The goal of this project is to define the network of Arabidopsis proteins responsible for covalent attachment of the small protein ubiquitin (Ub) to specific cellular targets. The attachment of Ub to other proteins is highly selective and plays an integral role in numerous developmental and metabolic processes in plants. Although addition of Ub was originally discovered as a mechanism to promote protein degradation, recently other functions have become apparent, including roles in DNA repair, lysosomal catabolism, intracellular trafficking, and the regulation of chromatin structure and transcription. Several enzymes are needed for attachment of Ub to targets, but among these the Ub-protein ligases (or E3s) are the crucial enzymes that control both target selectivity and the nature of the Ub linkage. Genome analysis now shows that Arabidopsis may express over 1,500 E3 components, making E3s one of the largest functional protein groups in plants (~5% of the proteome). The diversity of E3s is unique to the plant kingdom, and the large size of this family indicates it presents a versatile mechanism for controlling cellular processes. This versatility makes it of significant interest relative to plant evolution and adaptation to the environment, as well as holding potential for future plant genetic engineering. This project will expand successful collaborative efforts to define E3 proteins families and their targets in Arabidopsis, using the large battery of methods, reagents and mutants, as well as a variety of new genomic and structure-based approaches. The project will continue to identify and annotate the Arabidopsis E3 protein families and define their biochemical activities. Their importance to Arabidopsis physiology, development and survival will be analyzed by reverse genetic analyses and further studied by expression profiling, localization using GFP-E3 fusions, and strategies to identify partners or substrates (yeast-two-hybrid and co-immunoprecipitation). The project will continue to develop protein microarrays as a high-throughput method to unravel the network of proteins involved in E3 action. Further substrates of ubiquitylation will be identified and then confirmed genetically and by global protein stability assays. The relationships between E3s and their partner E2s will be assessed by substrate-specific in vitro assays and genetic analyses. 3-D models for how targets are recognized by representative E3s will be developed by X-ray crystallographic analyses. Finally, the project will transfer knowledge gained from Arabidopsis to other plant species to help define E3 evolution, to understand why plants are so reliant on this post-translational modification, and to identify E3 orthologs in crops, which can then be exploited to improve agricultural productivity.
Broader Impact: The program will provide high level training for postdoctoral researchers and graduate students. A mentoring plan is in place to involve trainees specifically in design of the research, and to develop career opportunities. Students and postdocs will present their work at annual project meetings of the investigators, exposing them to the interdisciplinary aspects of the work. In addition to training graduate students and postdoctoral fellows, under-represented minority undergraduates will also be part of the discovery process through a summer research program partnering with the University of West Alabama. This project aims to train, in a highly interdisciplinary context, the next generation of professionals to continue these efforts, and to expose under-represented minorities to plant research.
