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

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The ubiquitin (Ub)/26S proteasome proteolytic pathway plays an integral role in the growth, development, homeostasis, and defense of plants by selectively removing abnormal polypeptides and short-lived regulatory proteins. In this pathway, proteins are first covalently tagged with Ubs; these substrates are then degraded by the 26S proteasome with the concomitant release of the Ubs for reuse. The Ub-protein ligases (or E3s) determine the specificity of Ub conjugation and hence control the selectivity of the system. Their importance in plants is best demonstrated by the fact that the Arabidopsis genome encodes over 900 different E3s. Preliminary genetic analyses of just a few have identified important roles for E3s in hormone and stress responses, light signaling, circadian rhythms, enzymatic regulation, and pattern formation.To help define the depth and breath of ubiquitination in plants, this project will analyze the Arabidopsis E3 families in detail. Bioinformatic approaches will be used to assemble the E3s into subfamilies. For representative examples, protein interaction techniques will assign each with appropriate accessory factors and biochemical assays will confirm that each has Ub-ligase activity. Their expression patterns and locations will be examined by DNA microarrays and by analysis of GFP-E3 fusions. Yeast two-hybrid and mass spectrometric techniques will identify possible substrates. Functions will be further explored by the phenotypic analysis of appropriate Arabidopsis mutants. All information will be released to the scientific community in a freely accessible public web site (http://www.hort.wisc.edu/vierstra/vierstra1.htm). The results generated will form an essential framework for understanding E3 diversity, help reveal specific functions for each E3 type, and will develop a database of proteins whose abundance is affected by Ub-mediated proteolysis. Collectively, the project will provide comprehensive information on a large and important cluster of Arabidopsis genes/proteins that ultimately can be used to devise new strategies to alter proteolysis when it interferes with crop productivity