Soluble proteins can be targeted and inserted into the Endoplasmic Reticulum (ER) co-translationally or post-translationally, Co-translational translocation requires a ribonucleoprotein complex. Signal Recognition Particle (SRP), to target the ribosome-nascent chain complex to the ER-localised translocon. The targeting and insertion of membrane-bound proteins into the ER is more complex. One class of membrane proteins have the transmembrane domain near their C-terminus (tail-anchor proteins), and whilst the other classes of membrane proteins have had their mode of insertion elucidated, the insertion mechanism of these proteins remains unknown. This thesis deals with two areas of protein targeting in <i>Saccharomyces cerevisiae</i>. First the mode of insertion of Ufe1p, a tail-anchor protein, was examined by use of a variety of reporters. This work identified a region N-terminal to the transmembrane domain that is required for correct localisation of the protein. Second, the role of the SRP component, Srp72p was examined, Srp72p is essential for SRP function, but its role has not been determined. A screen was set up to identify conditional mutants of <i>SRP72</i>. In addition, deletions and point mutations were created to analyse the roles of conserved residues within the protein. Mutations affecting a conserved region of the protein towards the C-terminus were found to confer slight defects in SRP-dependent translocation. Intracellular localisation of the mutant proteins was identical to that of the wild-type protein. A mini-screen performed on two mutants identified multicopy suppressors of translocation defects. Finally, preliminary comparison of the binding affinities of SRP containing mutated Srp72p or wild-type SRP with the SRP receptor revealed a subtle difference.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:666169 |
| Date | January 2001 |
| Creators | McDonald, James Christopher |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12609 |
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