Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List

Full text release has been delayed at the author's request until June 01, 2029

ETD Abstract Container

Abstract Header

Genetic Studies of Skp1-Cullin1-F-box E3 Ligase Mediated Ubiquitylation Pathways in Arabidopsis

Li, Yang
http://orcid.org/0000-0002-2606-4681
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1710947210929608

Abstract Details

2024, Doctor of Philosophy (PhD), Ohio University, Plant Biology (Arts and Sciences).
Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex or through autophagy-mediated degradation. The S-Phase Kinase-Associated Protein 1 (Skp1), cullin (CUL)1, and F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants. Since the FBX proteins recognize and determine the specificity of SCF substrates, much effort has made to characterize their genomic, physiological, and biochemical roles in the past two decades of functional genomic studies. The co-immunoprecipitation failed to capture transient interaction while the yeast two-hybrid only test protein interaction in yeast cell instead of plant tissue. Therefore, the sheer number and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this research, first, from the proteomic level, I developed and tested a proximity labeling and co-immunoprecipitation approach for determining the proteome of active FBX proteins and their associated complexes in Arabidopsis. We demonstrate that ASK1-TurboID is not fully functioning, which led us to discover a novel antagonism between biotinylation and ubiquitylation in regulating protein stability in vivo. This discovery lowers the effectiveness of proximity labeling in ubiquitylation studies. Second, from the FBX level, I characterized a new FBX protein that is involved in reproductive development. The mutant was created using CRISPR/Cas9. And it was confirmed that mutation of this FBX gene impacted seed development. The potential substrates were identified and confirmed using yeast two-hybrid and split-luciferase assay. Third, from the substrate level, I labeled phytochrome A (phyA), a master red and far-red photoreceptor in plants, with a genetically engineered biotin ligase, TurboID, in vivo for searching putative unknown FBX proteins that may target phyA for degradation. I succeeded to apply phyA-TurboID fusion for complementing the far-red response of phyA-211 null mutant allele, suggesting an efficacy of PL in characterizing single ubiquitylation pathways. This study provided a new evidence about the limitation of PL in ubiquitylation studies, discovered a new antagonistic pathway of biotinylation, and developed effective genetic materials and theoretical guidance for future proximity labeling based characterization of ubiquitylation pathways.
Zhihua Hua (Advisor)
198 p.
Plant Biology; Plant Sciences
TurboID, ubiquitylation, ASK1, E3 ligase, F-box, EDF1, SKIP31, phyA

Recommended Citations

Citations

  • Li, Y. (2024). Genetic Studies of Skp1-Cullin1-F-box E3 Ligase Mediated Ubiquitylation Pathways in Arabidopsis [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1710947210929608

    APA Style (7th edition)

  • Li, Yang. Genetic Studies of Skp1-Cullin1-F-box E3 Ligase Mediated Ubiquitylation Pathways in Arabidopsis. 2024. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1710947210929608.

    MLA Style (8th edition)

  • Li, Yang. "Genetic Studies of Skp1-Cullin1-F-box E3 Ligase Mediated Ubiquitylation Pathways in Arabidopsis." Doctoral dissertation, Ohio University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1710947210929608

    Chicago Manual of Style (17th edition)

ohiou1710947210929608
© 2024, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.