A haploproficient interaction of the transaldolase paralogue NQM1 with the transcription factor VHR1 affects stationary phase survival and oxidative stress resistance
© Michel et al.; licensee BioMed Central. 2015
Received: 22 September 2014
Accepted: 21 January 2015
Published: 11 February 2015
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.
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.
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.
Eukaryotic cells possess a variety of different strategies to maintain homeostasis, and adjust metabolism upon changes in environment and to survive stress situations such as oxido-reductive stress, osmotic turbulences, exposure to toxins, temperature imbalances or fluctuating nutrient concentrations [1-5]. This cellular flexibility is a requirement for a successful ageing process. It has thus become common view that cellular stress response mechanisms are key players in gerontology. Transcriptional regulation is important in cellular adaptation to both stress and ageing, and many stress responsive genes and regulatory systems have been identified [6-9]. The transcriptional response varies between stress conditions, but many gene sets and control mechanisms overlap and play a role in multiple situations. Key proteins required to establish the transcriptional stress response include the transcription factors Yap1p, Msn2/4p, Hsf1p, Hog1p/Hot1p [10-12]. These transcriptional regulators allow the cell to react directly to the applied stress condition and induce effective defense mechanisms such as molecular chaperones and anti-oxidant proteins. However, transcriptional regulation does not explain the full spectra of cellular adaptations to changing environments and nutrition [13-15]. A presumably equally important, complementary strategy to react to environmental turbulences is to adjust cellular metabolism. A challenge for the cell in stress situations is to maintain the canonical function of metabolism, which is to continuously provide metabolites and cofactors such as ATP and NAD(P)H, required for the cell to survive and to continue proliferation upon the end of the stress situation. In particular, the ancient metabolic routes of glycolysis and the pentose phosphate pathway, amino acid and polyamine synthesis are at the centre of cellular metabolism, and have been shown to be involved in establishing fast and effective strategies in responding to situations like oxidative stress [4,5,16-18].
Genome duplication was one of the triggers that facilitated evolutionary adaptations to improve functionality of metabolism in stress situations. In budding yeast, a whole genome duplication event has been dated approximately 100 million years ago . This created the basis for the evolution of isozymes that obtained specific functions during the stress response [20,21]. A diversification of enzyme function in stress situations has been reported for isozymes of isocitrate dehydrogenases [22,23], alcohol dehydrogenases , sirtuins [25,26], nutrient transporters  and glycolytic enzymes. The latter includes the most downstream glycolytic enzyme pyruvate kinase (isozymes PYK1 and PYK2), where the low-activity orthologue PYK2 is induced under conditions of high respiratory activity and protects the cells from oxidative damage .
Here we present evidence that NQM1 , a paralogue of the pentose phosphate pathway (PPP) enzyme transaldolase TAL1, as well as its regulating transcription factor VHR1  are required during osmotic and oxidative stress, and affect survival of yeast cells upon long time starvation. We identified a heterozygous deletion of both genes through enrichment in a haploproficiency screen targeting genes previously implicated in chronological lifespan . In subsequent analysis we find that NQM1 is induced upon nutrient limiting-, osmotic- and oxidative stress conditions and that this induction is dependent on the presence of VHR1. Finally, we show that the two genes cooperate in achieving oxidative stress tolerance of wild type cells.
Yeast strains and cultivation
All yeast strains used in this study are derived from the parent strain S288C. Wild type reference strains are haploid BY4741 (MAT a) or diploid BY4743 (MAT a/ MAT α) and were derived from the international knock out collection  obtained from EUROSCARF (Frankfurt, Germany; a complete list of deletion mutants used in this study is given as Additional file 1: Table S1).
