A haploproficient interaction of the transaldolase paralogue NQM1 with the transcription factor VHR1 affects stationary phase survival and oxidative stress resistance

Abstract

Background: Studying the survival of yeast in stationary phase, known as chronological lifespan, led to the identification of molecular ageing factors conserved from yeast to higher organisms. To identify functional interactions among yeast chronological ageing genes, we conducted a haploproficiency screen on the basis of previously identified long-living mutants. For this, we created a library of heterozygous Saccharomyces cerevisiae double deletion strains and aged them in a competitive manner.

Results: Stationary phase survival was prolonged in a double heterozygous mutant of the metabolic enzyme non-quiescent mutant 1 (NQM1), a paralogue to the pentose phosphate pathway enzyme transaldolase (TAL1), and the transcription factor vitamin H response transcription factor 1 (VHR1). We find that cells deleted for the two genes possess increased clonogenicity at late stages of stationary phase survival, but find no indication that the mutations delay initial mortality upon reaching stationary phase, canonically defined as an extension of chronological lifespan. We show that both genes influence the concentration of metabolites of glycolysis and the pentose phosphate pathway, central metabolic players in the ageing process, and affect osmolality of growth media in stationary phase cultures. Moreover, NQM1 is glucose repressed and induced in a VHR1 dependent manner upon caloric restriction, on non-fermentable carbon sources, as well as under osmotic and oxidative stress. Finally, deletion of NQM1 is shown to confer resistance to oxidizing substances.

Conclusions: The transaldolase paralogue NQM1 and the transcription factor VHR1 interact haploproficiently and affect yeast stationary phase survival. The glucose repressed NQM1 gene is induced under various stress conditions, affects stress resistance and this process is dependent on VHR1. While NQM1 appears not to function in the pentose phosphate pathway, the interplay of NQM1 with VHR1 influences the yeast metabolic homeostasis and stress tolerance during stationary phase, processes associated with yeast ageing.

Figure 1

Haploproficiency of a VHR1/NQM1 double heterozygous mutant in late stationary phase. A) Generation of a heterozygous haplotype library. The kanMX4 marker present in the systematic knock-out strains was replaced by the LEU2 in 82 MAT a strains, and these were cross-mated in 96-well plates with the corresponding MAT α collection. After 2 days mating on YPD, double mutants were selected on synthetic media lacking leucine and supplemented with G418. B) The double heterozygous mutant library was combined with the wild type strain BY4743 in a 1:1 ratio and grown in independent duplicates at 30°C for 32 days in synthetic complete media (SC). Viability was monitored every 2nd day by serial dilution plating C) Viability of cells during stationary phase in the double mutant library (green line), wild type within a competitive pool (grey line) or the mutants within the competitive pool (red line). The wild type lost viability in competitive pools at day 26, the double mutants at day 32. The arrow marks time point of clonal selection. These trends in linear scaling are illustrated in Additional file 3, Figure S7, the trends of separate pools in Additional file 4: Figure S8. D) Identification of surviving haplotypes. 78% of genotypes were unique, while the remaining cells were double-heterozygous for Δvhr1 and Δnqm1 E) Gene Ontology (GO) Term analyses of surviving double heterozygous yeast mutants on the basis of their single gene deletion.

Figure 2

Chronological ageing of haploid Δvhr1 , Δnqm1 and Δvhr1/Δnqm1 cells. Survival rates were determined in quadruplicate stationary cultures by spotting serial dilutions on synthetic complete media. Error bars +/− SD. Right panel: Number of colony forming cells on day 14. The values represent the amount of cells that are able to form colonies (clonogenicity) on fresh agar media. Error bars +/− SEM. Student’s t-test significance values p < 0.05 = *, p < 0.01 = **.

