| Ubiquitin-protein conjugation is a multistep process. Activated ubiquitin is transferred from E1 to the thiol of a ubiquitin conjugating enzyme (E2 protein). Some E2s can directly transfer ubiquitin to certain targets. However, the formation of conjugates that are competent in degradation usually requires a third enzyme, ubiquitin-protein ligase or E3. Ubiquitination allows recognition and therefore degradation of the target protein by the 26S proteosome.; Degradation is especially efficient when a multiubiquitin chain is ligated to the target. Ubiquitin moieties in the best-studied chains are linked by isopeptide bonds between lysine 48 and glycine 76 of successive ubiquitins. A specific mammalian ubiquitin conjugating enzyme, E2-25K, catalyzes the ubiquitination of ubiquitin itself, thereby forming free multiubiquitin chains in vitro. This E2 is the focus of this thesis.; Members of the E2 protein family are conserved within and across species. All E2s have a conserved core region which contains the active site cysteine. Some E2s also have C-terminal "extensions". These extensions are thought to mediate conjugative specificity and biological activity.; Although the core region of E2-25K resembles the corresponding region of a family of essential yeast E2s, the C-terminus of E2-25K is quite divergent from the C-terminus of UBC1 (UBC4 and 5 lack tails). E2-25K has no known substrate other than ubiquitin itself. While the C-terminus of E2-25K is necessary for its extremely high specificity for ubiquitin itself as a conjugation target, the tail was not sufficient for such activity. Core residues are also critical either independently or through interactions with the tail.; To provide information relevant to E2-25K's potential role in proteolysis, we also investigated the expression of this protein in various tissues and in differentiating erythroid cells. We also sought to correlate the level of this E2 in murine erythroleukemia cells with the intracellular level of the known product of E2-25K catalysis, multiubiquitin chains. These quantitative analyses bear directly on the potential mechanisms for E2-25K function. |
