| Project/Area Number |
02454147
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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 |
General medical chemistry
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| Research Institution | Kumamoto University |
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Principal Investigator |
MORI Masataka Kumamoto University Medical School, Professor, 医学部, 教授 (40009650)
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| Co-Investigator(Kenkyū-buntansha) |
MORI Shunsuke Kumamoto University Medical School, Assistant Professor, 医学部, 助手 (70230068)
MORI Kaoru (MURAKAMI) Kumamoto University Medical School, Assistant Professor, 医学部, 助手 (20230063)
滝口 正樹 熊本大学, 医学部, 講師 (40179578)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1991: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1990: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | Precursor proteins / Intracellular transport / Mitochondrial biogenesis / Cytosolic factors / PBF (presequence binding factor) / Ornithine transcarbamylase / Hsp70 / Presequence / Presequence binding factor(PBF) |
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| Research Abstract |
Ornithine transcarbamylase (OTC), a mitochondrial matrix enzyme of the urea cycle, is initially synthesized on cytosolic free ribosomes as a larger precursor pOTC with an NH_2-terminal presequence of 32 amino acid residues. pOTC is released into a cytosolic pool and is imported into the mitochondrial matrix with a half life of 1-2 min. We established an in vitro import system in which the purified pOTC was imported into isolated mitochondria. Using this system, we showed that a cytosolic protein factor (s) in the reticulocyte lysate is required for the import of the purified pOTC. A protein factor that binds to pOTC but not to mature OTC and was named presequence binding factor (PBF), was purified from the lysate. The purified PBF migrated as a single polypeptide of about 50, 000 Da on SDS-PAGE. On sucrose gradients, pOTC and PBF cosedimented as a complex of 7S. The purified PBF markedly stimulated the import of the purified pOPC into the mitochondria. PBF-stimulated pOTC import was further enhanced by hsp70 purified from yeast ; the hsp70 alone had little effect. Thus, PBF binds to the presequence portion of the precursor and may hold it in a transport-competent form in cooperation with hsp70. We next asked whether PBF is also involved in transport of other mitochondrial proteins. The precursor for aspartate aminotransferase synthesized in the PBF-depleted lysate failed to be imported into the mitochondria. Readdition of the purified PBF to the depleted lysate fully restored the import. Therefore, PBF appears to be involved in transport of a set of mitochondrial proteins having presequences.
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