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Systematic lipidomic analysis of yeast protein kinase and phosphatase mutants reveals novel insights into regulation of lipid homeostasis.

da Silveira Dos Santos AX, Riezman I, Aguilera-Romero MA, David F, Piccolis M, Loewith R, Schaad O, Riezman H - Mol. Biol. Cell (2014)

Bottom Line: Our approach successfully identified known kinases involved in lipid homeostasis and uncovered new ones.By clustering analysis, we found connections between nutrient-sensing pathways and regulation of glycerophospholipids.We also found several new candidates for the regulation of sphingolipid homeostasis, including a connection between inositol pyrophosphate metabolism and complex sphingolipid homeostasis through transcriptional regulation of AUR1 and SUR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Geneva CH-1211, Switzerland National Centre of Competence in Research "Chemical Biology,", University of Geneva, Geneva CH-1211, Switzerland.

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Workflow of lipid analysis. The lipid profile of yeast knockouts of protein kinase or phosphatase gene was systematically generated. Each strain was grown in standard conditions and processed in independent biological duplicates. Lipids were extracted according to different protocols and subjected to mass spectrometry analysis. Glycerophospholipids (GPLs) and sphingolipids (SLs) were analyzed by electrospray ionization mass spectrometry (ESI-MS) using multiple reaction monitoring. Sterols were analyzed using gas chromatography coupled to mass spectrometry (GC-MS). MS signal intensities were converted to relative concentrations based on standard curves of internal standards spiked in the samples before lipid extraction. Data were normalized and analyzed as described in Materials and Methods and the Supplemental Information.
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Figure 1: Workflow of lipid analysis. The lipid profile of yeast knockouts of protein kinase or phosphatase gene was systematically generated. Each strain was grown in standard conditions and processed in independent biological duplicates. Lipids were extracted according to different protocols and subjected to mass spectrometry analysis. Glycerophospholipids (GPLs) and sphingolipids (SLs) were analyzed by electrospray ionization mass spectrometry (ESI-MS) using multiple reaction monitoring. Sterols were analyzed using gas chromatography coupled to mass spectrometry (GC-MS). MS signal intensities were converted to relative concentrations based on standard curves of internal standards spiked in the samples before lipid extraction. Data were normalized and analyzed as described in Materials and Methods and the Supplemental Information.

Mentions: To assess the effect of a missing kinase or phosphatase on the lipid profile of S. cerevisiae, we systematically analyzed the lipidome of knockout strains of nonessential kinases and phosphatases (Supplemental Table S1). Strains were grown in rich media and harvested at early exponential growth phase. Using class-specific lipid extractions followed by mass spectrometry, our lipidomic approach covered most of the major lipid classes in yeast (Figure 1; Ejsing et al., 2009; Guan et al., 2010). For phospholipid and sphingolipid analysis, we used multiple-reaction monitoring mass spectrometry (MRM-MS), which allows a robust analysis of lipids of interest with high selectivity and sensitivity (Guan et al., 2010). We monitored the signals of hundreds of lipid species (Supplemental Table S2), whose relative quantification was achieved using appropriate internal standards that were spiked into the sample before lipid extraction. For sterols, we used gas chromatography coupled to mass spectrometry (GC-MS). Ergosterol was identified by its pertinent fragment ions and retention time (Guan et al., 2010).


Systematic lipidomic analysis of yeast protein kinase and phosphatase mutants reveals novel insights into regulation of lipid homeostasis.

da Silveira Dos Santos AX, Riezman I, Aguilera-Romero MA, David F, Piccolis M, Loewith R, Schaad O, Riezman H - Mol. Biol. Cell (2014)

Workflow of lipid analysis. The lipid profile of yeast knockouts of protein kinase or phosphatase gene was systematically generated. Each strain was grown in standard conditions and processed in independent biological duplicates. Lipids were extracted according to different protocols and subjected to mass spectrometry analysis. Glycerophospholipids (GPLs) and sphingolipids (SLs) were analyzed by electrospray ionization mass spectrometry (ESI-MS) using multiple reaction monitoring. Sterols were analyzed using gas chromatography coupled to mass spectrometry (GC-MS). MS signal intensities were converted to relative concentrations based on standard curves of internal standards spiked in the samples before lipid extraction. Data were normalized and analyzed as described in Materials and Methods and the Supplemental Information.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4196872&req=5

Figure 1: Workflow of lipid analysis. The lipid profile of yeast knockouts of protein kinase or phosphatase gene was systematically generated. Each strain was grown in standard conditions and processed in independent biological duplicates. Lipids were extracted according to different protocols and subjected to mass spectrometry analysis. Glycerophospholipids (GPLs) and sphingolipids (SLs) were analyzed by electrospray ionization mass spectrometry (ESI-MS) using multiple reaction monitoring. Sterols were analyzed using gas chromatography coupled to mass spectrometry (GC-MS). MS signal intensities were converted to relative concentrations based on standard curves of internal standards spiked in the samples before lipid extraction. Data were normalized and analyzed as described in Materials and Methods and the Supplemental Information.
Mentions: To assess the effect of a missing kinase or phosphatase on the lipid profile of S. cerevisiae, we systematically analyzed the lipidome of knockout strains of nonessential kinases and phosphatases (Supplemental Table S1). Strains were grown in rich media and harvested at early exponential growth phase. Using class-specific lipid extractions followed by mass spectrometry, our lipidomic approach covered most of the major lipid classes in yeast (Figure 1; Ejsing et al., 2009; Guan et al., 2010). For phospholipid and sphingolipid analysis, we used multiple-reaction monitoring mass spectrometry (MRM-MS), which allows a robust analysis of lipids of interest with high selectivity and sensitivity (Guan et al., 2010). We monitored the signals of hundreds of lipid species (Supplemental Table S2), whose relative quantification was achieved using appropriate internal standards that were spiked into the sample before lipid extraction. For sterols, we used gas chromatography coupled to mass spectrometry (GC-MS). Ergosterol was identified by its pertinent fragment ions and retention time (Guan et al., 2010).

Bottom Line: Our approach successfully identified known kinases involved in lipid homeostasis and uncovered new ones.By clustering analysis, we found connections between nutrient-sensing pathways and regulation of glycerophospholipids.We also found several new candidates for the regulation of sphingolipid homeostasis, including a connection between inositol pyrophosphate metabolism and complex sphingolipid homeostasis through transcriptional regulation of AUR1 and SUR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Geneva CH-1211, Switzerland National Centre of Competence in Research "Chemical Biology,", University of Geneva, Geneva CH-1211, Switzerland.

Show MeSH
Related in: MedlinePlus