Insertion studies of model transmembrane segments into bacterial and eukaryotic membranes

Cells are encapsulated by a biological membrane in order to separate the cell interior from the surrounding environment. Different lipids and proteins compose the membrane and present a semi-permeable barrier for the diffusion of ions and molecules across the lipid bilayer. Membrane proteins also mediate the passage of signals between the interior and the exterior of the cell. To ensure the proper functioning of membrane proteins, it is essential that nascent membrane proteins are correctly integrated into the lipid bilayer to be able to fold and oligomerize. In this thesis, an engineered protein containing two natural transmembrane segments followed by an additional test segment, has been used as a model protein to study (i) sequence requirements for translocon-mediated insertion of the test segment, (ii) dynamics of nascent membrane proteins undergoing translocon-mediated insertion and (iii) to carry out an extensive mutagenesis scan to identify critical residues in the mammalian arrest peptide Xbp1 that enhances translational stalling in the ribosome. This provides a toolbox of arrest peptides with different stalling strengths that will be useful for force measurements on nascent protein chains. At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.