Yeast was cultivated as detailed previously  at 30°C either in liquid or on solid (agar) yeast-extract peptone dextrose complex media (YPD: 20 g/l peptone, 10 g/l yeast extract, 20 g/l agar, 2% glucose) or in synthetic complete media (SC: 6,8 g/l yeast nitrogen base with ammonium sulfate, 0.59 g/l complete supplement mixture (CSM-ADE-HIS-LEU-TRP-URA, MP Biomedicals), 10 mg/ml adenine, 20 mg/ml uracil, 20 mg/ml histidine, 60 mg/ml leucine, 40 mg/ml tryptophan, 20 g/l agar) or synthetic media lacking the indicated amino acids or nucleobases. As sole carbon source, glucose (2%, 0.5%, 0.05%), galactose (2%), ethanol (3% / 0.1% glucose) or glycerol (3% / 0.1% glucose) was used.
Generation of a MAT a / MAT α heterozygous double gene deletion collection
Plasmids used in the generation of mutant strains
Cloning of LEU2
Cloning of natMX4
Cloning of hphMX4
The 82 marker-exchanged knock-out strains were then replicated into 9 × 96-well plates containing 50 μl YPD, whereas 85 MAT α knock-out strains were replicated into one 96-well plate containing 50 μl YPD and incubated at 30°C for 24 hours. 10 MAT α strains were combined each and then transferred into replicates of the MAT a plates. 9 plates with the MAT a / MAT α mixture were replicated on solid YPD media and incubated at 30°C for 2 days allowing mating to occur. Finally, diploids were selected on SC-Leu + G418 (800 μg/ml) and subsequently pooled.
Haploproficient screening and chronological ageing experiments
Competitive haploproficiency screens were conducted by inoculating 100 ml synthetic complete media (SC) + 2% glucose with overnight cultures of the wild type strain BY4743 and the library of heterozygous double mutants at an OD600 = 0.3 in a 1:1 ratio. Cultures were incubated at 30°C for 32 days on a shaking platform. Viability was determined every 2nd to 4th day by plating serial dilutions in triplicates onto SC media, allowing all cells to grow and onto SC-Leu, selecting for double mutants only. Colonies were counted and expressed as CFU/ml.
Non-competitive stationary phase survival experiments were conducted in quadruplicates by setting an overnight culture of the haploid strains to an OD600 = 0.3 in 100 ml SC media. Yeast was grown until stationary phase (2 days) and viability was recorded for 14 days.
Isolation and sequencing of molecular barcodes from double heterozygous mutants
Primer sequences used in strain generation, verification, sequencing
5′ - 3′ sequence
kanMX4::LEU2 - fwd
Replace genomic kanMX4 with LEU2 (from pACT2)
kanMX4::LEU2 - rev
Replace genomic kanMX4 with LEU2 (from pACT2)
LEU2 – fwd
Downtag amplification and sequencing in double mutants
pFA6 – fwd
Downtag sequencing primer (kanMX4)
pFA6 – rev
Uptag sequencing primer (kanMX4)
Barcode forward primer
Barcode reverse primer
Replace genomic TAL1 with hphMX4 (from pAG32)
Replace genomic TAL1 with hphMX4 (from pAG32)
Replace genomic NQM1 with natMX4 (from pAG25)
Replace genomic NQM1 with natMX4 (from pAG25)
Yeast cultivation and extraction for metabolomics analysis
Sugar phosphate intermediates were extracted and quantified as described earlier : Yeast strains were grown in synthetic complete media and were collected during logarithmic growth at an OD600 of 1.5 ± 0.05 using a rapid cold methanol quenching procedure . Lysis of cells was achieved by FastPrep-24 cycles (MP Biomedicals, 3 × 20 s, 6.5 m/s) in organic extraction buffer (200 μl of 75:25 acetonitrile:water containing 0.2% formic acid). After a second extraction step with the same amount of water the supernatants of both steps were combined, dried (SpeedVac concentrator), resuspended in 100 μl water:acetonitrile (93:7) and then subjected to LC-MS/MS analysis. All solvents were used in HPLC grade or higher.
