Nishiyama Ken-ichi, Tokuda Hajime
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.
Biochem Biophys Res Commun. 2009 Dec 18;390(3):920-4. doi: 10.1016/j.bbrc.2009.10.078. Epub 2009 Oct 22.
A functional in vitro integration system for an integral membrane protein, SecG, comprising an efficient translation system supplemented with inverted membrane vesicles (IMV) was developed. When SecG was synthesized in the presence of IMV prepared from a DeltasecG strain (DeltaSecG IMV), the synthesized SecG was recovered with the IMV. A population of SecG was resistant to urea extraction, indicating that the synthesized SecG was integrated into DeltaSecG IMV. Addition of signal recognition particle and its receptor (SRP) and SecA caused an increase in the amount of the urea-resistant form of SecG. When IMV into which SecG had been integrated were subjected to the translocation assay, the translocation activity was found to be significantly stimulated compared with for DeltaSecG IMV. Moreover, when SRP and SecA had been supplemented, the translocation activity nearly recovered to the level in IMV prepared from the wild type strain. These results indicate that the in vitro synthesized SecG could be functionally integrated into DeltaSecG IMV with the help of SRP and SecA. We also present evidence that the membrane targeting and integration of SecG is stimulated by externally added SecA and SecG itself.
开发了一种用于整合膜蛋白SecG的功能性体外整合系统，该系统包括补充有反向膜泡（IMV）的高效翻译系统。当在由DeltasecG菌株制备的IMV（DeltaSecG IMV）存在下合成SecG时，合成的SecG与IMV一起回收。一部分SecG对尿素提取具有抗性，这表明合成的SecG已整合到DeltaSecG IMV中。添加信号识别颗粒及其受体（SRP）和SecA导致抗尿素形式的SecG量增加。当将已整合SecG的IMV进行转位测定时，发现与DeltaSecG IMV相比，转位活性受到显著刺激。此外，当补充了SRP和SecA时，转位活性几乎恢复到野生型菌株制备的IMV中的水平。这些结果表明，体外合成的SecG可以在SRP和SecA的帮助下功能性地整合到DeltaSecG IMV中。我们还提供证据表明，外部添加的SecA和SecG本身会刺激SecG的膜靶向和整合。
