Arabidopsis 2010: Functional Analysis of the Ubiquitin-Protein Ligase (E3) Families in Arabidopsis

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The goal of this project is to describe the network of Arabidopsis proteins responsible for the covalent attachment of ubiquitin (Ub) and to develop research tools for their analysis. The post-translational conjugation of one or more Ubs to selected intracellular proteins plays an integral role in numerous growth, developmental, and metabolic processes in plants via its ability to modify the function and/or half-life of its targets. Whereas its main function is to commit proteins for degradation by the 26S proteasome, other functions have become apparent more recently, including roles in DNA repair, lysosomal catabolism, intracellular trafficking, and the regulation of transcription. Among the enzymes responsible for Ub conjugation, the Ub-protein ligases (or E3s) are the crucial elements that control both target selectivity and the nature of the Ub linkage. Arabidopsis appears to contain over 1,300 E3 components, making this collection one of the largest functional groups in this plant (~5% of the proteome). This project is directed toward defining the E3 families and their targets in Arabidopsis, using the large battery of methods, reagents and mutants assembled during a previous award. The Arabidopsis E3 protein families will be further annotated and their biochemical activities will be defined. Potential functions will be explored by expression studies with DNA microarrays and by localization using GFP-E3 fusions. Interaction strategies (yeast-two-hybrid and co-immunoprecipitation) using individual E3s as bait will be exploited to identify potential targets and various accessory factors. High-throughput protein microarrays will identify targets, using individual E3s or their target-recognition modules as probes. The processes controlled by ubiquitination will be explored by the phenotypic analysis in bulk of large collections of E2 and E3 mutants. Affinity approaches coupled with tagged Ubs will be employed to isolate ubiquitinated proteins from Arabidopsis. These conjugates will then be identified by various mass spectrometric techniques to provide an extensive library of Ub targets. And finally, the web-accessible PlantsUBQ database (http://plantsubq.genomics.purdue.edu) of genes, mutants, targets, and other information pertinent to ubiquitination will be expanded for broad scientific exposure. Broader Impact: The results generated by this study will form an essential framework for understanding E3 diversity, help reveal specific functions for each E3 type, and likely assign function to a number of uncharacterized Arabidoposis genes, thus helping define the genome of this plant. Ultimately, the results will provide comprehensive information on the role of this post-translational modification in plants that ultimately can be used to devise new strategies to improve crop productivity. In addition to the training of graduate students and postdoctoral fellows, a main educational goal will be the inclusion of minority undergraduates in the discovery process as part of a summer research program.