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2003 Fiscal Year Final Research Report Summary

Genetic and biochemical dissection of the 26S proteasomes

Research Project

Project/Area Number 13480225
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Molecular biology
Research InstitutionThe University of Tokyo

Principal Investigator

AKIO Toh-c  The University of Tokyo, Graduate School of Science, Department of Biological Sciences, Professor, 大学院・理学系研究科, 教授 (90029249)

Co-Investigator(Kenkyū-buntansha) YOKOSAWA Hideyoshi  Hokkaido University, Graduate School of Pharmaceutical Sciences, Department of Biochemistry, Professor, 大学院・薬学系研究科, 教授 (90012765)
Project Period (FY) 2001 – 2003
Keywords26S proteasome / budding yeast / RPN7 / RPN5 / polyubiguitinated SicI
Research Abstract

The yeast 26S proteasome, a ca. 200kDa protein complex, consists of 20S proteasome and the 19S regulatory particle. This protein complex abundantly exists in the yeast cells, but because of its large size, it was difficult to purify holoenzyme intact. During this project, we devised a quick and easy method to purify the 26S proeasome; we constructed yeast strains, one of whose proteasome subunits, Rpnl, Rpnl 1, and Prel, had been tagged with 3xFlag and proteasomes produced in these strains were pulled down with anti-Hag antibody. By exploiting this purification method, we characterized proteasomes produced by rpnZ temperature-sensitive mutants and found that a new subcomplex existed in the extract of the rpn7-3 mutant cells. A major obstacle for biochemical study of the 26S proteasome is the absence of proper substrate, polyubiquitinated protein. In this study, we developed a method to produce polyubiquitinated Sici by in vitro reaction by using Rps5 as an E3 enzyme. A key idea is to introduce a PY motif, that attract Rsp5, into the Sici sequence. We confirmed that polyLibiquitinated Sici is a very good substrate of' the 26S proteasome.

  • Research Products

    (54 results)

All Other

All Publications (54 results)

