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Studying the ArfGAP-Dependent Conformational Changes in SNAREs

  • Fernanda Rodriguez
  • Anne Spang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 619)

Abstract

Vesicle SNAREs (v-SNAREs) are included with high fidelity into each transport vesicle generated in the cell. These SNAREs determine the fate of vesicles, as they are the key factors deciding with which compartment a particular vesicle will fuse. The mechanism of high fidelity inclusion of SNAREs into transport vesicles is very difficult to study in vivo. Therefore, we use in vitro assays aiming to recapitulate SNARE uptake into vesicles. One of the key assays is a pull-down with SNARE tails fused to GST in the presence or absence of ArfGAPs and coat components such as the small GTPase Arf1 and coatomer. This in vitro assay allowed us to show that the ArfGAPs Glo3 and Gcs1 can induce a conformational change in SNAREs. Protease protection assays were used to confirm the conformational change and can also be used to address the question about the nature of the different conformations in SNARE proteins.

Key words

SNAREs Glo3p Arf1p conformational changes SNARE binding assay protease protection assays 

Notes

Acknowledgments

The work on ArfGAPs and SNAREs in the Spang lab is funded by the University of Basel and the Swiss National Science Foundation.

References

  1. 1.
    Kirchhausen, T. (2002) Clathrin adaptors really adapt. Cell 109(4), 413–416.CrossRefPubMedGoogle Scholar
  2. 2.
    Spang, A. (2002) ARF1 regulatory factors and COPI vesicle formation. Curr Opin Cell Biol 14(4), 423–427.CrossRefPubMedGoogle Scholar
  3. 3.
    Lewis, S.M., Poon, P.P., Singer, R.A., Johnston, G.C., Spang, A. (2004) The ArfGAP Glo3 is required for the generation of COPI vesicles. Mol Biol Cell 15(9), 4064–4072.CrossRefPubMedGoogle Scholar
  4. 4.
    Lee, S.Y., Yang, J.S., Hong, W., Premont, R.T., Hsu, V.W. (2005) ARFGAP1 plays a central role in coupling COPI cargo sorting with vesicle formation. J Cell Biol 168(2), 281–290.CrossRefPubMedGoogle Scholar
  5. 5.
    Rein, U., Andag, U., Duden, R., Schmitt, H.D., Spang, A. (2002) ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat. J Cell Biol 157(3), 395–404.CrossRefPubMedGoogle Scholar
  6. 6.
    Schindler, C., Spang, A. (2007) Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18p/NSF. Mol Biol Cell 18(8), 2852–2863.CrossRefPubMedGoogle Scholar
  7. 7.
    Poon, P.P., Cassel, D., Spang, A., Rotman, M., Pick, E., Singer, R.A., Johnston, G.C. (1999) Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function. EMBO J 18(3), 555–564.CrossRefPubMedGoogle Scholar
  8. 8.
    Kahn, R.A., Clark, J., Rulka, C., Stearns, T., Zhang, C.J., Randazzo, P.A., Terui, T., Cavenagh, M. (1995) Mutational analysis of Saccharomyces cerevisiae ARF1. J Biol Chem 270(1), 143–150.CrossRefPubMedGoogle Scholar
  9. 9.
    Paris, S., Béraud-Dufour, S., Robineau, S., Bigay, J., Antonny, B., Chabre, M., Chardin, P. (1997) Role of protein-phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor Arno. J Biol Chem 272(35), 22221–22226.CrossRefPubMedGoogle Scholar
  10. 10.
    Springer, S., Schekman, R. (1998) Nucleation of COPII vesicular coat complex by endoplasmic reticulum to Golgi vesicle SNAREs. Science 281(5377), 698–700.CrossRefPubMedGoogle Scholar
  11. 11.
    Albert, S., Gallwitz, D. (1999) Two new members of a family of Ypt/Rab GTPase activating proteins. Promiscuity of substrate recognition. J Biol Chem 274(47), 33186–33189.CrossRefPubMedGoogle Scholar
  12. 12.
    Poon, P.P., Cassel, D., Huber, I., Singer, R.A., Johnston, G.C. (2001) Expression, analysis, and properties of yeast ADP-ribosylation factor (ARF) GTPase activating proteins (GAPs) Gcs1 and Glo3. Methods Enzymol 329, 317–324.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Fernanda Rodriguez
    • 1
  • Anne Spang
    • 1
  1. 1.Biozentrum, Growth and DevelopmentUniversity of BaselBaselSwitzerland

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