A Method for Sporulating Budding Yeast Cells That Allows for Unbiased Identification of Kinase Substrates Using Stable Isotope Labeling by Amino Acids in Cell Culture

Abstract

Abstract Quantitative proteomics has been widely used to elucidate many cellular processes. In particular, stable isotope labeling by amino acids in cell culture (SILAC) has been instrumental in improving the quality of data generated from quantitative high-throughput proteomic studies. SILAC uses the cell’s natural metabolic pathways to label proteins with isotopically heavy amino acids. Incorporation of these heavy amino acids effectively labels a cell’s proteome, allowing the comparison of cell cultures treated under different conditions. SILAC has been successfully applied to a variety of model organisms including yeast, fruit flies, plants, and mice to look for kinase substrates as well as protein–protein interactions. In budding yeast, several kinases are known to play critical roles in different aspects of meiosis. Therefore, the use of SILAC to identify potential kinase substrates would be helpful in the understanding the specific mechanisms by which these kinases act. Previously, it has not been possible to use SILAC to quantitatively study the phosphoproteome of meiotic Saccharomyces cerevisiae cells, because yeast cells sporulate inefficiently after pregrowth in standard synthetic medium. In this study we report the development of a synthetic, SILAC-compatible, pre-sporulation medium (RPS) that allows for efficient sporulation of S. cerevisiae SK1 diploids. Pre-growth in RPS supplemented with heavy amino acids efficiently labels the proteome, after which cells proceed relatively synchronously through meiosis, producing highly viable spores. As proof of principle, SILAC experiments were able to identify known targets of the meiosis-specific kinase Mek1.