Cerletti N, Böhni P C, Suda K
J Biol Chem. 1983 Apr 25;258(8):4944-9.
The cytoplasmically made subunit V was isolated from enzymically active yeast cytochrome c oxidase and its NH2-terminal amino acid sequence was determined to be (formula; see text) In order to exclude that this NH2 terminus had been generated by proteolysis during the lengthy isolation of the subunit, subunit V was directly immunoprecipitated from yeast cells that had been pulse-labeled with [35S]methionine; radiochemical sequencing revealed methionine at position 12, in agreement with the sequence given above. When the precursor to subunit V was synthesized in vitro in the presence of either [35S]methionine, [3H]leucine, or [3H]histidine and then incubated either with isolated yeast mitochondria or the partially purified matrix protease (Böhni, P. C., Daum, G., and Schatz, G. (1983) J. Biol. Chem. 258, 4937-4943), it was converted to a polypeptide co-migrating with mature subunit V on dodecyl sulfate-polyacrylamide gels. Radiochemical sequence analysis of the processed in vitro product showed that it contained histidine, leucine, and methionine in positions 4, 6, and 12, respectively, exactly as the authentic mature protein. In contrast, the unprocessed precursor contained methionine only at position 9, but not at position 12; thus, the precursor has a NH2 terminus different from the mature polypeptide. Similarly, if the in vitro synthesized cytochrome b2 precursor is incubated with isolated mitochondria, it is converted to a polypeptide which co-migrates with mature cytochrome b2 and, like the latter, contains leucine and methionine in positions 4 and 6, respectively. These data show that isolated yeast mitochondria convert the precursors to polypeptides which have the NH2 terminus of the authentic mature polypeptides. In the case of cytochrome c oxidase subunit V, correct NH2-terminal processing was also demonstrated with the purified matrix protease.
从具有酶活性的酵母细胞色素c氧化酶中分离出胞质合成的亚基V，并确定其NH2末端氨基酸序列为（分子式；见正文）。为了排除该NH2末端是在亚基长时间分离过程中由蛋白水解产生的可能性，从用[35S]甲硫氨酸进行脉冲标记的酵母细胞中直接免疫沉淀亚基V；放射化学测序显示第12位为甲硫氨酸，与上述序列一致。当在[35S]甲硫氨酸、[3H]亮氨酸或[3H]组氨酸存在的情况下体外合成亚基V的前体，然后与分离的酵母线粒体或部分纯化的基质蛋白酶一起孵育（Böhni, P. C., Daum, G., and Schatz, G. (1983) J. Biol. Chem. 258, 4937 - 4943）时，它在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上转化为与成熟亚基V共迁移的多肽。对体外加工产物的放射化学序列分析表明，它在第4、6和12位分别含有组氨酸、亮氨酸和甲硫氨酸，与 authentic成熟蛋白完全相同。相比之下，未加工的前体仅在第9位含有甲硫氨酸，而在第12位没有；因此，前体的NH2末端与成熟多肽不同。同样，如果将体外合成的细胞色素b2前体与分离的线粒体一起孵育，它会转化为与成熟细胞色素b2共迁移的多肽，并且与后者一样，在第4和6位分别含有亮氨酸和甲硫氨酸。这些数据表明，分离的酵母线粒体将前体转化为具有 authentic成熟多肽NH2末端的多肽。在细胞色素c氧化酶亚基V的情况下，用纯化的基质蛋白酶也证明了正确的NH2末端加工。
