| Project/Area Number |
04454174
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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 |
Pathological medical chemistry
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| Research Institution | Kumamoto University |
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Principal Investigator |
MORI Masataka Kumamoto University, School of Medicine Professor, 医学部, 教授 (40009650)
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| Co-Investigator(Kenkyū-buntansha) |
TERADA Kazutoyo School of Medicine Assistant Professor, 医学部, 助手 (00253724)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1993: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1992: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | Mitochondrial biogenesis / Ornithine transcarbamylase / Precursor protein / Presequence / PBF / Cytosolic factor / ミトコンドリア蛋白質前駆体 / プレシークエンス |
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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 recombinant 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 about50,000 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 pOTC into the mitochondria. PBF-stimulated pOTC import w
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as 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. The precursor for aspartate aminotransferase and malate dehydrogenase 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. On the other hand, depletion of PBF from the lysate had little effect on the import of 3-oxoacyl-CoA thiolase that has no cleavable presequence. These results indicate that PBF-dependent and -independent pathways of mitochondrial protein import do exist. Proteins of the latter pathway including 3-oxoacyl-CoA thiolase may skip a PBF-dependent step and bind directly to the "presequence receptor" on the mitochondrial surface. cDNAs for mouse and human PBF were isolated based on partial amino acid sequences of the purified PBF.Mouse PBF was highly expressed in Escherichia coli and was purified to near homogeneity. Characterization of the recombinant PBF is underway. Less
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