| F-box proteins serve as crucial gateway proteins to ensure that proteins destined for destruction are directed to the appropriate machinery, i.e. the proteasome. Through variable protein interaction motifs, F-box proteins harness fated proteins, or substrates, to multi-subunit SCF E3 ligases where they are modified by addition of polyubiquitin chains, which the proteasome recognizes as signals for proteolysis. Although a handful of F-box proteins have known substrates, including transcription factors and cell-cycle control proteins, the majority of F-box proteins have no known substrates.;Recently, an interesting link between proteolysis and transcription factor activation has been observed. The simplistic model that transcription factor abundance equates to transactivating potency is challenged by evidence showing instability enhances potency. Further experiments to clarify the relationship between stability and transactivating potential are hampered by deficiency of model substrates and lack of knowledge of transcription factor stabilities.;Therefore, in this dissertation, we undertook a global approach to identify novel F-box protein: substrate combinations, focusing on transcription factors as candidate substrates. By systematically analyzing the steady-state levels of 140 S. cerevisiae transcription factors in 12 F-box protein deletion strains, we identified Mbf1 as a putative target of F-box protein Skp2. We determined that Mbf1 is relatively stable under normal growth conditions and that its stability has little effect on target gene expression, indicating that proteolytic control of stable transcription factors does not contribute to activation potency. Future work on other stable and unstable transcription factors will clarify this relationship. Accordingly, we provide a profile of transcription factor half-lives which delineates the entire set of yeast transcription factors into groups of highly unstable and stable proteins. Finally, the limited number of substrates identified in this screen suggests that bulk levels of transcription factors are not subject to SCF-mediated degradation under normal growth conditions. |
