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

该项目的目标是描述负责泛素（Ub）共价附着的拟南芥蛋白网络，并开发用于分析它们的研究工具。翻译后，一个或多个Ubs与选定的细胞内蛋白结合，通过其改变靶标的功能和/或半衰期的能力，在植物的许多生长、发育和代谢过程中起着不可或缺的作用。虽然其主要功能是将蛋白质提交给26S蛋白酶体降解，但最近其他功能也变得明显，包括DNA修复，溶酶体分解代谢，细胞内运输和转录调节。在负责Ub偶联的酶中，Ub蛋白连接酶（或E3s）是控制靶标选择性和Ub连锁性质的关键因素。拟南芥似乎含有超过1300个E3成分，使其成为该植物中最大的功能群之一（约占蛋白质组的5%）。该项目旨在确定拟南芥中的E3家族及其靶点，使用在之前的奖项中组装的大量方法，试剂和突变体。拟南芥E3蛋白家族将进一步注释，并确定其生化活性。潜在的功能将通过DNA微阵列的表达研究和GFP-E3融合物的定位来探索。以单个E3s为诱饵，采用酵母-双杂交和免疫共沉淀的互作策略来鉴定潜在靶点和各种辅助因子。高通量蛋白质微阵列将识别目标，使用单个e3或其目标识别模块作为探针。泛素化控制的过程将通过对大量E2和E3突变体的表型分析来探索。结合标记Ubs的亲和方法将用于从拟南芥中分离泛素化蛋白。然后，这些共轭物将通过各种质谱技术进行鉴定，以提供广泛的Ub靶标库。最后，万维网上可访问的PlantsUBQ数据库（http://plantsubq.genomics.purdue.edu）的基因、突变体、靶标和其他与泛素化有关的信息将得到扩展，以广泛的科学曝光。更广泛的影响：本研究产生的结果将形成理解E3多样性的基本框架，有助于揭示每种E3类型的特定功能，并可能为许多未表征的拟南芥基因分配功能，从而帮助定义该植物的基因组。最终，这些结果将为这种翻译后修饰在植物中的作用提供全面的信息，最终可用于设计提高作物生产力的新策略。除了培养研究生和博士后之外，一个主要的教育目标是将少数民族本科生纳入发现过程，作为暑期研究项目的一部分。

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.