The Auxin-Inducible Degron as a system to study essential genes in the Budding Yeast Saccharomyces cerevisiae

The study of essential genes is challenging in any species. Historically, Saccharomyces cerevisiae (yeast) has been used because of the ability of diploids to sporulate and generate mutant haploids; other methods, such as the GAL1 promoter system, Tet-on/Tet-off systems, and temperature-sensitive mutants, have also been used. However, these systems have disadvantages, such as the use of non-preferred carbon sources, potential cell toxicity, and non-physiological expression levels. In this work, we present the auxin-inducible degron (AID) as an alternative system to study essential genes in yeast. AID leverages a C-terminal degron tag to recruit protein ubiquitination machinery and to trigger rapid proteolysis. This system offers several advantages, including rapid degradation upon auxin addition, straightforward tagging of essential genes, use of the native promoter, and minimal metabolic disruption. We demonstrate the utility of the AID system to study large and small G proteins in multiple pathways. We study an essential activator (Cdc25) in the small G protein-mediated sugar-sensing pathway and an essential large G protein (Gpa1) in the pheromone-signaling pathway. We also apply the system to investigate the effects of human oncogenic mutations in the G proteins Gq and G11, using the yeast homolog GPA1. An unexpected result of this study was that, while C-terminal tags typically disrupt the coupling of G proteins to their activating receptor, AID-tagged Gpa1 appears fully functional.