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SNAREs pp 175-189 | Cite as

Analysis of the Role of Sec3 in SNARE Assembly and Membrane Fusion

  • Kunrong Mei
  • Peng Yue
  • Wei Guo
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are highly conserved and tightly regulated by a variety of factors. The exocyst complex is one of the multi-subunit tethering complexes and functions in the tethering of the secretory vesicles to the plasma membrane. We have found that the yeast Sec3, a subunit of the exocyst, binds to the t-SNARE protein Sso2 and promotes its interaction with another t-SNARE protein, Sec9. Here, we describe the structural analysis and in vitro membrane fusion assays, by which we found that Sec3 binding leads to a conformational change within Sso2, and facilitates SNARE assembly and the membrane fusion.

Key words

Sec3 Soluble N-ethylmaleimide-sensitive factor attachment protein receptor Sso2 Sec9 Structure analysis Liposome Membrane fusion 

Notes

Acknowledgments

We thank Shelby Wilkinson for helpful suggestions. We also thank the Gang Dong lab for solving the structure of the Sec3N-Sso2 complex. The work in W. G. lab is supported by National Institutes of Health R01 grant GM111128.

Reference

  1. 1.
    Rothman JE (1994) Mechanisms of intracellular protein transport. Nature 372:55–63CrossRefGoogle Scholar
  2. 2.
    Sutton RB, Fasshauer D, Jahn R, Brunger AT (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2. 4 Å resolution. Nature 395:347–353CrossRefGoogle Scholar
  3. 3.
    Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F et al (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92:759–772CrossRefGoogle Scholar
  4. 4.
    Protopopov V, Govindan B, Novick P, Gerst JE (1993) Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiae. Cell 74:855–861CrossRefGoogle Scholar
  5. 5.
    Brennwald P, Kearns B, Champion K, Keränen S, Bankaitis V, Novick P (1994) Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell 79:245–258CrossRefGoogle Scholar
  6. 6.
    Aalto MK, Ronne H, Keränen S (1993) Yeast syntaxins Sso1p and Sso2p belong to a family of related membrane proteins that function in vesicular transport. EMBO J 12:4095–4104CrossRefGoogle Scholar
  7. 7.
    Ferro-Novick S, Jahn R (1994) Vesicle fusion from yeast to man. Nature 370:191–193CrossRefGoogle Scholar
  8. 8.
    Nicholson KL, Munson M, Miller RB, Filip TJ, Fairman R, Hughson FM (1998) Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p. Nat Struct Biol 5:793–802CrossRefGoogle Scholar
  9. 9.
    Munson M, Chen X, Cocina AE, Schultz SM, Hughson FM (2000) Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly. Nat Struct Biol 7:894–902CrossRefGoogle Scholar
  10. 10.
    TerBush DR, Maurice T, Roth D, Novick P (1996) The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 15:6483–6494CrossRefGoogle Scholar
  11. 11.
    Guo W, Grant A, Novick P (1999) Exo84p is an exocyst protein essential for secretion. J Biol Chem 274:23558–23564CrossRefGoogle Scholar
  12. 12.
    Hsu SC, Ting AE, Hazuka CD, Davanger S, Kenny JW, Kee Y et al (1996) The mammalian brain rsec6/8 complex. Neuron 17:1209–1219CrossRefGoogle Scholar
  13. 13.
    Grote E, Carr CM, Novick P (2000) Ordering the final events in yeast exocytosis. J Cell Biol 151:439–452CrossRefGoogle Scholar
  14. 14.
    He B, Guo W (2009) The exocyst complex in polarized exocytosis. Curr Opin Cell Biol 21:537–542CrossRefGoogle Scholar
  15. 15.
    Wu B, Guo W (2015) The exocyst at a glance. J Cell Sci 128:2957–2964CrossRefGoogle Scholar
  16. 16.
    Yue P, Zhang Y, Mei K, Wang S, Lesigang J, Zhu Y et al (2017) Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion. Nat Commun 8:14236CrossRefGoogle Scholar
  17. 17.
    Scott BL, Van Komen JS, Liu S, Weber T, Melia TJ, McNew JA (2003) Liposome fusion assay to monitor intracellular membrane fusion machines. Methods Enzymol 372:274–300CrossRefGoogle Scholar
  18. 18.
    Kreye S, Malsam J, Sollner TH (2008) In vitro assays to measure SNARE-mediated vesicle fusion. Methods Mol Biol 440:37–50CrossRefGoogle Scholar
  19. 19.
    Ollivon M, Lesieur S, Grabielle-Madelmont C, Paternostre M (2000) Vesicle reconstitution from lipid-detergent mixed micelles. Biochim Biophys Acta 1508:34–50CrossRefGoogle Scholar
  20. 20.
    Schaffner W, Weissmann C (1973) A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56:502–514CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of BiologyUniversity of PennsylvaniaPhiladelphiaUSA

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