism Vesicular Transport Proteins/chemistry,metabolism
Chemicals
Lipids Liposomes Molecular Chaperones Phosphatidylcholines Phosphatidylserines Qa-SNARE Proteins Qb-SNARE Proteins Qc-SNARE Proteins R-SNARE Proteins SEC17 protein, S cerevisiae SNARE Proteins Saccharomyces cerevisiae Proteins Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins Vesicular Transport Proteins Adenosine Triphosphatases SEC18 protein, S cerevisiae
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Mima Joji
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3844, USA.
Hickey Christopher M
Xu Hao
Jun Youngsoo
Wickner William
References (58)
58 references, click to expand
  1. Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion.
    Biophys J. 2006 Mar 1;90(5):1661-75 PMID: 16339880
  2. Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).
    EMBO J. 1996 Jul 1;15(13):3296-305 PMID: 8670830
  3. Excess vacuolar SNAREs drive lysis and Rab bypass fusion.
    Proc Natl Acad Sci U S A. 2007 Aug 21;104(34):13551-8 PMID: 17699614
  4. Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids.
    Nat Struct Mol Biol. 2006 Mar;13(3):209-17 PMID: 16491093
  5. Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles.
    J Cell Biol. 2004 Dec 20;167(6):1087-98 PMID: 15611334
  6. A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion.
    J Cell Biol. 1998 Jan 12;140(1):61-9 PMID: 9425154
  7. Oligomerization of fusogenic peptides promotes membrane fusion by enhancing membrane destabilization.
    Biophys J. 2004 Jan;86(1 Pt 1):272-84 PMID: 14695269
  8. Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle.
    Cell. 2002 Feb 8;108(3):357-69 PMID: 11853670
  9. Sec18p and Vam7p remodel trans-SNARE complexes to permit a lipid-anchored R-SNARE to support yeast vacuole fusion.
    EMBO J. 2007 Dec 12;26(24):4935-45 PMID: 18007597
  10. Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling.
    Curr Biol. 2003 Sep 16;13(18):1636-40 PMID: 13678596
  11. Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity.
    Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2701-6 PMID: 16469845
  12. Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion.
    Nat Struct Mol Biol. 2006 Apr;13(4):323-30 PMID: 16565726
  13. SNAREs--engines for membrane fusion.
    Nat Rev Mol Cell Biol. 2006 Sep;7(9):631-43 PMID: 16912714
  14. Compartmental specificity of cellular membrane fusion encoded in SNARE proteins.
    Nature. 2000 Sep 14;407(6801):153-9 PMID: 11001046
  15. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.
    J Cell Biol. 1999 Aug 23;146(4):741-54 PMID: 10459010
  16. Modulation of poly(ethylene glycol)-induced fusion by membrane hydration: importance of interbilayer separation.
    Biochemistry. 1992 Mar 17;31(10):2653-61 PMID: 1547208
  17. Genomic analysis of homotypic vacuole fusion.
    Mol Biol Cell. 2002 Mar;13(3):782-94 PMID: 11907261
  18. Fluorescence assay for phospholipid membrane asymmetry.
    Biochemistry. 1991 Dec 24;30(51):11819-27 PMID: 1751498
  19. Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion.
    EMBO J. 2001 Aug 1;20(15):4035-40 PMID: 11483507
  20. Physiological levels of diacylglycerols in phospholipid membranes induce membrane fusion and stabilize inverted phases.
    Biochemistry. 1989 May 2;28(9):3703-9 PMID: 2751991
  21. Topological restriction of SNARE-dependent membrane fusion.
    Nature. 2000 Sep 14;407(6801):194-8 PMID: 11001058
  22. SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis.
    EMBO J. 2001 May 1;20(9):2202-13 PMID: 11331586
  23. Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs.
    Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12571-6 PMID: 10535963
  24. Selective activation of cognate SNAREpins by Sec1/Munc18 proteins.
    Cell. 2007 Jan 12;128(1):183-95 PMID: 17218264
  25. Snares and Munc18 in synaptic vesicle fusion.
    Nat Rev Neurosci. 2002 Aug;3(8):641-53 PMID: 12154365
  26. Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion.
    EMBO J. 2005 May 18;24(10):1775-86 PMID: 15889152
  27. Use of resonance energy transfer to monitor membrane fusion.
    Biochemistry. 1981 Jul 7;20(14):4093-9 PMID: 7284312
  28. New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion.
    J Cell Biol. 2000 Oct 30;151(3):551-62 PMID: 11062257
  29. Munc18-1 binds directly to the neuronal SNARE complex.
    Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2697-702 PMID: 17301226
  30. Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes.
