Combining deep-sequencing, proteomics, phosphoproteomics and functional screens to discover novel regulators of sphingolipid homeostasis.

Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules. SL levels must be tightly regulated as imbalances affect cellular function and contribute to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is maintained as well as uncovering new regulators is required for understanding lipid biology and for identifying new targets for therapeutic interventions. Here we combine multiple omics technologies to identify the changes of the transcriptome, proteome and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin. Surprisingly we find that SL depletion triggers only minor changes in the expression and levels of regulatory proteins involved in SL homeostasis. However dramatic regulation occurs at the level of the phosphoproteome suggesting that maintaining SL homeostasis demands rapid responses. To discover which of the phosphoproteomic changes are required for the cell’s first-line response to SL depletion we combined the results with systematic growth screens for genes required during growth in myriocin when either mutated or overexpressed. By following the rate of SL biosynthesis in those candidates that are both essential for growth during myriocin and are phosphorylated in response to the drug we uncovered new regulators of SL homeostasis.