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Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles

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SNAREs

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

Abstract

Fluorescence de-quenching can be used to analyze membrane lipid mixing during an in vitro fusion reaction. Here we describe a method to measure lipid mixing using vacuolar membranes purified from the yeast Saccharomyces cerevisiae. Labeling the isolated organelles with rhodamine-phosphatidylethanolamine allows to reveal ATP-dependent lipid mixing through fluorescence de-quenching in a spectrofluorometer. Combining this assay with content mixing indicators, such as the fusion-dependent maturation of a luminal vacuolar phosphatase, then permits the detection of hemifusion intermediates and the analysis of the requirements for fusion pore opening.

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Authors and Affiliations

  1. Département de Biochimie, Université de Lausanne, Epalinges, Switzerland

    Massimo D’Agostino & Andreas Mayer

Authors
  1. Massimo D’Agostino
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  2. Andreas Mayer
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Correspondence to Andreas Mayer .

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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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D’Agostino, M., Mayer, A. (2019). Assay of Lipid Mixing and Fusion Pore Formation in the Fusion of Yeast Vacuoles. 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_16

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  • DOI: https://doi.org/10.1007/978-1-4939-8760-3_16

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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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