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).
|Date of Award||31 Dec 2010|
- The University of Manchester
|Supervisor||Stephen High (Supervisor)|
- Membrane integration