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Combining Deep Sequencing, Proteomics, Phosphoproteomics, and Functional Screens To Discover Novel Regulators of Sphingolipid Homeostasis

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† Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
‡ Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands
§ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel
∥ Department of Chemical Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
*M.S.: E-mail: [email protected]
*S.L.: E-mail: [email protected]
Cite this: J. Proteome Res. 2017, 16, 2, 571–582
Publication Date (Web):November 14, 2016
https://doi.org/10.1021/acs.jproteome.6b00691
Copyright © 2016 American Chemical Society
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    Abstract

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    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.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00691.

    • Table S-1. Transcriptomic analysis of yeast incubated in YPD medium containing 150 or 300 ng/mL of myriocin. Table S-2. Quantitative proteomic analysis of yeast after myriocin inhibition from 5 to 180 min. Table S-3. Quantitative phosphoproteomic analysis of yeast after myriocin inhibition from 5 to 180 min. Table S-4. Gene Ontology analysis (process, function, component) of significantly regulated phosphosites for each cluster analysis. Table S-5. Functional analysis of cell viability after deletion or overexpression of the corresponding gene in myriocin-containing medium. (XLSX)

    • Figure S-1. Transcriptomic workflow. Figure S-2. Effect of sphingolipid synthesis inhibition on yeast growing in various concentrations of myriocin. Figure S-3. Qualitative evaluation of cell viability in response to low-dose of myriocin. Figure S-4. Protein expression heatmap of proteins involved in (de)phosphorylation processes. Figure S-5. Individual dynamic profile of Sec16 phosphorylation. (PDF)

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