Stress-activated protein kinase pathway functions to support protein synthesis and translational adaptation in response to environmental stress in fission yeast

Abstract

The stress-activated protein kinase (SAPK) pathway plays a central role in coordinating gene expression in response to diverse environmental stress stimuli. We examined the role of this pathway in the translational response to stress in Schizosaccharomyces pombe. Exposing wild-type cells to osmotic stress (KCl) resulted in a rapid but transient reduction in protein synthesis. Protein synthesis was further reduced in mutants disrupting the SAPK pathway, including the mitogen-activated protein kinase Wis1 or the mitogen-activated protein kinase Spc1/Sty1, suggesting a role for these stress response factors in this translational control. Further polysome analyses revealed a role for Spc1 in supporting translation initiation during osmotic stress, and additionally in facilitating translational adaptation. Exposure to oxidative stress (H2O2) resulted in a striking reduction in translation initiation in wild-type cells, which was further reduced in spc1- cells. Reduced translation initiation correlated with phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in wild-type cells. Disruption of Wis1 or Spc1 kinase or the downstream bZip transcription factors Atf1 and Pap1 resulted in a marked increase in eIF2alpha phosphorylation which was dependent on the eIF2alpha kinases Hri2 and Gcn2. These findings suggest a role for the SAPK pathway in supporting translation initiation and facilitating adaptation to environmental stress in part through reducing eIF2alpha phosphorylation in fission yeast.

FIG. 1.

Effects of osmotic and oxidative stress on protein synthesis in wild-type (wt) and spc1-m13 cells. (A) [³⁵S]methionine incorporation in wild-type (TH9) and spc1-m13 (TH123) cells under osmotic stress. Cells were treated with 0.6 M KCl for times indicated and were labeled for 20 min prior to harvesting. Radiolabeled proteins were visualized by SDS-PAGE, followed by autoradiography (top panel), and quantitated using the Quantity One software. Coomassie stain of above gel showed the total protein levels loaded in each lane (bottom panel). (B) Graph representing the quantification of three independent experiments as described above. (C) [³⁵S] methionine incorporation in wild-type (TH9) and spc1-m13 (TH123) cells under oxidative stress. Cells were treated with 1 mM H₂O₂ for times indicated and were labeled for 20 min prior to harvesting. Radiolabeled proteins were visualized by SDS-PAGE, followed by autoradiography (top panel), and quantitated using the Quantity One software. Coomassie stain of above gel showed the total protein levels loaded in each lane (bottom panel). (D) Graph representing the quantification of three independent experiments as described above. (E) Cell viability of wild-type (black squares) and spc1-m13 (gray triangles) cells following exposure to 1 mM H₂O₂ for the times indicated. Values represent an average of three independent experiments.

FIG. 2.

Effects of osmotic and oxidative stresses on protein synthesis and viability in wis1⁻ cells. (A) [³⁵S] methionine incorporation levels in wis1⁻ (TH815) cells. Cells were subjected to 0.6 M KCl or 1 mM H₂O₂ and labeled for 20 min prior to harvesting. [³⁵S]methionine incorporation levels were determined by SDS-PAGE analysis (top panel). Coomassie stain of above gel showing total protein levels is presented (bottom panel). (B) Graph representing the quantification of above gel. Standard deviations do not exceed 10% of the mean value. (C) Cell viability of wild-type (black squares), and wis1⁻ (black circles) cells following exposure to 1 mM H₂O₂ for the times indicated. Values represent an average of three independent experiments.

FIG. 3.

Polysome profile analysis of wild-type (wild-type) and spc1-m13 cells under stress conditions. Exponentially growing cultures of wild-type (TH9) and spc1-m13 (TH123) cells in YE5S were incubated in 0.6 M KCl (A) or 1 mM H₂O₂ (B) for the time indicated. Samples were collected and polysome profile analysis performed following velocity sedimentation of whole cell extracts on sucrose gradients (7 to 47%). Fractions were scanned at 254 nm and absorbance profiles are shown (from 0 to 1.0) with sucrose concentrations increasing from left to right, as shown (gray gradients). The positions of 80S ribosomes and polysomes are indicated. The graphs represent the quantification of the polysome-to-monosome ratio of wild-type and spc1-m13 cells after exposure to 0.6 M KCl (left graph) and to 1 mM H₂O₂ (right graph). Standard deviations do not exceed 10% of the mean value.

FIG. 4.

Elevated eIF2α phosphorylation occurs in spc1-m13 cells during oxidative stress. (A) Wild-type (TH9) or spc1-m13 (TH123) cells were incubated at 30°C in the presence of 1 mM H₂O₂ for the times indicated. Total protein extracts were analyzed by immunoblotting using an antibody that specifically recognizes phosphorylated eIF2α(eIF2α-P) or antiserum that recognizes total eIF2 (eIF2α). (B) Wild-type (TH9), wis1⁻ (TH 815), spc1-m13 (TH123), atf1⁻ (TH393), pap1⁻ (TH 454), and atf1⁻ pap1⁻ (TH474) cells were incubated for 20 min at 30°C in the absence (left panel) or presence of 1 mM H₂O₂ (right panel). Western blotting was performed as described above.

FIG. 5.

Effects of eIF2 kinase deletion on eIF2α phosphorylation and viability in wild-type and sty1-1 cells during oxidative stress. (A) eIF2α phosphorylation in wild-type (wt) eIF2 kinase deleted and sty1-1 cells during oxidative stress. Wild-type (TH9), sty1-1 (TH 1740), hri1Δ hri2Δ (TH2019), hri2Δ gcn2Δ (TH2021), sty1-1 hri1Δ (TH2025), sty1-1 hri1Δ hri2Δ (TH2026), sty1-1 hri2Δ (TH2028), sty1-1 gcn2Δ (TH2029), and sty1-1 hri2Δ gcn2Δ (TH2031) were incubated at 30°C in the absence or presence of 1 mM H₂O₂. Total protein extracts were analyzed by immunoblotting using an antibody that specifically recognizes phosphorylated eIF2α (eIF2α-P) or antiserum that recognizes total eIF2 (eIF2α). (B) Sensitivity of eIF2 kinases and Spc1/Sty1 mutants to oxidative stress. Tenfold serial dilutions of wild-type (TH9), sty1-1 (TH1740), sty1-1 hri2Δ gcn2Δ (TH2031), hri2Δ gcn2Δ (TH2021), hri1Δ hri2Δ (TH2019), and sty1-1 hri1Δ hri2Δ (TH2026) were plated on YE5S medium (control) and YE5S medium containing 2 mM H₂O₂ (as indicated) and incubated for 5 days at 30°C.