Regulation of the unfolded protein response in yeast by oxidative stress

Abstract

In the unfolded protein response (UPR), Ire1 activates Hac1 to coordinate the transcription of hundreds of genes to mitigate ER stress. Recent work in Caenorhabditis elegans suggests that oxidative stress inhibits this canonical Ire1 signalling pathway, activating instead an antioxidant stress response. We sought to determine whether this novel mode of UPR function also existed in yeast, where Ire1 has been best characterized. We show that the yeast UPR is also subject to inhibition by oxidative stress. Inhibition is mediated by a single evolutionarily conserved cysteine, and affects both luminal and membrane pathways of Ire1 activation. In yeast, Ire1 appears dispensable for resistance to oxidative stress and, therefore, the physiological significance of this pathway remains to be demonstrated.

Keywords: Ire1; arsenic; cysteine; oxidative stress; unfolded protein response.

Figure 1.. Inhibition of the UPR by oxidative stress.

A) Relative protein abundance of UPR targets Kar2, Lhs1, and Pdi1 at 0, 1, and 4 h after treatment with sodium arsenite (1 mM). The data were generated using a quantitative mass spectrometry-based proteomic approach [16]. Pre2 is a proteasome subunit and a transcriptional target of the Rpn4 proteotoxic stress response which is best known for controlling proteasome abundance [16]. Error bars represent standard deviations from triplicate cultures. B) Splicing of the Ire1 target HAC1 in response to treatment with sodium arsenite (1 mM) or tunicamycin (5 μg/ml) for 1 hour, as determined by RT-PCR. Spliced and unspliced forms of HAC1 are indicated. ACT1 serves as a loading control (lower panel). C) Inhibition of tunicamycin-induced (5 μg/ml) or DTT-induced (1.5 mM) HAC1 splicing by concurrent treatment with sodium arsenite (1 mM), as determined by RT-PCR. Chemical treatment was for one hour. Lower panel, ACT1 loading control. D) Inhibition of tunicamycin-induced (5 μg/ml) expression of Kar2 (BiP) protein by concurrent treatment with sodium arsenite (1 mM) for one hour. Whole cell extracts were prepared and analyzed by SDS–PAGE followed by immunoblot with anti-Kar2 antibody (upper panel) or anti-Pgk1 antibody (lower panel; loading control). There was no tunicamycin-induced expression of Kar2 in the ire1Δ mutant. Images were quantitated using NIH Image J, and the relative expression of Kar2 and Pgk1 is indicated as a percentage of the untreated wild-type control (i.e. lane). E) Inhibition of tunicamycin-induced (5 μg/ml) or DTT-induced (1.5 mM) HAC1 splicing by concurrent treatment with hydrogen peroxide (5 mM), as determined by RT-PCR. Chemical treatment was for one hour. Lower panel, ACT1 loading control.

Figure 2.. Cysteine-832 mediates oxidative stress-induced inhibition of Ire1.

A) Sequence alignment of the critical region of Ire1 from S. cerevisiae, C. elegans, and humans. Cysteine-832 is highlighted in yellow. Cysteine-748 is highlighted in cyan. Asterisks, identical residues; double dots, highly similar residues; single dots, similar residues. B-C) The ire1-C832S mutant is refractory to arsenic-induced inhibition of HAC1 splicing, as determined by RT-PCR. HAC1 splicing was induced by either DTT (1.5 mM; panel B) or tunicamycin (5 μg/ml; panel C). Arsenite was used at 1 mM. Treatment was for one hour. Lower panels, ACT1 controls. D) The ire1-C832S mutant is refractory to arsenic-induced inhibition of transcription of the HAC1 target KAR2, as determined by RT-PCR. Treatment was with tunicamycin (5 μg/ml) and/or arsenite (1 mM) for one hour. Lower panel, ACT1 control. Images were quantitated using NIH Image J, and the relative expression of KAR2 and PGK1 is indicated as a percentage of the respective untreated control. E) The ire1-C832S mutant is refractory to hydrogen peroxide-induced inhibition of HAC1 splicing, as determined by RT-PCR. HAC1 splicing was induced by tunicamycin (5 μg/ml). Hydrogen peroxide was used at 5 mM. Chemical treatment was for one hour. Lower panel, ACT1 control.

Figure 3.. An Adjacent Cysteine (748) Does not Contribute to Arsenic-Induced Inhibition of Ire1.

A) Structural features of S. cerevisiae Ire1 (PDB: 3FBV). Cysteines 748 and 832 are indicated in blue. Cyan, catalytic loop. Green, activation loop. B) Inhibition of DTT-induced (1.5 mM) HAC1 splicing by concurrent treatment with sodium arsenite (1 mM) persists in the ire1-C748S mutant, as determined by RT-PCR. Chemical treatment was for one hour. Lower panel, ACT1 loading control.

Figure 4.. The ire1-C832S Mutant Does not Show Obvious Phenotypes or Alteration of MAP Kinase Activation

A) Growth of wild-type and ire1Δ strains expressing an empty vector, IRE1 wild-type, and ire1-C832S, as indicated. Cells were spotted in three-fold serial dilutions onto plates containing no drug, tunicamycin (1 μg/ml), or sodium arsenite (1 mM) and cultured for 2-4 days at 30°C. B) Arsenite-induced activation of the Hog1 is unaffected in the ire1Δ mutant. Cells were treated with sodium arsenite (1 mM) for hour. Whole cell extracts were prepared and analyzed by SDS-PAGE followed by immunoblotting for phosphorylated and total Hog1. Lower panel, Pgk1 (loading control). A parallel treatment with osmotic stress (0.4 M NaCl for 5 min) confirms the assignment of the phosphorylated Hog1 species. Parallel analysis of the hog1Δ strain confirms the accuracy of the Hog1 antibodies.