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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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Activation of Hsf1 by heat shock is mimicked by excess Sir2 and improved by the NAD+ precursor.(A) Wild-type BY4741 cells harboring HSE2-LacZ plasmid were transformed with an empty vector (−) or a centromeric pSIR2 plasmid (+). Cells grown at 30°C either exponentially (EG) or to stationary-phase (SP) were either incubated for 20 min at 30°C (−) or subjected to a 20 min HS at 42°C (+). (B) Wild-type BY4741 cells harboring HSE2-LacZ plasmid were transformed with an empty vector (−) or a pSIR2 plasmid (+). Cells grown at 30°C to the indicated growth phase were incubated for 30 min with (+) or without (−) NR (10 µM) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min heat shock (HS) at 42°C (+). (C) Activity in SP yeast from (B) drawn to a smaller scale. Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.
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pone-0111505-g007: Activation of Hsf1 by heat shock is mimicked by excess Sir2 and improved by the NAD+ precursor.(A) Wild-type BY4741 cells harboring HSE2-LacZ plasmid were transformed with an empty vector (−) or a centromeric pSIR2 plasmid (+). Cells grown at 30°C either exponentially (EG) or to stationary-phase (SP) were either incubated for 20 min at 30°C (−) or subjected to a 20 min HS at 42°C (+). (B) Wild-type BY4741 cells harboring HSE2-LacZ plasmid were transformed with an empty vector (−) or a pSIR2 plasmid (+). Cells grown at 30°C to the indicated growth phase were incubated for 30 min with (+) or without (−) NR (10 µM) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min heat shock (HS) at 42°C (+). (C) Activity in SP yeast from (B) drawn to a smaller scale. Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.

Mentions: To substantiate the role of Sir2 in Hsf1 activation by heat shock, we expressed in wild-type yeast excess SIR2 from a plasmid. Clearly, in exponentially-growing yeast excess Sir2 mimicked the effect of heat shock and there was no further increase in Hsf1 activity by heat shock (Figures 7A and S3A). However, while the NAD+ precursor NR had no effect on Hsf1 activity in exponentially-growing naive yeast (Figures 7B and S3B; see also Figure 5), NR exerted increased Hsf1 activity in cells expressing excess SIR2 (Figures 7B and S3B). We next tested the effect of excess SIR2 and NR also in stationary-phase cells, and, again, while NR by itself had no effect (Figures 7B and S3B) and excess SIR2 by itself increased Hsf1 activity by two-fold (Figures 7 and S3), supplementing stationary-phase yeast expressing excess SIR2 with NR increased Hsf1 activity nearly four-fold, yet there was no additional effect of heat shock (Figures 7B,C and S3 B,C).


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)

Activation of Hsf1 by heat shock is mimicked by excess Sir2 and improved by the NAD+ precursor.(A) Wild-type BY4741 cells harboring HSE2-LacZ plasmid were transformed with an empty vector (−) or a centromeric pSIR2 plasmid (+). Cells grown at 30°C either exponentially (EG) or to stationary-phase (SP) were either incubated for 20 min at 30°C (−) or subjected to a 20 min HS at 42°C (+). (B) Wild-type BY4741 cells harboring HSE2-LacZ plasmid were transformed with an empty vector (−) or a pSIR2 plasmid (+). Cells grown at 30°C to the indicated growth phase were incubated for 30 min with (+) or without (−) NR (10 µM) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min heat shock (HS) at 42°C (+). (C) Activity in SP yeast from (B) drawn to a smaller scale. Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111505-g007: Activation of Hsf1 by heat shock is mimicked by excess Sir2 and improved by the NAD+ precursor.(A) Wild-type BY4741 cells harboring HSE2-LacZ plasmid were transformed with an empty vector (−) or a centromeric pSIR2 plasmid (+). Cells grown at 30°C either exponentially (EG) or to stationary-phase (SP) were either incubated for 20 min at 30°C (−) or subjected to a 20 min HS at 42°C (+). (B) Wild-type BY4741 cells harboring HSE2-LacZ plasmid were transformed with an empty vector (−) or a pSIR2 plasmid (+). Cells grown at 30°C to the indicated growth phase were incubated for 30 min with (+) or without (−) NR (10 µM) prior to the heat shock. Cells were either incubated further for 20 min at 30°C (−) or subjected to a 20 min heat shock (HS) at 42°C (+). (C) Activity in SP yeast from (B) drawn to a smaller scale. Hsf1 activity was measured as β-galactosidase specific activity. The data are mean plus standard error of at least 3 independent experiments.
Mentions: To substantiate the role of Sir2 in Hsf1 activation by heat shock, we expressed in wild-type yeast excess SIR2 from a plasmid. Clearly, in exponentially-growing yeast excess Sir2 mimicked the effect of heat shock and there was no further increase in Hsf1 activity by heat shock (Figures 7A and S3A). However, while the NAD+ precursor NR had no effect on Hsf1 activity in exponentially-growing naive yeast (Figures 7B and S3B; see also Figure 5), NR exerted increased Hsf1 activity in cells expressing excess SIR2 (Figures 7B and S3B). We next tested the effect of excess SIR2 and NR also in stationary-phase cells, and, again, while NR by itself had no effect (Figures 7B and S3B) and excess SIR2 by itself increased Hsf1 activity by two-fold (Figures 7 and S3), supplementing stationary-phase yeast expressing excess SIR2 with NR increased Hsf1 activity nearly four-fold, yet there was no additional effect of heat shock (Figures 7B,C and S3 B,C).

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