Protein import into mitochondria and its disorders

70 kDa胞质热休克蛋白（hsp 70）在前体蛋白向线粒体输入中的作用被假定为与（i）前体蛋白在胞质中的解折叠、（ii）维持输入能力状态和（iii）前体蛋白与基质hsp 70合作通过接触位点的解折叠和运输有关。我们研究的作用，胞质热休克蛋白70家族成员在进口的鸟氨酸转氨甲酰酶前体（pOTC）到大鼠肝线粒体，使用体外进口系统和抗体对热休克蛋白70。使用hsc 70（70-kDa热休克同源蛋白）特异性单克隆抗体和多克隆抗体与hsc 70和hsp 70反应的免疫印迹分析表明，hsc 70是唯一的或主要的兔网织红细胞裂解液中的hsp 70家族成员。当在输入测定之前添加时，hsc 70抗体不抑制pOTC输入。然而，当在抗体存在下合成pOTC，然后进行输入测定时，pOTC输入显著降低。当前体在通过用hsc 70抗体缀合的Sepharose处理而去除hsc 70的裂解物中合成时，pOTC输入也减少。在pOTC合成过程中，通过再加入纯化的小鼠hsc 70，这种减少几乎完全恢复。在pOTC合成后和仅在输入测定期间再加入hsc 70是无效的。因此，一旦pOTC的输入能力丧失，hsc 70就无法恢复。新合成的pOTC失去进口能力的hsc 70的情况下，有点更快地比在其存在。这些结果表明，在pOTC合成过程中需要hsc 70，而在输入线粒体过程中不需要hsc 70。hsc 70可能与pOTC多肽结合并将其维持在输入感受态形式。

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.