LC-MS/MS measurements of sugar phosphates
1.5 μl of the extracted samples were injected on a C8 column (Agilent Zorbax SB-C8 Rapid Resolution HD, 2.1×100 mm, 1.8 μm) for separation with a gradient that started with isocratic flow at acetonitrile:water (12:88) for 3.5 min, a subsequent ramp to 38:62 acetonitrile:water for 2.5 min, an 30 sec washing step at 42% acetonitrile, followed by re-equilibration for 30 sec. This totaled in a cycle time of 7.5 min. 750 mg/l octylammonium acetate was added to all buffers as ion pairing reagent . Analytes were quantified in MRM mode with an online coupled triple quadrupole mass spectrometer (Agilent 6460). Each metabolite was identified by similar retention time and fragmentation behavior and compared with externally measured commercially available standards. Optimal SRM transitions, ionization and fragmentation energies as well as ion source parameters can be found in . Masshunter software (Agilent) was used for peak integration, external calibration (with repetitively measured standard dilution curves), absolute quantification and initial data analysis. All further data analysis was performed with R (http://www.r-project.org/).
Determination of osmolality
Osmolality was determined by freezing point depression in a Knauer Osmometer (Knauer) from aliquots of 1 ml taken from the yeast cultures at the indicated time points throughout the chronological aging experiment. Yeast cultures were centrifuged twice at 16.000 × g for 2 min. 200 μl of the media were transferred to a vial and measured in triplicates.
Stress tests addressing oxidative stress tolerance (with menadione and H2O2), salt and osmotic stress (NaCl, sorbitol) in liquid cultures or on solid plates were induced by adding the stressors to the given final concentration to the growth media as described previously .
Overnight cultures were diluted to an OD600 = 0.15 and grown until mid-log phase (OD600 = 0.8 – 1.0) in SC + 2% glucose (for menadione, H2O2, NaCl, sorbitol treatment) and in SC with either 2% glucose, 2% galactose, 3% ethanol + 0.1% glucose or 3% glycerol + 0.1% glucose. In time course experiments (menadione, H2O2, NaCl, sorbitol treatment), samples were taken on the specified time points.
Primer sequences used in qRT-PCR
Total yeast RNA was isolated using the RNAeasy Mini Kit (Qiagen). Two μg total yeast RNA was transcribed into cDNA using a 12–18 oligo dT primer and Moloney Murine Leukemia virus (M-MuLV) reverse transcriptase (NEB) according to the manufacturer’s instructions. qRT-PCR was performed on a Prism 7900HT cycler (Applied Biosystems) with gene-specific primers (Table 3) and Maxima SYBR Green/Rox qPCR Master Mix (Thermo Scientific). The relative expression ratio of the target genes was normalized to the geometric mean of the three endogenous reference genes (UBA4, ATG27, TAF10) following the method of Pfaffl et al. .
