In this thesis, the mechanism of endoplasmic reticulum localization of Saccharomyces cerevisiae alpha1,2-mannosidase is studied. alpha1,2-mannosidase is a type II membrane glycoprotein localized in the endoplasmic reticulum, yet it does not have any of the known endoplasmic reticulum localization signals. / Immunofluorescence studies show that the endoplasmic reticulum localization of alpha1,2-mannosidase depends on the Golgi protein Rer1p, since in rer1-deleted cells, alpha1,2-mannosidase migrates to the vacuoles. Furthermore, alpha1,2-mannosidase acquires Golgi-specific carbohydrate modification. These results show that the steady state endoplasmic reticulum localization of alpha1,2-mannosidase involves recycling from the Golgi apparatus. The transmembrane domain of alpha1,2-mannosidase is important for endoplasmic reticulum localization since fusing it to the Golgi protein Kre2p results in the endoplasmic reticulum localization of the chimera in an Rer1p-dependent manner. Mutation of the polar residues in the transmembrane domain do not affect endoplasmic reticulum localization of alpha1,2-mannosidase, nor Rer1p-dependent recycling, indicating that the polar residues are not important for these processes. alpha1,2-Mannosidase and Rer1p interact, determined using the split-ubiquitin system, a genetic method adapted to study membrane protein interactions in vivo. Therefore, the transmembrane domain of alpha1,2-mannosidase mediates recycling from the Golgi apparatus in a mechanism that involves interaction with the Golgi protein Rer1p. / When solubilized and subjected to gel filtration analysis, endogenous alpha1,2-mannosidase is eluted on Sephacryl S-200 as twice the molecular weight of the purified recombinant enzyme lacking its transmembrane domain. Immunoprecipitation studies show that alpha1,2-mannosidase can form a homodimer. Furthermore, mutation of the asparagine residue at position 3, or the tyrosine residues at positions 20 and 21, prevents dimerization.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84293 |
| Date | January 2003 |
| Creators | Massaad, Michel Jean |
| Contributors | Herscovics, Annette (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 Biochemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002031497, proquestno: AAINQ98323, Theses scanned by UMI/ProQuest. |
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