A Yeast Model for Studying Pyruvate Kinase
(A) PYK expression levels. Quantification of a Pyk1p (upper panel)- or Pyk2p (lower panel)-specific peptide in Δpyk1Δpyk2 yeast expressing TEF_pr-PYK1, TEF_pr-PYK2, CYC_pr-PYK1, or CYC_pr-PYK2. Normalized peak intensities of >3 MRM transitions are presented as the relative expression level (%) compared to the concentrations in the wild-type strain BY4741. For both isoforms, levels from the CYC1 promoter (CYC_pr) were around 20% of the expression levels from the TEF1 (TEF_pr) promoter. Error bars, ±SD from normalization to three reference peptides.
(B) Pyruvate kinase activity of BY4741 (WT) and TEF_pr-PYK1, TEF_pr-PYK2, CYC_pr-PYK1, or CYC_pr-PYK2 yeast. Error bars, ±SD.
(C) Overview on the yeast models with varying PYK activity.

Activation of Respiration Does Not Increase ROS Levels, but It Does Increase Oxidant Resistance
(A) Superoxide levels are not increased upon the activation of oxidative metabolism. ROS levels were determined by assaying DHE fluorescence in exponentially growing yeast in SCGluc and SCGal media (upper panel) or in yeast with varying PYK activity (lower panel). Error bars, ±SD. See also Figure 6B.
(B) Oxidant resistances increase with low PYK activity. BY4741 and yeast strains with varying PYK activity were spotted in triplicates on media containing diamide, cumene hydroperoxide (CHP), tert-butyl hydroperoxide (TBH), hydrogen peroxide (H₂O₂), menadione, or juglone. Pictures taken from spot tests were analyzed with CellProfiler, values indicate the ratio of spot intensity of the indicated strain to the wild-type control. Error bars, ±SD. See also Figure S1.
(C) Low PYK activity increases growth capacity in the presence of oxidants. Yeast models with different PYK activity were grown with our without oxidants and analyzed spectrophotometrically. Values indicate the doubling time relative to the nontreated control culture. Error bars, ±SD.
(D) Increased survival of yeast with low PYK activity in oxidant media. TEF_pr-PYK1 and CYC_pr-PYK1 yeast was incubated in YPD and in YPD supplemented with 3 mM diamide for 24 hr, plated, and formed colonies counted. Error bars, ±SD.

The PYK-PEP-TPI Feedback Loop Stabilizes ROS Levels by Activating the Pentose Phosphate Pathway
(A) Overview on the PPP and glycolysis.
(B) Cells with low PYK activity have increased concentrations of PPP intermediates. Sugar phosphates were extracted from exponential cultures and quantified by LC-MRM. All measured PPP intermediates—g6p (glucose 6-phosphate), f6p (fructose 6-phosphate), 6pg (6-phosphogluconate), r5p (ribose 5-phosphate), rib5p (ribulose 5-phosphate), x5p (xylulose 5-phosphate), s7p (sedoheptulose 7-phosphate), and e4p (erythrose 4-phosphate)—as well as TPI substrates dhap (dihydroxyacetone phosphate) and gly3p (glyceraldehyde 3-phosphate) were increased in yeast with low PYK activity grown in glucose (upper panel) and galactose media (lower panel). Reproducibility of sugar phosphate quantification is demonstrated by linear regression (R² 0.989) (lower panel left). See also Table S1.

Synchronization of Redox and Energy Metabolism by Pyruvate Kinase
Low PYK activity increases respiration. At the same time, the PYK substrate PEP accumulates. This stimulates the PPP by feedback inhibition of TPI, which in turn prevents ROS accumulation during respiration.

PYK Activity Regulates Respiration
(A) Growth deficits caused by low PYK activity are rescued on galactose. Strains were grown overnight, diluted to an OD₆₀₀ of 3.0, and spotted as serial dilutions (1:1, 1:5, 1:25, 1:125, and 1:625) on YPD (glucose) and YPGal (galactose).
(B) Galactose rescue of low PYK activity requires a functional respiratory chain. Yeast strains with deficient mitochondrial DNA (ρ0) were grown alongside with controls (ρ+) on YP media with the indicated carbon sources. Galactose did not compensate for the growth deficits caused by low PYK activity in ρ0 yeast.
(C) Regulation of oxidative metabolism's mRNA expression by PYK and galactose. COX1, COX2, COX3, and CIT1, implicated in oxidative energy metabolism, were analyzed by qRT-PCR. Expression values are given as fold change compared to TEF_pr-PYK1 yeast, relative to glucose grown yeast (upper panel). Changes in mRNA expression of the same transcripts in yeast shifted to galactose for 1 hr, or permanently, activating oxidative metabolism (lower panel). Both conditions caused a similar mRNA expression fingerprint. Error bars, ±SD.
(D) Oxygen consumption increases with low PYK activity. Oxygen consumption in logarithmically growing wild-type yeast and PYK mutants was determined on glucose (upper panel) and galactose media (lower panel), and cell numbers were determined with an electric field multichannel cell counting system (CASY). Overall oxygen consumption was three times increased in galactose, low PYK activity increased oxygen uptake under both conditions. Error bars, ±SD.

