AUTHOR=Kozlic Aida , Winter Nikola , Telser Theresia , Reimann Jakob , Rose Katrin , Nehlin Lilian , Berckhan Sophie , Sharma Gunjan , Dambire Charlene , Boeckx Tinne , Holdsworth Michael J. , Bachmair Andreas TITLE=A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions JOURNAL=Frontiers in Plant Science VOLUME=12 YEAR=2022 URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.806129 DOI=10.3389/fpls.2021.806129 ISSN=1664-462X ABSTRACT=

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.