Abstract
Phosphorylation of proteins is a key posttranslational modification in cellular signaling, regulating many aspects of cellular responses. We used a quantitative, integrated, phosphoproteomics approach to characterize the cellular responses of the yeast Saccharomyces cerevisiae to the fatty acid oleic acid, a molecule with broad human health implications and a potent inducer of peroxisomes. A combination of cryolysis and urea solubilization was used to minimize the opportunity for reorientation of the phosphoproteome, and hydrophilic interaction liquid chromatography and IMAC chemistries were used to fractionate and enrich for phosphopeptides. Using these approaches, numerous phosphorylated peptides specific to oleate-induced and glucose-repressed conditions were identified and mapped to known signaling pathways. These include several transcription factors, two of which, Pip2p and Cst6p, must be phosphorylated for the normal transcriptional response of fatty acid-responsive loci encoding peroxisomal proteins. The phosphoproteome data were integrated with results from genome-wide assays studying the effects of signaling molecule deletions and known protein-protein interactions to generate a putative fatty acid-responsive signaling network. In this network, the most highly connected nodes are those with the largest effects on cellular responses to oleic acid. These properties are consistent with a scale-free topology, demonstrating that scale-free properties are conserved in condition-specific networks.
Highlights
Phosphorylation of proteins is a key posttranslational modification in cellular signaling, regulating many aspects of cellular responses
Hydrophilic interactionbased chromatography, and a combination of LC-MS2 and multistage activation (MSA) approaches, we identified a global data set of proteins that are differentially phosphorylated upon transition from a glucose- to a fatty acid (FA)-stimulated state
We sought to complement these initial efforts by developing a robust methodology to quantitatively interrogate phosphoproteomes and apply this approach to enumerate changes of the phosphorylated proteome corresponding to a shift from glucose- (2% glucose in YPB) to oleic acid (0.2% oleic acid, 0.5% Tween 40 in YPB)-grown S. cerevisiae
Summary
Cell Growth and Media—A single colony of BY4742 arg4⌬ lys1⌬ cells was grown overnight in 100 ml of rich medium to an A600 of 1.0 and seeded into two 1-liter cultures of a minimal yeast medium (0.17% yeast nitrogen base without ammonium sulfate or amino acids, 0.5% ammonium sulfate) containing a full complement of amino acids and supplemented with 20 mg/liter isotopically normal or heavy arginine (13C6,15N4; Isotec) and lysine (13C6,15N2; Isotec). Peptides were eluted with 2–3 min washes of 50 mM Na2HPO4, pH 8.4 acidified to pH 3 with 5 l of 100% formic acid; flash frozen in liquid nitrogen; and dried. This cutoff returned 15,123 spectra containing 148 decoy peptides for a false discovery rate (FDR) of 1% (FDR ϭ (148/(15,271 Ϫ 148) ϭ 0.0099) for phosphopeptides The selection of this cutoff was based on the accuracy to error plotted for all spectral results shown in supplemental Fig. S1 that returned an FDR of 0.002% for all peptides (both phosphorylated and non-phosphorylated) identified. Cells were pelleted by centrifugation and resuspended in YPBO (0.3% yeast extract, 0.5% peptone, 0.5% potassium phosphate buffer, pH 6.0, 0.5% Tween 40, 0.2% oleic acid), and samples were collected at 0, 30, and 90 min. Deletion strains were obtained from the commercially available yeast deletion library
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