  • [Publications] Y.Takahashi, T.Kahyo, A.Toh-e, H.Yasuda, Y.Kikuchi: "Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as adaptor between conjugating enzyme and substrates."J.Biol.Chem.. 276. 48973-48977 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Takahashi, A.Toh-e, Y.Kikuchi: "A novel factor required for the SUMO1/Smt3 conjugation of yeast septins."Gene. 275. 223-231 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] A.Toh-e, T.Oguchi: "Defects in glycosylphosphatidylinositol (GPI) anchor sunthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisiae."Gene Genet.Sys.. 76. 393-410 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Iwase, A.Toh-e: "Nis1 encoded by YNL078W : a new neck protein of Saccharomyces cerevisiae."Gene Genet.Sys.. 76. 335-343 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Yoshida, A.Toh-e: "Regulation of the localization of Dbf2 and Mob1 during cell division of Saccharomyces cerevisiae."Gene Genet.Sys.. 76. 141-147 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] A.Toh-e, M.Nishizawa: "Structure and function of cyclin-dependent Pho85 kinase of Saccharomyces cerevisiae."J.Gen.Appl.Microbiol.. 47. 107-117 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] A.Toh-e, K.Asakawa, S.Yoshida: "Spindle checkpoint in budding yeast."J.Microbiol.. 39. 1-10 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Asakawa, S.Yoshida, F.Otake, A.Toh-e: "A novel functional domain of Cdc15 kinase is required for its interaction with Tem1 GTPase in Saccharomyces cerevisiae."Genetics. 157. 1437-1450 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] J.Takeuchi, A.Toh-e: "Genetic dissection of the yeast 26S proteasome : cell cycle defects caused by the Δrpn9 mutation."Biochimie. 83. 333-340 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] E.Bon, P.Recordor-Navarro, M.Iwase, A.Toh-e, M.Aigle: "A network of proteins around Rsv167 and Rvs161, two proteins related to actin cytoskeleton."Yeast. 16. 1229-1241 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Uesono, A.Toh-e: "Transient inhibition of translation initiation by osmotic stress."J.Biol.Chem.. 277. 13848-13855 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Miyoshi, R.Tsuji, H.Yoshida, Y.Maki, A.Wada, Matsui, Y., A.Toh-e, K.Mizuta: "Normal assembly of 60S risosomal subunits is required for the signaling in response to a secretory defect in Saccharomyces cerevisiae."J.Biol.Chem.. 277. 18334-18339 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Yoshida, K.Asakawa, A.Toh-e: "Mitotic exit network controls the localization of Cdc14 to the spindle pole body in Saccharomyces cerevisiae."Curr.Biol.. 12. 944-950 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Saeki, A.Saitoh, A.Toh-e, H.Yokosawa: "Ubiquitin-like proteins and Rpn10 play cooperative roles in ubiquitin -dependent proteolysis."Biochem.Biophys.Res.Commun.. 293. 986-992 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] D.Kaida, H.Yashiroda, A.Toh-e, Y.Kikuchi: "Yeast Whi2 and Psr1-phosphatase form a complex and regulate STRE-mediated gene expression."Genes Cells. 7. 547-552 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Yoshida, A.Toh-e: "Budding yeast Cdc5 phosphoryulates Net1 and assists Cdc14 release from the nucleolus."Biochem.Biophys.Res.Commun.. 294. 687-691 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] D.Morita, K.Miyoshi, Y.Matsui, A.Toh-e, H.Shinkawa, T.Miyakawa, K.Mizuta: "Rpf2p, an evolutionalily conserved protein, interacts with ribosomal protein L11 and is essential for processing of 27SB pre-rRNA to 25S rRNA and 60S ribosomal subunit assembly in Saccharmyces cerevisiae."J.Biol.Chem.. 277. 28780-28786 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Asakawa, A.Toh-e: "A defect in KAP104 alleviates the requirement of the MEN (mitotic exit network) gene function in Saccharomyces cerevisiae."Genetics. 162. 1545-1556 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Itoh, A.Watabe, A.Toh-e, Y.Matsui: "Complex formation with the class V myosin Myo2p is essential for Ypt11p, a rab-type small GTPase, to facilitate the Myo2p function for mitochondorial distribution in the budding yeast."Mol.Cell.Biol.. 22. 7744-7757 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] K.Nakano, J.Imai, R.Arai, A.Toh-e, Y.Matsui, I.Mabuchi: "The small GTPase Rho3 and the diaphanous/forming For3 function in polarized cell growth in fission yeast."J.Cell Sci.. 115. 4629-4639 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] A.Toh-e, T.Oguchi: "Genetic characterization of genes encoding enzymes catalyzing addition of phosphoethanolamine to the glycerolphosphatidylinositol anchor in Saccharomyces cerevisiae."Gene.Genet.Sys.. 77. 309-322 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Saeki, T.Sone, A.Toh-e, H.Yokosawa: "Identification of ubiquitin-like protein binding subunits of the 26S proteasomes."Biochem.Biophys.Res.Commun.. 296. 813-819 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Tone, A.Toh-e: "Noblpis required for biogenesis of the 26S proteaome and degraded upon its maturation in Saccharomyces cerevisiae."Genes Dev.. 16. 3142-3157 (2002)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] S.Yoshida, R.Ichihashi, A.Toh-e: "Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast."J.Cell Biol.. 161. 889-897 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Takahashi, A.Toh-e, Y.Kikuchi: "Comparative analysis of yeast PIAS- type SUMO ligases in vitro and in vivo."J.Biochem.. 133. 415-422 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] D.Kaida, A.Toh-e, Y.Kikuchi: "Rsp5-Bul1/2 complex is necessary for theHSE-mediated gene expression in budding yeast."Biochem.Biophys.Res.Commun.. 306. 1037-1041 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] N.Sato, H.Kawahara, A.Toh-e, T.Maeda: "Phosphorelay-regulate degradation of the yeast Ssk1p resonse regulator by the ubiquitin-proteasome system."Mol.Cell.Boil.. 23. 6662-6671 (2003)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Takahashi, T.Kahyo, A.Toh-e, H.Yasuda, Y.Kikuchi: "Yeast UlI1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as adaptor between conjugating enzyme and substrates."J. Biol.Chem.. 276. 