EAGER: Optimization of proximity labeling approach to understand composition and regulation of the plant ubiquitin ligases interactome

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The goal of this project is to use a novel technology to identify proteins that interact with a component of the cellular protein degradation machinery. The protein degradation machine best known as the Ubiquitin Proteasome System (UPS) has emerged to be at the center of virtually every biological process. UPS-mediated protein degradation is a highly regulated process and failure to degrade specific proteins in a timely fashion will have a deleterious effect on the organism. In plants, UPS control numerous processes including growth and development, hormone signaling, and responses to biotic and abiotic stress. Plants are known to adapt the UPS to facilitate cellular changes required to respond to and tolerate adverse environmental conditions. Since the mechanisms underlying fundamental biological processes such as responses to environmental changes are often conserved in plants, our findings from this project will be broadly applicable across different plant species. Furthermore, the project will impact science and education beyond the immediate goals by: (1) providing mentorship, research experiences, and novel platform for undergraduate students and senior researchers, (2) propagating the importance of plant sciences in the larger community, and (3) by advancing the knowledge in cutting edge approaches of the emerging plant functional proteomic field.Despite the advances made in the field of proteolytic regulation in plants, little is known about the composition and regulation of plant ubiquitin (Ub) ligase enzymes, in particular under diverse environmental conditions. While increasing number of genomic studies implicate the critical role for Ub ligase in regulating biotic and abiotic stress signaling, comprehensive studies at the protein levels remain unmapped. A full understanding of UPS processes in plants demands the identification of Ub system targets and other regulators (e.g., deubiquitinating enzymes, ubiquitin-like and ubiquitin associated proteins). Thus far, only very limited attempts at the protein levels have been made to characterize Ub ligases from plant cells because of technical challenges in capturing dynamic Ub ligase complexes. Therefore, in this EAGER project, we will specifically: (1) Develop and optimize proximity labeling tools to isolate Cullin-RINGs (CRLs) Ub ligases and their interactors in planta and, (2) Use optimized methods and materials to isolate specific Ub ligase interactomes under normal growth conditions. In addition, we will use the MAX2 F-box protein as a test case to demonstrate the utility of the proximity labeling approach in capturing interactors of MAX2 in response to strigolactone hormone treatment.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.