Abstract
The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that facilitate the insertion of membrane proteins. Depletion of YidC in E. coli leads to a specific defect in the functional assembly of major energy transducing complexes such as the F1F0 ATPase and cytochrome bo3 oxidase. Here we report on the in vitro reconstitution of the membrane insertion of the CyoA subunit of cytochrome bo3 oxidase. Efficient insertion of in vitro synthesized pre-CyoA into proteoliposomes requires YidC, SecYEG, and SecA and occurs independently of the proton motive force. These data demonstrate that pre-CyoA is a substrate of a novel pathway that involves both SecYEG and YidC.
Highlights
We have shown that in E. coli the functional assembly of major energy-transducing complexes such as the Hϩ-translocating F1F0 ATPase and cytochrome bo3 oxidase is strongly affected by the depletion of YidC [11]
In vitro experiments demonstrate that the membrane insertion of F0c is solely mediated by YidC [4], establishing a novel route for membrane insertion of authentic E. coli membrane proteins, which involves only YidC
YidC interacts with the SecYEG complex, and cross-linking approaches have shown that it contacts the TMs of newly inserted membrane proteins [5,6,7,8]
Summary
Strains and Plasmids—E. coli strain SF100 was used for the isolation of inner membrane vesicles (IMVs) and for overexpression of SecYEG and YidC [14]. The S135 lysate was prepared from E. coli MC4100. Plasmids pBSKftsQ [15] and pET27bCyoA In Vitro Transcription, Translation, and Insertion Reaction—In vitro transcription was performed using the RiboMax kit (Promega) with plasmids pBSKftsQ and pET27bCyoA as templates. Other Methods—IMVs containing overproduced SecYEG or YidC were isolated as described [16]. Proteoliposomes were analyzed by SDS-PAGE and silver staining to verify the reconstituted levels of SecYEG and YidC [14]. Functional levels of reconstituted SecYEG and YidC were verified by proOmpA translocation [14] and Foc membrane insertion [4] assays, respectively. SecA was removed from the S135 lysate by immunodepletion and verified by immunoblotting using monoclonal SecA antibodies [15]
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