Ueda Haruko, Yokota Etsuo, Kuwata Keiko, Kutsuna Natsumaro, Mano Shoji, Shimada Tomoo, Tamura Kentaro, Stefano Giovanni, Fukao Yoichiro, Brandizzi Federica, Shimmen Teruo, Nishimura Mikio, Hara-Nishimura Ikuko
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.).
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.)
Plant Physiol. 2016 Feb;170(2):867-80. doi: 10.1104/pp.15.01172. Epub 2015 Dec 18.
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.
内质网（ER）由动态变化的小管和潴泡组成。在动物和酵母中，同型内质网膜融合由膜相关的发动蛋白样GTP酶（分别为atlastin和Sey1p）介导。在拟南芥中，另一种发动蛋白样GTP酶，即根毛缺陷3（RHD3），被认为是一种内质网膜融合蛋白，但缺乏直接证据。在此，我们表明RHD3具有内质网膜融合活性，其C末端的磷酸化可增强该活性。通过外源添加GTP、ATP和F-肌动蛋白，从烟草（Nicotiana tabacum）培养细胞的胞质溶胶和微粒体部分以RHD3依赖的方式重建了内质网网络。接下来，我们建立了一个用拟南芥内质网囊泡形成内质网小管的体外检测系统，其中添加GTP会导致内质网囊泡形成内质网囊。随后对该系统施加剪切力会以RHD依赖的方式触发内质网囊形成小管。出乎意料的是，在没有剪切力的情况下，丝氨酸/苏氨酸激酶处理会触发RHD3依赖的小管形成。质谱分析表明，RHD3在C末端的多个丝氨酸和苏氨酸残基处被磷酸化。一种针对RHD3 C末端肽的抗体消除了激酶触发的小管形成。当丝氨酸簇被删除或丝氨酸残基被丙氨酸残基取代时，激酶处理对小管形成没有影响。激酶处理诱导了RHD3的寡聚化。对于atlastin或Sey1p，尚未报道膜融合的磷酸化依赖性调节或寡聚化。综上所述，我们提出RHD3的磷酸化刺激寡聚化增强了内质网膜融合以形成内质网网络。
