Glucose triggers apoptosis through Ras

We show that glucose addition to galactose-grown tps1∆ cells triggers rapid and dramatic activation of Ras, as opposed to the modest activation observed in wild type cells (Fig. 2a). The GTP loading state on Ras in the tps1∆ strain, 10 min after glucose addition, is similar to that in a strain expressing the constitutively active Ras2val19 oncogene equivalent (Fig. 2a). A Ras2val19 strain, however, has no detectable growth inhibition on glucose medium10. Deletion of hexokinase 2 abolishes glucose-induced hyperactivation of Ras (Fig. 2a), which is consistent with its complete suppression of the glucose growth defect of the tps1∆ strain30. Whereas the Ras2val19 strain shows normal growth on glucose, it grows with strongly reduced rate on poor carbon sources, like galactose, and dies in stationary phase10. More recent work has shown that hyperactive Ras causes programmed cell death/apoptosis in non-growing yeast cells11. Hence, we reasoned that the combination of aberrant growth initiation due to the glycolytic deregulation and hyperactivation of Ras in tps1∆ cells may cause apoptosis and that this actually might be the real cause for the unusually high glucose sensitivity of tps1∆ cells29 rather than the deregulation of glycolysis per se. Determination of classical read-outs for apoptosis in yeast gave results consistent with this hypothesis. Glucose addition to tps1∆ cells, as opposed to wild type cells, caused rapid release of cytochrome c from the mitochondria (Fig. 2b), exposure of phosphatidylserine at the plasma membrane (Fig. 2c) and generation of reactive oxygen species (ROS) (Fig. 2d). These results are consistent with recent evidence that the Tps1 protein acts as an anti-apoptotic factor in yeast31. The drop in the cytochrome c level in the wild-type cells might be caused by glucose repression of the CYC1 gene32. It is absent in the tps1∆ strain because that strain is deficient in glucose-induced signaling28. The inability to detect a decrease in the cytochrome c content of the mitochondria in the tps1∆ strain concomitant with the appearance of cytochrome c in the supernatant, is likely due to the fact that the amount appearing in the supernatant is much smaller than the amount remaining in the mitochondria.

Fig. 2 Glucose triggers activation of Ras and apoptosis in tps1∆ cells. a Ras-GTP level before and 10 min after addition of glucose (glu) to cells grown on galactose (gal). The strain expressing Ras2 val19 was grown on glucose. Total Ras = Ras-GDP + Ras-GTP. Quantification of the signals as Ras-GTP level/total Ras level compared to the ratio for wild type or tps1∆ on galactose set at 1.0. The values were determined through quantification by densitometry and are expressed as relative arbitrary units. b Specific cytochrome c release from the mitochondria after addition of glucose to wild type and tps1∆ strain. M, mitochondrial fraction; SN, post-mitochondrial supernatant; Anti-CyC, anti-cytochrome c antibody; Anti-Cox2, cytochrome oxidase 2 antibody; C: control cytochrome c or cytochrome oxidase 2. c Annexin-V-FITC/propidium iodide staining 2h after addition of glucose to cells grown on galactose. Green colored fluorescence originates from annexin staining, red colored fluorescence represents propidium iodide binding to DNA of death cells. The scale bar is 250 μm. d Reactive oxygen species (ROS) accumulation 2 h after addition of glucose to cells grown on galactose. ROS was stained with the dye dihydrorhodamine 123. DIC, differential interference contrast image. The scale bar is 125 μm Full size image

