Activation of Gcn2 in response to different stresses

Abstract

All organisms have evolved pathways to respond to different forms of cellular stress. The Gcn2 kinase is best known as a regulator of translation initiation in response to starvation for amino acids. Work in budding yeast has showed that the molecular mechanism of GCN2 activation involves the binding of uncharged tRNAs, which results in a conformational change and GCN2 activation. This pathway requires GCN1, which ensures delivery of the uncharged tRNA onto GCN2. However, Gcn2 is activated by a number of other stresses which do not obviously involve accumulation of uncharged tRNAs, raising the question how Gcn2 is activated under these conditions. Here we investigate the requirement for ongoing translation and tRNA binding for Gcn2 activation after different stresses in fission yeast. We find that mutating the tRNA-binding site on Gcn2 or deleting Gcn1 abolishes Gcn2 activation under all the investigated conditions. These results suggest that tRNA binding to Gcn2 is required for Gcn2 activation not only in response to starvation but also after UV irradiation and oxidative stress.

Fig 1. Inhibition of translation does not prevent Gcn2 activation after UV irradiation in fission yeast.

Fig 2. Mutating the tRNA-binding sites of Gcn2 abolishes activation in response to UVC irradiation.

A Alignment of the HisRS-like domain of budding yeast and fission yeast Gcn2. B The indicated strains were irradiated with 1100 J/m² and samples were taken at the indicated times after irradiation. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading.

Fig 3. Gcn1 is required for Gcn2 activation after UVC irradiation.

A The indicated strains were starved for leucine as described in Materials and Methods. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading. B eIF2α phosphorylation after UVC-irradiation in wild-type, gcn2Δ and gcn1Δ cells. Exponentially growing cells of the indicated strains were irradiated as described in Materials and methods and [23]. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading. C preRC loading in gcn1Δ cells. Cells carrying a cdc10-M17 mutation and GFP-tagged Mcm2 were grown in EMM medium, arrested in G1 by shifting them to 36°C for 4 h, released from the G1 block and irradiated with 1100 J/m² UVC. The percentage of cells containing chromatin-bound Mcm2:GFP was determined. The delay was calculated as the time difference between irradiated and unirradiated cells at reaching 70% of maximal preRC loading. D Prototroph wild-type cells were grown to mid-log phase in EMM medium. Supplements were added to 80 mg/l for 30 min before UV irradiation. Samples were taken immediately after irradiation. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading.

Fig 4. Gcn1 is required for Gcn2 activation after H₂O₂- treatment.

eIF2α phosphorylation after H₂O₂ treatment in wild-type, gcn2Δ and gcn1Δ cells. The indicated strains were grown in EMM medium and treated with H₂O₂ at the concentrations shown, for 15 minutes. eIF2α phosphorylation was detected by immunoblotting, α-tubulin levels are shown to check even loading.