Moore K E, Dalbey R E, Wickner W
Molecular Biology Institute, University of California, Los Angeles 90024.
J Bacteriol. 1988 Sep;170(9):4395-8. doi: 10.1128/jb.170.9.4395-4398.1988.
Leader peptidase is an integral protein of the Escherichia coli cytoplasmic membrane whose topology is known. We have taken advantage of this knowledge and available mutants of this enzyme to develop a genetic test for a cell-free protein translocation reaction. We report that leader peptidase inserted into inverted plasma membrane vesicles in its correct transmembrane orientation. We have examined the in vitro membrane assembly characteristics of a variety of leader peptidase mutants and found that domains required for insertion in vivo are also necessary for insertion in vitro. These data demonstrate the physiological validity of the in vitro insertion reaction and strengthen the use of this in vitro protein translocation reaction for the dissection of this complex sorting pathway.
前导肽酶是一种拓扑结构已知的大肠杆菌细胞质膜整合蛋白。我们利用这一知识以及该酶的可用突变体，开发了一种用于无细胞蛋白质转运反应的遗传学检测方法。我们报告称，前导肽酶以其正确的跨膜方向插入到倒置的质膜囊泡中。我们研究了多种前导肽酶突变体的体外膜组装特性，发现体内插入所需的结构域在体外插入时也是必需的。这些数据证明了体外插入反应的生理有效性，并加强了利用这种体外蛋白质转运反应来剖析这一复杂分选途径的应用。