Figure 3

Metabolic profiles of Δvhr1, Δnqm1, Δvhr1/Δnqm1 deletion strains. The sugar phosphate metabolic profile of haploid Δvhr1, Δnqm1, Δvhr1/Δnqm1 deletion strains (left panel), and the corresponding diploid heterozygous mutants (right panel). Metabolite concentrations were normalized to wild type and color-coded. n = 3, G6P/F6P = glucose 6-phosphate/fructose 6-phosphate, F1,6BP = fructose 1,6-bisphosphate, G3P = glyceraldehyde 3-phosphate, DHAP = dihydroxyacetone phosphate, 1,3BPG = 1,3-bisphospoglycerate, 2PG/3PG = 2-phosphoglycerate/3-phosphoglycerate, PEP = phosphoenolpyruvate, Pyr = pyruvate, 6PG = 6-phosphogluconate, RI5P/X5P = ribulose 5-phosphate/xylulose-5-phosphate, R5P = ribose-5-phosphate, S7P = sedoheptulose 7-phosphate, E4P = erythrose 4-phosphate. The metabolites S7P, E4P, 1,3-BPG have not been quantified, and G6P/F6P, 2PG/3PG, RI5P/X5P have been quantified as sum.

Figure 4

NQM1 mRNA levels are induced by caloric restriction and on non-fermentable carbon sources. A) Induction of NQM1 on non-fermentable carbon sources and calorie restriction in wild type, Δnqm1 and Δtal1 deletion mutants. Expression of NQM1 mRNA levels is induced on non-fermentable carbon sources (ethanol, glycerol) and on galactose, indicating that NQM1 is subject to glucose repression. TAL1 level instead are increased on ethanol only. B) Induction of NQM1 on non-fermentable carbon sources and calorie restriction in wild type and Δvhr1 mutant. NQM1 induction is not suppressed by the deletion of Δvhr1, but strongly reduced. Samples for qRT-PCR have been generated in liquid media containing the indicated carbon source. Cells were harvested at mid-log phase (OD₆₀₀ = 0.8–1.0). n = 3, Error bars, ± SD.

Figure 5

NQM1 mRNA expression is dependent on osmolarity. A) Osmolality was determined in triplicates by freezing-point depression. The grey bar at 233.75mOsmol/kg refers to the synthetic complete media osmolality prior to the experiment. All yeast strains depict a high osmolality at the start of the experiment which decreases during the chronological ageing below the control value (blank media) and remains stable until the end of the aging. Wild type (WT) and Δnqm1 cells show a similar trend, whereas the decrease is stronger in the double mutant and diminished in Δvhr1. B) + C) mRNA expression of NQM1 during the osmotic stress response in wild type and Δvhr1 mutant yeast. Time courses of NQM1 were generated by quantitative real-time-polymerase chain reaction (qRT-PCR) in wild type and Δvhr1 yeast treated with NaCl or sorbitol for 0, 0.5, 1, and 2 hours. Upon NaCl treatment, NQM1 was severely induced in both wild type and Δvhr1 mutant yeast (Figure 5B). Upon sorbitol treatment, the induction pattern of NQM1 was similar in wild type and Δvhr1 mutant yeast, albeit the magnitude in the Δvhr1 mutant was reduced (Figure 5C). All experiments were performed in triplicates. Samples for qRT-PCR have been generated in liquid media containing glucose (2%) as sole carbon source. NaCl, sorbitol was added to induce osmotic shock upon cells reached mid-log phase (OD₆₀₀ = 0.8-1.0). Error bars, ± SD.

Figure 6

NQM1 Expression and growth phenotypes of NQM1/VHR1 mutants under oxidative stress. A) NQM1 mRNA expression in response to treatment with the oxidant menadione. Wild type and Δvhr1 mutant yeast induced NQM1 expression over time. B) H₂O₂ induces NQM1 in both wild type and Δvhr1 mutant; qRT-PCR was conducted on cells grown in liquid media containing glucose (2%) as sole carbon source. Oxidants were added to induce oxidative stress at mid-log growth phase (OD₆₀₀ = 0.8–1.0). C) Oxidant tolerance spot tests on media containing different concentrations of the oxidant menadione, H₂O₂ and diamide. Δvhr1 and Δvhr1/Δnqm1 mutants exhibit increased tolerance to menadione, Δnqm1 to H₂O₂, whereas the double mutants Δvhr1/Δtal1, Δnqm1/Δtal1 and the triple mutant Δvhr1/Δnqm1/Δtal1 were sensitive to this oxidant. Δvhr1 and Δvhr1/Δnqm1 mutants were sensitive to diamide, while all Δtal1 mutants were resistant to this oxidant.