Deletion of Ras2 in the tps1∆ strain caused partial recovery of growth on glucose (Fig. 3a). Overexpression of PDE2, which encodes the high-affinity cAMP phosphodiesterase and is known to counteract phenotypes caused by hyperactive Ras33, restored growth on glucose even better (on solid medium: Fig. 3a; in liquid medium: Supplementary Fig. 1). This may be due to the fact that overexpression of Pde2 lowers the cAMP level in the cells even more than deletion of Ras2 and thus leads to lower PKA activity. Our results are consistent with overactive Ras playing a role in the glucose growth defect of the tps1∆ strain. Interestingly, both in the tps1∆ and tps1∆ ras2∆ strain a very similar deregulation of glycolysis after addition of glucose was present, including hyperaccumulation of Fru1,6bisP and depletion of ATP, but the metabolite profile started to recover in the tps1∆ ras2∆ strain after about 5 h to finally reach the same profile as observed in wild type cells (Fig. 3b). The difference was most striking in the ATP level, which stayed close to zero in the tps1∆ strain, but recovered to the same level as in wild type cells in the tps1∆ ras2∆ and tps1∆ pPDE2 strains (Fig. 3b). This indicates remarkable robustness and flexibility of the glycolytic pathway in yeast and contradicts that the strong perturbation of metabolite homeostasis is causing the glucose growth defect. The results suggest that the induction of programmed cell death, caused by overactivation of the Ras-cAMP-PKA pathway, is the true cause of the inability of the tps1∆ strain to grow on glucose, rather than the glycolytic deregulation, which is usually thought to be the main cause34. The onset of the normalization of glycolytic metabolite homeostasis a few hours after addition of glucose in the tps1∆ ras2∆ and tps1∆ pPDE2 strains (Fig. 3b) might suggest that an additional time-dependent change in initial glucose catabolism occurs, which allows the cells to recover normal glycolytic flux and intermediate levels.

Fig. 3 Restoration of growth on glucose and glycolytic metabolite levels in the tps1∆ strain with downregulation of the Ras-cAMP-PKA pathway. a Growth of tps1∆ and suppressor strains on galactose and different concentrations of glucose. Five-fold dilutions were spotted. b Intracellular Glu6P, Fru1,6bisP and ATP concentration before and after addition of 100 mM glucose. The short-term measurements (0–10 min) were performed with non-growing cells incubated in 25 mM MES buffer (pH 6.0), while the long-term measurements (0–30 h) were performed with cells growing in minimal galactose medium Full size image

Fru1,6bisP triggers activation of Ras

To investigate whether one or more of the hyperaccumulated glycolytic metabolites in the tps1∆ strain acted as the trigger for hyperactivation of Ras, we made use of nystatin-permeabilized wild type yeast spheroplasts35. These are cells of which the cell wall has been largely removed and the plasma membranes permeabilized to allow passage of all small molecules. Because of their fragility the permeabilized cell sacs were not washed and the small molecules leaked into the medium were thus only strongly diluted. Addition of glycolytic metabolites to permeabilized spheroplasts revealed strong activation of Ras by Fru1,6bisP at the higher physiological concentrations (4–8 mM; Fig. 4a, b), similar to that observed with physiological concentrations of GTP (Fig. 4b). DHAP and GAP also caused activation but only in unphysiologically high concentrations (1–5 mM; Fig. 4a, b). Other glycolytic intermediates, and glucose or Tre6P did not cause activation (Fig. 4a).

Fig. 4 Activation of Ras by Fru1,6bisP. a Ras-GTP level 3 min after addition of 5 mM of different glycolytic metabolites to permeabilized yeast spheroplasts. Glu6P, glucose-6-phosphate; Tre6P, trehalose-6-phosphate; Fru6P, fructose-6-phosphate; Fru1,6bisP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate. Quantification of the signals as Ras-GTP level/total Ras level compared to the ratio for the water control set at 1.0. The values were determined through quantification by densitometry and are expressed as relative arbitrary units. b Activation of Ras in permeabilized spheroplasts with different concentrations of GTP, Fru1,6bisP, DHAP and GAP. Quantification of the signals as Ras-GTP level/total Ras level compared to the ratio for the water control set at 1.0. The values were determined through quantification by densitometry and are expressed as relative arbitrary units. c Ras-GTP level before and 10 min after addition of 100 mM glucose (glu) to cells grown on glycerol +2.5 mM glucose (gly). Quantification of the signals as Ras-GTP level/total Ras level compared to the ratio for tps1∆ on glycerol set at 1.0. The values were determined through quantification by densitometry and are expressed as relative arbitrary units. d Intracellular ATP, Glu6P, and Fru1,6bisP concentration before and after addition of 100 mM glucose to cells grown in glycerol +2.5 mM glucose Full size image

