In mammalian cells newly synthesised proteins are translocated across the ER membrane and their subsequent folding is facilitated by an array of folding factors present in the lumen. These include the lectins calreticulin and calnexin, which form complexes with ERp57 to generate glycoprotein specific molecular chaperones. ERp57 is a member of the protein disulphide isomerase (PDI) family and its binding to ER lectins can be reconstituted in vitro. I have exploited this approach to define the regions of ERp57 that are necessary and sufficient for its specific interaction with calreticulin and calnexin. Truncated forms of ERp57, chimeric proteins containing various domains of ERp57 and PDI (which does not interact with calreticulin) and ERp57 b' domain point mutants have been constructed. By analysing the interactions of ERp57 derivatives with calreticulin using both cross-linking and binding assays I have been able to provide detailed insights into the molecular basis for the specific assembly of these components within the ER lumen. My results indicate that the b and b' domains of ERp57 are necessary, but not sufficient for binding to both calreticulin and calnexin. The more stringent binding assay revealed that the a' domain of ERp57 significantly enhanced binding to biotin-tagged calreticulin. The ERp57 C-terminal extension also increased binding to biotin-tagged calreticulin, perhaps by playing a role in the overall stability of the ERp57. In addition, the ERp57 b' domain point mutants show that certain amino acids in this domain, in particular residues F280, V283 and F299, may be crucial for binding to calreticulin, consistent with the principal lectin-binding site being located in the b' domain. However, the binding region clearly extends into other domains, in particular the b and a' domains.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606749 |
Date | January 2003 |
Creators | Russell, Sarah J. |
Publisher | University of Manchester |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Source | http://www.manchester.ac.uk/escholar/uk-ac-man-scw:77484 |
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