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SNAREs pp 303–322Cite as

Reconstituted Proteoliposome Fusion Mediated by Yeast SNARE-Family Proteins

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1860))

Abstract

Membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-family proteins is an essential process for intracellular membrane trafficking in all eukaryotic cells, which delivers proteins and lipids to their appropriate subcellular membrane compartments such as organelles and plasma membrane. The molecular basis of SNARE-mediated membrane fusion has been revealed by studying fusion of reconstituted proteoliposomes bearing purified SNARE-family proteins and chemically defined lipid species. This chapter describes the detailed experimental protocols for (1) purification of recombinant SNARE-family and SM (Sec1/Munc18-family) proteins in the yeast Saccharomyces cerevisiae; (2) preparation of reconstituted proteoliposomes bearing purified yeast SNARE proteins; and (3) developing an assay to monitor lipid mixing between reconstituted SNARE-bearing proteoliposomes. Lipid mixing assays for reconstituted SNARE-bearing proteoliposomes are useful for evaluating the intrinsic capacity of SNARE-family proteins to directly catalyze membrane fusion and to determine the specificity of membrane fusion.

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Acknowledgments

This work was supported by the Program to Disseminate Tenure Tracking System from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) and Grants-in-Aid for Scientific Research from MEXT to J.M.

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Authors and Affiliations

  1. Institute for Protein Research, Osaka University, Osaka, Japan

    Joji Mima

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  1. Joji Mima
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Correspondence to Joji Mima .

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Editors and Affiliations

  1. Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Rutilio Fratti

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Mima, J. (2019). Reconstituted Proteoliposome Fusion Mediated by Yeast SNARE-Family Proteins. In: Fratti, R. (eds) SNAREs. Methods in Molecular Biology, vol 1860. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8760-3_20

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  • DOI: https://doi.org/10.1007/978-1-4939-8760-3_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8759-7

  • Online ISBN: 978-1-4939-8760-3

  • eBook Packages: Springer Protocols

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