To evaluate whether Fru1,6bisP is responsible for glucose-induced hyperactivation of Ras in the tps1∆ strain, we constructed tps1∆ strains with additional single or double deletion of the PFK1 and PFK2 genes, encoding phosphofructokinase 1. Glucose-induced activation of Ras was reduced in cells of the tps1∆ pfk1∆ and tps1∆ pfk2∆ strains, and abolished in cells of the tps1∆ pfk1∆ pfk2∆ strain (Fig. 4c). This would be consistent with the hyperaccumulation of Fru1,6bisP in tps1∆ cells being responsible for hyperactivation of Ras. However, upon measurement of the glycolytic metabolites, the tps1∆ pfk1∆ and tps1∆ pfk2∆ strains were already deficient in glucose-induced hyperaccumulation of Fru1,6bisP and consistently showed a much higher accumulation of Glu6P than the tps1∆ strain (Fig. 4d). Glu6P was also higher than in the corresponding pfk1∆ and pfk2∆ strains (Fig. 4d).

The presence of partial Ras activation without Fru1,6bisP accumulation in the tps1∆ pfk1∆ and tps1∆ pfk2∆ strains suggests that there may be a second mechanism responsible for glucose-induced hyperactivation of Ras in the tps1∆ strain and/or that the hyperaccumulation of Glu6P in these strains in some way triggers a residual activation. Although the complete absence of Ras hyperactivation in the tps1∆ pfk1∆ pfk2∆ strain seems to contradict this possibility, the very poor growth of this strain, requiring feeding with both a respiratory carbon source and a low level of glucose, may have compromised the second activation mechanism. It can also not be excluded that one of the glucose signaling pathways modifies Cdc25, for instance through phosphorylation, to render it more sensitive to Fru1,6bisP. This could explain why even in the absence of an increase in the level of Fru1,6bisP, it would still be able to trigger activation of Ras after addition of glucose.

Fru1,6bisP acts through the Ras GEF Cdc25 in yeast

We next investigated whether Fru1,6bisP caused direct activation of Ras or acted through one of its regulators, the guanine nucleotide exchange proteins Cdc25 and Sdc25, or the GTPase activating proteins Ira1 and Ira2. For that purpose we constructed several strains with combinations of tps1∆ and deletions in the genes encoding these regulators. Deletion of both CDC25 and SDC25 is lethal36, while deletion of IRA1 and IRA2 causes hyperactivation of Ras37. The ira1∆ ira2∆ cdc25∆ sdc25∆ strain retains enough Ras activity for growth (Fig. 5a). All quadruple tps1∆ strains with at least one wild-type GEF factor, Cdc25, or Sdc25, were unable to grow on glucose, whereas absence of both GEF factors in the quintuple deletion strain tps1∆ cdc25∆ sdc25∆ ira1∆ ira2∆ restored to some extent growth on glucose medium (Fig. 5a, Supplementary Fig. 2). This suggests that glucose-induced hyperactivation of Ras through the GEF factors is a major cause of the glucose sensitivity of the tps1∆ strain. The tps1∆ ira1∆ ira2∆ strain showed already a high Ras-GTP level on galactose medium, which made it difficult to evaluate any further glucose-induced increase (Fig. 5b). On the other hand, in the quadruple deletion strains, tps1∆ sdc25∆ ira1∆ ira2∆ and tps1∆ cdc25∆ ira1∆ ira2∆, the rapid glucose-induced hyperactivation of Ras was largely abolished, indicating requirement of both GEF factors. The absence of Ras hyperactivation in these strains may be restricted to the short-term response, since both strains were still unable to grow on glucose (Fig. 5a). Unexpectedly, we found that the quintuple deletion strain, tps1∆ cdc25∆ sdc25∆ ira1∆ ira2∆, showed a higher basal activity of Ras on galactose medium, which, however, did not increase further upon addition of glucose (Fig. 5b). Although these experiments and the interpretation of the results are complicated because of the essential character of the GEF factors, the high basal Ras activity caused by deletion of the GAP proteins, and the unexpectedly higher basal level of Ras-GTP in the quintuple deletion strain, the results are consistent with a role of the GEF factors in mediating glucose-induced hyperactivation of Ras in the tps1∆ strain. This was confirmed by the observation that deletion of SDC25 and especially CDC25 in the tps1∆ strain caused partial growth recovery on glucose (Supplementary Fig. 3). This result also suggests that Fru1,6bisP exerts its activating effect mainly through Cdc25.

