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. 2019 Jun;18(3):e12921.
doi: 10.1111/acel.12921. Epub 2019 Feb 20.

Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH

Laura Dolz-Edo  1 , Margaretha van der Deen  1 , Stanley Brul  1 , Gertien Jacoba Smits  1
Affiliations

Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH

Laura Dolz-Edo et al. Aging Cell. 2019 Jun.

Abstract

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pHc ) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pHc and the central nutrient signalling cAMP-Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pHc correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pHc showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.

Keywords: Saccharomyces cerevisiae; cAMP; calorie restriction; chronological lifespan; glucose; intracellular pH.

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Conflict of interest statement

None Declared.

Figures

Figure 1
Figure 1
Genetic manipulation of PKA activity modulates pHc upon glucose depletion. Mutants with overactive PKA activity (green) have reduced pHc and low PKA activity mutants (red) an increased pHc when glucose is depleted from the media. (a) BY4741 (black), bcy1Δ (green) and DAmP CYR1 (red) strains were grown in microplates and OD600 (filled symbols) and pHc (open symbols) were monitored. A representative example is shown. (b) and (c) summarise pHc during exponential growth (b) and after glucose depletion (c) for the set of PKA mutants analysed (See Materials and Methods for details). Representative OD600 and pHc curves for each strain can be found in Supporting information Figure S1. Data represent average ± standard deviation (SD) of at least three biological replicates per strain
Figure 2
Figure 2
Addition of cAMP during exponential growth promotes cytosolic acidification upon subsequent glucose depletion. (a) OD600 (diamonds) and pHc (circles) were monitored during growth of the parental strain BY4741. 20 mM cAMP (closed symbols) or water (open symbols) were added to the cultures after 4 hr of growth (arrow). A representative example is shown. (b) Comparison of the pHc at the end of the growth curve (18 hr) for the cAMP treatments indicated. Data are averages ±SD of three biological replicates
Figure 3
Figure 3
Manipulation of PKA activity before glucose depletion modulates pHc upon glucose starvation. (a, c) Exponentially growing BY4741 (a) or TPK1astpk2∆tpk3∆ (c) cultures were subjected to the treatments indicated in the adjacent table and transferred to a microplate to monitor pHc as described in Materials and Methods. Data show a representative result. Pre‐treatments indicate incubation for 90 min prior to starvation with 20 mM cAMP (a) or 2 µM 1NM‐PP1 (c), while cAMP during starvation in (a) indicates the cells were washed with and starved in media without glucose containing 20 mM cAMP. (b, d) pHc 60 min after starvation for the indicated treatments as in (a) or (c), respectively. Data show averages ±SD of three biological replicates. Significance was tested using two‐way ANOVA with matching for both factors and Bonferroni's multiple comparisons test
Figure 4
Figure 4
The initial concentration of glucose in the media regulates starvation pHc via PKA. (a) pHc after glucose depletion (calculated as in Figure 1c) of BY4741 cultures after growth at the indicated initial concentrations of glucose (glc). A representative growth and pHc curve can be found in Supporting information Figure S7. (b) pHc dynamics of BY4741 cultures starved for glucose after growth at the indicated initial concentrations of glucose (c) Cell viability after three days of glucose starvation as in panel B. (d–g) Cytosolic pH 60 min after glucose starvation for BY4741 (d), TPK1as tpk2Δ tpk3Δ without (e) or with (f) 1NM‐PP1 pre‐treatment as in Figure 3c–d, and bcy1Δ (g). Data shown are averages ±SD of three biological replicates. Significance was tested using one‐way ANOVA with matching with Bonferroni's correction
Figure 5
Figure 5
Cytosolic acidification correlates with viability upon glucose starvation. (a, b) pHc dynamics of BY4741 cultures starved for glucose (glc) at different extracellular pH (pHex) in the absence (a; DMSO) or presence (b) of ebselen. (c, d) pHc dynamics of TPK1astpk2∆tpk3∆ cultures starved for glucose at different pHex after a control treatment with DMSO (c) or a treatment with 0.2 µM of 1NM‐PP1 (d) for 90 min prior to starvation. (e, f) Cell viability after three days of glucose starvation for (e) BY4741 cultures as in a and b or (f) for TPK1astpk2∆tpk3∆ cultures as in c and d. Grey bars represent control treatments. Black bars represent ebselen treatment (e) or 1NM‐PP1 pre‐treatment (f). Data shown are averages ±SD of three biological replicates
Figure 6
Figure 6
Model for the glucose‐dependent control of survival via PKA and pHc proposed in this work. (a) Schematic representation of the data. Solid lines represent glucose concentrations ([glc]), and dashed lines represent pHc measurements. Growth (OD600) is represented by a black dotted line. (b) Model derived from the data. During growth, glucose levels activate PKA quantitatively. When glucose is depleted from the media, pHc decreases according to the level of PKA activity. Orange represents high glucose concentrations, which, via induction of high PKA activity, trigger a low pHc upon glucose depletion and limit survival. Green represents low glucose, which leads to lower PKA activity and thus increases pHc upon glucose depletion and survival
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