1999 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 Category	Grant-in-Aid for Scientific Research on Priority Areas (B)
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)	原口徳子郵政省通信総合研究所, 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 / Importin / CaMキナーゼIV / ノックアウトマウス / 細胞内情報伝達 / 遺伝子破壊 / 核局在化シグナル / 核膜孔複合体
Research Abstract	(1)Importin αの多様性に着目した研究マウスにおいては、importin αファミリーは大きく3つのサブグループに分類できることが知られており、その中の1つであるNPI-1と通称されている分子は、脳の神経細胞で強い発現が見られる。そこで、個体レベルで見た場合、それぞれのimportin α分子がどのような役割分担をしているかを明らかにする目的でNPI-1遺伝子ノックアウトマウスの作成を開始した。現在、遺伝子破壊用ベクターの作成を完了し、それをES細胞に導入することができた。 (2)細胞種特異的な核蛋白質輸送機構の解明 Calcium/calmodulin-dependent protein kinase type IV(CaM kinase IV)を指標にして研究を進めた。大腸菌で発現させたレコンビナントGFP-CaM kinase IVを培養細胞の細胞質にインジェクションしたところ、COS7細胞では速やかに核に移行したのに対し、HeLa細胞では核への集積がほとんど見られなかった。次に、ジギトニンで処理したCOS7セミインタクト細胞を用いたin vitro核蛋白質輸送実験系を利用して解析を進めたところ、Ehrlich腹水癌細胞およびマウス脳からの細胞抽出液に依存してCaM kinase IVの核への移行が見られたが、脳からの抽出液の方がより強い輸送活性が認められた。CaM kinase IVの核への移行は、importin αのIBB(importin β binding)domainやimportin βの核膜孔複合体結合領域によっては阻害されないことから、importin α/βが関与する輸送経路ではない、新規の経路で輸送されていることが示唆された。以上の結果を考え合わせると、CaM kinase IVは、importin βファミリー分子に依存しない、未知の輸送因子によって核内へ輸送されており、その輸送活性が細胞種によって何らかの形で調節されるという、細胞種特異性のある新規輸送系が存在する可能性が示唆された。

Research Products

(16 results)

All Other

All Publications (16 results)

[Publications] Tachibana, T.: "Up-regulation of nuclear protein import by nuclear localization signal sequences in living cells"FEBS Lett.. 442. 235-240 (1999)
[Publications] Matsuoka, Y.: "Identification and characterrization of nuclear pore subcomplexes in mitotic extract of human somatic cells"Biochem. Biophys. Res. Commun.. 254. 417-423 (1999)
[Publications] Kose, S.: "β-Subunit of nuclear pore-targeting complex (importin-β) can be exported from the nucleus in a Ran-independent manner"J. Biol. Chem.. 274. 2946-3952 (1999)
[Publications] Hieda, M.: "A monoclonal antibody to the C-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells"J. Cell. Biol.. 144. 645-655 (1999)
[Publications] Kamei, Y.: "Three distinct classes of the α subunit of the nuclear pore-targeting complex (importin-α) are differentially expressed in adult mouse tissues"J. Histochem. Cytochem.. 47. 363-372 (1999)
[Publications] Kikuchi, N.: "Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase"J. Biochem.. 125. 487-494 (1999)
[Publications] Yokoya, F.: "β-Catenin can be transported into the nucleus in a Ran-unassisted manner"Mol. Biol. Cell. 10. 1119-1131 (1999)
[Publications] Nagoshi, E.: "Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor occurs through the direct interaction of importin β with HTH-Zip"Mol. Biol. cell. 10. 2221-2233 (1999)
[Publications] Takagi, M.: "Chmadrin : a novel Ki-67 antigen-related perichromosomal protein possibly implicated in higher order chromatin structure"J. Cell Sci.. 112. 2463-2472 (1999)
[Publications] Sahara, S.: "Acinus, a caspase-3-activated protein responsible for apoptotic chromatin condensation"Nature. 401. 168-173 (1999)
[Publications] Ohshima, T.: "CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mine"Biochem. Biophys. Res. Commun.. 264. 144-150 (1999)
[Publications] Kose, S.: "Distinct energy requirement for nuclear import and export of importin β in living cells"FEBS Lett.]. 463. 327-330 (1999)
[Publications] Yoneda, Y.: "Nucleocytoplasmic protein transport and recycling of Ran"Cell Struct. Funct.. 24. 425-433 (1999)
[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.. (in press).
[Publications] Tachibana, T.: "Recycling of importin α from the nucleus is suppressed by loss of RCC1 fuction in living mammalian cells"Cell Struct Funct.. (in press).

1999 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] Tachibana, T.: "Up-regulation of nuclear protein import by nuclear localization signal sequences in living cells"FEBS Lett.. 442. 235-240 (1999)

[Publications] Matsuoka, Y.: "Identification and characterrization of nuclear pore subcomplexes in mitotic extract of human somatic cells"Biochem. Biophys. Res. Commun.. 254. 417-423 (1999)

[Publications] Kose, S.: "β-Subunit of nuclear pore-targeting complex (importin-β) can be exported from the nucleus in a Ran-independent manner"J. Biol. Chem.. 274. 2946-3952 (1999)

[Publications] Hieda, M.: "A monoclonal antibody to the C-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells"J. Cell. Biol.. 144. 645-655 (1999)

[Publications] Kamei, Y.: "Three distinct classes of the α subunit of the nuclear pore-targeting complex (importin-α) are differentially expressed in adult mouse tissues"J. Histochem. Cytochem.. 47. 363-372 (1999)

[Publications] Kikuchi, N.: "Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase"J. Biochem.. 125. 487-494 (1999)

[Publications] Yokoya, F.: "β-Catenin can be transported into the nucleus in a Ran-unassisted manner"Mol. Biol. Cell. 10. 1119-1131 (1999)

[Publications] Nagoshi, E.: "Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor occurs through the direct interaction of importin β with HTH-Zip"Mol. Biol. cell. 10. 2221-2233 (1999)

[Publications] Takagi, M.: "Chmadrin : a novel Ki-67 antigen-related perichromosomal protein possibly implicated in higher order chromatin structure"J. Cell Sci.. 112. 2463-2472 (1999)

[Publications] Sahara, S.: "Acinus, a caspase-3-activated protein responsible for apoptotic chromatin condensation"Nature. 401. 168-173 (1999)

[Publications] Ohshima, T.: "CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mine"Biochem. Biophys. Res. Commun.. 264. 144-150 (1999)

[Publications] Kose, S.: "Distinct energy requirement for nuclear import and export of importin β in living cells"FEBS Lett.]. 463. 327-330 (1999)

[Publications] Yoneda, Y.: "Nucleocytoplasmic protein transport and recycling of Ran"Cell Struct. Funct.. 24. 425-433 (1999)

[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.. (in press).

[Publications] Tachibana, T.: "Recycling of importin α from the nucleus is suppressed by loss of RCC1 fuction in living mammalian cells"Cell Struct Funct.. (in press).

米田悦啓大阪大学, 医学系研究科, 教授 (80191667)