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).
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:677716 |
| Date | January 2010 |
| Creators | Leznicki, Pawel |
| Contributors | High, Stephen |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-biogenesis-of-tailanchored-membrane-proteins-at-the-endoplasmic-reticulum(70d89000-57e0-4f5f-97dc-381abc1b0da4).html |
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