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Preparation of Proteoliposomes with Purified TMEM16 Protein for Accurate Measures of Lipid Scramblase Activity

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Intracellular Lipid Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1949))

Abstract

The distribution of different lipid species between the two leaflets is tightly regulated and underlies the concerted action of distinct catalytic entities. While flippases and floppases establish membrane asymmetry, scramblases randomize the lipid distribution and play pivotal roles during blood clotting, apoptosis, and in processes such as N-linked glycosylation of proteins. The recent discovery of TMEM16 family members acting as scramblases has led to an increasing demand for developing protocols tailored for TMEM16 proteins to enable functional investigations of their scrambling activity. Here we describe a protocol for the expression, purification, and functional reconstitution of TMEM16 proteins into preformed liposomes and measurement of their scrambling activity using fluorescence-labeled lipid derivatives. The reconstitution involves extrusion of liposomes through a membrane, destabilization of liposomes using Triton X-100, and stepwise detergent removal by adsorption on styryl-beads. The scrambling assay is based on the selective bleaching of nitrobenzoxadiazol fluorescent lipids on the outer leaflet of liposomes by the membrane-impermeant reducing agent sodium dithionite. The assay allows conclusions on the substrate specificity and on the kinetics of the transported lipids as shown with the example of a Ca2+-activated TMEM16 scramblase from the fungus Nectria haematococca (nhTMEM16).

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Acknowledgments

We thank Prof. Raimund Dutzler (University of Zurich) for support of this work and critically reading the manuscript.

Competing Financial Interests

The authors declare that they have no competing financial interests.

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Authors and Affiliations

  1. Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen, Switzerland

    Janine Denise Brunner & Stephan Schenck

Authors
  1. Janine Denise Brunner
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  2. Stephan Schenck
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Corresponding author

Correspondence to Janine Denise Brunner .

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Editors and Affiliations

  1. Institut de Pharmacologie Moléculaire et Cellulaire, Université Côte d’Azur and CNRS, Valbonne, France

    Guillaume Drin

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Brunner, J.D., Schenck, S. (2019). Preparation of Proteoliposomes with Purified TMEM16 Protein for Accurate Measures of Lipid Scramblase Activity. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9136-5_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9135-8

  • Online ISBN: 978-1-4939-9136-5

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