Wolfe P B, Wickner W, Goodman J M
J Biol Chem. 1983 Oct 10;258(19):12073-80.
The nucleotide sequence of the leader peptidase structural gene from Escherichia coli has been determined. The gene codes for a protein of 323 amino acid residues with a calculated Mr = 35,994, in agreement with the apparent molecular weight of leader peptidase (37,000) determined from gel electrophoresis in sodium dodecyl sulfate. In addition, the amino acid composition predicted from the DNA sequence matches that of the purified enzyme. Leader peptidase synthesized in a cell-free transcription-translation system and isolated from cell membranes have identical apparent molecular weights on sodium dodecyl sulfate-polyacrylamide gels. The presence of methionine and cysteine in the NH2-terminal tryptic peptide shows that no more than three amino acid residues have been removed from the primary translation product predicted from the DNA sequence. These results indicate that leader peptidase is synthesized and assembled into the membrane without proteolytic removal of a leader peptide. Leader peptidase is shown to be largely found in the inner membrane of overproducing strains. The majority of the polypeptide chain is exposed on the outer surface of the inner membrane. It is anchored by a membrane-spanning segment near the NH2 terminus. This orientation agrees with the functional orientation of leader peptidase observed in artificial membrane vesicles.
已测定了大肠杆菌前导肽酶结构基因的核苷酸序列。该基因编码一种由323个氨基酸残基组成的蛋白质，计算所得的分子量为35,994，这与通过十二烷基硫酸钠凝胶电泳测定的前导肽酶的表观分子量（37,000）一致。此外，根据DNA序列预测的氨基酸组成与纯化酶的氨基酸组成相匹配。在无细胞转录-翻译系统中合成并从细胞膜中分离出的前导肽酶，在十二烷基硫酸钠-聚丙烯酰胺凝胶上具有相同的表观分子量。NH2末端胰蛋白酶肽中存在甲硫氨酸和半胱氨酸，这表明从DNA序列预测的初级翻译产物中去除的氨基酸残基不超过三个。这些结果表明，前导肽酶在合成后组装到膜中，没有经过前导肽的蛋白水解去除过程。结果表明，前导肽酶主要存在于过量表达菌株的内膜中。多肽链的大部分暴露在内膜的外表面。它通过靠近NH2末端的跨膜区段锚定。这种取向与在人工膜泡中观察到的前导肽酶的功能取向一致。
