Clathrin-mediated endocytosis (CME) plays a central role in the regulation of multiple cellular processes such as uptake of nutrients, recycling of housekeeping receptors and transporters, as well as for cell surface removal and downregulation of signaling receptors. Once endocytosed, cargo passes through early endosomes where sorting mechanisms traffic the cargo to the recycling pathway or to degradation in the lysosome. The general objectives of this doctoral research were to identify and characterize new players of the clathrin-mediated trafficking pathway to reveal differences between the abundant components of the trafficking machinery in two tissues, and to examine the mechanisms of endosomal sorting. / We used subcellular proteomics to reveal the differences in components of clathrin-coated vesicles (CCVs) isolated from brain and liver and to identify new molecules participating in clathrin trafficking. We demonstrated that the ratio between the clathrin adaptor proteins AP-1 and AP-2 is different in brain and liver, which indicates differential functions between the two tissues. We also discovered that clathrin-light chains, which have been proposed for many years to be regulatory proteins in the assembly of CCVs, were less abundant relative to clathrin-heavy chain in liver and in non-brain tissues compared to brain. / We identified a new DnaJ domain-containing protein, receptor-mediated endocytosis protein 8 (RME-8) that was detected in liver CCVs specifically. Further characterization revealed that the RME-8 DnaJ domain binds to the chaperone heat-shock cognate 70 (Hsc70) in an ATP-dependent manner. RME-8 is a ubiquitously expressed protein that tightly associates with endosomes, and its depletion causes intracellular trafficking defects. Moreover, we demonstrated that RME-8 depletion also leads to a decrease in levels of epidermal growth factor receptor (EGFR), as a result of an increase in EGFR degradation. RME-8 knock-down causes decreased EGFR levels even in cancer cells lines where EGFR is generally protected from degradation. / Globally this doctoral project revealed new insights on specialized functions for c1athrin-mediated trafficking in different tissues and allowed the identification and characterization of a novel protein implicated in sorting decisions occurring on endosomes.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.115684 |
| Date | January 2008 |
| Creators | Girard, Martine. |
| 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 (Division of Neuroscience.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 003129995, proquestno: AAINR66306, Theses scanned by UMI/ProQuest. |
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