48973-48977 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Takahashi, A.Toh-e, Y.Kikuchi: "A novel factor required for the SUMO1/Smt3 conjugation of yeast septins."Gene. 275. 223-231 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] A.Toh-e, T.Oguchi: "Defects in glycosylphosphatidylinositol (GPI) anchor sunthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisiae."Gene Genet. Sys.. 76. 393-40 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] M.Iwase, A.Toh-e: "Nisi encoded by YNLO78W: a new neck protein of Saccharomyces cerevisiae."Gene Genet. Sys.. 76. 335-343 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] S.Yoshida, A.Toh-e: "Regulation of the l ; ocalization of Dbf2 and Mobi during cell division of Saccharomyces cerevisiae."Gene Genet. Sys.. 76. 141-147 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] A.Toh-e, M.Nishizawa: "Structure and function of cyclin-dependent Pho85 kinase of Saccharornyces cerevisiae."J. Gen. Appl. Microbiol.. 47. 107-117 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] A.Toh-e, K.Asakawa, S.Yoshida: "Spindle checkpoint in budding yeast."J. Microbiol.. 39. 1-10 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Asakawa, S.Yoshida, F.Otake, A.Toh-e: "A novel functional domain of Cdc15 kinase is required for its interaction with Tem 1 GTPase in Saccharomyces cerevisiae."Genetics. 157. 1437-1450 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] J.Takeuchi, A.Toh-e: "Genetic dissection of the yeast 26S proteasome: cell cycle defects caused by the Δrpn9 mutation."Biochimie. 83. 333-340 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] E.Bon, P.Recordor-Navarro, M.Lwase, A.Toh-e, M.Aigle: "A network of proteins around Rsv 167 and Rvs 161, two proteins related to actin cytoskeleton."Yeast. 16. 1229-1241 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Uesono, A.Toh-e: "Transient inhibition of translation initiation by osmotic stress."J. Biol. Chem.. 277. 13848-13855 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Miyoshi, R.Tsuji, H.Yoshida, Y.Maki, A.Wada, Matsui, Y., A.Toh-e, K.Mizuta: "Normal assembly of 60S risosomal subunits is required for the signaling in response to a secretoly defect in Saccliaromyces cerevisiae."J. Biol. Chem.. 277. 18334-18339 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] S.Yoshida, K.Asakawa, A.Toh-e: "Mitotic exit network controls the localization of Cdcl4 to the spindle pole body in Saccharomyces cerevisiae."Curr. Biol.. 12. 944-950 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Saeki, A.Saitoh, A.Toh-e, H.Yokosawa: "Ubiquitin-like proteins and RpnlO play cooperative roles in ubiquitin-dependent proteol ysis."Biochem. Biophys. Res. Comnnin.. 293. 986-992 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] D.Kaida, H.Yashiroda, A.Toh-e, Y.Kikuchi: "Yeast Whi2 and Psrl-phosphatase form a complex and regulate STRE-mediated gene expression."Genes Cells. 7. 547-552 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] S.Yoshida, A.Toh-e: "Budding yeast Cdc5 phosphoryulates Neti and assists Cdc14 release from the nucleolus."Bi ochem. Biophys. Res. Commun.. 294. 687-691 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] D.Morita, K.Miyoshi, Y.Matsui, A.Toh-e, H.Shinkawa, T.Miyakawa, K.Mizuta: "Rpf2p, an evolutiona1ily conserved protein, interacts with ribosomal protein L11 and is essential for processing of 27SB pre-rRNA to 25S rRNA and 60S ribosomal subunit assembly in Saccharmyces cerevisiae."J.Biol. Chem.. 277. 28780-28786 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Asakawa, A.Toh-e: "A defect in KAP1O4 alleviates the requirement of the MEN (mitotic exit network) gene function in Saceharomyces cerevisiae."Genetics. 162. 1545-1556 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T.Itoh, A.Watabe, A.Toh-e, Y.Matsui: "Complex formation with the class V myosin Myo2p is essential for Yptillp, a rab-type small GTPase, to facilitate the Myo2p function for mitochondorial distribution in the budding yeast."Mol. Cell. Biol.. 22. 7744-7757 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] K.Nakano, J.Imai, R.Arai, A.Toh-e, Y.Matsui I.Mabuchi: "Fhe small GTPase Rho3 and the diaphanous/forming For3 function in polarized cell growth in fission yeast."J. Cell Sci.. 115. 4629-4639 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] A.Toh-e T.Oguchi: "Genetic characterization of genes encoding enzymes catalyzing addition of phosphoethanolamine to the glycerolphosphatidylinositol anchor in Saccharomyces cerevisiae."Gene. Genet. Sys.. 77. 309-322 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Saeki, T.Sone, A.Toh-e, H.Yokosawa: "Identification of ubiquitin-like protein binding subunits of the 26S proteasomes."Biochem. Biophys. Res. Commun.. 296. 813-819 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Tone, A.Toh-e: "Noblpis required for biogenesis of the 26S proteaome and degraded upon its maturation in Saccharomyces cerevisiae."Genes Dev.. 16. 3142-3157 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] S.Yoshida, R.Ichihashi, A.Toh-e: "Ras recruits mitotic exit regulator Ltei to the bud cortex in budding yeast."J. Cell Biol.. 161. 889-897 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Takahashi, A.Toh-e, Y.Kikuchi: "Comparative analysis of yeast PIAS-type SUMO ligases in vitro and invo."J. Biochem.. 133. 415-422 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] D.Kaida, A.Toh-e, Y.Kikuchi: "Rsp5-Bull/2 complex is necessary for theHSE-mediated gene expression in budding yeast."Biochem. Biophys. Res. Commun.. 306. 1037-1041 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] N.Sato, H.Kawahara, A.Toh-e, T.Maeda: "Phosphorelay-regulate degradation of the yeast Ssklp resonse regulator by the ubiquitin -proteasome system."Mol. Cell. Boil.. 23. 6662-6671 (2003)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2005-04-19  

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