Protein import into mitochondria and its disorders

70 kDa胞浆热休克蛋白(HSP70)在前体蛋白进入线粒体中的作用被推测与(I)前体蛋白在胞浆中的展开，(Ii)维持输入能力状态，以及(Iii)前体蛋白的解离和通过接触部位与基质HSP70的运输有关。我们使用体外导入系统和抗HSP70的抗体，研究了胞质HSP70家族成员在鸟氨酸转氨基甲酸酶前体(POTC)导入大鼠肝线粒体中的作用。用hsc70(70 kDa热休克相关蛋白)特异性单抗和同时与hsc70和HSP70反应的多克隆抗体进行免疫印迹分析表明，hsc70是兔网织红细胞裂解物中HSP70家族成员的唯一或主要形式。在进口检测前加入hsc70抗体并不能抑制POTC进口。然而，当POTC在抗体存在下合成并进行进口检测时，POTC的进口显著减少。在hsc70抗体偶联琼脂糖凝胶处理去除hsc70的裂解物中合成前体时，POTC的进口也减少了。在POTC合成过程中，通过重现纯化的小鼠hsc70，这种还原几乎完全恢复。Hsc70在POTC合成后的重现和仅在进口实验中的重现是无效的。因此，一旦POTC失去进口能力，hsc70就无法恢复。新合成的POTC在没有hsc70的情况下失去进口能力的速度比存在hsc70的速度要快一些。这些结果表明，hsc70在POTC合成过程中是必需的，而在输入线粒体时不是必需的。Hsc70可能与POTC多肽结合，并将其维持在一个具有进口能力的来源。

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.