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. Expression of this large enzyme profits from the eukaryotic folding catalysis provided by budding yeast, and from the presence of endogenous ubiquitin activating enzyme. While yeast encodes a ubiquitin ligase, Ubr1, that is functionally related to PRT6, a strain with deletion of the UBR1 gene offers a background-free host. Two different substrates were analyzed. One was a model substate, and the other one a natural substrate fused to a reporter. Two different methods were compared for assessment of protein stability. A method based on internal standardization via tandem fluorescent timer measurement turned out to be complementary to standardization based on cell culture density.

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 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.