Regulation of yeast central metabolism by enzyme phosphorylation

摘要

AbstractAs a frequent post-translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino-acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 α subunit Pda1 and the newly identified phosphoregulated glycerol-3-phosphate dehydrogenase Gpd1 and phosphofructose-1-kinase complex β subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes.SynopsisA strategy is presented that combines metabolic fluxes with targeted phosphoproteomics measurements to drive testable hypotheses for the functionality of post-translational regulation in S. cerevisiae central metabolism.Discovery-driven mass spectrometry phosphoproteomics identified 35 differentially phosphorylated enzymes of yeast central metabolism.Phosphoenzymes are predominant in upper glycolysis, around the pyruvate node and in carbohydrate storage pathways.A targeted phosphoproteomics method was developed to quantify total, phospho and non-phosphoprotein directly from crude cell extracts.Correlation of phosphoprotein levels with metabolic fluxes across conditions provided functional evidence for five novel phosphoregulated enzymes.Functional follow-ups demonstrated the inhibitory role of phosphorylation in controlling metabolic fluxes realised by Gpd1, Pda1 and Pfk2.