| Project/Area Number |
09044070
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Field |
Structural biochemistry
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| Research Institution | Nagoya University |
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Principal Investigator |
ENDO Toshiya Nagoya University, Faculty of Science, Professor, 大学院・理学研究科, 教授 (70152014)
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| Co-Investigator(Kenkyū-buntansha) |
SCHULTZ Peter G. Univ. of California, Dept. of Chemistry, Professor, バークレイ校, 教授
NISHIKAWA Shun-ichi Nagoya University, Faculty of Science, Research Associate, 大学院・理学研究科, 助手 (10252222)
YOSHIHISA Toru Nagoya University, R. C. M. S., Associate Professor, 物質科学国際研究センター, 助教授 (60212312)
SCHULTZ Pete カリフォルニア大学バークレイ校, 教授
SHULTZ Peter カリフォルニア大学, バークレイ校, 教授
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥5,500,000 (Direct Cost: ¥5,500,000)
Fiscal Year 1998: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1997: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | translocation across the membranes / mitichondria / chloroplasts / suppressor tRNA method / photocrosslinking / unnatural amino acid |
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| Research Abstract |
Most mitochondrial proteins are synthesized as precursor proteins in the cytosol and imported into mitochondria with the aid of protein translocation machineries in the outer and the inner membranes called the TOM complex and the TIM complex, respectively. In the present study, artificially aminoacylated suppressor tRNAs were used to introduce photoreactive unnatural amino acids into model mitochondrial precursor proteins to map interactions between precursor proteins and translocation machineries along the import pathway.
A model precursor protein, pSu9-DHFR, was arrested at two distinct stages, stage A (accumulated at 0℃) and stage B (accumulated at 30℃), in the translocation across the outer membranes and interactions between the arrested precursor protein and TOM proteins were analyzed at high resolution not achieved previously. Although the stage-A and the stage-B intermediates were previously assigned to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B while remains folded at stage A. After dissociation from the TOM complex, translocation of the stage-B intermediate, but not of the stage-A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. These results indicate that translocation of the presequence and unfolding of the mature domain are not necessarily coupled. We have also applied the approach of site-specific photocrosslinking to the process of carrier proteins transport to the mitochondrial inner membrane and of protein translocation across the chloroplast envelope membranes.
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