Development of a functional in vitro integration system for an integral membrane protein, SecG.
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.
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan. unishiy@mail.ecc.u-tokyo.ac.jp
Ken-ichi
K
Nishiyama
Hajime
H
Tokuda
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D014176:Protein Biosynthesis; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D018271:Signal Recognition Particle / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C088616:SecG protein, E coli; D018271:Signal Recognition Particle; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins