Abstract
Phosphoinositides are key players from which the various membranes of the cells acquire their identity. The relative accumulation of these low-abundant anionic phospholipids in the cytosolic leaflet of the plasma membrane and of various organelles generates a landmark code, responsible for the selective recruitment of extrinsic proteins at given membranes. One of the key players in the protein/lipid interaction at the plasma membrane in plant cells, is phosphatidylinositol 4-phosphate (PI4P), which patterns the recruitment of effector proteins from the plasma membrane to organelles along the endocytic pathway. Here we describe a fast assay to assess the requirement of PI4P for membrane localization of extrinsic membrane proteins in vivo. This system relies on perturbing PI4P distribution in plant cells via the action of a PI4P phosphatase that depletes the pool of PI4P at a given membrane. This system efficiently decreases PI4P levels, and can therefore be easily used to assess requirement of PI4P (and electrostatics) for the targeting of extrinsic membrane proteins to the plasma membrane or endosomes. Ultimately, this system could also be extended to test the phosphatase activity in planta of enzymes putatively involved in PI4P metabolism.
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References
Monnard PA, Walde P (2015) Current ideas about Prebiological compartmentalization. Life (Basel) 5:1239–1263
Platre MP, Jaillais Y (2017) Anionic lipids and the maintenance of membrane electrostatics in eukaryotes. Plant Signal Behav 12:e1282022
Zhu Y, Hu L, Zhou Y, Yao Q, Liu L, Shao F (2010) Structural mechanism of host Rab1 activation by the bifunctional legionella type IV effector SidM/DrrA. Proc Natl Acad Sci U S A 107:4699–4704
Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H (2009) Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of legionella pneumophila. J Biol Chem 284:4846–4856
Del Campo CM, Mishra AK, Wang YH, Roy CR, Janmey PA, Lambright DG (2014) Structural basis for PI(4)P-specific membrane recruitment of the legionella pneumophila effector DrrA/SidM. Structure 22:397–408
Simon ML, Platre MP, Marques-Bueno MM, Armengot L, Stanislas T, Bayle V, Caillaud MC, Jaillais Y (2016) A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants. Nat Plants 2:16089
Platre MP, Noack LC, Doumane M, Bayle V, Simon MLA, Maneta-Peyret L, Fouillen L, Stanislas T, Armengot L, Pejchar P, Caillaud MC, Potocky M, Copic A, Moreau P, Jaillais Y (2018) A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes. Dev Cell 45(465–80):e11
Lemmon MA (2008) Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol 9:99–111
Noack LC, Jaillais Y (2017) Precision targeting by phosphoinositides: how PIs direct endomembrane trafficking in plants. Curr Opin Plant Biol 40:22–33
Noack LC, Pejchar P, Sekeres J, Jaillais Y, Potocky M (2019) Transient gene expression as a tool to monitor and manipulate the levels of acidic phospholipids in plant cells. Methods Mol Biol 1992:189–199
Guo S, Stolz LE, Lemrow SM, York JD (1999) SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases. J Biol Chem 274:12990–12995
Hammond GR, Machner MP, Balla T (2014) A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J Cell Biol 205:113–126
Simon ML, Platre MP, Assil S, van Wijk R, Chen WY, Chory J, Dreux M, Munnik T, Jaillais Y (2014) A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis. Plant J 77:322–337
Moravcevic K, Mendrola JM, Schmitz KR, Wang YH, Slochower D, Janmey PA, Lemmon MA (2010) Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipids. Cell 143:966–977
Acknowledgments
We thank Yvon Jaillais (RDP laboratory, ENS LYON, France) for comments on the manuscript, as well as Romain Boisseau (OBEE department, University of Montana, USA) and Antoine Grissot (Department of Vertebrate Ecology and Zoology, University of Gdansk, Poland) for their advice concerning the statistical analysis. This work was supported by Seed Fund ENS LYON-2016 and Junior Investigator grant ANR-16-CE13-0021. M.D. is funded by a fellowship from the French Ministry of Higher Education.
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Doumane, M., Caillaud, MC. (2020). Assessing Extrinsic Membrane Protein Dependency to PI4P Using a Plasma Membrane to Endosome Relocalization Transient Assay in Nicotiana benthamiana. 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_9
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DOI: https://doi.org/10.1007/978-1-0716-0767-1_9
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