title: Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis.
abstract: The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Haruko
authors.initials: H
authors.lastname: Ueda
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.) yokota@sci.u-hyogo.ac.jp ihnishi@gr.bot.kyoto-u.ac.jp.
authors.forename: Etsuo
authors.initials: E
authors.lastname: Yokota
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Keiko
authors.initials: K
authors.lastname: Kuwata
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Natsumaro
authors.initials: N
authors.lastname: Kutsuna
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Shoji
authors.initials: S
authors.lastname: Mano
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Tomoo
authors.initials: T
authors.lastname: Shimada
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tamura
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Giovanni
authors.identifier: http://orcid.org/0000-0002-2744-0052
authors.initials: G
authors.lastname: Stefano
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Fukao
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Federica
authors.initials: F
authors.lastname: Brandizzi
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Teruo
authors.initials: T
authors.lastname: Shimmen
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.).
authors.forename: Mikio
authors.initials: M
authors.lastname: Nishimura
authors.affiliation: Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (H.U., To.S., K.T., I.H.-N.); Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan (E.Y., Te.S.); Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan (K.K.); Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan (N.K.); Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (S.M., M.N.); MSU-DOE Plant Research Laboratory and Department of Plant Biology, Michigan State University, Michigan 48824 (G.S., F.B.); and Department of Bioinformatics, Ritsumeikan University, Kusatsu 525-8577, Japan (Y.F.) yokota@sci.u-hyogo.ac.jp ihnishi@gr.bot.kyoto-u.ac.jp.
authors.forename: Ikuko
authors.identifier: http://orcid.org/0000-0001-8814-1593
authors.initials: I
authors.lastname: Hara-Nishimura
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D001681:Biological Assay; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D055503:Protein Multimerization; D012694:Serine / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D010748:Phosphopeptides; D012694:Serine; D006160:Guanosine Triphosphate; D011494:Protein Kinases; D019204:GTP-Binding Proteins; C464507:RHD3 protein, Arabidopsis