Fig 2.

Hac1 is required to induce GCREs. (A) Expression of GCRE6::lacZ was determined in the haploid Σ1278b wild-type strain (WT) (RH3409) as well as in Δhac1 (RH3363), Δgcn4 (RH3410), and Δgcn4 Δhac1 (RH3411) mutant strains each carrying a chromosomally integrated GCRE6::lacZ reporter. Expression was measured under different nutritional conditions. Starting from one overnight culture, strains were diluted into fresh medium and further cultivated for 6 to 24 h in the respective media before specific β-galactosidase activities were assayed. Cultures were grown to log phase in YNB under nonstarvation conditions [light gray bars, YNB (6 h)]. Amino acid starvation and ER stress conditions were induced by addition of either 10 mM 3AT [dark gray bars, +3AT (8 h)] or 1 μg/ml tunicamycin [black bars, +Tm (6 h)]. For nitrogen starvation (N-starv), yeast cells were washed twice with 2% glucose before incubation for 24 h in minimal medium containing only 50 μM ammonium sulfate as the sole nitrogen source [white bars, N-starv (24 h)]. Units of specific β-galactosidase activities are shown in nanomoles per minutes per milligram. The bars represent the mean values of at least three independent measurements. As additional control, the haploid Σ1278b wild-type strain (WT) (RH2816), as well as Δhac1 (RH3351), Δgcn4 (RH2676), and Δgcn4 Δhac1 (RH3402) mutant strains, was transformed with the UPRE-CYC1-lacZ reporter gene carried on a multicopy vector (pMCZ-Y). Starting from one overnight culture, strains were diluted into fresh medium and further cultivated for 6 h in the respective media before specific β-galactosidase activities were assayed. Either cultures were grown to log phase in YNB medium [light gray bars, YNB (6 h)], or ER stress conditions were induced by the addition of 1 μg/ml tunicamycin for 2 h [black bars, YNB (4 h) + Tm (2 h)]. Units of specific β-galactosidase activities are shown in nanomoles per minutes per milligram. The bars represent the mean values based on quadruplicate determinations of at least three independent transformants. (B) The diploid wild-type yeast strain (WT) (RH3421), as well as homo- and heterozygous Δhac1/Δhac1 (RH3422), Δhac1/HAC1 (RH3423), Δgcn4/Δgcn4 (RH2398), Δgcn4/GCN4 (RH3424), Δgcn4/Δgcn4 Δhac1/Δhac1 (RH3350), and Δgcn4/GCN4 Δhac1/HAC1 (RH3425) mutant strains each carrying a chromosomally integrated GCRE6::lacZ reporter, was grown to log phase in YNB in the absence (light gray bars, YNB) or presence (dark gray bars, +3AT) of 10 mM 3AT before specific β-galactosidase activities were assayed. As a further control, the diploid yeast strains indicated on the abscissa were transformed with the UPRE-CYC1-lacZ reporter gene (pMCZ-Y) and transformants were grown in the presence or absence of tunicamycin before being used for β-galactosidase assays.