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

Abstract

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.