A haploproficient interaction of the transaldolase NQM1 and the transcription factor VHR1 enriches cells at a late stage of stationary phase survival
In order to be able to select for diploids and to identify double heterozygous strains by means of barcode sequencing, we replaced the kanMX4 gene in all MAT a strains by the LEU2 marker (Figure 1A). After generation of the library containing up to 6970 bona fide dual heterozygous mutants, we inoculated this set of strains together with the diploid wild type BY4743. Viability of the population was followed for 32 days at 30°C by determining CFU/ml by spotting serial dilutions on synthetic complete (SC) and SC media lacking leucine (SC-Leu) to distinguish the double mutants from the wild type cells (Figure 1B). This measure was used as indicator whether the population contained haplotypes with increased stationary phase survival (Figure 1C, and Additional file 3: Figure S7 (linear scaling), Additional file 4: Figure S8, (for trends in separate pools)). We observed a strong decrease in viability during the first days of stationary phase growth; with day 3 (Figure 1C) the cells reached a plateau phase lasting until the 18th day. Then, cells started to lose viability rapidly and nearly linearly until the 30th day. At day 26, the wild type strain within the cultures had largely lost viability, but double mutants still produced viable colonies (Figure 1C). Thus, strains in the heterozygous library expressed prolonged stationary phase survival. From the surviving population, we randomly isolated 62 clones (31 from two parallel experiments). Via barcode sequencing , 39 haplotypes could be identified unambiguously which did lack two genes (Additional file 2: Table S2). 78% of the isolated strains possessed a private (unique) genotype (Figure 1D). These deleted genes were enriched for (overlapping) Gene ontology (GO) process terms protein metabolic process, meiosis, regulation of biological activity, cell wall organization, cellular components terms macromolecular complex, non-membrane-bounded organelle and GO function term catalytic activity (Figure 1E) when considering the deletant genes as background set (Additional file 1: Table S1). The remaining cells (22%) however corresponded to one sole enriched genotype. These strains were mainly isolated from one of the two experiments (Additional file 2: Table S2) and heterozygously deleted for the genes VHR1 and NQM1, encoding for a transcription factor and an alternative transaldolase.
VHR1/NQM1 gene knock-outs possess increased clonogenicity at a late stage of stationary phase survival
Altered concentration of glycolytic and PPP metabolites in Δvhr1 and Δnqm1 strains and in their double mutants
Profiles of the heterozygous mutants were different to those of the haploid strains, but also here the metabolic profile of the double mutant was more similar to that of the wild type strain. Heterozygous deletion of VHR1 or NQM1 increased the concentration of PPP intermediates 6PG, RI5P/X5P and R5P, while VHR1/Δvhr1 NQM1/Δnqm1 yeast instead displayed nearly wild type level concentrations of the PPP intermediates (Figure 3, right panel). This effect was also observed for metabolites of lower glycolysis, where the double mutant did not replicate the concentration changes of both single mutants. Hence, while both the NQM1 and VHR1 deletions affected the concentration of glycolytic and PPP intermediates, the double deletion reversed some of the effects, and yielded opposite concentration effects in other cases (Figure 3).
NQM1 is induced upon caloric restriction in a VHR1 dependent manner
The results instead implied that NQM1 could be a glucose repressed gene. We tested the effects of glucose caloric restriction on its expression levels. Upon lowering the media glucose concentration from 2% over 0.5% to 0.05% mRNA levels of NQM1 were induced up to 50-fold (Figure 4A, lower panels). In contrast, TAL1 mRNA levels were not affected by glucose restriction, and a Δtal1 did not influence NQM1 expression. Thus, gene expression of paralogues is regulated independently from each other, and NQM1 but not TAL1, is a glucose repressed gene.
As we had identified NQM1 in concert with the transcription factor VHR1 (Figure 1), we next addressed their genetic interplay upon the nutritional shift. We altered carbon sources and glucose concentration in the Δvhr1 mutant and monitored NQM1 levels in comparison to equally treated wild type cells. The absence of VHR1 did not prevent the NQM1 induction on non-fermentable carbon sources and upon glucose restriction. However, the expression levels of NQM1 reached only 1/4th of the level compared to wild type cells (Figure 4B). Hence, NQM1 is inducible on non - fermentable carbon sources and by glucose starvation. This function is independent to the canonical transaldolase TAL1, but requires the presence of the transcription factor VHR1 for achieving wild-type expression levels.
NQM1 is induced upon osmotic stress
However, we could confirm that osmotic stressors induce NQM1 expression. We treated cells with NaCl and sorbitol, and consistent with the previous findings , NQM1 mRNA level were dramatically induced up to 750-fold in the cells treated with NaCl and sorbitol (Figure 5B, C). The Δvhr1 deletion did not significantly affect the NQM1 gene expression on NaCl. On sorbitol however, a result analogous to glucose restriction was obtained: Deletion of VHR1 did not prevent the induction of NQM1, but in the absence of this transcription factor, NQM1 gene expression did not any longer reach wild type levels (Figure 5C).
