Studies of mitochondrial protein import factors in mammals

线粒体蛋白质的合成、转位、加工和折叠的整个过程涉及存在于细胞质、线粒体膜和基质中的因子。研究了哺乳动物线粒体前蛋白输入过程中的胞浆和外膜因子。为了研究分子伴侣的作用，我们开发了一个从兔网织红细胞裂解物中去除和再扩增分子伴侣的系统。利用该系统，我们发现参与线粒体前蛋白输入的是hsc70和DJ2(HSDJ/HDJ-2)，而不是DJ1(Hsp40/HDJ-1)。最近发现的Dj3(cpr3/DNJ3/HIRIP4/rdj2)在前蛋白输入中与DJ2一样活跃。我们分离了人Tom20和Tom22，并研究了它们在体外和培养细胞中的作用。我们发现Tom20和Tom22的胞浆结构域对前蛋白结合是重要的，而Tom22的膜间隙结构域并不重要。免疫共沉淀实验表明，Tom20和Tom22通过胞浆结构域形成了复合体。发现Tom34主要存在于胞浆中，部分存在于线粒体外膜中，在真菌中没有明显的对应蛋白，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.