Optimization of Sic1-Cln2-based phosphodegron tag for inducible protein degradation

Abstract

Precise regulation of protein abundance in the cell would be instrumental in both research and industry. A variety of tools exists for the regulation of protein expression at the transcriptional level. However, there is a shortage of tools that would allow control over already expressed proteins. In this study, we exploit the native cell cycle regulation system of Saccharomyces cerevisiae to develop a system for targeted inducible protein degradation. Using sequence elements that modulate cyclin-dependent kinase (CDK) substrate specificity, we generated a set of phosphodegron tags that have diverse effects on degradation rates of the tagged proteins. The control over the system is provided by conditional expression of the stabilized version of cyclin Clb3, which drives CDK-dependent phosphorylation of the tag, and F-box protein Grr1, which recognizes phosphorylated tag and label it for further degradation. In the course of this work, we have designed a set of phosphodegron tags that can be used for the regulation of the protein abundance in the cell and its maintenance at desirable levels that can be useful for the industrial production of value-added products. In addition, we discovered that Cks1 priming effect on the multisite phosphorylation is not only distance-, but, likely, also context-dependent which might be of interest for fundamental research.