The yeast <i>Saccharomyces cerevisiae</i> utilises many transport pathways for the efficient an accurate segregation of proteins to the compartments of the cell in which they function. Of these, the secretory pathway is responsible for the localisation of proteins to the endoplasmic reticulum (ER), Golgi apparatus, endocytic and vacuolar compartments and the cell surface. The first step along this pathway is the targeting of nascent polypeptides to the ER membrane. For many proteins, the signal recognition particle (SRP), a cytosolic ribonucleoprotein, and its cognate receptor (SR) on the ER membrane are responsible for this step. SRP and the SR direct proteins to the translocon, which forms an aqueous channel through which the nascent proteins are translocated or from which they are then integrated into the ER membrane. The aim of this study was to dissect incompletely understood interactions that occur at the ER membrane between SRP, SR and the translocation and to reconstitute the SRP-dependent translocation reaction with purified proteins. In particular, through 2-hybrid analysis and pull-down assays a novel interaction was identified between SR and the major translocon component Sec61p. To facilitate the study of this and other interactions, attempts were made to reconstitute the SRP-dependent targeting pathway with SR and translocon purified from yeast. It was demonstrated that SRP-dependent translocation could be reconstituted with solubilised yeast ER membrane proteins. SR was purified and shown to be functional, and the translocation reaction was shown to be stimulated by the presence of the ER-lumenal chaperone Kar2p. Preliminary experiments were also carried out that suggested that the purified translocon was active; indicating that the goal of reconstituting SRP-dependent translocation with purified components is attainable.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:661188 |
| Date | January 2002 |
| Creators | Robb, Allison |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14291 |
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