Analysis of higher plant N-degron pathway components and substrates via expression in S. cerevisiae.

Heterologous expression of enzymes can generate a background-free environment that facilitates investigation of enzyme properties, for instance to focus on particular isoforms in case of gene families, or on individual splicing variants. If a proper host can be found, in vivo assays are often simpler than overexpression and purification, followed by in vitro measurements, would be. We expressed plant ubiquitin ligase PRT6 in the budding yeast Saccharomyces cerevisiae for studies on activity and substrate preferences.

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

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 in a 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.