2000 Fiscal Year Annual Research Report

核蛋白質輸送担体importinβを利用した核膜孔複合体の分子構築の解析

Research Project

Project/Area Number	12480215
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)	今本尚子大阪大学, 構造遺伝学研究センター, 助教授 (20202145)
Keywords	importin β / 核蛋白質輸送 / 核膜孔複合体 / X線結晶構造 / ヌクレオポリン
Research Abstract	核-細胞質間の物質輸送は,真核細胞内の情報交換を担う重要なシステムであり,分子レベルでのメカニズム解明が急速に進んできたが,蛋白質が,核膜に存在する関門である核膜孔をどのように方向性を持って通過していくのかはほとんど理解されていない.本研究では,それ自身で核膜孔を両方向性に通過する性質を持つimportin βに着目し,蛋白質を通過させるという観点から,核膜孔複合体の分子構築を解明することを目的として,以下の研究を進めた.先ず,importin βのX線結晶構造解析のデータや,種を越えて保存されているアミノ酸配列などを参考にして,importin βの核膜孔通過に必須の領域の内,Ranに結合する側のヘリックス構造の部分や,その反対側のヘリックス構造の部分に人工的に点変異を導入した変異型importin βを構築した.分子表面に現れていると予測される複数のアミノ酸を選び,それらをすべてアラニンに置換した.レコンビナント変異型importin βを培養細胞の細胞質あるいは核にインジェクションし,それらの挙動を観察したところ,Ranに結合する側のヘリックスに変異を導入した蛋白質(以下,4B5Bと呼ぶ)は,野生型importin βに比較して,核への集積が顕著であった.一方,Ranとの結合には直接関係しない側のヘリックス構造に変異を導入した蛋白質(以下,5A6Aと呼ぶ)は,野生型に比べて核への集積が低下していた.この結果は,セミインタクト細胞を用いたin vitro系においても再現が見られた.以上より,5A6Aの変異型importin βは,核から細胞質への通過の能力は野生型と同程度に保たれているが,細胞質から核への移行活性は低下していることが推測され,4B5Bの変異型importin βは,全く逆の活性変化を起こしていることが予測された.これらの変異蛋白質と核膜孔複合体構成因子(ヌクレオポリン)との親和性を計測することにより,蛋白質の核膜孔通過における方向性の鍵を握るヌクレオポリンが決定される可能性があると思われる.

Research Products

(15 results)

All Other

All Publications (15 results)

[Publications] Haraguchi,T.: "Live fluorescence imaging reveals early recruitment of emerin, LBR, HP1β and RanBP2 to reforming functional nuclear envelope."J.Cell Sci.. 113. 779-794 (2000)
[Publications] Matsubae,M.: "Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein."FEBS Lett.. 468. 171-175 (2000)
[Publications] Tachibana,T.: "Recycling of importin α from the nucleus is suppressed by loss of RCC1 function in living mammalian cells."Cell Struct.Funct.. 25. 95-103 (2000)
[Publications] Ikuta,T.: "Nucleocytoplasmic shuttling of the aryl hydrocarbon receptor."J.Biochem.. 127. 503-509 (2000)
[Publications] Tanabe,K.: "The small GTP-binding protein TC10 promotes growth cone formation and nerve elongation in neuronal cells, and its transcription is induced during nerve regeneration in rat."J.Neurosci.. 20. 4138-4144 (2000)
[Publications] Takeda,K.: "Characterization of human herpesvirus 7 U27 gene product and identification of its nuclear localization signal."Virol.. 272. 394-401 (2000)
[Publications] Lee,S.J.: "The adoption of a twisted structure of importin-β is essential for the protein-protein interaction required for nuclear transport."J.Mol.Biol.. 302. 251-264 (2000)
[Publications] Sueishi,M.: "The FHA domain of Ki-67 antigen interacts with a novel kinesin-like protein Hklp2."J.Biol.Chem.. 275. 28888-28892 (2000)
[Publications] Yoneda,Y.: "Nucleocytoplasmic protein traffic and its significance to cell function."Genes Cells. 5. 777-787 (2000)
[Publications] Yoneda,Y.: "New steps toward the nucleocytoplasmic traffic of macromolecules."Cell Struct.Funct.. 25. 241-242 (2000)
[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.. (in press). (2001)
[Publications] Akakura,S.: "A role for Hsc70 in regulating nucleo-cytoplasmic transport of a temperature-sensitive p53 (p53Val135)."J.Biol.Chem.. (in press). (2001)
[Publications] Kurisaki,A.: "Transforming growth factor-β induces nuclear import of Smad3 in an importin-β1 and Ran-dependent manner."Mol.Biol.Cell. (in press). (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.. (in press). (2001)

2000 Fiscal Year Annual Research Report

核蛋白質輸送担体importinβを利用した核膜孔複合体の分子構築の解析

Principal Investigator

米田 悦啓 大阪大学, 医学系研究科, 教授 (80191667)

Research Products

[Publications] Haraguchi,T.: "Live fluorescence imaging reveals early recruitment of emerin, LBR, HP1β and RanBP2 to reforming functional nuclear envelope."J.Cell Sci.. 113. 779-794 (2000)

[Publications] Matsubae,M.: "Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein."FEBS Lett.. 468. 171-175 (2000)

[Publications] Tachibana,T.: "Recycling of importin α from the nucleus is suppressed by loss of RCC1 function in living mammalian cells."Cell Struct.Funct.. 25. 95-103 (2000)

[Publications] Ikuta,T.: "Nucleocytoplasmic shuttling of the aryl hydrocarbon receptor."J.Biochem.. 127. 503-509 (2000)

[Publications] Tanabe,K.: "The small GTP-binding protein TC10 promotes growth cone formation and nerve elongation in neuronal cells, and its transcription is induced during nerve regeneration in rat."J.Neurosci.. 20. 4138-4144 (2000)

[Publications] Takeda,K.: "Characterization of human herpesvirus 7 U27 gene product and identification of its nuclear localization signal."Virol.. 272. 394-401 (2000)

[Publications] Lee,S.J.: "The adoption of a twisted structure of importin-β is essential for the protein-protein interaction required for nuclear transport."J.Mol.Biol.. 302. 251-264 (2000)

[Publications] Sueishi,M.: "The FHA domain of Ki-67 antigen interacts with a novel kinesin-like protein Hklp2."J.Biol.Chem.. 275. 28888-28892 (2000)

[Publications] Yoneda,Y.: "Nucleocytoplasmic protein traffic and its significance to cell function."Genes Cells. 5. 777-787 (2000)

[Publications] Yoneda,Y.: "New steps toward the nucleocytoplasmic traffic of macromolecules."Cell Struct.Funct.. 25. 241-242 (2000)

[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.. (in press). (2001)

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

[Publications] Kurisaki,A.: "Transforming growth factor-β induces nuclear import of Smad3 in an importin-β1 and Ran-dependent manner."Mol.Biol.Cell. (in press). (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.. (in press). (2001)

米田悦啓大阪大学, 医学系研究科, 教授 (80191667)