Cooperative regulation of DOG2, encoding 2-deoxyglucose-6-phosphate phosphatase, by Snf1 kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade in stress responses of Saccharomyces cerevisiae

Abstract

We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of DOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of DOG2 gene expression were increased in a mig1Delta mutant, while the derepression of DOG2 was not observed in a snf1Delta mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Delta, hog1Delta, and snf1Delta. However, the osmotic induction was completely abolished in both the snf1Delta pbs2Delta mutant and the snf1Delta hog1Delta mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.

FIG. 1

Diagram of lacZ-LEU2 insertion in the DOG2 locus. Adenine (A) of the translational initiation codon (ATG) of the DOG2 gene was taken as +1. MBS, Mig1p-binding site; STRE, stress response element.

FIG. 2

(A) Effect of glucose starvation on derepression of DOG2-lacZ gene. Cells were cultured in YPD medium until the OD₆₁₀ reached approximately 0.8 to 1.0. After cultivation, the cells were collected by centrifugation, resuspended in fresh YPD medium (white bars) or fresh YEP medium without glucose (1% yeast extract, 2% peptone [pH 5.5]) (black bars), and cultured for another 1 h. Strains used were as follows: wild type (WT), SET8-1-C, mig1 mutant, SCMG1; snf1 mutant, SCS1. Data are a summary of three independent experiments (mean ± standard deviation). (B) Regulation of DOG2-lacZ expression by Msn2p and Msn4p. Cells were cultured in YPD medium until the OD₆₁₀ reached approximately 0.8 to 1.0, and 0.6 mM t-BHP (black bars) or solid NaCl (final concentration, 0.3 M) was added. Cells were cultured for another 1 h, and cell extracts were prepared to measure β-galactosidase activity. Strains used are as follows: wild type (WT), SET8-1-C; msn2 mutant, SCM2; msn4 mutant, SCM4; msn2/4 mutant, SCM24. Data are a summary of three independent experiments (mean ± standard deviation).

FIG. 3

Regulation of DOG2-lacZ expression by Snf1p kinase and HOG-MAP kinase cascade. Cells were cultured in YPD medium until the OD₆₁₀ reached approximately 0.8 to 1.0, and 0.6 mM t-BHP or solid NaCl (final concentration, 0.3 M) was added. Cells were cultured for another 1 h, and β-galactosidase activity was measured. Strains used are as follows: wild type (WT), SET8-1-C; pbs2 mutant, SCP2; hog1 mutant, SCH1; snf1 mutant, SCS1; snf1/pbs2 mutant, SCSP12; snf1/hog1 mutant, SCSH11. Data are a summary of three independent experiments (mean ± standard deviation).

FIG. 4

Effects of disruption of DOG2 and DOG1 on susceptibility to various stresses. (A) Cells were grown in fructose medium (2% fructose, 0.67% yeast nitrogen base without amino acids supplemented with appropriate amino acids and bases; pH 5.5) at 28°C with shaking for 16 h. A small portion of the culture was transferred to fresh fructose medium containing 0.4 mM t-BHP or 0.1% 2-deoxyglucose (2-deGlc) and cultured at 28°C to monitor the OD₆₁₀. Strains used are as follows: open triangles, YPH252 (wild type); open circles, SET8-1-C (dog2); closed circles, SCDG1 (dog1 dog2). (B) Cells (10⁵ cells/ml) were spotted (5 μl) on YPD agar plates with or without 0.9 M KCl and incubated at 28°C for 2 days. Strains used are as follows: wild type (WT), YPH252; dog2, SET8-1-C; dog1 dog2, SCDG1; pbs2, YPB2; hog1, YHG1.