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Immunopurification of Intact Endosomal Compartments for Lipid Analyses in Arabidopsis

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Plant Endosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2177))

Abstract

Endosomes play a major role in various cellular processes including cell–cell signaling, development and cellular responses to environment. Endosomes are dynamically organized into a complex set of endomembrane compartments themselves subcompartmentalized in distinct pools or subpopulations. It is increasingly evident that endosome dynamics and maturation is driven by local modification of lipid composition. The diversity of membrane lipids is impressive and their homeostasis often involves crosstalk between distinct lipid classes. Hence, biochemical characterization of endosomal membrane lipidome would clarify the maturation steps of endocytic routes. Immunopurification of intact endomembrane compartments has been employed in recent years to isolate early and late endosomal compartments and can even be used to separate subpopulations of early endosomes. In this section, we will describe the immunoprecipitation protocol to isolate endosomes with the aim to analyze the lipid content. We will detail a procedure to identify the total fatty acid and sterol content of isolated endosomes as a first line of lipid identification. Advantages and limitations of the method will be discussed as well as potential pitfalls and critical steps.

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Acknowledgments

This work is supported by the French National Research agency (ANR) grant “caLIPSO” to Y.B (ANR-18-CE13-0025) and Overseas Research Fellowship granted from Japan Society for Promotion of Science (JSPP) to Y.I. We gratefully acknowledge Pierre Van Delft for helpful discussions and support for lipidomic analyses (Plateforme MetaboHUB-Bordeaux) (ANR-11-INBS-0010). The authors would like to warmly acknowledge Natasha Raikhel (Distinguished Professor of Plant Cell Biology, Institute for Integrative Genome Biology, University of California Riverside, USA) for making available the SYP61-CFP Arabidopsis line and the support she originally provided to analyze lipid content in immunopurified TGN fraction. We thank Patrick Moreau and Sebastien Mongrand for helpful comments and critical reading of the manuscript.

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Author notes

  1. Yoko Ito and Magali Grison contributed equally to this work.

Authors and Affiliations

  1. Laboratoire de Biogenèse Membranaire, UMR 5200, CNRS, Université de Bordeaux, Villenave d’Ornon, France

    Yoko Ito, Magali Grison, Nicolas Esnay, Laetitia Fouillen & Yohann Boutté

  2. Department of Biological Sciences, BioDiscovery Institute, University of North Texas, Denton, TX, USA

    Nicolas Esnay

  3. MetaboHub-Metabolome Facility of Bordeaux, Functional Genomics Center, Bordeaux, France

    Laetitia Fouillen

Authors
  1. Yoko Ito
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  2. Magali Grison
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  3. Nicolas Esnay
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  4. Laetitia Fouillen
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  5. Yohann Boutté
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Corresponding authors

Correspondence to Magali Grison or Yohann Boutté .

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Editors and Affiliations

  1. Department of Botany and Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA

    Marisa S. Otegui

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Ito, Y., Grison, M., Esnay, N., Fouillen, L., Boutté, Y. (2020). Immunopurification of Intact Endosomal Compartments for Lipid Analyses in Arabidopsis. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 2177. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0767-1_11

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  • DOI: https://doi.org/10.1007/978-1-0716-0767-1_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0766-4

  • Online ISBN: 978-1-0716-0767-1

  • eBook Packages: Springer Protocols

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