Chu T W, Grant P M, Strauss A W
J Biol Chem. 1987 Sep 15;262(26):12806-11.
Arginine residues in the transit peptides of mitochondrial precursors are proposed to be important for uptake into mitochondria. To study this further, we have used cassette mutagenesis to create site-specific amino acid replacements within the transit peptide of rat mitochondrial malate dehydrogenase. Plasmids containing mutant sequences were expressed in vitro and tested in a mitochondrial uptake system utilizing isolated rat liver mitochondria. Substitution for arginine at position 14 with asparagine, glutamine, or alanine decreased the relative import level by 20-30% compared to the wild-type sequence when assayed in 1-h uptake experiments. Although lysine substitution did not alter import, substitution with glutamic acid decreased import by 40%. Alanine substitution for arginines at both positions 14 and 15 also dramatically decreased import. Uptake was partially restored in this mutant when positive charge was inserted at a new location within the transit peptide. Time course experiments showed that the initial rates of import were decreased in these mutants, as were the relative amounts of incorporated protein. These results were best explained by the loss of positive charge following amino acid substitutions for the arginine residues and suggest that the role of the charge is to enhance the efficiency of membrane translocation.
线粒体前体转运肽中的精氨酸残基被认为对线粒体摄取很重要。为了进一步研究这一点，我们使用盒式诱变在大鼠线粒体苹果酸脱氢酶的转运肽内产生位点特异性氨基酸替换。含有突变序列的质粒在体外表达，并在利用分离的大鼠肝线粒体的线粒体摄取系统中进行测试。在1小时摄取实验中测定时，用天冬酰胺、谷氨酰胺或丙氨酸替换第14位的精氨酸，与野生型序列相比，相对导入水平降低了20-30%。虽然赖氨酸替换没有改变导入，但用谷氨酸替换则使导入降低了40%。用丙氨酸替换第14位和第15位的精氨酸也显著降低了导入。当在转运肽内的新位置插入正电荷时，该突变体的摄取部分恢复。时间进程实验表明，这些突变体的初始导入速率降低，掺入蛋白的相对量也降低。这些结果最好用精氨酸残基氨基酸替换后正电荷的丧失来解释，表明电荷的作用是提高膜转运效率。
