| Project/Area Number |
09460043
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | University of Tsukuba |
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Principal Investigator |
YAMANE Kunio UNIVERSITY OF TSUKUBA, INSTITUTE OF BIOLOGICAL SCIENCES, PROFESSOR., 生物科学系, 教授 (20013336)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAMURA Kouji UNIVERSITY OF TSUKUBA, INSTITUTE OF BIOLOGICAL SCIENCES, ASSISTANT PROFESSOR., 生物科学系, 講師 (40212097)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥13,500,000 (Direct Cost: ¥13,500,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥8,400,000 (Direct Cost: ¥8,400,000)
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| Keywords | Bacillus subtilis / Protein secretion / SRP / Signal peptide / Ffh / SRP homologous / SecA / 枯草菌Bacillus subtilis / SPR 相同因子 / SRP / 分泌蛋白質 / SRP相同因子 / 蛋白質分泌機構 / 枯草菌 / 蛋白質分泌 / SRP受容体 / Sec蛋白質 / 蛋白質・蛋白質相互作用 |
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| Research Abstract |
Bacillus subtilis secretes high levels of extracellular enzymes and generates a heat-resistant endospore under poor nutrient conditions.
To understand the protein secretion pathway of Bacillus subtilis, we analyzed the interaction of Ffh and SecA, as well as the passage of presecretory proteins from Ffh to SecA. Precursors of a-amylase and b-lactamase fusion proteins having signal peptide of B. subtilis alkaline protease (pAprE-BlaH6) or penicillin binding protein 5* (pPBP5*-BlaH6) accumulated in the absence of SecA or Ffh. This suggests that SRP- and Sec-protein secretion pathway co-operate to secret proteins in B. subtilis. The SRP of B. subtilis consists of scRNA, Ffh and HBsu. Ffh functions as a central protein for the recognition and targeting of presecretory proteins. Ffh binds to pAprE-BlaH6 and pPBP5*-BlaH6 in vitro, but not to their mature form. SecA also binds to both pAprE-BlaH6 and pPBP5*-BlaH6. SecA-precursor complex formation was enhanced 15 to 30 fold when the precursors and Ffh were initially incubated followed by SecA addition, but not vice versa. Moreover precursors that bind to Ffh transferred to SecA. Direct interaction of Ffh and SecA was shown by the ligand affinity blotting, 6xHis tag purification and immuno-electron microscopy. These results indicate that SecA and Ffh interact to from a single protein secretion pathway including other proteins. Proteome analysis of extracellular proteins using two-dimensional electrophoresis revealed that most secretory proteins are translocated into culture media and the interspace of the mother cell and prespore by the protein secretion pathway.
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