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Biosynthesis pathway of GPI anchored proteins.

Research Project

Project/Area Number 13480194
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionOsaka University

Principal Investigator

KINOSHITA Taroh  Osaka University, Research Institute for Microbial Diseases, Professor, 微生物病研究所, 教授 (10153165)

Co-Investigator(Kenkyū-buntansha) OHISHI Kazuhito  Osaka University, Research Institute for Microbial Diseases, Research Associate, 微生物病研究所, 助手 (60273702)
MURAKAMI Yoshiko  Osaka University, Research Institute for Microbial Diseases, Technical Official, 微生物病研究所, 教務職員 (00304048)
NAGAMUNE Kisaburo  Osaka University, Research Institute for Microbial Diseases, Research Associate, 微生物病研究所, 助手 (90314418)
MAEDA Yusuke  Osaka University, Research Institute for Microbial Diseases, Associate Professor, 微生物病研究所, 助教授 (00294124)
Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2002: ¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 2001: ¥8,000,000 (Direct Cost: ¥8,000,000)
Keywordspost trarnslational modification / endoplasmic reticulum / glycolipid / GPI anchor / biosynthesis / gene cloning / 塘脂質
Research Abstract

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a prolacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GOI. It was known that the GPI transamidase is a complex containing GAAI and GPI8 To further characterize GPI transamidase, we affinity-purified the enzyme complex and found two new components PIG-S and PIG-T. To determinc roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins. We established Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the responsible gene, termed PIG-U, that encodes a 435-amino-acid hydrophobic protein. GPI transamidase complex affnity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U as well as four other known components. Cells lacking PIG-U normally formed complexes of four other components but had no ability to cleave the GPI attachment signal peptide. Therefore, these results demonstrated that GPI transamidase consists of five proteins. We also demonstrated that two subunits, GPI8 and GPI-T, from a functionally important disulfide bond through conserved cysteine residues.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] Maeda, Yusuke: "PIG-M transfers the first mannose to glycosyiphosphatidylinositol on the lumenal side of the ER"EMBO J.. 20. 250-261 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ohishi, Kazuhito: "PIG-S and PIG-T, essential for GPI-anchor attachment to proteins, form a complex with GAA1 and GPI8"EMBO J.. 20. 4088-4098 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Vainauskas, S.: "Structural requirements for the recruitment of Gaal into a functional glycosylphosphatidylinositol transamidase complex"J.Biol.Chem.. 277. 30535-30542 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hong, Y.: "Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum a-toxin"EMBO J.. 21. 5047-5056 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hong, Y.: "Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins"Mol.Biol.Cell. (in press). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ohishi, Kazuhito: "Two subunits of glycosylphophatidylinositol transamidase GPI8 and PIG-T form a functionally imtermolecular disulfode brifge"J.Biol.Chem.. 278. 13959-13967 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Maeda, Y.: "PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER."EMBOJ. 20. 250-261 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ohishi,K.: "PIG-S and PIG-T, essential for GPI-anchor attachment to proteins, from a complex with GAA1 and GPI8."EMBOJ. 20. 4088-4098 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Vainauskas, S.: "Structural requirements for the recruitment of Gaal into a functional glycosylphosphatidylinositol transamidase complex."J. Biol. Chem.. 277. 30535-30542 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hong, Y: "Requirement of N-giycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum α-toxim."EMBOJ. 21. 5047-5056 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Hong, Y: "Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchor to proteins."Mol. Biol. Cell. in press.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ohishi, K.: "Two subunits of glycosylphosphatidylinositol taransamidase, GPI8 and PIG-T, from a functionally important intermolecular disulfide bridge"J.Biol.Chem.. 278. 13959-13967 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Vainauskas, S.: "Structural requirements for the recruitment of Gaal into a functional glycosylphosphatidylinositol transamidase complex"J. Biol. Chem.. 277. 30535-30542 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Hong, Y: "Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum a-toxin"EMBO J.. 21. 5047-5056 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Hong, Y: "Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins"Mol. Biol. Cell. (in press). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Ohishi, K.: "Two subunits of glycosylphosphatidylinositol transamidase GPI8 and PIG-T form a functionally important intermolecular disulfide bridge"J. Biol. Chem.. (in press). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Maeda, Yusuke: "PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER."EMBO Journal. 20. 250-261 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Ohishi, Kazuhito: "PIG-S and PIG-T, essential for GPI-anchor attachment to proteins, form a complex with GAA1 and GPI8"EMBO Journal. 20. 4088-4098 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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