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.
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.
Nicolas
N
Lebesgue
Department of Molecular Genetics, Weizmann Institute of Science , Rehovot 7610001, Israel.|Department of Chemical Biology, Weizmann Institute of Science , Rehovot 7610001, Israel.
Márton
M
Megyeri
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.
Alba
A
Cristobal
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.
Arjen
A
Scholten
Department of Molecular Genetics, Weizmann Institute of Science , Rehovot 7610001, Israel.
Silvia G
SG
Chuartzman
Department of Molecular Genetics, Weizmann Institute of Science , Rehovot 7610001, Israel.
Yoav
Y
Voichek
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.
Richard A
RA
Scheltema
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.
Shabaz
S
Mohammed
Department of Chemical Biology, Weizmann Institute of Science , Rehovot 7610001, Israel.
Anthony H
AH
Futerman
Department of Molecular Genetics, Weizmann Institute of Science , Rehovot 7610001, Israel.
Maya
M
Schuldiner
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.
Albert J R
0000-0002-2405-4404
AJ
Heck
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.
Simone
S
Lemeer
D000935:Antifungal Agents / Q000494:pharmacology; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000378:metabolism; D000071183:Autophagy-Related Proteins / Q000235:genetics* / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D059014:High-Throughput Nucleotide Sequencing; D006706:Homeostasis / Q000187:drug effects / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D013107:Sphingolipids / Q000037:antagonists & inhibitors / Q000096:biosynthesis
C434065:ATG9 protein, S cerevisiae; D000935:Antifungal Agents; D000071183:Autophagy-Related Proteins; D005229:Fatty Acids, Monounsaturated; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; D016282:Aspartic Acid Endopeptidases; C527928:Gvp36 protein, S cerevisiae; C001996:thermozymocidin
Saccharomyces cerevisiae; label-free quantification; mass spectrometry; myriocin; phosphorylation; proteomics; sphingolipid metabolism