NQM1 limits oxidant tolerance
Then, we tested whether cells deficient in VHR1, NQM1 or TAL1 would exhibit altered resistance to oxidants or reductants. Stress resistance was assayed by serial dilution spot testing for survival of a set of single, double- and triple deletion mutants comprising the genes VHR1, NQM1, TAL1 on the oxidants diamide, H2O2, menadione, t-butyl hydroperoxide, cumene hydroperoxide and the reductants N-acetyl-cysteine, DTT, reduced glutathione. These experiments were conducted on media containing glucose, galactose, ethanol or glycerol. On glucose media, we identified a different tolerance of NQM1 and VHR1 mutants to menadione, H2O2 and diamide (Figure 6C): Δvhr1 and Δvhr1/Δnqm1 cells were resistant to menadione, Δnqm1 to H2O2, whereas the Δvhr1/Δtal1 mutant, the double transaldolase knock-out Δnqm1/Δtal1 and in particular the triple knock-out strain Δvhr1/Δnqm1/Δtal1 were sensitive to H2O2 (Figure 6C). The response to the thiol-oxidizing substance diamide differed to peroxide stresses. Δvhr1 and Δvhr1/Δnqm1 yeast were sensitive to diamide, while all TAL1 mutants, Δtal1, Δnqm1/Δtal1 and Δvhr1/Δnqm1/Δtal1 mutants were resistant. Stress tests with the corresponding diploid, heterozygous strains did not detect difference to the wild type (Additional file 5: Figure S9). Taken together, NQM1 is induced by oxidative stress in a partially VHR1 dependent manner. NQM1 and VHR1 mutants exhibit a series of stress resistance and sensitivity phenotypes. This indicates that the interplay of the two genes is required during a native anti-oxidant stress response.
In this study we describe a competitive haploproficiency screening to identify genetic interactions that occur during yeast chronological ageing. We identified increased clonogenicity in late stationary phase of yeast mutants heterozygously deleted for NQM1 and VHR1, encoding for the yeast transaldolase paralogue (NQM1) and a transcription factor (VHR1). Since a competition experiment among ~ 7000 double heterozygous deletion mutants reflects a complex system, we are limited to speculate about the dynamics that occurred during the course of the 30 day-long lasting stationary experiment. Previously, several studies showed that multiple factors like apoptosis , acetic acid accumulation  can affect the survival of yeast upon chronological aging. The latter could play a role in the selection dynamics of heterozygous mutants. Acetic acid has been proposed to be a major regulator of chronological lifespan  and has been linked to nutrient  and Ras signaling . This advantage of the heterozygous double mutant was however also confirmed in individual, haploid mutants. Also in these experiments, the phenotype did express as higher clonogenicity of the double mutant culture at late time-points, right before the stationary cultures lost viability. The phenotype did however not express as decreased mortality upon yeast entering stationary phase. It is thus possible that the double mutant maintained longer viability in stationary phase by being more efficient in adaptive re-growth . We are thus not considering this phenotype as an increase in chronological lifespan in the canonical sense, but refer to it as increased survival at late stationary phase. Being a different phenotype to a chronological lifespan extension, this phenotype could however cover an alternative feature of cellular survival; the ability to persist nutrient limiting conditions is an essential feature in the natural environment, and can provide significant population advantages.
