An experimental study of the regulation of glycolytic oscillations in yeast

Abstract

We have studied oscillating glycolysis in the strain BY4743 and isogenic strains with deletions of genes encoding enzymes in glycolysis, mitochondrial electron transport and ATP synthesis. We found that deletion of the gene encoding the hexokinase 1 isoform does not affect the oscillations while deletion of the gene encoding the hexokinase 2 isoform results in oscillations with smaller amplitude. The latter is associated with an almost 50% decrease in hexokinase activity. Deletions in the genes encoding the a-and b-subunits of phosphofructokinase abolish the oscillations entirely. This loss in oscillatory activity is associated with a fourfold decrease in phosphofructokinase activity. Deletions of genes encoding subunits of the F 1 F 0 ATPase also inhibit the oscillations in accordance with earlier studies using for example inhibitors. Finally, we identified an apparently new control point involving the mitochondrial cytochrome c oxidase. The latter is difficult to explain as oscillatory activity entails 100% inhibition of this enzyme. The mitochondria of this strain seem to have normal F 1 F 0 ATPase activity. Overall these results support earlier experimental and model studies suggesting that in addition to processes within glycolysis also processes outside this pathway contribute to the control of the oscillatory behaviour.

The dynamics of intracellular water constrains glycolytic oscillations in Saccharomyces cerevisiae

“…However, it is worth noting that when grown in a different laboratory, this strain shows NADH oscillations with a reduced amplitude 43 . When grown in our laboratory, this strain was previously shown to have a much-reduced glycolytic flux 44 , which could explain why no oscillations were observed. For the other 24 strains, it was not possible to establish a correlation between the glycolytic flux and the amplitude or frequency of the oscillations.…”

“…S13 ). This fact, along with the observations that i) most mutations in proteins directly associated with glycolysis do not inhibit oscillations 43 , 44 and ii) several mutations in proteins that are usually not considered in the context of glycolysis do inhibit the oscillations, makes a mechanism based on local mass action kinetics as the sole explanation very unlikely. Instead, these observations suggest that glycolysis, at least in part, is controlled by a cell-wide property (i.e., the physical state of water), which through the A-I mechanism can transfer a response to the relevant glycolytic enzymes.…”

“…The yeast strains used in this study are listed in Table S2 . The strains, all obtained from EUROSCARF (Frankfurtam Main, Germany), were grown as described previously 44 . Before use, the cells were washed and resuspended in 100 mM potassium phosphate to a cell density of 10% (w/v) and starved for 3 h on a rotary shaker at 30 °C.…”

Yeast Phosphofructokinase-1 Subunit Pfk2p Is Necessary for pH Homeostasis and Glucose-dependent Vacuolar ATPase Reassembly

“…The concentration of phosphofructokinase-1 reaction product is 10 -15-fold lower in the pfk2⌬ mutant compared with wild-type cells; it is only 2-fold lower in pfk1⌬ (40). In addition, the NADH spike triggered by readdition of glucose is reduced or absent in pfk2⌬, but not in pfk1⌬ (55,56).…”

Is a constant low-entropy process at the root of glycolytic oscillations?

“…The yeast strain used in this study was the S. cerevisiae BY4743 strain, which was obtained from EUROSCARF (Frankfurt am Main, Germany). Cells were grown essentially as described previously [28,29], i.e., under semi-aerobic conditions at 30°C, on a rotary shaker, 180 rpm, in a medium containing 10 g l −1 glucose, 6.7 g l −1 yeast nitrogen base (Bacto) and 100 mM potassium phthalate (Sigma-Aldrich, Steinhem, Germany) at pH 5.0. The medium was further supplemented with 60 mg l −1 histidine, 60 mg l −1 methionine, 80 mg l −1 leucine, 80 mg l −1 lysine and 80 mg ml −1 uracil.…”