Phosphoenolpyruvate Accumulates in Cells with Low PYK Activity and Inhibits Triosephosphate Isomerase
(A) HILIC-MRM quantification of PEP (left panel) Standard addition of PEP to whole-cell methanol/water yeast extracts; and quantification of PEP separated by HILIC using multiple reaction monitoring (MRM). Quantification is demonstrated by linear regression, R² vales of >0.97 were obtained. (Right panel) HILIC-MRM quantification of PEP in TEF_pr-PYK1, CYC_pr-PYK1, and CYC_pr-PYK2 yeast; PEP is strongly accumulated in yeast with low PYK activity. Error bars, ±SD, n = 3.
(B) PEP inactivates yeast and mammalian TPI. Michaelis-Menten kinetics were determined for yeast TPI (left panel), human TPI (middle panel), and rabbit muscle TPI (right panel). To determine v_max and K_m, the TPI substrate gly3p was added in incremental doses (left y and lower x axis, blue; y values are normalized to total cellular protein [left and middle panel] or to the purified protein [right panel]). For all TPI isozymes, v_max is highlighted with a green dot in the saturation curve; K_m values are given in μM (right y and upper x axis, black). Inhibition of TPI activity demonstrated by addition of PEP in incremental concentrations; IC₅₀ values and the inhibitory constant K_i are given in μM, the Kcat in Mol^∗s⁻¹.
(C) Inactivation of pathogenic TPI alleles by PEP. TPI activity was assayed in transgenic yeast expressing human TPI (WT) or indicated pathogenic TPI alleles without or in the presence of 900 μmol PEP. Error bars, ±SD. Values within bars indicate the absolute enzyme activity in μmol/(min^∗μg protein).
(D) PYK activity does not change oxidant resistance in yeast expressing TPI_Ile170Val. The wild-type control, transgenic yeast expressing either PYK1 or PYK2, and either human TPI or human TPI_Ile170Val were spotted as serial dilutions onto SC media without or with diamide (left panel). Spot growth on different concentrations was analyzed with CellProfiler and normalized to the strain with highest PYK activity (right panel). Low PYK activity increased diamide tolerance if expressed in combination with human TPI, but there were no differences in TPI_Ile170Val-expressing yeast.

The PYK-PEP-TPI Feedback Loop Protects Cells from ROS-Induced Damage during Respiration
(A) Low PYK activity in Δzwf1 yeast does not protect against oxidants. The first enzyme involved in the irreversible NADPH producing oxidative PPP branch (ZWF1) was deleted, and PYK strains were tested for diamide resistance (left panels). Densitometric processing of (A), including more oxidant concentrations (right panels). Deletion of ZWF1 prevented the increase in redox tolerance in yeast with low PYK activity.
(B) ROS levels increase in respiring cells when the PPP is deficient. ROS levels in ZWF1 and Δzwf1 yeast with varying PYK activity were determined with DHE, which preliminarily detects superoxide (left panel), and DCFDA, which detects H₂O₂ (right panel). Low PYK activity did not increase DHE and DCFDA oxidation in wild-type, but in Δzwf1 yeast. Error bars, ±SD.
(C) Low PYK activity increases protein carbonylation in Δzwf1 yeast. Protein extracts (7.5 μg) were analyzed by oxyblotting (left panel) and extracts controlled with Coomassie staining (right panel). ZWF1 and Δzwf1 are juxtaposed images from the same blot/gel. Low PYK activity strongly increased carbonylation in Δzwf1 yeast.
(D) Mitochondrial damage in Δzwf1 cells with low PYK activity. PYK models expressing Aco1-eGFP were analyzed for mitochondria morphology. Strains wild-type for ZWF1 contain typical tubular mitochondria. In combination with low PYK activity, Δzwf1 caused mitochondrial fragmentation gradually increasing with low PYK activity; Aco1p indicated 100% mitochondrial network fragmentation in Δzwf1 CYC_pr-PYK2 yeast.