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The molecular basis for ERp57/calreticulin complex formation
Sarah J. Russell
[Thesis].University of Manchester;2003.
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Abstract
In mammalian cells newly synthesised proteins are translocated across the ER membraneand their subsequent folding is facilitated by an array of folding factors present in thelumen. These include the lectins calreticulin and calnexin, which form complexes withERp57 to generate glycoprotein specific molecular chaperones. ERp57 is a member of theprotein disulphide isomerase (PDI) family and its binding to ER lectins can bereconstituted in vitro. I have exploited this approach to define the regions of ERp57 thatare necessary and sufficient for its specific interaction with calreticulin and calnexin.Truncated forms of ERp57, chimeric proteins containing various domains of ERp57 andPDI (which does not interact with calreticulin) and ERp57 b??? domain point mutants havebeen constructed. By analysing the interactions of ERp57 derivatives with calreticulinusing both cross-linking and binding assays I have been able to provide detailed insightsinto the molecular basis for the specific assembly of these components within the ERlumen.My results indicate that the b and b??? domains of ERp57 are necessary, but notsufficient for binding to both calreticulin and calnexin. The more stringent binding assayrevealed that the a??? domain of ERp57 significantly enhanced binding to biotin-taggedcalreticulin. The ERp57 C-terminal extension also increased binding to biotin-taggedcalreticulin, perhaps by playing a role in the overall stability of the ERp57. In addition, theERp57 b??? domain point mutants show that certain amino acids in this domain, in particularresidues F280, V283 and F299, may be crucial for binding to calreticulin, consistent withthe principal lectin-binding site being located in the b??? domain. However, the bindingregion clearly extends into other domains, in particular the b and a??? domains.