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SNAREs pp 277-288 | Cite as

An In Vitro Assay of Trans-SNARE Complex Formation During Yeast Vacuole Fusion Using Epitope Tag-Free SNAREs

  • Youngsoo Jun
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

SNARE complexes assembled between fusing membranes (in trans) are the core machinery driving lipid bilayer merger. Thus, an assay monitoring the formation of these trans-SNARE complexes is essential for SNARE-mediated membrane fusion studies. Homotypic yeast vacuole fusion is an important model system for such studies. Although several assays measuring trans-SNARE complex formation are available to study yeast vacuole fusion, most use SNAREs conjugated with epitope tags, which may affect the function of SNAREs or even the formation of trans-SNARE complexes. Here, I describe an assay for trans-SNARE complex formation during yeast vacuole fusion that does not require epitope-tagged SNAREs.

Key words

Trans-SNARE complex Yeast Vacuole Membrane fusion In vitro assay 

References

  1. 1.
    Jahn R, Lang T, Südhof TC (2003) Membrane fusion. Cell 112:519–533CrossRefGoogle Scholar
  2. 2.
    Wickner W, Schekman R (2008) Membrane fusion. Nat Struct Mol Biol 15:658–664CrossRefGoogle Scholar
  3. 3.
    Jahn R, Scheller RH (2006) SNAREs--engines for membrane fusion. Nat Rev Mol Cell Biol 7:631–643.  https://doi.org/10.1038/nrm2002CrossRefPubMedGoogle Scholar
  4. 4.
    Fasshauer D, Sutton RB, Brunger AT, Jahn R (1998) Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc Natl Acad Sci U S A 95:15781–15786CrossRefGoogle Scholar
  5. 5.
    Sutton RB, Fasshauer D, Jahn R, Brunger AT (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395:347–353.  https://doi.org/10.1038/26412CrossRefPubMedGoogle Scholar
  6. 6.
    Weber T, Zemelman BV, McNew JA et al (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92:759–772CrossRefGoogle Scholar
  7. 7.
    Ungermann C, Sato K, Wickner W (1998) Defining the functions of trans-SNARE pairs. Nature 396:543–548.  https://doi.org/10.1038/25069CrossRefPubMedGoogle Scholar
  8. 8.
    Nichols BJ, Ungermann C, Pelham HR et al (1997) Homotypic vacuolar fusion mediated by t- and v-SNAREs. Nature 387:199–202.  https://doi.org/10.1038/387199a0CrossRefPubMedGoogle Scholar
  9. 9.
    Wickner W (2010) Membrane fusion: five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles. Annu Rev Cell Dev Biol 26:115–136.  https://doi.org/10.1146/annurev-cellbio-100109-104131CrossRefPubMedGoogle Scholar
  10. 10.
    Wickner W, Haas A (2000) Yeast homotypic vacuole fusion: a window on organelle trafficking mechanisms. Annu Rev Biochem 69:247–275.  https://doi.org/10.1146/annurev.biochem.69.1.247CrossRefPubMedGoogle Scholar
  11. 11.
    Ostrowicz CW, Meiringer CTA, Ungermann C (2008) Yeast vacuole fusion: a model system for eukaryotic endomembrane dynamics. Autophagy 4:5–19CrossRefGoogle Scholar
  12. 12.
    Wickner W (2002) Yeast vacuoles and membrane fusion pathways. EMBO J 21:1241–1247.  https://doi.org/10.1093/emboj/21.6.1241CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Haas A, Conradt B, Wickner W (1994) G-protein ligands inhibit in vitro reactions of vacuole inheritance. J Cell Biol 126:87–97CrossRefGoogle Scholar
  14. 14.
    Jun Y, Wickner W (2007) Assays of vacuole fusion resolve the stages of docking, lipid mixing, and content mixing. Proc Natl Acad Sci U S A 104:13010–13015.  https://doi.org/10.1073/pnas.0700970104CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Collins KM, Wickner WT (2007) Trans-SNARE complex assembly and yeast vacuole membrane fusion. Proc Natl Acad Sci U S A 104:8755–8760.  https://doi.org/10.1073/pnas.0702290104CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Jun Y, Xu H, Thorngren N, Wickner W (2007) Sec18p and Vam7p remodel trans-SNARE complexes to permit a lipid-anchored R-SNARE to support yeast vacuole fusion. EMBO J 26:4935–4945.  https://doi.org/10.1038/sj.emboj.7601915CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Pieren M, Schmidt A, Mayer A (2010) The SM protein Vps33 and the t-SNARE H(abc) domain promote fusion pore opening. Nat Struct Mol Biol 17:710–717.  https://doi.org/10.1038/nsmb.1809CrossRefPubMedGoogle Scholar
  18. 18.
    Wada Y, Nakamura N, Ohsumi Y, Hirata A (1997) Vam3p, a new member of syntaxin related protein, is required for vacuolar assembly in the yeast Saccharomyces cerevisiae. J Cell Sci 110(Pt 11):1299–1306PubMedGoogle Scholar
  19. 19.
    Darsow T, Rieder SE, Emr SD (1997) A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole. J Cell Biol 138:517–529CrossRefGoogle Scholar
  20. 20.
    Daniel Gietz R, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Meth Enzymol 350:87–96.  https://doi.org/10.1016/S0076-6879(02)50957-5CrossRefPubMedGoogle Scholar
  21. 21.
    Scott JH, Schekman R (1980) Lyticase: endoglucanase and protease activities that act together in yeast cell lysis. J Bacteriol 142:414–423PubMedPubMedCentralGoogle Scholar
  22. 22.
    Shen SH, Chrétien P, Bastien L, Slilaty SN (1991) Primary sequence of the glucanase gene from Oerskovia xanthineolytica. Expression and purification of the enzyme from Escherichia coli. J Biol Chem 266:1058–1063PubMedGoogle Scholar
  23. 23.
    Slusarewicz P, Xu Z, Seefeld K et al (1997) I2B is a small cytosolic protein that participates in vacuole fusion. Proc Natl Acad Sci U S A 94:5582–5587CrossRefGoogle Scholar
  24. 24.
    Starai VJ, Jun Y, Wickner W (2007) Excess vacuolar SNAREs drive lysis and Rab bypass fusion. Proc Natl Acad Sci U S A 104:13551–13558.  https://doi.org/10.1073/pnas.0704741104CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Rak A, Fedorov R, Alexandrov K et al (2000) Crystal structure of the GAP domain of Gyp1p: first insights into interaction with Ypt/Rab proteins. EMBO J 19:5105–5113.  https://doi.org/10.1093/emboj/19.19.5105CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Thorngren N, Collins KM, Fratti RA et al (2004) A soluble SNARE drives rapid docking, bypassing ATP and Sec17/18p for vacuole fusion. EMBO J 23:2765–2776.  https://doi.org/10.1038/sj.emboj.7600286CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Ko Y-J, Lee M, Kang K et al (2014) In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion. Proc Natl Acad Sci U S A 111:7677–7682.  https://doi.org/10.1073/pnas.1400036111CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Haas A (1995) A quantitative assay to measure homotypic vacuole fusion in vitro. Methods Cell Sci 17:283–294.  https://doi.org/10.1007/BF00986234CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Life Sciences, Cell Logistics Research Center, and Silver Health Bio Research CenterGwangju Institute of Science and TechnologyGwangjuRepublic of Korea

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