Evidence of a new role for the high-osmolarity glycerol mitogen-activated protein kinase pathway in yeast: regulating adaptation to citric acid stress

Abstract

Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite minor activation of glycerol biosynthesis, the inhibitory effect of citric acid was not due to an osmotic shock. HOG1 negatively regulated the expression of a number of proteins in response to citric acid stress, including Bmh1p. Evidence suggests that BMH1 is induced by citric acid to counteract the effect of amino acid starvation. In addition, deletion of BMH2 rendered cells sensitive to citric acid. Deletion of the transcription factor MSN4, which is known to be regulated by Bmh1p and Hog1p, had a similar effect. HOG1 was also required for citric acid-induced up-regulation of Ssa1p and Eno2p. To counteract the cation chelating activity of citric acid, the plasma membrane Ca(2+) channel, CCH1, and a functional vacuolar membrane H(+)-ATPase were found to be essential for optimal adaptation. Also, the transcriptional regulator CYC8, which mediates glucose derepression, was required for adaptation to citric acid to allow cells to metabolize excess citrate via the tricarboxylic acid (TCA) cycle. Supporting this, Mdh1p and Idh1p, both TCA cycle enzymes, were up-regulated in response to citric acid.

FIG. 1.

Growth of S. cerevisiae BY4741 is inhibited in the presence of citric acid. (a) Cultures of S. cerevisiae BY4741 were grown to late exponential phase in ME medium alone (pH 3.5) and in the presence of 200, 300, and 400 mM citric acid (CA). (b) Serial dilutions of mid-exponential-phase cultures of S. cerevisiae BY4741 were spotted onto ME agar plates at pH 3.5 with and without the presence of 200, 300, and 400 mM citric acid. Plates were incubated for 48 h at 30°C before the plates were photographed.

FIG. 2.

Identification of changes in protein expression induced in S. cerevisiae BY4741 due to growth in the presence of 300 mM citric acid, pH 3.5. Total soluble proteins were separated by IEF over the pH range 4.5 to 5.5. Proteins were prepared from control cells (grown in ME medium [pH 3.5]) and cells grown in ME medium (pH 3.5) plus 300 mM citric acid (CA). The positions of Ado1p (protein 1), Gpp2p (protein 2), hypothetical ORF Ylr301wp (protein 3), His7p (protein 4), Sti1p (protein 5), Ura2p (protein 6), and Pdc1p (protein 7) are indicated. Molecular masses (in kilodaltons) are shown on the y axis, and pI values are shown on the x axis. A representative result from two replicate experiments is shown. No ID, no protein identification.

FIG. 3.

Identification of changes in protein expression induced in S. cerevisiae BY4741 due to growth in the presence of 300 mM citric acid, pH 3.5. Total soluble proteins were separated by IEF over the pH range 4 to 7. Proteins were prepared from control cells (grown in ME medium [pH 3.5]) and cells grown in ME medium (pH 3.5) plus 300 mM citric acid (CA). The positions of Ssa1p (protein 8), Vma4p (protein 9), Rps0ap (protein 10), Gpp1p (protein 11), Ipp1p (protein 12), Eno2p (protein 13), Ssb2p (protein 14), and Hsp26p (protein 15) are indicated. Molecular masses (in kilodaltons) are shown on the y axis, and pI values are shown on the x axis. A representative result of two replicate experiments is shown.

FIG. 4.

Identification of changes in protein expression induced in S. cerevisiae BY4741 due to growth in the presence of 300 mM citric acid, pH 3.5. Total soluble proteins were separated by IEF over the pH range 6 to 11. Proteins were prepared from control cells grown in ME medium [pH 3.5]) and cells grown in ME medium (pH 3.5) plus 300 mM citric acid (CA). The positions of Atp1p (protein 16), Imd3p (protein 17), Ilv5p (protein 18), Mdh1p (protein 19), Sis1p (protein 20), Aro3p (protein 21), and Idh1p (protein 22) are indicated. Molecular masses (in kilodaltons) are shown on the y axis, and pI values are shown on the x axis. A representative result of two replicate experiments is shown.

