OsTIR1F74G Expression Controls Basal and Auxin-Induced Degradation Rates of the AID System in Yeast

Abstract

The auxin-inducible degron (AID) system has been widely used to conditionally and dynamically deplete proteins in yeast. In this system, the plant hormone auxin promotes OsTIR1-mediated degradation of proteins carrying an auxin-inducible degron tag. However, "basal" degradation of AID-tagged proteins in the absence of auxin has hampered work with essential proteins due to defective or non-viable strains. A second-generation AID system based on the OsTIRF47G mutant was recently introduced to overcome the limitations of basal degradation in budding yeast. However, it remained unclear to what extent the use of OsTIRF47G eliminates basal degradation and how it impacts auxin-induced degradation. Here, by performing a quantitative characterization of the basal and auxin-induced degradation dynamics using OsTIR1F74G in budding yeast, we find that basal degradation is still detectable and that it depends on OsTIR1F74G expression levels. We show that also the auxin-induced degradation rates and auxin-induced steady-state concentrations of AID-tagged proteins depend on OsTIR1F74G expression levels, in addition to the type of auxin used. Lastly, we showcase how increased basal degradation of AID-tagged functional proteins can impair cell growth and lead to unwanted phenotypes. We anticipate that our findings will guide future applications of the AID system in budding yeast by helping researchers select appropriate OsTIR1F74G expression levels, particularly in studies focused on precise control of degradation dynamics.