Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase

J Biol Chem. 2017 Jul 14;292(28):11751-11759. doi: 10.1074/jbc.M117.787341. Epub 2017 May 24.

Abstract

The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole-S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins.

Keywords: Gpd1p; Ptc5p; Ptc6p; Ptc7p; Saccharomyces cerevisiae; mitochondria; phosphoproteomics; phosphorylation; protein phosphatase 2C (PP2C); pyruvate dehydrogenase complex (PDC).

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Biochemistry / methods
  • Conserved Sequence
  • Databases, Protein
  • Enzyme Activation
  • Gene Knockout Techniques
  • Immunoprecipitation
  • Phosphoprotein Phosphatases / genetics
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation
  • Phylogeny
  • Protein Processing, Post-Translational*
  • Proteomics / methods
  • Pyruvate Dehydrogenase Complex / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Pyruvate Dehydrogenase Complex
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • PTC6 protein, S cerevisiae
  • Phosphoprotein Phosphatases