Rassow J, Guiard B, Wienhues U, Herzog V, Hartl F U, Neupert W
Institut für Physiologische Chemie, Zellbiologie und Physikalische Biochemie der Universität München, Federal Republic of Germany.
J Cell Biol. 1989 Oct;109(4 Pt 1):1421-8. doi: 10.1083/jcb.109.4.1421.
Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.
通过研究由酵母细胞色素b2前体的NH2末端167个氨基酸和完整的小鼠二氢叶酸还原酶组成的融合蛋白的导入，对前体蛋白通过线粒体易位接触位点的过程进行了研究。粗糙脉孢菌的分离线粒体很容易导入融合蛋白。在甲氨蝶呤存在的情况下，导入停止，一种跨越易位接触位点的两个线粒体膜的稳定中间体积累。该中间体中完整的二氢叶酸还原酶部分位于外膜外部，经基质加工肽酶切割后剩余的细胞色素b2部分的136个氨基酸残基跨越外膜和内膜。去除甲氨蝶呤导致保留在接触位点的中间体导入基质。因此，线粒体外膜表面的去折叠是通过易位接触位点的前提条件。跨膜中间体被用于估计易位位点的数量。每毫克线粒体蛋白70皮摩尔中间体时达到饱和。计算得出，这个数量的易位中间体大约占据外膜总表面积的1%。通过形态测定确定的外膜和内膜之间紧密接触的面积约占外膜总表面积的7%。中间体的积累抑制了其他前体蛋白的导入，这表明不同的前体蛋白使用共同的易位接触位点。我们得出结论，蛋白质转运到线粒体的机制存在于数量有限的接触位点。