    Nat Cell Biol. 2001 Jul;3(7):613-8 PMID: 11433291
  31. Mosaic organization of the endocytic pathway.
    Exp Cell Res. 2002 Jan 1;272(1):8-14 PMID: 11740860
  32. Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes.
    J Cell Biol. 2002 May 13;157(4):645-55 PMID: 11994317
  33. A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.
    Cell. 1993 Nov 5;75(3):409-18 PMID: 8221884
  34. A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion.
    Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9402-7 PMID: 10944212
  35. The diversity of Rab proteins in vesicle transport.
    Curr Opin Cell Biol. 1997 Aug;9(4):496-504 PMID: 9261061
  36. HOPS proofreads the trans-SNARE complex for yeast vacuole fusion.
    Mol Biol Cell. 2008 Jun;19(6):2500-8 PMID: 18385512
  37. Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p.
    EMBO J. 2006 Apr 19;25(8):1579-89 PMID: 16601699
  38. Trans-SNARE complex assembly and yeast vacuole membrane fusion.
    Proc Natl Acad Sci U S A. 2007 May 22;104(21):8755-60 PMID: 17502611
  39. Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae.
    J Bacteriol. 1991 Mar;173(6):2026-34 PMID: 2002005
  40. Rabs and their effectors: achieving specificity in membrane traffic.
    Proc Natl Acad Sci U S A. 2006 Aug 8;103(32):11821-7 PMID: 16882731
  41. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.
    Nature. 1998 Sep 24;395(6700):347-53 PMID: 9759724
  42. Lipid mixing during membrane aggregation and fusion: why fusion assays disagree.
    Biochemistry. 1987 Dec 15;26(25):8435-42 PMID: 3442666
  43. Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion.
    Mol Biol Cell. 2000 Mar;11(3):807-17 PMID: 10712501
  44. Diacylglycerol and its formation by phospholipase C regulate Rab- and SNARE-dependent yeast vacuole fusion.
    J Biol Chem. 2004 Dec 17;279(51):53186-95 PMID: 15485855
  45. Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs.
    Science. 2004 Apr 16;304(5669):435-8 PMID: 15044754
  46. SNAREpins: minimal machinery for membrane fusion.
    Cell. 1998 Mar 20;92(6):759-72 PMID: 9529252
  47. Reversible, cooperative reactions of yeast vacuole docking.
    EMBO J. 2006 Nov 15;25(22):5260-9 PMID: 17082764
  48. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs.
    Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15781-6 PMID: 9861047
  49. Class C Vps protein complex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion.
    Mol Cell. 2000 Sep;6(3):661-71 PMID: 11030345
  50. Sec1p directly stimulates SNARE-mediated membrane fusion in vitro.
    J Cell Biol. 2004 Oct 11;167(1):75-85 PMID: 15466482
  51. Liposome fusion assay to monitor intracellular membrane fusion machines.
    Methods Enzymol. 2003;372:274-300 PMID: 14610819
  52. Selective formation of Sed5p-containing SNARE complexes is mediated by combinatorial binding interactions.
    Mol Biol Cell. 2001 Mar;12(3):521-38 PMID: 11251068
  53. Vam3p structure reveals conserved and divergent properties of syntaxins.
    Nat Struct Biol. 2001 Mar;8(3):258-64 PMID: 11224573
  54. Functional architecture of an intracellular membrane t-SNARE.
    Nature. 2000 Sep 14;407(6801):198-202 PMID: 11001059
  55. The synaptic SNARE complex is a parallel four-stranded helical bundle.
    Nat Struct Biol. 1998 Sep;5(9):765-9 PMID: 9731768
  56. Yeast vacuole fusion: a model system for eukaryotic endomembrane dynamics.
    Autophagy. 2008 Jan;4(1):5-19 PMID: 17932463
  57. A fluorescence assay to monitor vesicle fusion and lysis.
    J Biol Chem. 1982 Dec 10;257(23):13892-5 PMID: 6815181
  58. Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion.
    Biochim Biophys Acta. 2000 Jul 31;1467(1):227-43 PMID: 10930525
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
1460-2075
DOI
10.1038/emboj.2008.139
Published
2008-08-06
Epub
2008-00-24
Pages
2031-42
Language
English
Region
England
NLM ID
8208664
PMCID
PMC2516887
Subset
IM
Grants
NIGMS NIH HHS · R01 GM023377 · United States
NIGMS NIH HHS · T32 GM008704 · United States
NIGMS NIH HHS · T32GM08704 · United States
NIGMS NIH HHS · GM23377 · United States
External Links
PubMed source DOI: 10.1038/emboj.2008.139 PMC Full Text View journal details
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