Project/Area Number |
06454180
|
Research Category |
Grant-in-Aid for General Scientific Research (B)
|
Allocation Type | Single-year Grants |
Research Field |
Pathological medical chemistry
|
Research Institution | Kumamoto University |
Principal Investigator |
MORI Masataka Kumamoto University, School of Medicine, Professor, 医学部, 教授 (40009650)
|
Co-Investigator(Kenkyū-buntansha) |
TERADA Kazutoyo Kumamoto University, School of Medicine, Assistant Professor, 医学部, 助手 (00253724)
|
Project Period (FY) |
1994 – 1995
|
Project Status |
Completed (Fiscal Year 1995)
|
Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1995: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1994: ¥4,400,000 (Direct Cost: ¥4,400,000)
|
Keywords | Mitochondrial import / Precursor protein / Ornithine transcarbamylase / Hsp70 / Hsc70 / Molecular chaperon / ミトコンドリア / タンパク質前駆体 / Hsp70 / Hsc70 / サイトソル因子 |
Research Abstract |
The roles of the 70-kDa cytosolic heat shock protein (hsp70) in import of precursor proteins into the mitochondria were postulated to be related to (i) unfolding of precursor proteins in the cytosol, (ii) maintenance of the import-competent state, and (iii) unfokding and transport of precursor proteins through contact sites, in cooperation with matrix hsp70. We examined roles of cytosolic hsp70 family members in import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria, using an in vitro import system and antibodies against hsp70. Immunoblot analysis using an hsc70 (70-kDa heat shock cognate protein) -specific monoclonal antibody and a polyclonal antibody that reacts with both hsc70 and hsp70 showed that hsc70 is the only or major from of hsp70 family members in the rabbit reticulocyte lysate. The hsc70 antibody did not inhibit pOTC import when added prior to import assay. However when pOTC was synthesized in the presence of the antibody and then subjected to import assay, pOTC import was markedly decreased. pOTC import was also decreaced when the precursor was synthesized in the lysate depleted for hsc70 by treatment with hsc70 antibody-conjugated Sepharose. This reduction was almost completely restored by readdition of purified mouse hsc70 during pOTC synthesis. The readdition of hsc70 after pOTC synthesis and only during the import assay was not effective. Thus, once import competence of pOTC was lost, hsc70 was ineffective for restoration. Newly synthesized pOTC lost import competence in the absence of hsc70 somewhat more rapidly than in its presence. These results indicate that hsc70 is required during pOTC synthesis and not during import into the mitochondria. hsc70 presumably binds to pOTC polypeptide and maintains it in an import-competent from.
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