Association of constitutive hyperphosphorylation of Hsf1p with a defective ethanol stress response in Saccharomyces cerevisiae sake yeast strains

Abstract

Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains.

Fig 1

HSE-mediated gene expression activities in laboratory and sake yeast strains. (A) β-Galactosidase activities of the HSE-pCYC1-lacZ reporter in K701 AUR1::AUR1-C-HSE-pCYC1-lacZ (■) and X2180 AUR1::AUR1-C-HSE-pCYC1-lacZ (○) under sake brewing conditions. The data represent average values ± the standard deviations (SD) of two independent experiments. *, significantly higher than the value on day 1 (Student t test, P < 0.05). (B) β-Galactosidase activities of the HSE-pCYC1-lacZ reporter in K701 AUR1::AUR1-C-HSE-pCYC1-lacZ and X2180 AUR1::AUR1-C-HSE-pCYC1-lacZ under nonstress (■) or 8% ethanol stress (□) conditions. The data represent average values ± the SD of four independent experiments. (C) β-Galactosidase activities of the HSE-pCYC1-lacZ reporter in K701 AUR1::AUR1-C-HSE-pCYC1-lacZ and X2180 AUR1::AUR1-C-HSE-pCYC1-lacZ under nonstress (■) or heat shock (41°C, □) conditions. The data represent average values ± the SD of four independent experiments.

Fig 2

Immunoblot analyses of Hsf1p in laboratory and sake yeast strains. (A) Phosphorylation states of Hsf1p in X2180 (upper) and K701 (lower) during sake fermentation. The rightmost lanes show the same samples as day 9 of K701 (upper) and day 21 of X2180 (lower) as controls. (B) Phosphorylation states of Hsf1p under 8% ethanol stress in X2180 (upper) and K701 (lower). (C) Effects of phosphatase treatment on Hsf1p. The cell extracts of X2180 and K701 under nonstress and 8% ethanol stress conditions were incubated in the absence (left panel) or presence (right panel) of calf intestinal alkaline phosphatase (CIAP) and subjected to immunoblot analysis. The white dashed line represents the position of furthest migration of the dephosphorylated forms of Hsf1p. (D) Phosphorylation states of Hsf1p under heat shock (41°C) in X2180 (upper) and K701 (lower). White triangles, gray triangles, and asterisks indicate hypophosphorylated, hyperphosphorylated, and superhyperphosphorylated forms of Hsf1p, respectively, whose positions were determined by comparison with the control samples analyzed in the same gel. The arrows indicate the direction of electrophoretic migration.

Fig 3

Exchange of ScHSF1 or K7HSF1 alleles has no apparent effects on Hsf1p-mediated stress responses. (A) Mutation points in the K7HSF1 allele revealed by whole-genome sequencing of K7 (http://nribf1.nrib.go.jp/SYGD). White circles, arrows, and the asterisk indicate nonsynonymous SNPs, other SNPs, and an adenine-nucleotide insertion, respectively. (B) β-Galactosidase activities of the HSE-pCYC1-lacZ reporter in X2180 Δhsf1 AUR1::AUR1-C-HSE-pCYC1-lacZ and K701 Δhsf1 AUR1::AUR1-C-HSE-pCYC1-lacZ background strains expressing ScHSF1 or K7HSF1 under nonstress (■) and 8% ethanol stress (□) conditions. The data represent average values ± the SD of three or more independent experiments. (C) Phosphorylation states of Hsf1p in X2180 Δhsf1 and K701 Δhsf1 background strains expressing ScHSF1 or K7HSF1 under nonstress and 8% ethanol stress conditions. The same symbols as in Fig. 2 are used to show the phosphorylation states of Hsf1p, whose respective positions were determined by comparison with the control samples analyzed in the same gel. The arrow indicates the direction of electrophoretic migration.

Fig 4

Phosphorylation states of Hsf1p in wild-type cells and the indicated phosphatase gene disruptants in a BY4743 background under nonstress and 8% ethanol stress conditions. The same symbols as in Fig. 2 are used to indicate the phosphorylation states of Hsf1p. The arrows indicate the direction of electrophoretic migration.

Fig 5

Loss of PPT1 is related to the marked hyperphosphorylation of Hsf1p. (A) Schematic representation of the PPT1 locus in laboratory and sake strains revealed by whole-genome sequencing of K7 (1). Black, white, and gray symbols indicate ORFs, tRNA genes, and a retrotransposon and LTRs, respectively. Numbers indicate the chromosomal positions of gene replacement by a Ty2 element. (B) Phosphorylation states of Hsf1p under 8% ethanol stress conditions in the laboratory, classic sake, and modern sake strains. The same symbols as in Fig. 2 are used to indicate the phosphorylation states of Hsf1p, whose respective positions were determined by comparison with the control samples analyzed in the same gel. The arrow indicates the direction of electrophoretic migration.

Fig 6

Effects of the loss of PPT1 on the HSE-mediated stress response and fermentation properties in strains BY4743, BY4743 Δppt1, and K701. (A) β-Galactosidase activities of the HSE-pCYC1-lacZ reporter in BY4743 AUR1::AUR1-C-HSE-pCYC1-lacZ, BY4743 Δppt1 AUR1::AUR1-C-HSE-pCYC1-lacZ, and K701 AUR1::AUR1-C-HSE-pCYC1-lacZ background strains transformed with a PPT1 expression construct (+ PPT1) or empty vector (+ Vector) under nonstress (■) and 8% ethanol stress (□) conditions. The data represent average values ± the SD of three or more independent experiments. (B) Sake fermentation profiles of BY4743 (gray line) and BY4743 Δppt1 (black line). Total carbon dioxide emission during fermentation progression was monitored using a Fermograph II (Atto), as previously described (42). Averaged data from three independent experiments are shown. *, significantly higher than the wild type (Student t test, P < 0.05).