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
and uncovering new regulators is required for understanding lipid
biology and for identifying new targets for therapeutic interventions.
Here we combine omics technologies to identify the changes of the
transcriptome, proteome, and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin.
Surprisingly, while SL depletion triggers important changes in the
expression of regulatory proteins involved in SL homeostasis, the
most 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 overlaid
our omics results with systematic growth screens for genes required
during growth in myriocin. By following the rate of SL biosynthesis
in those candidates that are both affecting growth and are phosphorylated
in response to the drug, we uncovered Atg9, Stp4, and Gvp36 as putative
new regulators of SL homeostasis.