Fig. 5 Evaluation of requirement for the GEF and GAP regulators of Ras. a Growth on galactose and glucose. Five-fold dilutions were spotted. b, c Ras-GTP level before and 10 min after addition of glucose (glu) to cells grown on galactose (gal). Quantification of the signals as Ras-GTP level/total Ras level compared to the ratio for wild type (b) or tps1∆ (c) on galactose set at 1.0. The values were determined through quantification by densitometry and are expressed as relative arbitrary units Full size image

We have also performed similar experiments in the wild-type strain background. This confirmed the much weaker and more variable glucose-induced activation of Ras in the wild type strain (which may be due to transient oscillation in the response; Fig. 5c). Deletion of IRA1 and IRA2 caused a dramatic increase in Ras-GTP both in galactose and glucose incubated cells (Fig. 5c), which made it impossible to evaluate reliably any possible further increase in Ras-GTP in the presence of glucose. Deletion of CDC25 or SDC25 caused a partial reduction of the very high Ras-GTP level, while double deletion of CDC25 and SDC25 caused a further reduction in the Ras-GTP level (Fig. 5c). In none of the latter strains was a significant difference between the galactose and glucose condition observed, consistent with the idea that the glucose activation effect on Ras acts through its Cdc25 and Sdc25 GEF factors.

Since previous work had shown that the highly conserved C terminus of Cdc25 was required for glucose activation of cAMP synthesis38, 39, we focused on this part of Cdc25 to identify amino acid residues of which mutagenesis might abolish Fru1,6bisP activation of Ras in permeabilized spheroplasts and also restore growth of the tps1∆ mutant on glucose. We selected for site-directed mutagenesis a highly conserved region with several positively charged residues (Fig. 6a) (aa 1478–1501 in Cdc25) that could possibly be involved in the formation of a binding site for the negatively charged Fru1,6bisP molecule. Interestingly, another positively charged residue, R1122 in yeast Cdc25, which corresponds to K602 in human Sos1, resides in the vicinity of this region in the 3D structure of the human Sos1 catalytic domain40. The R1122/K602 residue is located itself in a small conserved region. Figure 6a shows the 3D structure of Sos1 in association with Ras and the position in space of selected amino acid residues in the cleft formed by the alpha helix I of Sos1 and the switch 1 region of Ras, thus in the area where the two proteins interact. The amino acid residues K602, R962, and K963 of Sos1, are located within this cleft (Fig. 6a).

Fig. 6 Conserved residues in the C terminus of Cdc25 and Sos1 are required for Fru1,6bisP activation of Ras. a Alignment of the conserved region in the Ras GEF factors, HsSos1 and HsSos2 from Homo sapiens and ScCdc25 and ScSdc25 from Saccharomyces cerevisiae. Crystal structure of the complex between human Sos1 (green) and human Ras (purple) (PD entry: 1BKD40). The arrow points to the cleft, which contains the conserved region. Enlarged view on the 3D structure surrounding the relevant residues in Sos1 and Cdc25 located in the conserved region. b, d Ras-GTP level 3 min after addition of 5 mM Fru1,6bisP or 1 mM GTP as positive control, to permeabilized yeast spheroplasts from strains expressing mutant ScCdc25 (b) or HsSos1 (d) alleles. Western blots were quantified and the Ras-GTP levels are shown relative to the level observed after addition of GTP. Values represent average (number of replicates shown above each graph), error bars represent standard deviations. Sidak’s multiple comparison test was used for evaluation of statistical significance ((NS) identifies p-values greater than 0.05, (*) identifies p-values between 0.01 and 0.05, (**) between 0.01 and 0.001, (***) between 0.001 and 0.0001, (****) identifies p-values lower than 0.0001). c Recovery of growth on glucose in the tps1∆ sdc25∆ strain expressing Cdc25R1122D,K1491E instead of wild type Cdc25 Full size image

