Fig 6. Snf1 downregulates the protein level of Ssk1.

(A, B) Genetic interaction between the ypd1Δ, sln1Δ and reg1Δ mutations. Diploid ypd1Δ/YPD1 reg1Δ/REG1 and sln1Δ/SLN1 reg1Δ/REG1 yeast cells were sporulated, dissected on YPD plates and the meiotic products were incubated for 4 days at 25°C. Each genotype was shown in the right panel. Wild-type cells were labeled with W. The ypd1Δ, sln1Δ, and reg1Δ mutations were labeled with y, s, and r, respectively. (C) Effects of hyperosmotic stress on Ssk1 expression. Wild-type (WT) cells harboring Myc-tagged SSK1 were grown at 25°C until exponential phase and treated with 0.4 M sodium chloride (NaCl) for the indicated time. Extracts prepared from each cell were immunoblotted with anti-Myc (Ssk1-Myc) and anti-Mcm2 antibodies. The intensities of Ssk1-Myc were measured and normalized to Mcm2 level. The values are plotted as the fold change from cells at the time of NaCl addition. The data show mean ± SEM (n = 4). The statistical difference was determined by Student’s t-test. ns, not significant. (D) Effects of the snf1Δ mutation on ER stress-induced upregulation of Ssk1. Wild-type (WT) and snf1Δ mutant strains harboring Myc-tagged SSK1 were grown at 25°C until exponential phase and treated with 4 mM dithiothreitol (DTT) for the indicated time. Immunoblot was performed as described in (C). The intensities of Ssk1-Myc were measured and normalized to the Mcm2 level. The values are plotted as the fold change from wild-type cells at the time of DTT addition. The data show mean ± SEM (n = 4). *P < 0.05 as determined by Student’s t-test. (E) Effects of the reg1Δ and snf1Δ mutations on DTT-induced upregulation of Ssk1. Wild-type (WT) and reg1Δ and reg1Δ snf1Δ mutant strains harboring Myc-tagged SSK1 were analyzed as described in (D). The data show mean ± SEM (n = 4). *P < 0.05 and **P < 0.01 as determined by Student’s t-test. (F) Effects of the snf1Δ and reg1Δ mutations on tunicamycin-induced upregulation of Ssk1. Wild-type (WT) and reg1Δ and reg1Δ snf1Δ mutant strains harboring Myc-tagged SSK1 were grown at 25°C until exponential phase and treated with 2 μg/ml tunicamycin (TM) for the indicated time. Immunoblot was performed as described in (C). (G) Effects of the ssk1Δ and snf1Δ mutations on ER stress-induced Hog1 activation. Wild-type (WT) and ssk1Δ, and snf1Δ ssk1Δ mutant strains were grown at 25°C until exponential phase and treated with 4 mM dithiothreitol (DTT) for the indicated time. Extracts prepared from each cell were immunoblotted with anti-phospho-p38 (P-Hog1) and anti-Hog1 antibodies. (H) ER stress sensitivity in the snf1Δ ssk1Δ mutants. Wild-type (WT) and ssk1Δ, snf1Δ, and snf1Δ ssk1Δ mutant strains were spotted onto YPD medium lacking or containing 1 or 1.5 μg/ml tunicamycin (TM) and incubated at 25°C.