Date of Award
Arabidopsis, diglycine, SCF, ubiquitin
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
The regulation of the temporal and spatial location of proteins is paramount in maintaining a properly-functioning cell. The control of protein abundance and localization is mediated, in part, by post-translational modifications. One such notable modification is protein ubiquitination. Protein ubiquitination involves the covalent attachment of the small, ubiquitously-expressed and highly-conserved protein ubiquitin (Ub) to a substrate protein. The function of the Ub tag is dependent on the type and topology of the Ub chain, and can direct proteins for degradation by the 26S proteasome, or may serve other non-proteolytic functions. Protein ubiquitination is achieved by three enzyme complexes: the E1 Ubactivating enzyme, the E2 Ub-conjugating enzyme, and the E3 Ub ligase enzyme. A particular subclass of E3 ligases is the SCF subclass, a multi-subunit complex comprising at least four components. Of these components, the variable F-box protein is responsible for substrate specificity of the SCF. Interestingly, the genome of the plant model species Arabidopsis thaliana is known or predicted to encode for roughly 700 F-box proteins, in contrast to the yeast and human genomes that encode for only 11 and 69 F-box proteins, respectively. In addition, more than 5% of the Arabidopsis genome encodes for components for the Ub-proteasome pathway (UPS). Given this potential complexity, it is surprising that the catalogue of known ubiquitinated proteins in Arabidopsis is very small. The most high-throughput approaches thus far have only identified about a thousand candidate proteins. Furthermore, unbiased genetic surveys of mutants with impaired plant-specific patterning and development have identified alleles of genes involved in the UPS. To address the complexity of the ubiquitination machinery in Arabidopsis, a more comprehensive list of ubiquitinated proteins must be obtained. To that end, the major objective of this work is to expand this catalogue, through the adaptation and use of approaches employed in other systems for the high-throughput identification of ubiquitinated proteins. Using a diglycine-scanning-based approach, we have identified over 600 novel candidate ubiquitinated proteins with their associated ubiquitination sites. Of the candidates identified in this study, we further studied PATL1, a novel cellplate associated protein, whose expression was found to be modulated by auxin. Our studies towards the characterization of PATL1 suggests that its degradation is sensitive to 26S proteasome inhibition, but not to inhibition of the vacuole. Further, BiFC and co-IP experiments suggest that PATL1 may act as a homodimer. Lastly, I worked towards adapting the BioID system for biotin-based proximity labeling in plant. My results suggest that the plant system is amenable to the use of this system for study, though further work is required for its full-scale implementation in Arabidopsis. With further refinement, the tools described in this study may prove to be useful in expanding the catalogue of ubiquitinated proteins in Arabidopsis, and shed light on the prominence of protein ubiquitination as a key regulator of plant patterning and development.
Roberto, Don Karl A., "Profiling protein ubiquitination in Arabidopsis thaliana" (2017). Electronic Theses and Dissertations. 7293.