We first mutagenized the positively charged residues R1122 and K1491 in Cdc25 to negatively charged aspartate and glutamate residues, respectively. In addition, we constructed the T1490P allele of Cdc25 in order to disturb the structure of the alpha helix39 in this region. In permeabilized spheroplasts of strains expressing Cdc25R1122D,K1491E or Cdc25T1490P instead of wild-type Cdc25, addition of Fru1,6bisP in a physiological concentration of 5 mM no longer caused activation of Ras as opposed to addition of GTP (Fig. 6b). This indicates that the residues R1122 and K1491 are in some way important for Fru1,6bisP activation of Ras, either because of direct interaction with Fru1,6bisP or maintenance of a proper Cdc25 conformation, and that also the structure of the alpha helix surrounding T1490 is important for a similar reason.

The activity of Sdc25 was assumed negligible since it is repressed during exponential growth in rich media36. The strains carrying the Cdc25 mutated alleles in the absence of Sdc25 were viable, showing that the alleles were functional. Interestingly, a tps1∆ sdc25∆ strain expressing Cdc25R1122D,K1491E instead of wild-type Cdc25 displayed strong recovery of growth on glucose, supporting the requirement of these residues for Fru1,6bisP activation of Ras in vivo (Fig. 6c). We did not observe such recovery of growth on glucose with a strain expressing Cdc25T1490P instead of wild-type Cdc25 (Supplementary Fig. 4a). However, such a strain displayed reduced trehalose and glycogen levels, suggesting the presence of constitutively activated PKA (Supplementary Fig. 4b). This may be explained by Thr1490 serving as a PKA target site for feedback inhibition of PKA on Cdc25, as previously suggested39 or may be due to another effect caused by the distortion of the alfa-helix structure.

Next, we assessed whether human Sos1 expressed in yeast is also responsive to the same type of glycolytic stimulation as yeast Cdc25. Interestingly, addition of Fru1,6bisP in a physiological concentration of 5 mM to permeabilized yeast spheroplasts expressing only the guanine nucleotide exchange factor region of human Sos1 (residues 553–1024), caused a clear activation of Ras similar to that of GTP (Fig. 6d). Wild type Sos1 has the INFSKRR962K sequence. However, the yeast strain expressing the complete Sos1 protein grew poorly on galactose. The mutant protein R962T, which has the same INFSKRTK sequence as in Cdc25, also supported activation of Ras with Fru1,6bisP and GTP (Fig. 6d). The strain expressing this protein showed normal growth on galactose, consistent with the threonine being a target of PKA feedback inhibition. Activation of Ras in permeabilized spheroplasts with Fru1,6bisP, but not GTP, was absent for the mutant alleles Sos1K602E, K963E and Sos1R962P (Fig. 6d), which have the equivalent mutations to those mentioned above for yeast Cdc2540. The single-mutation K963E did not yet abolish Fru1,6bisP activation of Ras. These results show that Sos1 is able to mediate activation of Ras by Fru1,6bisP and that the corresponding amino acid residues or combinations to those in Cdc25 are also important for the activation.

There are several possibilities for the mechanism by which interaction of Fru1,6bisP with Cdc25/Sos1 might stimulate activation of Ras. It might alleviate one of the limiting steps in the Cdc25/Sos1-Ras nucleotide exchange cycle, for instance the binding of Ras to Cdc25/Sos1, the release of GDP, the incorporation of GTP, the dissociation of Cdc25/Sos1 from Ras or the shielding of Ras from the action of the GTPase activating proteins Ira1,2/NF1.

