title: Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.
abstract: The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3.
authors.affiliation: Laboratory of Developmental Cell Biology, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Present address: Laboratory of Plant Molecular Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Fujimoto
authors.affiliation: RIKEN Center for Advanced Photonics, Live Cell Molecular Imaging Research Team, Extreme Photonics Research Group, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan Present address: Department of Molecular Cell Biology, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Suda
authors.affiliation: INRA, UMR1318, Institut Jean-Pierre Bourgin, Saclay Plant Sciences, F-78000 Versailles, France AgroParisTech, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France.
authors.forename: Samantha
authors.initials: S
authors.lastname: Vernhettes
authors.affiliation: Laboratory of Developmental Cell Biology, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan RIKEN Center for Advanced Photonics, Live Cell Molecular Imaging Research Team, Extreme Photonics Research Group, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakano
authors.affiliation: Laboratory of Developmental Cell Biology, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Japan Science and Technology Agency (JST), PRESTO, 4-1-8 Honcho Kawaguchi, Saitama, 332-0012 Japan tueda@bs.s.u-tokyo.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueda
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism*; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001152:Arsenicals / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D005964:Glucosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007038:Hypotonic Solutions / Q000494:pharmacology; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology*; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport / Q000187:drug effects; D013843:Thiazines / Q000494:pharmacology; D013997:Time Factors
chemical_list: D029681:Arabidopsis Proteins; D001152:Arsenicals; C434263:CGA 325615; D002867:Chromones; D002966:Clathrin; D007038:Hypotonic Solutions; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D013843:Thiazines; C029341:oxophenylarsine; D049452:Green Fluorescent Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C516179:At5g05170 protein, Arabidopsis; D005964:Glucosyltransferases; D058539:Phosphatidylinositol 3-Kinase; D019870:1-Phosphatidylinositol 4-Kinase
keywords: CESA; Cellulose synthase; Endocytosis; Membrane trafficking; Phosphoinositides