A proteomics approach to inner membrane biogenesis in Escherichia coli

In this thesis, the role of the Escherichia coli YidC protein in membrane biogenesis is investigated with a focus on subunits of respiratory complexes. A directed proteomics approach was applied to investigate the effect of YidC depletion on components of the respiratory chains expressed under anaerobic conditions. It was found that YidC depletion under these conditions caused a cessation in growth and that a number of respiratory complexes were negatively affected. This was further investigated, where metabolic labeling with 15N/14N was employed to study the membrane proteome-wide changes upon YidC depletion under both aerobic and anaerobic respiratory conditions. This allowed the identification of putative YidC substrates and the in vitro insertion requirements of subunit K of complex I were characterized. It was found that NuoK absolutely requires the Sec translocase and YidC for insertion. Essential membrane-embedded negative charges contained in NuoK were shown to confer YidC-dependence. Using the mechanosensitive channel of large conductance, MscL, it was shown further that in vitro translation and membrane insertion systems can be exploited to produce fully active oligomeric protein complexes in a cell-free system. Since YidC has been shown to interact with the SecDFYajC complex the effect of SecDFYajC depletion on the membrane proteome of E. coli was also investigated. The findings are discussed in the context of our current knowledge of the Oxa1/Alb3/YidC family of proteins with particular focus on the diverse functioning of YidC in membrane biogenesis in E. coli.