| The ubiquitination of intracellular proteins is essential for eukaryotic cell regulation because it allows cells to selectively target proteins for an array of cellular fates, including degradation by the 26S proteasome. Substrate ubiquitination occurs through the sequential action of the E1, E2, and E3 enzymes. The E3s, or ubiquitin-protein ligases, recognize appropriate substrates and facilitate transfer of activated ubiquitin from the E2s to the target proteins. SCF complexes are E3s that use F-box proteins to deliver targets to a core ligase activity provided by the Skp1, Cullin1 and Rbx1 subunits. The F-box subunit binds the Skp1 subunit through a degenerate N-terminal ∼60-amino-acid F-box motif and the target protein via C-terminal protein-protein interaction domains.; Using published F-box motifs as queries, I identified ∼700 potential F-box genes in Arabidopsis thaliana, making this gene superfamily one of the largest known in plants. The encoded F-box proteins cluster phylogenically into 5 major families. The proteins have diverse C-terminal domains that presumably participate in substrate recognition. This diversity suggests that individual F-box proteins may have distinct functions and/or substrates. Representatives of all the major families interact in yeast two-hybrid experiments with members of the Arabidopsis SKP1 family, supporting their classification as F-box proteins. Collectively, these results suggest that Arabidopsis has exploited the SCF E3 complex as a major route for cellular regulation.; To further understand the roles of SCF E3s in Arabidopsis, I initiated the reverse genetic analysis of two F-Box proteins, EIN3-binding F-box (EBF)-1 and -2. I found that EBF1 and 2 work coordinately in SCF complexes to repress ethylene action. Mutations in either gene induce hypersensitivity to exogenous ethylene. Both proteins interact directly with Ethylene-Insensitive (EIN)-3, a transcriptional regulator important for ethylene signaling. Levels of EIN3 are increased in mutants affecting either EBF1 or 2, suggesting that the corresponding SCF complexes work together to ubiquitinate EIN3. Double ebf1 ebf2 mutants display a substantial arrest of seedling growth and have elevated EIN3 levels in the absence of ethylene. Collectively, my results show that the SCFEBF1/EBF2 -dependent ubiquitination and subsequent removal of EIN3 is critical for proper ethylene signaling and growth in plants. |
