Studies of mitochondrial protein import factors in mammals

线粒体蛋白质的合成、其移位、加工和折叠的整个过程涉及存在于细胞质、线粒体膜和基质隔室中的因子。研究了哺乳动物中参与线粒体前蛋白输入的胞浆因子和外膜因子。为了研究分子伴侣的作用，我们建立了一个从兔网织红细胞裂解液中去除分子伴侣并重新加入的系统。利用这个系统，我们发现，hsc 70和dj 2（HSDJ/hdj-2），而不是dj 1（hsp 40/hdj-1），参与线粒体前蛋白输入。最近鉴定的dj 3（cpr 3/DNJ 3/HIRIP 4/rdj 2）在前蛋白输入中与dj 2一样活跃。分离人Tom 20和Tom 22，并在体外和培养细胞中研究其作用。我们发现，Tom 20和Tom 22的胞质结构域，但不是Tom 22的膜间空间结构域，是重要的前蛋白结合。免疫共沉淀实验表明，Tom 20和Tom 22通过胞质结构域形成复合物。Tom 34主要存在于细胞质中，部分存在于线粒体外膜中，在真菌中没有明显的对应物，它参与了前蛋白的输入，这些结果表明，从低等真核生物到高等动物，线粒体蛋白的输入机制是高度保守的，但也存在一些差异。

The whole process of synthesis of mitochondrial proteins, their translocation, processing and folding involves factors present in the cytosol, mitochondrial membranes and matrix compartments. Cytosolic and outer membrane factors involved in mitochondrial preprotein import in mammals were studied. To investigate the roles of molecular chaperones, we developed a system of chaperone depletion from and readdition to rabbit reticulocyte lysates. Using this system, we found that hsc70 and dj2 (HSDJ/hdj-2), not dj1 (hsp40/hdj-1), are involved in mitochondrial preprotein import. Recently identified dj3 (cpr3/DNJ3/HIRIP4/rdj2) was as active as dj2 in preprotein import. Human Tom20 and Tom22 were isolated and their roles were studied in vitro and in cultured cells. We revealed that the cytosolic domains of both Tom20 and Tom22, but not the intermembrane space domain of Tom22, are important for preprotein binding. Coimmunoprecipitation experiments showed that Tom20 and Tom22 form a complex via the cytosolic domains. Participation of Tom34 which is present largely in the cytosol and partly in the mitochondrial outer membrane and has no apparent counterpart in fungi, in preprotein import was found. These results indicate that the mechanism of mitochondrial protein import is highly conserved from lower eukaryotes to higher animals but some differences exist.