A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

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

doi:10.3389/fpls.2021.806129

A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

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

Authors

Aida Kozlic

Nikola Winter

Theresia Telser

Jakob Reimann

Katrin Rose

Lilian Nehlin

Sophie Berckhan

Gunjan Sharma

Charlene Dambire

Tinne Boeckx

Professor MICHAEL HOLDSWORTH michael.holdsworth@nottingham.ac.uk
Professor of Crop Science

Andreas Bachmair

Contributors

Gunjan Sharma
Researcher

Charlene Dambire
Researcher

Sophie Berckhan
Researcher

Professor MICHAEL HOLDSWORTH michael.holdsworth@nottingham.ac.uk
Researcher

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.

Journal Article Type	Article
Acceptance Date	Dec 7, 2021
Online Publication Date	Jan 7, 2022
Publication Date	Jan 7, 2022
Deposit Date	Jan 7, 2022
Publicly Available Date	Jan 10, 2022
Journal	Frontiers in Plant Science
Electronic ISSN	1664-462X
Publisher	Frontiers Media SA
Peer Reviewed	Peer Reviewed
Volume	12
Article Number	806129
DOI	https://doi.org/10.3389/fpls.2021.806129
Keywords	Plant Science
Public URL	https://nottingham-repository.worktribe.com/output/7169669
Publisher URL	https://www.frontiersin.org/articles/10.3389/fpls.2021.806129/full