title: The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes.
abstract: The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan; Departments of Animal Sciences and Agricultural Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Shibano
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mamada
authors.affiliation: Departments of Animal Sciences and Agricultural Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Hakuno
authors.affiliation: Departments of Animal Sciences and Agricultural Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Taira
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000032:analysis / Q000378:metabolism; D019556:COS Cells; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D034882:Lamins / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D060066:Protein Interaction Maps; D029867:Xenopus Proteins / Q000032:analysis / Q000378:metabolism*; D014982:Xenopus laevis / Q000196:embryology / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000032:analysis / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D034882:Lamins; D008565:Membrane Proteins; C539763:Nemp1 protein, Xenopus; D009687:Nuclear Proteins; D029867:Xenopus Proteins; C000607185:Nemp1 protein, mouse; D020931:ran GTP-Binding Protein