Ubiquitin is a highly conserved 8 kDa protein whose many cellular functions are mediated by its covalent ligation to other proteins. Conjugation of ubiquitin is a multistep process involving a ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (UBCs or E2s), and ubiquitin protein ligases (E3s). The multi-ubiquitination of substrates marks them for degradation by the 26S proteasome. / Previously, rat UBC4 isoforms homologous to S. cerevisiae UBC4/UBC5 were cloned and characterized (Wing and Jain, 1995) (Wing et al., 1996). The UBC4-1 isoform is highly expressed in the testis, and the UBC4-testis isoform is induced in round spermatids. This thesis describes the identification and characterization of E3s interacting with these UBC4 isoforms expressed in the rat testis. / First, the isolation and characterization of a novel E3 from rat testis extracts, E3Histone, is described. E3Histone mediates conjugation of ubiquitin to histones in a UBC4-dependent manner. Interestingly, E3Histone was immunodepleted by antibodies against Cdc27, a subunit of the a&barbelow;naphase-p&barbelow;romoting c&barbelow;omplex (APC), an E3 which plays a critical role in the regulation of the cell cycle. However, E3Histone and the APC are distinct complexes. Gel filtration resolved the 600 kDa E3Histone from the 1500 kDa APC. E3Histone interacts preferentially with UBC4, whereas the APC interacts preferentially with UbcH10 and shows specificity for the substrate cyclin. E3Histone and the APC may be members of a newly-recognized family of combinatorial E3s that share some common core subunits, such as CDC27, yet possess distinct subunits that confer upon them their respective E2 and substrate specificities. In addition, E3Histone activity was detected in extracts from various purified germ cells. Induction of UBC4 may lead to the increased ubiquitination of histories and together with E3 Histone may play a role in the chromatin condensation that occurs during spermatid maturation. / Secondly, the characterization of a HECT domain E3, Rat100, is described. UBC4-1 and UBC4-testis were found to transfer ubiquitin to Rat100 in vitro. Immunoblotting showed that Rat100 has a molecular weight of 300 kDa, and that the developmental and cell-specific expression of Rat100 correlates with that of UBC4. The induction of Rat 100 may playa role in the activation of ubiquitin-dependent proteolysis during spermatogenesis.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.36825 |
| Date | January 2000 |
| Creators | Oughtred, Rose W. |
| Contributors | Wing, Simon (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 (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001771314, proquestno: NQ69914, Theses scanned by UMI/ProQuest. |
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