2001 Fiscal Year Annual Research Report

細胞質から核への蛋白質輸送の分子機構:その多様性とエネルギー要求性

Research Project

Project/Area Number	11237202
Research Institution	Osaka University
Principal Investigator	米田悦啓大阪大学, 医学系研究科, 教授 (80191667)
Co-Investigator(Kenkyū-buntansha)	原口徳子独立行政法人通信総合研究所, 関西先端研究センター・生物情報研究室, 主任研究員
Keywords	核蛋白質輸送 / CaM kinase IV / importin α / importin β / 核膜孔複合体 / green fluorescent protein (GFP) / セミインタクト細胞
Research Abstract	核蛋白質輸送の多様性を知る目的で、神経細胞では主として核に局在しているが、それ以外の細胞、例えば、甲状腺傍濾胞細胞では細胞質全体に局在しており、細胞種によって異なる局在を示す分子である、Ca/calmodulin-dependent protein kinase type IV(CaM kinase IV)に着目し、その輸送機構の解明を進めた。先ず、COS7細胞ではCaM kinase IVが核に移行できることがわかったので、GFPを融合させた様々な欠失変異体をCOS7細胞に発現させ、CaM kinase IVの核内移行に必須の領域の同定を進めた。その結果、N末端側の58-80番目のアミノ酸残基の部分にCaM kinase IVを核へ移行させる活性があることがわかった。また、この部分は、分子の構造上、常に分子表面に露出していない可能性が示唆され、どのようなメカニズムで分子表面に露出され、輸送装置と出会うようになるのかという点も興味深い研究対象であることがわかってきた。一方、これまで核蛋白質と輸送担体であるimportinβとをつなぐアダプター分子として知られていたimportinαが、それ自身単独で核内に移行するという現象をはじめて見出した。このimportinα単独の核膜孔通過は、酵母からヒトまで種を越えて保存されていること、importinβの輸送経路と競合しないことも明らかとなった。さらに、その挙動をin vitro系でリアルタイムで観察したところ、importinβの挙動とは全く異なり、個々の核ごとで蓄積する速度に大きな差があるという興味深い結果が得られた。このことから、これまで多様性はないと考えられていた核膜孔複合体の構造あるいは構成成分がそれぞれの核によって多様性がある可能性が示唆されるとともに、蛋白質の核膜孔通過に少なくとも2つ以上の経路がある可能性が示された。また、importinαは、細胞を紫外線で照射した時に、importinβとは独立した形で速やかに核内に集積することがわかったが、この現象は、importinαが単独で核内に移行する生理的意義の理解につながる可能性があり、興味深い。

Research Products
(14 results)

All Other

All Publications (14 results)

[Publications] Shimizu, S.: "Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells"J.Cell Biol.. 152. 237-250 (2001)
[Publications] Nagoshi, E.: "Dimerization of the sterol regulatory element-binding protein-2 via the helix-loop-helix-leucine zipper domain is prerequisite for its nuclear localization mediated by importin β"Mol.Cell.Biol.. 21. 2779-2789 (2001)
[Publications] Jiang, C.-J.: "Molecular cloning of a novel importin α homologue from rice, by which constitutive photomorphogenic 1 (COP1) nuclear localization signal (NLS)-protein is preferentially nuclear imported"J.Biol.Chem.. 276. 9322-9329 (2001)
[Publications] Akakura, S.: "A role for Hsc70 in regulating nucleo-cytoplasmic transport of a temperature-sensitive p53 (p53Val135)"J.Biol.Chem.. 276. 14649-14657 (2001)
[Publications] Kurisaki, A.: "Transforming growth factor-b induces nuclear import of Smad3 in an importin-b1 and Ran-dependent manner"Mol.Biol.Cell. 12. 1079-1091 (2001)
[Publications] Hieda, M.: "Nuclear import of the U1A splicesome protein is mediated by importin α/β and Ran in living mammalian cells"J.Biol.Chem.. 276. 16824-16832 (2001)
[Publications] Takagi, M.: "A novel nucleolar protein, NIFK, interacts with the forkhead associated domain of Ki-67 antigen in mitosis"J.Biol.Chem.. 276. 25386-25391 (2001)
[Publications] Haraguchi, T.: "BAF is required for emerin assembly into the reforming nuclear envelope"J.Cell Sci.. 114. 4575-4585 (2001)
[Publications] Kobayashi, M.: "Nuclear localization of duplin, a β-catenin-binding protein, is essential for its inhibitory activity on the Wnt signaling pathway"J.Biol.Chem.. (in press). (2002)
[Publications] Shibata, S.: "Nucleocytoplasmic transport of proteins and poly(A)+RNA at the reconstituted Tpr-less nuclei in living mammalian cells"Genes Cells. (in press). (2002)
[Publications] Katahira, J.: "Complex formation between Tap and p15 affects binding to FG-repeat nucleoporins and nucleocytoplasmic shuttling"J.Biol.Chem.. (in press). (2002)
[Publications] Miyamoto, Y.: "Genomic organization and chromosomal localization of the importin a1 gene in the mouse"Gene. (in press). (2002)
[Publications] Matsuoka, Yu.: "A chromodomain-containing nuclear protein, MRG15 is expressed as a novel type of dendritic mRNA in neurons"Neurosci.Res.. (in press). (2002)
[Publications] Matsuoka, Yo.: "Bovine liver chromatin fraction contains actin polymerization activity inducing micronuclei formation when injected into prometaphase cultured cells"Cell Struct.Funct.. (in press). (2002)

