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Deteriorated stress response in stationary-phase yeast: Sir2 and Yap1 are essential for Hsf1 activation by heat shock and oxidative stress, respectively.

Nussbaum I, Weindling E, Jubran R, Cohen A, Bar-Nun S - PLoS ONE (2014)

Bottom Line: However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging.This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses.Rather, factors that participate in Hsf1 activation appear to be compromised.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response.

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Hsf1 response to oxidative stress is lost in stationary-phase yeast and depends on Yap1.(A) Wild-type BY4741 cells harboring the HSE2-LacZ plasmid grown exponentially at 30°C were incubated for 30 min with the indicated concentrations of H2O2. (B) Wild-type and Δyap1 BY4741 cells harboring the HSE2-LacZ plasmid grown at 30°C either exponentially (EG) or to stationary-phase (SP) were incubated for 30 min with (+) or without (−) H2O2 (3 mM) prior to heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min to heat shock (HS) at 42°C (+). Hsf1 activity was measured as β-galactosidase specific activity. The data are the mean plus standard error of at least 3 independent experiments. Kruskal-Wallis one way analysis of variance on ranks (pairwise multiple comparison with Tukey test) applied on data of EG cells in (B) indicates a statistically significant difference (p<0.001) between the activity in untreated cells and in cells exposed to HS or H2O2.
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pone-0111505-g004: Hsf1 response to oxidative stress is lost in stationary-phase yeast and depends on Yap1.(A) Wild-type BY4741 cells harboring the HSE2-LacZ plasmid grown exponentially at 30°C were incubated for 30 min with the indicated concentrations of H2O2. (B) Wild-type and Δyap1 BY4741 cells harboring the HSE2-LacZ plasmid grown at 30°C either exponentially (EG) or to stationary-phase (SP) were incubated for 30 min with (+) or without (−) H2O2 (3 mM) prior to heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min to heat shock (HS) at 42°C (+). Hsf1 activity was measured as β-galactosidase specific activity. The data are the mean plus standard error of at least 3 independent experiments. Kruskal-Wallis one way analysis of variance on ranks (pairwise multiple comparison with Tukey test) applied on data of EG cells in (B) indicates a statistically significant difference (p<0.001) between the activity in untreated cells and in cells exposed to HS or H2O2.

Mentions: Since stationary-phase yeast lost the Hsf1 response to heat shock (Figure 1) but maintained its response to glucose starvation (Figure 3), we tested in these cells the activation of Hsf1 by yet a third stressor, the oxidative stress. Exponentially-growing yeast harboring HSE2-LacZ were exposed for 30 min to increasing concentrations of H2O2 and the measured β-galactosidase activity showed that 3 mM H2O2 yielded maximal response (Figure 4A), a concentration that was used in subsequent experiments. Notably, the expression of neither Hsp26-GFP nor Btn2-GFP was significantly upregulated by H2O2 itself, although the heat shock response was augmented in the presence of this oxidant (Figure S2). The β-galactosidase activity revealed that in exponentially-growing yeast H2O2 activated Hsf1 by itself and the combination of H2O2 and heat shock generated a stronger activation (Figure 4B). This suggests that oxidative stress and heat shock utilize distinct activation pathways. Conversely, stationary-phase yeast responded neither to heat shock nor to H2O2 or to their combination (Figure 4B), reflecting deteriorated Hsf1 activation by both stresses.


Deteriorated stress response in stationary-phase yeast: Sir2 and Yap1 are essential for Hsf1 activation by heat shock and oxidative stress, respectively.

Nussbaum I, Weindling E, Jubran R, Cohen A, Bar-Nun S - PLoS ONE (2014)

Hsf1 response to oxidative stress is lost in stationary-phase yeast and depends on Yap1.(A) Wild-type BY4741 cells harboring the HSE2-LacZ plasmid grown exponentially at 30°C were incubated for 30 min with the indicated concentrations of H2O2. (B) Wild-type and Δyap1 BY4741 cells harboring the HSE2-LacZ plasmid grown at 30°C either exponentially (EG) or to stationary-phase (SP) were incubated for 30 min with (+) or without (−) H2O2 (3 mM) prior to heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min to heat shock (HS) at 42°C (+). Hsf1 activity was measured as β-galactosidase specific activity. The data are the mean plus standard error of at least 3 independent experiments. Kruskal-Wallis one way analysis of variance on ranks (pairwise multiple comparison with Tukey test) applied on data of EG cells in (B) indicates a statistically significant difference (p<0.001) between the activity in untreated cells and in cells exposed to HS or H2O2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214751&req=5

pone-0111505-g004: Hsf1 response to oxidative stress is lost in stationary-phase yeast and depends on Yap1.(A) Wild-type BY4741 cells harboring the HSE2-LacZ plasmid grown exponentially at 30°C were incubated for 30 min with the indicated concentrations of H2O2. (B) Wild-type and Δyap1 BY4741 cells harboring the HSE2-LacZ plasmid grown at 30°C either exponentially (EG) or to stationary-phase (SP) were incubated for 30 min with (+) or without (−) H2O2 (3 mM) prior to heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min to heat shock (HS) at 42°C (+). Hsf1 activity was measured as β-galactosidase specific activity. The data are the mean plus standard error of at least 3 independent experiments. Kruskal-Wallis one way analysis of variance on ranks (pairwise multiple comparison with Tukey test) applied on data of EG cells in (B) indicates a statistically significant difference (p<0.001) between the activity in untreated cells and in cells exposed to HS or H2O2.
Mentions: Since stationary-phase yeast lost the Hsf1 response to heat shock (Figure 1) but maintained its response to glucose starvation (Figure 3), we tested in these cells the activation of Hsf1 by yet a third stressor, the oxidative stress. Exponentially-growing yeast harboring HSE2-LacZ were exposed for 30 min to increasing concentrations of H2O2 and the measured β-galactosidase activity showed that 3 mM H2O2 yielded maximal response (Figure 4A), a concentration that was used in subsequent experiments. Notably, the expression of neither Hsp26-GFP nor Btn2-GFP was significantly upregulated by H2O2 itself, although the heat shock response was augmented in the presence of this oxidant (Figure S2). The β-galactosidase activity revealed that in exponentially-growing yeast H2O2 activated Hsf1 by itself and the combination of H2O2 and heat shock generated a stronger activation (Figure 4B). This suggests that oxidative stress and heat shock utilize distinct activation pathways. Conversely, stationary-phase yeast responded neither to heat shock nor to H2O2 or to their combination (Figure 4B), reflecting deteriorated Hsf1 activation by both stresses.

Bottom Line: However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging.This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses.Rather, factors that participate in Hsf1 activation appear to be compromised.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response.

Show MeSH
Related in: MedlinePlus