Aberrant Intracellular pH Regulation Limiting Glyceraldehyde-3-Phosphate Dehydrogenase Activity in the Glucose-Sensitive Yeast tps1 Δ Mutant

Abstract

Whereas the yeast Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH₄Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate (Fru1,6bisP) hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant.IMPORTANCE Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast tps1Δ mutant, which undergoes apoptosis in the presence of just a few millimolar glucose. Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate (Fru1,6bisP) in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.

Keywords: Saccharomyces cerevisiae; TPS1; glucose metabolism; glyceraldehyde-3-phosphate dehydrogenase; glycolysis; intracellular pH; trehalose-6-phosphate synthase.

FIG 1

Single TDH isoform deletions increase the glucose sensitivity of the tps1Δ strain but do not affect growth on glucose of the wild-type strain. Wild-type and tps1Δ cells and their respective TDH single-deletion derivatives were spotted on YP agar in serial 5-fold dilutions after pregrowth on 3% glycerol. Cells were spotted onto plates containing 220 mM ethanol, 325 mM glycerol, or 100 mM galactose (a) or increasing concentrations of glucose: 0.5, 1, 2.5, 5 or 10 mM (b). Pictures were taken after 3 days.

FIG 2

The deletion of TDH3 enhances Fru1,6bisP accumulation in the tps1Δ strain after the addition of glucose but not after addition of galactose. Fru1,6bisP accumulation was measured after addition of 100 mM glucose (closed symbols) or galactose (open symbols) to cells of the tps1Δ strain (circles) or tps1Δ tdh3Δ strain (triangles). Cells were pregrown on complete synthetic medium with 3% glycerol as carbon source.

FIG 3

The overexpression of TDH2 or TDH3 does not rescue the glucose growth defect of the tps1Δ strain. Plasmid-transformed cells of wild-type and tps1Δ strains were spotted in serial 5-fold dilutions on YP agar supplemented with 325 mM glycerol, 100 mM galactose, or 100 mM glucose (a) or different concentrations of glucose: 0.5, 1, 2.5, 5 or 10 mM (b). TDH2 and TDH3 were overexpressed from the p426 multicopy plasmid behind the strong constitutive TEF1 promoter. Strains containing the p426 plasmid without an insert served as controls. Pictures were taken after 3 days.

FIG 4

The in situ GAPDH activity is strongly compromised at low pH and not affected by Tre6P or Fru1,6bisP. GAPDH activity was measured in permeabilized spheroplasts of wild-type cells studied over a wide pH range (highlighted: physiological pH range from 6 to 7.5) (a) and wild-type compared to tps1Δ cells (b). GAPDH activity was measured in permeabilized wild-type cells at pH 6.8 in the absence or presence of increasing concentrations of Tre6P (2, 4, or 6 mM) (c) and Fru1,6bisP (2.5, 5, or 10 mM) (d). Statistical analysis was performed by one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test. No significant difference was observed between the samples with and without Tre6P or Fru1,6bisP. ns, not significant, P > 0.05.

FIG 5

Intracellular acidification correlates with Fru1,6bisP accumulation. (a) Intracellular pH (full lines without symbols) and Glu6P levels (full lines with circles) after addition of 100 mM glucose in wild-type (blue) and tps1Δ (red) strains. (b) Intracellular pH profile of wild-type (blue), tps1Δ (red), tps1Δ hxk2Δ (yellow), and tps1Δ snf1Δ (green) strains after addition of 100 mM glucose. Standard deviations were calculated from at least 9 technical repeats per strain. (c) Fru1,6bisP levels after addition of 100 mM glucose to wild-type (blue), tps1Δ (red), tps1Δ hxk2Δ (yellow), and tps1Δ snf1Δ (green) strains. Cells were always pregrown on complete synthetic medium supplemented with 2% galactose.

FIG 6

The prevention of intracellular acidification reduces Glu6P and Fru1,6bisP hyperaccumulation and partially counteracts metabolic deregulation in tps1Δ cells. Wild-type (blue) and tps1Δ (red) galactose-grown cells were resuspended in 200 mM Tris-HCl, pH 9. At time point zero, cells were provided with 100 mM glucose (full lines) or with 100 mM glucose and 200 mM NH₄Cl (dotted lines). Graphs represent the time course profiles of intracellular pH (a), extracellular pH (b), Glu6P level (c), Fru1,6bisP level (d), ATP level (e), and normalized level of 3PG (f).

FIG 7

Reduced glucose transport delays but does not prevent deregulation of glycolytic flux in tps1Δ cells. Relative uptake rate of 100 mM glucose in wild-type and tps1Δ cells in 200 mM Tris-HCl, pH 9, in the absence (black bars) or presence (gray bars) of 200 mM NH₄Cl (a) and tps1Δ (black bar) and tps1Δ hxt6,7,2,4,5Δ (gray bar) cells in complete synthetic medium (b). Metabolite profiles for Glu6P (c) and Fru1,6bisP (d) after addition of 100 mM glucose to galactose-grown tps1Δ (closed symbols) or tps1Δ hxt7,6,2,4,5Δ (open symbols) cells. Statistical analysis was performed by two-way ANOVA with Bonferroni’s correction (a) and an unpaired Student's t test (b). ***, P < 0.001.