Recent studies suggested that NQM1 has either a direct role in gluconeogenic metabolism or in the supply of pentose phosphate precursors required for amino acid or nucleic acid biosynthesis under conditions under which regular synthesis from glucose 6-phosphate is difficult or insufficient . Supporting parts of this assumption, we observed the induction of NQM1 on different carbon sources, caloric restriction and several stress conditions. NQM1 expression dominated over that of TAL1 on non-fermentable carbon sources (ethanol/glycerol), in the presence of osmotic stressors, and under caloric restriction (low glucose) conditions (the latter indicating that NQM1 is a glucose-repressed gene). However, we would like to note that NQM1 expression levels did not respond to the deletion of TAL1, and thus not to changes in total transaldolase and PPP activity. In addition, we did not find canonical transaldolase substrates to accumulate in Δnqm1 cells, and the oxidant resistance profile of Δtal1 and Δnqm1 strains were substantially different. Taken together these results indicate that despite its in vitro activity resembling Tal1p, NQM1 could have yet unknown physiological substrates, instead of participating in the PPP.
We found NQM1 induced under various stress conditions. This finding is consistent with a previous study focusing on the cumene hydroperoxide response, that identified a specific role of NQM1 in the cellular response to the oxidizing molecules  and that NQM1 is induced in those conditions . The induction of NQM1 after release of glucose repression and in stress situations was in part dependent on the VHR1 transcription factor (Figures 4 and 5). The DNA binding motif of Vhr1p is well studied, and it has been shown that the transcription factor is able to recognize bZIP-like DNA motifs and Gcn4-like motifs . We could however not detect such binding sites for the NQM1 locus using the Yeastract database, implying that the role of VHR1 in regulating NQM1 is likely indirect. Thus, further studies are needed to clarify the mechanisms and interactions which allow VHR1 to influence the gene expression level of NQM1.
Yeast growth does change the growth media composition. The availability of nutrients, carbon or nitrogen sources will decline with time, while other metabolites (the exometabolome) accumulate [63-66]. Yeast preferentially metabolizes glucose through anaerobic metabolism resulting in the production of ethanol and carbon dioxide . Changes in the concentration of soluble nutrients do affect the osmotic potential of the media. We detected a general decline in the media osmolality during in stationary phase survival (Figure 5A). Indicating that these changes reflect metabolic activity, this change in osmolality differed between wild type cells and the VHR1 and VHR1/NQM1 double mutants (Figure 5A). The changes in osmolality, albeit they were only moderate and thus unlikely a cause of toxicity, could however be recognized by mitogen activated protein (MAP) kinase pathways , in yeast represented by the high osmolarity glycerol (Hog1/Hot1) pathway . NQM1 has previously been shown to be induced during osmotic shock and that this induction was dependent upon Hog1/Hot1 . Our data is consistent with this observation; we find NQM1 strongly induced upon adding osmotic stressors (Figure 5B, C). Of note, the osmotic response that is triggered by Hog1p has been associated with increased lifespan by enhancing glycerol production from glycolytic intermediates . The mechanisms how double heterozygosity of NQM1 and VHR1 influences late stationary phase survival could thus potentially dependent on the induction of NQM1 through osmotic signaling.
In summary, the data presented here shows that combined deficiency in NQM1 and VHR1 confers haploproficiency and extends clonogenicity at a late stage of yeast in stationary phase. We find that NQM1 is a glucose repressed and stress induced isoform of the pentose phosphate pathway transaldolase TAL1, but is independently regulated and appears to have a different biochemical function compared to its paralogue. We find NQM1 to be induced under conditions of caloric restriction, osmotic stress and in the presence of some oxidative stressors, and that full induction of NQM1 requires the transcription factor VHR1. The detailed molecular mechanism by which NQM1 is implicated in stationary phase metabolism needs to be resolved in future studies. However, our data shows that NQM1 affects glucose metabolic activity in yeast stationary phase, under oxidative stress, osmotic stress and caloric restriction, and is thus implicated in difference facettes of the ageing process.
We thank the Max Planck Society, Wellcome Trust (RG 093735/Z/10/Z), the ERC (Starting grant 260809), and the Isaac Newton Trust for funding. Markus A Keller is supported by an Erwin Schroedinger postdoctoral fellowship (FWF, Austria, J 3341). Markus Ralser is a Wellcome Trust Research Career Development and Wellcome-Beit Prize fellow.
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