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Transient Expression of ESCRT Components in Arabidopsis Root Cell Suspension Culture-Derived Protoplasts

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The ESCRT Complexes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1998))

Abstract

Localization studies are important to understand the function of diverse proteins. The endosomal trafficking pathway is very complex, and a lot of proteins function in this pathway, primarily the endosomal sorting complexes required for transport (ESCRTs). Some of the ESCRT-related proteins or mutant variants cannot be stably expressed in planta due to the toxicity of their expression. Therefore, a transient expression system is necessary to study their function. Transient expression in protoplasts from Arabidopsis root cell-derived culture serves as a fast and reliable method for the expression and cell biological and biochemical analyses of otherwise toxic constructs.

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References

  1. Henne WM, Buchkovich NJ, Emr SD (2011) The ESCRT pathway. Dev Cell 21(1):77–91. https://doi.org/10.1016/j.devcel.2011.05.015

    Article  CAS  PubMed  Google Scholar 

  2. Paez Valencia J, Goodman K, Otegui MS (2016) Endocytosis and endosomal trafficking in plants. Annu Rev Plant Biol 67:309–335. https://doi.org/10.1146/annurev-arplant-043015-112242

    Article  CAS  PubMed  Google Scholar 

  3. Winter V, Hauser MT (2006) Exploring the ESCRTing machinery in eukaryotes. Trends Plant Sci 11(3):115–123. https://doi.org/10.1016/j.tplants.2006.01.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Korbei B, Moulinier-Anzola J, De-Araujo L, Lucyshyn D, Retzer K, Khan MA, Luschnig C (2013) Arabidopsis TOL proteins act as gatekeepers for vacuolar sorting of PIN2 plasma membrane protein. Curr Biol 23(24):2500–2505. https://doi.org/10.1016/j.cub.2013.10.036

    Article  CAS  PubMed  Google Scholar 

  5. Nagel MK, Kalinowska K, Vogel K, Reynolds GD, Wu Z, Anzenberger F, Ichikawa M, Tsutsumi C, Sato MH, Kuster B, Bednarek SY, Isono E (2017) Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3. Proc Natl Acad Sci U S A 114(34):E7197–E7204. https://doi.org/10.1073/pnas.1710866114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gao C, Luo M, Zhao Q, Yang R, Cui Y, Zeng Y, Xia J, Jiang L (2014) A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr Biol 24(21):2556–2563. https://doi.org/10.1016/j.cub.2014.09.014

    Article  CAS  PubMed  Google Scholar 

  7. Haas TJ, Sliwinski MK, Martinez DE, Preuss M, Ebine K, Ueda T, Nielsen E, Odorizzi G, Otegui MS (2007) The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell 19(4):1295–1312. https://doi.org/10.1105/tpc.106.049346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Babst M, Sato TK, Banta LM, Emr SD (1997) Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p. EMBO J 16(8):1820–1831. https://doi.org/10.1093/emboj/16.8.1820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Katsiarimpa A, Anzenberger F, Schlager N, Neubert S, Hauser MT, Schwechheimer C, Isono E (2011) The Arabidopsis deubiquitinating enzyme AMSH3 interacts with ESCRT-III subunits and regulates their localization. Plant Cell 23(8):3026–3040. https://doi.org/10.1105/tpc.111.087254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Spitzer C, Reyes FC, Buono R, Sliwinski MK, Haas TJ, Otegui MS (2009) The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in Arabidopsis and are required for plant development. Plant Cell 21(3):749–766. https://doi.org/10.1105/tpc.108.064865

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Spitzer C, Schellmann S, Sabovljevic A, Shahriari M, Keshavaiah C, Bechtold N, Herzog M, Muller S, Hanisch FG, Hulskamp M (2006) The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis. Development 133(23):4679–4689. https://doi.org/10.1242/dev.02654

    Article  CAS  PubMed  Google Scholar 

  12. Kolb C, Nagel MK, Kalinowska K, Hagmann J, Ichikawa M, Anzenberger F, Alkofer A, Sato MH, Braun P, Isono E (2015) FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol 167(4):1361–1373. https://doi.org/10.1104/pp.114.253377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  Google Scholar 

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Acknowledgments

This work conducted in the authors’ laboratory is supported by grants from the German Science Foundation (DFG) to E.I. (IS 221/4-1). The authors thank Prof. Takashi Ueda (NIBB, Japan) and Prof. Tomohiro Uemura (The University of Tokyo, Japan) for advices to optimize the protocol.

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Authors and Affiliations

  1. Plant Physiology and Biochemistry, Department of Biology, University of Konstanz, Konstanz, Germany

    Marie-Kristin Nagel, Karin Vogel & Erika Isono

Authors
  1. Marie-Kristin Nagel
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  2. Karin Vogel
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  3. Erika Isono
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Corresponding author

Correspondence to Erika Isono .

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Editors and Affiliations

  1. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ.Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France

    Emmanuel Culetto

  2. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ.Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France

    Renaud Legouis

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Nagel, MK., Vogel, K., Isono, E. (2019). Transient Expression of ESCRT Components in Arabidopsis Root Cell Suspension Culture-Derived Protoplasts. In: Culetto, E., Legouis, R. (eds) The ESCRT Complexes. Methods in Molecular Biology, vol 1998. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9492-2_12

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  • DOI: https://doi.org/10.1007/978-1-4939-9492-2_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9491-5

  • Online ISBN: 978-1-4939-9492-2

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