Osmotic stress-induced gene expression in Saccharomyces cerevisiae requires Msn1p and the novel nuclear factor Hot1p

Abstract

After a sudden shift to high osmolarity, Saccharomyces cerevisiae cells respond by transiently inducing the expression of stress-protective genes. Msn2p and Msn4p have been described as two transcription factors that determine the extent of this response. In msn2 msn4 mutants, however, many promoters still show a distinct rise in transcriptional activity upon osmotic stress. Here we describe two structurally related nuclear factors, Msn1p and a newly identified protein, Hot1p (for high-osmolarity-induced transcription), which are also involved in osmotic stress-induced transcription. hot1 single mutants are specifically compromised in the transient induction of GPD1 and GPP2, which encode enzymes involved in glycerol biosynthesis, and exhibit delayed glycerol accumulation after stress exposure. Similar to a gpd1 mutation, a hot1 defect can rescue cells from inappropriately high HOG pathway activity. In contrast, Hot1p has little influence on the osmotic stress induction of CTT1, where Msn1p appears to play a more prominent role. Cells lacking Msn1p, Msn2p, Msn4p, and Hot1p are almost devoid of the short-term transcriptional response of the genes GPD1, GPP2, CTT1, and HSP12 to osmotic stress. Such cells also show a distinct reduction in the nuclear residence of the mitogen-activated protein kinase Hog1p upon osmotic stress. Thus, Hot1p and Msn1p may define an additional tier of transcriptional regulators that control responses to high-osmolarity stress.

FIG. 1

Hot1p is structurally related to Msn1p and Gcr1p. Pairwise alignments of the C-terminal regions of Hot1p with Msn1p and Gcr1p are shown. Arrows delineate a region of 64 residues that has the highest similarity. The similarity between Hot1p and Msn1p extends further towards the C-terminal end.

FIG. 2

Hot1p is a nuclear protein. Logarithmically growing cells of strain VRY140 (HOT1-GFP) were examined by fluorescence or light microscopy under normal growth conditions and 5 min after addition of NaCl to a final concentration of 0.4 M. DIC, differential interference contrast.

FIG. 3

Role of Hog1p and Hot1p in the induction of expression of GPD1 and GPP2. Cells of the wild type (●) and of the hot1 (○), hog1 (×), and hot1 hog1 (■) mutants were exposed to 0.7 M NaCl at time zero. (A) Northern blotting; (B) quantification. The highest relative mRNA level in the wild type was set to 100.

FIG. 4

GPD1-lacZ induction is affected by the hot1 mutation. The mutant effect was tested by using a reporter consisting of a CYC1-lacZ construct lacking its own UAS fused to part (position −693 to −177) of the GPD1 promoter. Wild-type (●) and hot1 (○) cells were transformed with the pGPD1-lacZ plasmid and exposed to 0.7 M NaCl, and total RNA was probed for lacZ mRNA. The graph shows lacZ mRNA levels relative to those of IPP1; the highest relative mRNA level in the wild type was set to 100.

FIG. 5

Effects of combinations of hot1, msn1, msn2, and msn4 mutations on osmotic induction of GPD1, GPP2, CTT1, and HSP12. Cells of the wild type (●) and the msn1 (○), msn2 msn4 (×), msn1 msn2 msn4 (■), hot1 (□), hot1 msn1 (▴), hot1 msn2 msn4 (▵), and hot1 msn1 msn2 msn4 (⧫) mutants were exposed to osmotic stress (0.7 M NaCl) at time zero. (A) Northern blotting; (B) quantification. The highest relative mRNA level in the wild type was set to 100.

FIG. 6

Effects of combinations of hot1, msn1, msn2, and msn4 mutations on GPD1 and CTT1 expression. mRNA levels during continuous growth in rich glucose medium containing no NaCl (black bars), 0.5 M NaCl (grey bars), or 0.85 M NaCl (open bars) are shown. The highest relative mRNA level in the wild type (WT) was set to 100.

FIG. 7

Induction by heat shock of GPD1 and CTT1 expression in hot1, msn1, and msn2 msn4 mutants. Cells of the wild type (●) and the hot1 (○), msn1 (×), and msn2 msn4 (■) mutants were exposed to heat stress (37°C) at time zero. (A) Northern blotting; (B) quantification. The highest relative mRNA level in the wild type was set to 100.

FIG. 8

Suppression of the growth defect caused by a constitutively activated HOG pathway. The wild type and hot1, hog1, msn2 msn4, msn1, and gpd1 mutants were transformed with plasmid pGAL1::SSK2ΔN, encoding Ssk2p with its N-terminal inhibitory domain (amino acids 1 to 1172) deleted (30). Expression of this construct is induced by a shift to galactose medium. Transformants were grown to logarithmic phase in selective medium containing glucose, sedimented, washed with water, and diluted to an OD₆₀₀ of 1.0. Ten microliters of this suspension and of a 1:10 dilution were dropped on plates containing glucose or galactose.

FIG. 9

Hot1p, Msn1p, Msn2p, and Msn4p affect kinetics of Hog1p phosphorylation and nuclear residence. (A) Logarithmically growing cells of the wild type (●) and the hot1 msn1 (○), msn2 msn4 (×), and msn1 msn2 msn4 hot1 (■) mutants were transformed with pVR65-WT (HOG1-GFP) and examined in time courses by fluorescence microscopy under normal growth conditions and after addition of NaCl to a final concentration of 0.4 M. (B) Determination of the phosphorylation profile of Hog1-GFP. Samples were taken at the time points indicated, and protein extracts were prepared. Western blots were analyzed with anti-active p38 antibody (Ab) or with anti-GFP antibody to determine the protein level of Hog1-GFP.

FIG. 10

Hot1p affects glycerol production rate (A) and glycerol accumulation (B). Cells of the wild type (●) and the hot1 (○), msn2 msn4 (×), and hot1 msn2 msn4 (■) mutants were exposed to 0.7 M NaCl at time zero. The total glycerol production rate and intracellular glycerol accumulation were measured as described in Materials and Methods.

FIG. 11

(A) Growth of a hot1 strain is unaffected by 1.5 M sorbitol. Cells were grown to logarithmic phase and diluted to an OD₆₀₀ of 1.0. A 10-fold serial dilution series was plated on rich glucose medium containing either no osmolyte or 1.5M sorbitol. (B) Cells with HOT1 deleted are more sensitive to severe osmotic stress conditions. Logarithmically growing strains were incubated in the presence of 0.4 M NaCl for 0 (black bars), 15 (grey bars), or 60 (open bars) min before the final concentration of NaCl was increased to 1.7 M. After 20 h, the samples were appropriately diluted and plated at about 300 cells per plate. After 3 days, the total number of colonies was counted and expressed relative to that in the corresponding unstressed sample.