Residue-by-residue analysis of cotranslational membrane protein integration in vivo

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.

Automated summary

The study investigates the cotranslational biosynthesis of E. coli inner membrane proteins and reveals the complexities in membrane integration process, including protein folding in the ribosome exit tunnel and interactions between charged residues and the membrane. The results also suggest residue-specific interactions between transmembrane helices during the integration process, supporting the 'sliding' model of translocon-mediated membrane protein integration.