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. 2017 Jan 10;18(2):307-313.
doi: 10.1016/j.celrep.2016.12.049.

Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function

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Free PMC article

Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function

Xiao Guo et al. Cell Rep. .
Free PMC article

Abstract

Proper maintenance of mitochondrial activity is essential for metabolic homeostasis. Widespread phosphorylation of mitochondrial proteins may be an important element of this process; yet, little is known about which enzymes control mitochondrial phosphorylation or which phosphosites have functional impact. We investigate these issues by disrupting Ptc7p, a conserved but largely uncharacterized mitochondrial matrix PP2C-type phosphatase. Loss of Ptc7p causes respiratory growth defects concomitant with elevated phosphorylation of select matrix proteins. Among these, Δptc7 yeast exhibit an increase in phosphorylation of Cit1p, the canonical citrate synthase of the tricarboxylic acid (TCA) cycle, that diminishes its activity. We find that phosphorylation of S462 can eliminate Cit1p enzymatic activity likely by disrupting its proper dimerization, and that Ptc7p-driven dephosphorylation rescues Cit1p activity. Collectively, our work connects Ptc7p to an essential TCA cycle function and to additional phosphorylation events that may affect mitochondrial activity inadvertently or in a regulatory manner.

Keywords: Pptc7; Ptc7; citrate synthase; mitochondria; mitochondrial phosphorylation; phosphatase; phosphoproteomics.

Figures

Figure 1
Figure 1. Ptc7p phosphatase activity supports respiratory function
(A) Summary table of mitochondrial phosphatases, analyzed for protein phosphatase domains, matrix localization, non-association with a protein complex (e.g. PDH), and presence of a yeast ortholog. PPTC7 (highlighted) is the only phosphatase to meet all four criteria. (B) In vitro phosphatase activity assay of Ptc7p against pNPP with divalent cations Mn2+ and Mg2+ (mean ± SD, n=3). (C) In vitro pNPP phosphatase activity assay of WT Ptc7p and two mutants predicted to disrupt metal binding (mean ± SD, n=3). Coomassie staining (below) demonstrates comparable protein concentration and purity. (D) Maximum growth rate of WT and Δptc7 yeast in Ura media containing 3% glycerol (G) (mean ± SD, n=4). Rescue strains express a plasmid containing ptc7 (WT: wild type ptc7; D109A: catalytically inactive mutant of ptc7). (E) OCR of same cultures as in (D) grown in Ura media containing 2% dextrose (D) (mean ± SD, n=3). * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; N.S., not significant. See also Figure S1.
Figure 2
Figure 2. Quantitative phosphoproteomics identifies potential Ptc7p substrates
(A) Experimental workflow of phosphoproteomics. Peptides from three strains (WT, Δptc7, and Δptc7 + ptc7) were tagged with 8-plex TMT for isobaric quantification. (B) Fold changes in mitochondrial phosphoisoform abundances (log2ptc7/WT), normalized to total protein abundance, n=3) versus significance (−log10(p-value)). Grey area indicates significance threshold (log2ptc7/WT) > 1 and p-value < 0.05). Five highlighted phosphoisoforms are prioritized candidate Ptc7p substrates. Inset table summarizes quantified total and mitochondrial proteins or phosphoisoforms; up arrow indicates significantly increased changes; down arrow indicates significantly decreased changes. (C) Heat map of 19 mitochondrial phosphoisoforms whose abundances were significantly increased in Δptc7, and were restored to WT level or below in the rescue strain (Δptc7 + ptc7). * denotes mitochondrial matrix phosphosites conserved in higher eukaryotes. See also Figure S2, Table S1, and Table S2.
Figure 3
Figure 3. Phosphorylation of Cit1p at S462 disrupts enzyme function
(A) Serial dilutions of WT and Δcit1 yeast expressing various plasmids grown on glucose- or acetate-containing Ura plates. (B) Citrate synthase activity of Δcit1 lysate expressing EV, WT, S462A, or S462E Cit1p (mean ± SD, n=3). Statistics are relative to WT activity (lane #2). Inset shows immunoblot against FLAG (Cit1p-FLAG), or actin (loading control). (C) Kinetic curve of recombinant WT, S462A, or S462E Cit1p. Citrate synthase activity (μM/sec) is plotted versus concentration of OAA (μM). Inset shows comparable loading (Coomassie staining). Table shows calculated Vmax and Km for each Cit1p mutant. (D) Citrate synthase activity of lysate from WT or Δptc7 (mean ± SD, n=4). Rescue strains express a plasmid containing ptc7 (WT: wild type ptc7; D109A: catalytically inactive mutant of ptc7). (E) Citrate synthase activity of recombinant WT or phospho-S462 (pSer462) Cit1p treated with WT or D109A Ptc7p (mean ± SD, n=3). (F) Coomassie staining of a PhosTag gel loaded with samples in (E). * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; N.S., not significant. See also Figure S3.
Figure 4
Figure 4. Phosphorylation of Cit1p occurs at the dimer interface
(A–D) Structural interaction of residue 462 with L89 in the opposite chain with residue 462 modeled as (A) S (WT), (B) A, (C) pS (phospho-serine), and (D) E (based on structure PDB code 3ENJ). (E) Energy of computed models of A, pS, and E mutants at position 462, compared to WT, with analysis of pig and chicken structures and a yeast homology model. (F) Coomassie staining of native-PAGE loaded with recombinant WT (2x biological replicates), S462A, and S462E Cit1p. (G) FLAG immunoblot of native- and SDS-PAGE resolved lysates of Δcit1 expressing EV, or c-terminal FLAG-tagged WT, S462A, or S462E Cit1p. See also Figure S4.

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