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

泛素(Ub)/26S蛋白酶体蛋白分解途径通过选择性地去除异常多肽和短暂的调节蛋白，在植物的生长、发育、动态平衡和防御中发挥着不可或缺的作用。在这个途径中，蛋白质首先被UBS共价标记；然后这些底物被26S蛋白酶体降解，伴随着UBS的释放以便重复使用。Ub-蛋白连接酶(或E3)决定了Ub结合的特异性，从而控制了系统的选择性。拟南芥基因组编码了900多种不同的E3，这一事实最好地证明了它们在植物中的重要性。初步的遗传分析已经确定了E3在激素和胁迫反应、光信号、昼夜节律、酶调节和模式形成中的重要作用。为了帮助确定植物泛素化的深度和广度，本项目将详细分析拟南芥E3家族。生物信息学方法将被用来将E3组装成亚家族。对于有代表性的例子，蛋白质相互作用技术将为每一个赋予适当的辅助因子，生化分析将确认每一个都具有Ub-连接酶活性。它们的表达模式和位置将通过DNA微阵列和GFP-E3融合分析来检测。酵母双杂交和质谱学技术将确定可能的底物。功能将通过适当的拟南芥突变体的表型分析来进一步探索。所有信息将在可免费访问的公共网站(http://www.hort.wisc.edu/vierstra/vierstra1.htm).上向科学界发布所产生的结果将形成一个理解E3多样性的基本框架，有助于揭示每一种E3类型的特定功能，并将开发其丰度受到Ub介导的蛋白降解影响的蛋白质数据库。总而言之，该项目将提供关于一大群重要的拟南芥基因/蛋白质的全面信息，这些基因/蛋白质最终可以用来设计新的策略，在蛋白质分解干扰作物生产力时改变它。

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