Fig 3.

Tunicamycin-mediated ER stress represses GCN4 mRNA translation by a Hac1-independent mechanism. (A) Expression of a GCN4::lacZ fusion gene (p180) was measured in a haploid Σ1278b wild-type strain (WT) (RH2816) as well as in Δhac1 (RH3351), Δgcn4 (RH2676), and Δgcn4 Δhac1 (RH3402) mutant strains under different nutritional conditions. Bars depict means of at least three independent measurements of β-galactosidase activities. (B) Crude protein extracts were prepared from haploid Σ1278b wild-type cells (WT) (RH2816) as well as from Δgcn4 (RH2676) and Δhac1 (RH3351) mutant strains, respectively. Cells were grown either under normal conditions (untreated) or in the presence of ER stress conditions induced by 1 μg/ml tunicamycin (Tm). Additional amino acid starvation was obtained by adding 10 mM 3AT. Starting from one main culture with an OD₆₀₀ of ∼0.8 at 30°C, cultures were quartered and cultivated for a further 90 min under the indicated conditions. Protein levels of Hac1 and eIF2α-P were analyzed by immunoblotting using specific antibodies. eIF2α was used as a loading control.