FIG. 5.

Growth inhibition of S. cerevisiae BY4741 MATa (BY4741/a) and single deletion mutants of genes that constitute the HOG pathway or are known to regulate or be regulated by the HOG pathway. (a) Cultures were grown to late exponential phase in ME medium (pH 3.5) in the presence or absence of 300 mM citric acid. For each strain, growth inhibition is expressed as the percent inhibition of growth rate. This was calculated by measuring the growth rate in the presence or absence of 300 mM citric acid and expressing the additional inhibition seen in the citric acid-treated culture relative to the untreated culture as a percentage. Three replicate experiments were performed. The values are shown as the means ± standard deviations (error bars) of the three experiments. (b) Complementation of the Δhog1 citric acid-sensitive phenotype. Growth inhibition of S. cerevisiae BY4741 MATa (BY4741/a), Δhog1 mutant, and Δhog1 mutant carrying pRS313::HOG1 is shown on ME agar (pH 3.5) and ME agar (pH 3.5) plus 200, 300, and 400 mM citric acid (CA). A representative result is shown.

FIG. 6.

Western blot demonstrating the dual phosphorylation of Hog1p upon exposure to citric acid. Soluble protein extracts were prepared from S. cerevisiae BY4741 MATa (BY4741/a) and Δhog1, Δssk1, and Δpbs2 mutants with or without a 10-min exposure to 300 mM citric acid (CA) and 0.4 M NaCl in ME medium (pH 3.5). Dually phosphorylated forms of Hog1p were detected using anti-phospho-p38. To check that equal amounts of protein were loaded in the lanes, Hog1p was detected using a polyclonal anti-C-terminal Hog1p antibody. A representative result is shown.

FIG. 7.

Identification of citric acid-induced proteins whose expression was dependent on deletion of the HOG1 gene. Protein expression in wild-type (WT) S. cerevisiae BY4741 and Δhog1 mutant in the presence or absence of 300 mM citric acid (CA), pH 3.5, is shown. Total soluble proteins were separated by IEF over the pH range 4 to 7. Proteins were prepared from untreated (control) cells (grown in ME medium [pH 3.5]) and cells grown in ME medium (pH 3.5) plus 300 mM citric acid (CA). The positions of Bmh1p (protein 23), Pdb1p (protein 24), Ura1p (protein 25), Fba1p (protein 26), YDR533cp (protein 27), Gnd1p (protein 28), and Car1p (protein 29) are indicated. We also detected a protein whose expression was unaffected by exposure to citric acid alone but whose expression was repressed in the Δhog1 deletion mutant in the presence of citric acid. This protein was identified as Rps0bp (protein A). Molecular masses (in kilodaltons) are shown on the y axis, and pI values are shown on the x axis. A representative result of two replicate experiments is shown.

FIG. 8.

Identification of two citric acid-induced proteins whose expression is dependent on fully functional HOG1. Protein expression in wild-type (WT) S. cerevisiae BY4741 and Δhog1 mutant in the presence or absence of 300 mM citric acid (pH 3.5) is shown. Total soluble proteins were separated by IEF over the pH range 4 to 7. Proteins were prepared from untreated (control) cells (grown in ME medium [pH 3.5]) and cells grown in ME medium (pH 3.5) plus 300 mM citric acid (CA). The positions of Ssa1p (protein 8) and Eno2p (protein 13) are indicated. We also detected a protein whose expression was unaffected by exposure to citric acid alone but whose expression was repressed in the Δhog1 deletion mutant in the presence of citric acid. This protein was identified as Egd2p (protein B). Molecular masses (in kilodaltons) are shown on the y axis, and pI values are shown on the x axis. A representative result of two replicate experiments is shown.

FIG. 9.

Summary of our findings regarding the role of the HOG pathway in adaptation to citric acid stress in S. cerevisiae. Proteins and genes that are sensitive to the deletion of citric acid or that are up-regulated on the miniarray and proteome are indicated. Abbreviations: FBP, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; G3P, glucose-3-phosphate.