As nascent secretory and membrane proteins are inserted into the endoplasmic reticulum (ER), they are maintained in folding and/or assembly competent states by molecular chaperones including the Hsp70 and Hsp90 homologues, BiP and GRP94, and the lectin-like chaperones calnexin (CNX) and calreticulin (CRT). Folding is catalyzed by protein disulfide isomerase (PDI), its CNX (and CRT) associated homologue, ERp57, and protein prolyl isomerase (PPI). Moreover, N-linked glycoproteins benefit from a lectin-based "quality control apparatus" that ensures their correct folding or oligomeric assembly. Binding to these lectins occurs through oligosaccharide trimming from Glc 3Man9GlcNAc2 to the monoglucosylated form (Glc 1Man9GlcNAc2). Release and subsequent rebinding occurs though the hydrolysis and reglucosylation of the innermost glucose by glucosidase II and UDP-glucose glycoprotein:glucosyltransferase, respectively. This cyclical process, termed the "Calnexin Cycle", continues until their correct conformation is achieved. / The cloning and characterization of human glucosidase II is reported here. cDNAs for two splice variants of the catalytic alpha subunit and the beta subunit were isolated. Expression of the beta subunit was shown to be required for enzymatic activity, solubility and/or stability, and ER retention of the enzyme. Detailed kinetic analysis on recombinant alpha1/beta and alpha2/beta isoforms, using p-nitrophenyl alpha-D-glucopyranoside as a substrate, reveals that both exhibit kinetic profiles of a two binding site model, and share properties of catalysis and inhibition on this substrate. Moreover, similar rates of hydrolysis of the oligosaccharide substrates rules out the possibility that the two binding site kinetic model, first proposed by Alonso et al. (1999, Biochem J. 278:721--7), is the result of co-purified isoforms of glucosidase II that have different substrate specificities. / Also, an ER protein two-hybrid system, based on Ire1p and the unfolded protein response (UPR) pathway in Saccharomyces cerevisiae, was developed to examine and map the interactions between CNX/CRT and ERp57. Ire1p fusions with CNX and CRT were shown to interact specifically with ERp57, and as expected, PDI did not. Through deletion analysis, new roles were assigned to the proline-rich loop domains of CNX and CRT, and the non-catalytic B thioredoxin domain of ERp57 in mediating their heterodimerization.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37627 |
| Date | January 2001 |
| Creators | Pelletier, Marc-François. |
| Contributors | Thomas, David Y. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001846195, proquestno: NQ75669, Theses scanned by UMI/ProQuest. |
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