The natural osmolyte trehalose is a positive regulator of the heat-induced activity of yeast heat shock transcription factor

Abstract

In Saccharomyces cerevisiae, the intracellular concentration of trehalose increases rapidly in response to many environmental stresses, including heat shock. These high trehalose levels have been correlated with tolerance to adverse conditions and led to the model that trehalose functions as a chemical cochaperone. Here, we show that the transcriptional activity of Hsf1 during the heat shock response depends on trehalose. Strains with low levels of trehalose have a diminished transcriptional response to heat shock, while strains with high levels of trehalose have an enhanced transcriptional response to heat shock. The enhanced transcriptional response does not require the other heat-responsive transcription factors Msn2/4 but is dependent upon heat and Hsf1. In addition, the phosphorylation levels of Hsf1 correlate with both transcriptional activity and the presence of trehalose. These in vivo results support a new role for trehalose, where trehalose directly modifies the dynamic range of Hsf1 activity and therefore influences heat shock protein mRNA levels in response to stress.

FIG. 1.

Changes in trehalose levels during the heat shock response. Relative trehalose levels, determined by thin-layer chromatography and normalized to cell counts, are shown over the course of a 40°C heat shock for the control (YHN450), LT (YHN455), HT (YHN437), and VHT (YHN454) strains. (A) The LT strain (gray bars), which harbors a TPS1 deletion, has decreased levels of trehalose compared to the control strain (black bars). (B) The HT (gray bars) and VHT (white bars) strains, which harbor NTH1 deletions (nth1Δ P_ADH1-TPS1 P_ADH1-NTH1 and nth1Δ, respectively), have increased levels of trehalose compared to the control strain (black bars). Error bars show standard errors of the mean (SEM). *, P < 0.05; **, P < 0.01.

FIG. 2.

Reduced HSP mRNA levels are correlated with decreased trehalose levels. (A) Northern blot analysis showing HSP mRNA levels over the course of a 40°C heat shock for the LT (YHN455) and control (YHN450) strains. (B) Graphical representation of HSP26 mRNA levels, normalized to ACT1, shows that the LT strain (gray squares) has lower heat-induced transcript levels than the control strain (black circles). Error bars show SEM. *, P < 0.05; **, P < 0.01.

FIG. 3.

Elevated HSP mRNA levels are correlated with increased trehalose levels. (A) Northern blot analysis showing HSP mRNA levels over the course of a 40°C heat shock for the HT (YHN437), VHT (YHN454), and control (YHN450) strains. (B) Graphical representation of HSP26 mRNA levels, normalized to ACT1, shows that the HT (gray squares) and VHT (white squares) strains have higher heat-induced transcript levels than the control strain (black circles). Error bars show SEM. *, P < 0.05; **, P < 0.01.

FIG. 4.

The nth1 knockout strains behave differently during prolonged exposure to heat stress. The HT (YHN437), VHT (YHN454), and control (YHN450) strains were subjected to heat shocks of up to 2.5 h. (A) Trehalose levels of both the HT (gray bars) and VHT (white bars) strains are significantly increased over that of the control strain (black bars) at every time point. (B) Northern blot showing the levels of several HSP mRNAs during a prolonged heat shock. (C) Graphical representation of Northern blot of HSP26 mRNA normalized to ACT1 reveals strain differences between the HT (gray squares), VHT (white squares), and control (black circles) strains. Error bars show SEM. *, P < 0.05; **, P < 0.01.

FIG. 5.

Increased trehalose levels are unable to prevent recovery from heat stress. After a 60-min heat shock (HS) at 40°C, the HT (YHN437) and control (YHN450) strains were allowed to recover for up to 90 min at a constitutive temperature (30°C). (A) Trehalose levels remain high during recovery in the HT strain (gray bars), while trehalose levels decrease in the control strain (black bars). (B) Northern blot analysis showing HSP mRNA levels upon recovery from heat shock. (C) Graphical representation of HSP26 mRNA levels, normalized to ACT1, shows that the HT (gray squares) and control (black circles) strains have similar decreases in HSP mRNA levels. Error bars show SEM. *, P < 0.05; **, P < 0.01.

FIG. 6.

HSF1 is required for the trehalose-dependent increase in HSP mRNA levels during heat shock. Strains containing a tetracycline-repressible HSF1 allele were grown for 16 h in medium containing doxycycline, followed by heat shock for 60 min at 40°C. (A) Northern blot analysis showing HSP mRNA levels for the HT (YHN459) and control (YHN456) strains with the conditional tetracycline-repressible Hsf1 allele. (B) Graphical representation of HSP26 mRNA levels, normalized to ACT1, shows that the HT and control strains had a loss in heat-induced expression upon depletion of HSF1. Error bars show SEM. **, P < 0.01; +, present; −, absent.

FIG. 7.

The C-terminal activation domain is not required for a trehalose-dependent increase in HSP mRNA levels during heat shock. (A) Northern blot analysis showing HSP mRNA levels over the course of a 40°C heat shock for the HSFΔCAD HT (YHN2066) and HSFΔCAD control (YHN2063) strains containing an hsf1 (1-583) allele. (B) Graphical representation of HSP26 mRNA levels, normalized to ACT1, shows that the HSFΔCAD HT strain (gray squares) has higher heat-induced transcript levels than the HSFΔCAD control strain (black circles). Error bars show SEM. *, P < 0.05.

FIG. 8.

Changes in Hsf1 phosphorylation correlate with changes in trehalose-dependent transcriptional activity. Western blot analysis (A, C, E, G, and H) or immunoprecipitation-Western blot analysis (B, D, F, and I) was performed on the control strain (labeled C; YHN2020) or LT (YHN2058), HT (YHN2023), and VHT (YHN2022) strains carrying Myc-tagged HSF1. Cells were heat shocked and allowed to recover for the indicated times. Calf intestinal alkaline phosphatase (CIP) treatment (B, D, F, and I) confirmed that the shift in mobility of the Myc-HSF1 gene product was due to phosphorylation. +, present; −, absent.