| Project/Area Number |
63480497
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | Yamagata University School of Medicine |
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Principal Investigator |
TUBOI Syozo Yamagata University School of Medicine Professor, 医学部, 教授 (70004554)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Tamio Yamagata University School of Medicine Assistant, 医学部, 助手 (30206502)
ONO Hideyu Yamagata University School of Medicine Assistant Professor, 医学部, 助教授 (40160915)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1989: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1988: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Keywords | cytosolic factor / precursor of mitochondrial proteins / targeting factor / Import of mitochondrial proteins / receptor for precursor proteins / mitochondrial outermembrane / 延長ペプチド / 蛋白質輸入機構 / 熱ショック蛋白質 / オルニチンアミノ基転移酵素 |
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| Research Abstract |
Impoort of the peptide of the presequence of ornithine aminotransferase (the extrapeptide of OAT) into mitochondrial matrix was significantly stimulated by addition of a rabbit reticulocyte lysate, and a component of the lysate (the cytosolic factor) stimulating import of the extrapeptide was purified about 20,000 times by successive column chromatography on DEAE-cellulose and aminopentyl-Sepharose 4B. The extrapeptide and the cytosolic factor were shown to form a complex. The cytosolic factor was shown also to stimulate the import of the precursors of several mitochondrial proteins. The amounts of these precursors bound to the outer mitochondrial membrane were increased by this cytosolic factor, suggesting that the cytosolic factor participates in the recognition step in the import process of the precursor protein. Immunochemical studies showed that the cytosolic factor is different from the 70-kDa heat shock-related protein, which was identified as a factor required for the import of
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mitochondrial proteins in yeast. The cytosolic factor was also detected in the cytosol of rat liver cells, and a considerable amount of this factor was recovered from rat liver mitochondria by washing them with high salt buffer, suggesting that the cytosolic factor has affinity to the outer mitochondrial membrane and binds to its receptor on the membrane. From these results, we conclude that the cytosolic factor forms a complex with the precursor of mitochondrial protein and then this complex binds to the outer mitochondrial membrane, probably via the receptor of the cytosolic factor. The cytosolic factor was further purified to homogeneity from a rabbit reticulocyte lysate by affinity column chromatography using a synthetic peptide containing the "extrapeptide of OAT as a ligand. The molecular mass of the purified protein was estimated as 28 kDa by SDS-polyacrylamide gel electrophoresis. The import of precursors of ornithine aminotransferase and sulfite oxidase into mitochondria was inhibited by anti-28 kDa protein IgG raised in guinea pigs. This antibody also blocked the binding of these precursors to mitochondria. These results suggest that the 28 kDa protein is an essential component of the import machinery in the cytosol and that anti-28 kDa protein IgG blocked the binding of the precursor of ornithine aminotransferase to mitochondria, but not the penetration step. Therefore, the 28 kDa protein should be named the "targeting factor" for import to mitochondrial protein.
The receptor protein for the mitochondrial protein precursor synthesized in the cytosol was also extensively purified from the outer mitochondrial membrane by affinity column chromatography using a synthetic peptide containing the extrapeptide of OAT as a ligand. The purified fraction contained two major proteins with molecular masses of 52 kd and 29 kDa. Of these proteins, only the 29 kDa protein bound to the extrapeptide of OAT. Furthermore, anti-29 kDa protein Fab fragments inhibited the import of precursor of OAT into mitochondria, suggesting that the 29 kDa protein plays an essential role in the process of import of the mitochondrial protein precursor. Less
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