Membrane Protein Biogenesis in Escherichia coli : A proteomics approach

Abstract: In the bacterium Escherichia coli all proteins are synthesized in the cytoplasm. However, at least 25% of them then need to be exported to another cellular compartment where they exert their function. E. coli has several different targeting pathways to ensure the correct localization of its proteins. When a nascent chain emerges at the ribosomal tunnel exit the signal recognition particle (SRP) can bind to it. The ribosomal nascent chain -SRP- complex is then targeted either to the Sec-translocon or the YidC insertase at the cytoplasmic membrane. Integral cytoplasmic membrane proteins are inserted into the lipid bilayer whilst periplasmic and outer membrane proteins are translocated across the membrane. In order to be fully functional, proteins need to be both correctly localized and folded. In many cases, they also assemble into complexes with other proteins or co-factors. In my thesis I will present an improved protocol for two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) that makes it very suitable to study the biogenesis of integral cytoplasmic membrane proteins and especially the complexes in the cytoplasmic membrane. Using this and other methods I have studied the biogenesis of integral cytoplasmic membrane proteins and other exported proteins. This has been done on a global scale in mutant strains where the SRP-targeting pathway, the Sec-translocon and the integral cytoplasmic membrane chaperone/insertase YidC have been compromised.