The biogenesis of multispanning membrane proteins at the Sec61-translocon and their integration into the lipid bilayer of the endoplasmic reticulum

The key entry point of most membrane proteins into the lipid bilayer is the Sec61/SecYEG translocon, that mediates the transfer of hydrophilic sequences across the membrane and integration of mostly apolar a-helical transmembrane domains into the lipid bilayer. Three distinct integrations steps can be distinguished: (1) a first hydrophobic signal sequence targets the protein to the translocon, integrates itself into the membrane, and initiates translocation of the downstream polypeptide. (2) A subsequent hydrophobic segment laterally exits the translocon into the bilayer and thus stops further transfer. (3) The next hydrophobic sequence triggers re-integration into the translocon, re-initiating polypeptide transfer. Successive stop-transfer and reintegration sequences result in complex multispanning proteins. The major determinant of membrane topology appears to be the hydrophobicity of transmembrane domains. This has been best demonstrated for potential stop-transfer segments, suggesting a sequence-autonomous thermodynamic equilibration between the hydrophilic environment of the translocon and the apolar lipid phase.In this thesis, we analyzed in detail the hydrophobicity threshold for a potential re-integration TM domain downstream of different cytoplasmic loop sequences. Surprisingly, we discovered a strong dependence on the length of this cytoplasmic sequence. Short sequences are facilitating re-integration, while long ones seem to impede it. This demonstrates, that re-integration is not independent from the sequence-context. Further investigations revealed that loop sequences containing isolated folding domains, intrinsically disordered sequences, or sequences with a high affinity for chaperones enhance the reintegration efficiency, whereas those with low affinity to chaperones, and fragments of natural protein domains impair re-integration. We propose that the latter sequences, as they collapse to molten globules – i.e. near-native conformation of high compactness with already pronounced secondary structure and increased amount of hydrophobic residues on the surface area – compete with the translocon for interaction with the potential transmembrane segment. Our results thus define the environment of the nascent polypeptide chain when re-integration can occur and may serve as a guide in de novo membrane protein design. In a second part, we characterized the antiviral natural product cavinafungin as an inhibitor of signal peptidase for Dengue virus as well as host substrates, inhibiting biogenesis of viral proteins from a single precursor membrane polyprotein.