title: Regulation of yeast central metabolism by enzyme phosphorylation.
abstract: As 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.
authors.affiliation: Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Oliveira
authors.forename: Christina
authors.initials: C
authors.lastname: Ludwig
authors.forename: Paola
authors.initials: P
authors.lastname: Picotti
authors.forename: Maria
authors.initials: M
authors.lastname: Kogadeeva
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.forename: Uwe
authors.initials: U
authors.lastname: Sauer
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D055162:Biocatalysis; D002244:Carbon / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D020543:Proteome / Q000378:metabolism; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D002244:Carbon