De Vrije T, Tommassen J, De Kruijff B
Biochim Biophys Acta. 1987 Jun 12;900(1):63-72. doi: 10.1016/0005-2736(87)90278-1.
In order to reach their final destination, periplasmic and outer membrane proteins have to pass the cytoplasmic membrane of Escherichia coli cells. To study the transport of PhoE protein, we developed an in vitro transcription-translation and translocation system. In this in vitro system, the protein is synthesized as a larger precursor, which can be processed by purified leader peptidase. The precursor can be translocated into inverted inner membrane vesicles as judged by the protection against externally added protease. Only part of the translocated protein is in the processed mature form. Translocation can occur posttranslationally and requires both ATP and the protonmotive force for an optimal process. Upon incubation of vesicles with mature PhoE protein or precursor PhoE in the absence of ATP, the proteins are bound to the vesicles, but they are not translocated, since they are still sensitive to externally added protease.
为了到达其最终目的地，周质蛋白和外膜蛋白必须穿过大肠杆菌细胞的细胞质膜。为了研究PhoE蛋白的转运，我们开发了一种体外转录-翻译和转运系统。在这个体外系统中，该蛋白作为一个更大的前体被合成，它可以被纯化的前导肽酶加工。根据对外部添加蛋白酶的抗性判断，前体可以转运到翻转的内膜囊泡中。只有部分转运的蛋白是加工后的成熟形式。转运可以在翻译后发生，并且需要ATP和质子动力势才能实现最佳过程。在没有ATP的情况下，将囊泡与成熟的PhoE蛋白或PhoE前体一起孵育时，蛋白质会与囊泡结合，但它们不会被转运，因为它们仍然对外部添加的蛋白酶敏感。
