Molecular Systems Biology | |
Regulation of yeast central metabolism by enzyme phosphorylation | |
Ana Paula Oliveira1  Christina Ludwig1  Paola Picotti1  Maria Kogadeeva1  Ruedi Aebersold1  | |
[1] Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland | |
关键词: metabolic flux; metabolism; phosphoproteome; post‐translational regulation; selected reaction monitoring; | |
DOI : 10.1038/msb.2012.55 | |
来源: Wiley | |
【 摘 要 】
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. A 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.Abstract
Synopsis
【 授权许可】
CC BY-NC-SA
Copyright © 2012 EMBO and Macmillan Publishers Limited
Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.
【 预 览 】
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