Figure 6.

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.