Protein import into mitochondria and its disorders

70 kda的细胞质热休克蛋白（hsp70）在前体蛋白进入线粒体中的作用被认为与(i)细胞质中前体蛋白的展开，（ii）维持进口能力状态，以及（iii）与基质hsp70合作，通过接触位点展开和运输前体蛋白有关。我们利用体外导入系统和抗hsp70抗体，研究了胞浆hsp70家族成员在鸟氨酸转氨基甲酰基酶前体（pOTC）进入大鼠肝脏线粒体中的作用。使用hsc70 （70-kDa热休克同源蛋白）特异性单克隆抗体和与hsc70和hsp70反应的多克隆抗体进行免疫印迹分析表明，hsc70是兔网织细胞裂解液中唯一或主要的hsp70家族成员。在进口试验前添加hsc70抗体不抑制pOTC的进口。然而，当在抗体存在下合成poc并进行进口试验时，poc的进口明显减少。当用hsc70抗体偶联的Sepharose处理hsc70的裂解物中合成前体时，pOTC的进口也减少了。在poc合成过程中，通过读取纯化的小鼠hsc70，这种还原几乎完全恢复。hsc70在poc合成后和仅在进口试验期间的检测无效。因此，一旦pOTC的进口能力丧失，hsc70对恢复是无效的。新合成的poc在没有hsc70的情况下比有hsc70的情况下更快地丧失进口能力。这些结果表明，hsc70在pOTC合成过程中是必需的，而不是在进入线粒体的过程中。hsc70可能与pOTC多肽结合，并将其维持在具有进口能力的细胞中。

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.