Fru1,6bisP stimulates dissociation of the Ras/Sos1 complex

To study the interaction of Fru1,6bisP with Ras and its Ras GEF factor, we performed an in vitro binding assay (Octet Biolayer interferometry) using human H-Ras (residues 1–166) and the catalytic part of Sos1 (residues 564–1049), and analyzed the effect of Fru1,6bisP on the stability of the Ras/Sos1 complex. First, H-Ras molecules were loaded onto the biosensor, and Sos1 molecules were allowed to bind to H-Ras during the association phase. Next, the dissociation of the H-Ras/Sos1 complex was monitored in the presence of increasing concentrations of Fru1,6bisP. The biolayer interferometry measurements showed that Fru1,6bisP increases the dissociation rate of the H-Ras/Sos1 complex in a dose-dependent way (Fig. 7a). Plotting of the response (taken 150 s after addition of Fru1,6bisP) vs. the Fru1,6bisP concentration and fitting yields an apparent K D (K Dapp ) of 9.3 ± 0.3 mM (Fig. 7b), which is in accordance with physiological concentrations of Fru1,6bisP in both mammalian and S. cerevisiae cells41,42,43. As a control we also tested the effect of addition of glucose, Fru6P, DHAP and GAP. Glucose had no effect on the H-Ras/Sos1 complex, while Fru6P and DHAP produced a very small effect at non-physiological concentrations (K Dapp = 96 ± 40 mM and K Dapp = 120 ± 41 mM for Fru6P and DHAP, respectively; Supplementary Fig. 11). Addition of GAP had no effect on the Ras/Sos1 complex up to a concentration of 2 mM, but it led to a very fast decrease in signal due to unspecific dissociation of H-Ras from the biosensor at higher concentrations.

Fig. 7 Fru1,6bisP binds to Sos1 and disrupts the Sos1/H-Ras complex. a, Biolayer interferometry (BLI) measurements showing the disruption of the Sos1/H-Ras complex. His-tagged H-Ras was coupled to Ni2+-coated biosensors and loaded with 0.5 µM of non-tagged Sos1 (association phase). Subsequently, dissociation of Sos1 was monitored in buffer containing increasing concentrations of Fru1,6bisP (0, 2, 5, 10, 20, 50, and 100 mM as labeled on the curves). The rates of Sos1 dissociation are dependent on the Fru1,6bisP concentration. b Titration curve showing the influence of Fru1,6bisP concentration on Sos1/H-Ras complex dissociation. Plotting the BLI signal amplitude as shown in panel a after 150 s of complex dissociation (R; mean ± s.d.; n = 3 independent experiments) vs. the Fru1,6bisP concentration yields a binding curve that was fitted on a Langmuir binding model to obtain an apparent K D (K Dapp ) value (±s.e.). c Titration curves for binding of Fru1,6bisP to either Sos1 or H-Ras. Melting temperatures (T m ) of Sos1 and H-Ras at different ligand concentrations were obtained via thermal shift assays (n = 3 independent experiments). Plotting T m values (mean ± s.d.) vs. ligand concentration yielded binding curves that were fitted on a Langmuir binding model to obtain K D values (±s.e.) Full size image

We also performed additional biolayer interferometry (BLI) measurements monitoring the disruption of the Sos1/H-Ras complex by different intermediates of glycolysis and GTP (Supplementary Fig. 11). This showed that only Fru1,6bisP and to a lesser extent Fru6P enhanced the rate of Sos1 dissociation.

Fru1,6bisP directly binds to human Sos1

To find out whether the observed disruption of the H-Ras/Sos1 complex is due to direct binding of Fru1,6bisP to either H-Ras or Sos1, we determined the thermal stability (melting temperature, T m ) of both proteins in the presence of increasing amounts of Fru1,6bisP, using thermal shift assays44. While the thermal stability of H-Ras was quasi unaffected by addition of Fru1,6bisP, the thermal stability of Sos1 increased in the presence of Fru1,6bisP in a dose-dependent way indicating direct binding of Fru1,6bisP to Sos1 (Fig. 7c). Fitting the T m value vs. the Fru1,6bisP concentration on a Langmuir-binding model yielded a K D of 17.5 ± 2.2 mM. It should be noted that the latter K D value corresponds to the real thermodynamic K D for binding of Fru1,6bisP to Sos1, while the K Dapp for dissociation of the H-Ras/Sos1 complex also depends on kinetic parameters (rate of complex dissociation).

