| This study focuses on regulated protein degradation in budding yeast and plants. The mechanism of regulation is governed by controlling the attachment of ubiquitin moieties to proteins marked for degradation by the proteasome. The specificity of this attachment is derived from protein complexes known as ubiquitin ligases, and the best characterized ubiquitin ligase complex is the SCF (Skp-Cullin/Cdc53p-F-box protein). Biochemical analysis from mammalian cell culture lines has determined that the SCF requires the covalent attachment of the ubiquitin-like protein, Rub/Nedd8 to the cullin for robust activity. This study focuses on expanding the knowledge of the Rub proteins in Saccharomyces cerevisiae and Arabidopsis thaliana.; Primarily, two new targets for Rub1p conjugation in S. cerevisiae were identified, Rtt101p and Cul3p. Rub1p-modified forms of these cullins were lost when a lysine in a conserved region near the C-terminus of each protein was mutated, suggesting that these lysines are the sites of Rub1p-dependent modification in Rtt101p and Cul3p. Identification of additional Rub1p targets in S. cerevisiae indicates an expanded role for Rub in this organism, which previously was in question because of the wild-type phenotype of a rub1 null strain.; To study the role of RUB in a higher eukaryote, I switched to studying the RUB genes of Arabidopsis. The effects of reducing protein levels for two nearly identical Rub proteins, RUB1 and RUB2, were characterized. T-DNA insertional lines null for each of the single RUB-encoding loci have a wild-type phenotype. However, a double mutant was never recovered, supporting the hypothesis that the RUB genes were essential in Arabidopsis. To study these proteins further, RNAi-mediated silencing was used to reduce the expression of both RUB genes. Mature plants were severely dwarfed, seedlings were insensitive to the plant hormones auxin and ethylene, and dark-grown seedlings had a phenotype consistent with ethylene overproduction. Pursuing this novel phenotype, biochemical assays and RT-PCR implicate the stabilization of a family of ethylene biosynthetic proteins regulated by a new CUL3-based complex. Epitope-tagged version of all three RUB family members was used to identify targets. All three proteins attach to the original cullin in Arabidopsis, CUL1, and RUB1/2 attach to AtCUL3. |
