Abstract:
: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.
journal_name
Biochem Biophys Res Communjournal_title
Biochemical and biophysical research communicationsauthors
Nishiyama K,Tokuda Hdoi
10.1016/j.bbrc.2009.10.078subject
Has Abstractpub_date
2009-12-18 00:00:00pages
920-4issue
3eissn
0006-291Xissn
1090-2104pii
S0006-291X(09)02069-5journal_volume
390pub_type
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