Glucose activates Ras/MEK/ERK pathway in human cell lines

To assess whether the observed effect of Fru1,6bisP retains physiological relevance in human cells, we studied Ras, MEK, and ERK activation after the addition of glucose to glucose-starved HEK293T and Hela Kyoto cell lines. A 48 h glucose starvation period lowers the glycolytic flux drastically and thus also the Fru1,6bisP level41. Re-addition of glucose is known to elevate the intracellular concentration of Fru1,6bisP when glycolysis starts up. The results showed that glucose addition transiently activates Ras, as well as its downstream targets MEK and ERK, both in regular human cells and in cancer cells (Fig. 8a). The ratio of Ras-GTP over total Ras was quantified for better interpretation (Fig. 8b).

Fig. 8 Addition of glucose triggers activation of the Ras/MEK/ERK pathway in glucose-starved human HEK293T and Hela Kyoto cells. a Western blot analysis of Ras-GTP, P-MEK and P-ERK levels at different time points after glucose addition to HEK293T (left) and Hela Kyoto (right) cells. Samples at time point 0 were taken prior to glucose addition. Results are representative for at least three repetitions. b Quantification of Ras-GTP levels relative to total Ras signals, as depicted in a. All values are expressed relative to that of sample t = 0, after correction with the vinculin loading control Full size image

Absence of correlation with proliferation rate

We have also performed multiple experiments to evaluate whether there might be a direct relationship between the proliferation rate of yeast cells, the concentration of Fru1,6bisP and the Ras-GTP level. First, we have measured these three parameters using a series of carbon sources sustaining different proliferation rates. Although there was a tendency for the Ras-GTP level to be lower with lower growth rate, it was certainly not in proportion with the drastically reduced Fru1,6bisP level during growth on non-fermentable carbon sources compared to growth on fermentable sugars (Supplementary Fig. 5). Next, we starved yeast cells for nitrogen on a glucose-containing medium for 50 h and then re-initiated growth by adding a nitrogen source again. This resulted in a drastic drop in Fru1,6bisP and rapid recovery, respectively. However, there was no concomitant change in the level of Ras-GTP at all in the two conditions (Supplementary Fig. 6). We made also use of a yeast strain lacking the PFK26 and PFK27 genes, which encode phosphofructokinase 245. It is responsible for the synthesis of Fru2,6bisP, a potent allosteric activator of phosphofructokinase 1. The double-deletion strain had a strongly reduced level of Fru1,6bisP and a slight reduction in growth rate. However, its Ras-GTP level remained unchanged (Supplementary Fig. 7). Next, we investigated whether the slight reduction in growth rate of the pfk26∆ pfk27∆ strain could be suppressed by an increase in the activity of Ras. The latter was accomplished by single or double deletion of the IRA1 and IRA2 genes37 (Supplementary Fig. 8) or by expression of RAS2 val19 [10] (Supplementary Fig. 9). However, in none of the two cases was there any increase in the growth rate, neither on glucose nor on galactose medium. All these results indicate that there is no direct relationship between the Fru1,6bisP level and the Ras-GTP level in yeast cells with different proliferation rates. The absence of a clear correlation between Fru1,6bisP and Ras-GTP levels implies that other mechanisms, such as regulation of Ira1,2 activity, must also control the Ras-GTP level. Since yeast strains with constitutively high-Ras activity always showed a tendency for slower growth rates, we overexpressed RAS2 val19 with a multi-copy vector in different yeast strains. In all cases this led to a dramatic reduction in growth rate on glucose medium (Supplementary Fig. 10). This shows that RAS2 val19 is not only toxic under poor growth conditions and in stationary phase, but that very-high expression is also toxic under optimal growth conditions for yeast.