Characterization of SCF Ubiquitin-Ligase Subunits in Arabidopsis
Date of Award
William L. Crosby
Molecular biology, Cellular biology, Oncology
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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
The Arabidopsis thaliana genome encodes several families of polypeptides that are known or predicted to participate in the formation of the SCF-class of E3-ubiquitin ligase complexes. One such gene family encodes the Skp1-like class of polypeptide subunits, where 21 genes have been identified and are known to be expressed in Arabidopsis. The complexity of this family of Arabidopsis Skp1-like--or ASK --genes, together with the close structural similarity among its members, raises the prospect of significant functional redundancy among select paralogs. We have assessed the potential for functional redundancy within the ASK gene family by analyzing an expanded set of criteria that define redundancy with higher resolution. The criteria used include quantitative expression of locus-specific transcripts using qRT-PCR, assessment of the sub-cellular localization of individual ASK:YFP auto-fluorescent fusion proteins expressed in vivo, as well as the in planta assessment of individual ASK-F-box protein interactions using BiFC. The results indicated significant functional divergence of steadystate transcript abundance and protein-protein interaction specificity involving ASK proteins in a pattern that is poorly predicted by sequence-based phylogeny. The information emerging from this and related studies was used to functionally characterize using an RNAi approach complemented by phenotypical analysis. The observation of diverse phenotypes not only argues a high level of sub-functionalization has occurred throughout the ASK gene family, but also underscores the breadth of functions that this gene family plays throughout plant development. Transport Inhibitor Response (TIR1), is a member of a family of five Auxin-signaling F-box proteins (AFBs) and has been shown to act as the receptor for auxin binding and activation of the SCF TIR1 complex, leading to targeted protein degradation events involved in auxin perception. We provide evidence for homo-dimerization of TIR1 protein in planta together with a role for TIR1 homo-dimerization in the degradation of Aux/IAA proteins as part of the auxin-signaling pathway.
Dezfulian, Mohammad Haj, "Characterization of SCF Ubiquitin-Ligase Subunits in Arabidopsis" (2013). Electronic Theses and Dissertations. 4759.