2001 Fiscal Year Annual Research Report

細胞質から核への蛋白質輸送の分子機構:その多様性とエネルギー要求性

Principal Investigator

米田 悦啓 大阪大学, 医学系研究科, 教授 (80191667)

Research Products

[Publications] Shimizu, S.: "Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells"J.Cell Biol.. 152. 237-250 (2001)

[Publications] Nagoshi, E.: "Dimerization of the sterol regulatory element-binding protein-2 via the helix-loop-helix-leucine zipper domain is prerequisite for its nuclear localization mediated by importin β"Mol.Cell.Biol.. 21. 2779-2789 (2001)

[Publications] Jiang, C.-J.: "Molecular cloning of a novel importin α homologue from rice, by which constitutive photomorphogenic 1 (COP1) nuclear localization signal (NLS)-protein is preferentially nuclear imported"J.Biol.Chem.. 276. 9322-9329 (2001)

[Publications] Akakura, S.: "A role for Hsc70 in regulating nucleo-cytoplasmic transport of a temperature-sensitive p53 (p53Val135)"J.Biol.Chem.. 276. 14649-14657 (2001)

[Publications] Kurisaki, A.: "Transforming growth factor-b induces nuclear import of Smad3 in an importin-b1 and Ran-dependent manner"Mol.Biol.Cell. 12. 1079-1091 (2001)

[Publications] Hieda, M.: "Nuclear import of the U1A splicesome protein is mediated by importin α/β and Ran in living mammalian cells"J.Biol.Chem.. 276. 16824-16832 (2001)

[Publications] Takagi, M.: "A novel nucleolar protein, NIFK, interacts with the forkhead associated domain of Ki-67 antigen in mitosis"J.Biol.Chem.. 276. 25386-25391 (2001)

[Publications] Haraguchi, T.: "BAF is required for emerin assembly into the reforming nuclear envelope"J.Cell Sci.. 114. 4575-4585 (2001)

[Publications] Kobayashi, M.: "Nuclear localization of duplin, a β-catenin-binding protein, is essential for its inhibitory activity on the Wnt signaling pathway"J.Biol.Chem.. (in press). (2002)

[Publications] Shibata, S.: "Nucleocytoplasmic transport of proteins and poly(A)+RNA at the reconstituted Tpr-less nuclei in living mammalian cells"Genes Cells. (in press). (2002)

[Publications] Katahira, J.: "Complex formation between Tap and p15 affects binding to FG-repeat nucleoporins and nucleocytoplasmic shuttling"J.Biol.Chem.. (in press). (2002)

[Publications] Miyamoto, Y.: "Genomic organization and chromosomal localization of the importin a1 gene in the mouse"Gene. (in press). (2002)

[Publications] Matsuoka, Yu.: "A chromodomain-containing nuclear protein, MRG15 is expressed as a novel type of dendritic mRNA in neurons"Neurosci.Res.. (in press). (2002)

[Publications] Matsuoka, Yo.: "Bovine liver chromatin fraction contains actin polymerization activity inducing micronuclei formation when injected into prometaphase cultured cells"Cell Struct.Funct.. (in press). (2002)

米田悦啓大阪大学, 医学系研究科, 教授 (80191667)