Tail anchored (TA) proteins constitute an evolutionarily-conserved group of integral membrane proteins that are characterised by the presence of a single C-terminal transmembrane segment (TMS), which acts as both a membrane anchor and a targeting signal. In eukaryotes, TA-proteins localise to most intracellular membranes with the endoplasmic reticulum (ER) being the entry site for TA-proteins destined for the compartments of the secretory pathway and the plasma membrane. Notably, distinct routes for TA-protein delivery to the ER have been identified, and the pathway preference seems to be determined by a relative hydrophobicity of the TMS.In the present study I demonstrate that two major routes for TA-protein delivery to the ER membrane, the TRC40-dependent and “unassisted”/chaperone-mediated pathways, both rely on the action of cytosolic factors which are extremely flexible and can accommodate substrates with TMSs that have been extensively modified (Chapters 2.1 – 2.3). Moreover, the ability of PEGylated forms of the TRC40 client Sec61b to become membrane-integrated correlates very well with the calculated changes in free energy that are associated with its partitioning into a lipid bilayer, supporting a thermodynamics-driven mode of membrane insertion for TA-proteins (Chapter 2.1). The use of fluorescently-labelled recombinant cytochrome b5 (Cytb5), a model TA-protein exploiting the “unassisted”/chaperone-mediated pathway, strongly suggests the involvement of cytosolic components during its biogenesis, whilst the accessibility of novel cysteine residues to the reagent mPEG-5000 indicates a role for peripheral membrane proteins during Cytb5 membrane integration (Chapter 2.2). Importantly, pull down assays using recombinant TA-proteins as bait, followed by mass spectrometric analysis, allowed me to identify a number of cytosolic interacting partners of TA-proteins (Chapters 2.3 and 2.4). The function of one such a factor, Bat3, was further investigated, and it was found to act prior to TRC40 and facilitate the loading of TA-protein substrates onto this targeting factor (Chapter 2.3). Based on these results and available published data, a hypothetical protein-protein interaction network is presented, and I speculate about the role of individual components during TA-protein biogenesis (Discussion).

The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts. The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.

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. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>