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. 2011 Sep 30;333(6051):1891-4.
doi: 10.1126/science.1209126. Epub 2011 Aug 18.

Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response

Brooke M Gardner  1 , Peter Walter
Affiliations

Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response

Brooke M Gardner et al. Science. .

Abstract

The unfolded protein response (UPR) detects the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and adjusts the protein-folding capacity to the needs of the cell. Under conditions of ER stress, the transmembrane protein Ire1 oligomerizes to activate its cytoplasmic kinase and ribonuclease domains. It is unclear what feature of ER stress Ire1 detects. We found that the core ER-lumenal domain (cLD) of yeast Ire1 binds to unfolded proteins in yeast cells and to peptides primarily composed of basic and hydrophobic residues in vitro. Mutation of amino acid side chains exposed in a putative peptide-binding groove of Ire1 cLD impaired peptide binding. Peptide binding caused Ire1 cLD oligomerization in vitro, suggesting that direct binding to unfolded proteins activates the UPR.

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Figures

Fig. 1
Fig. 1
An unfolded protein co-immunoprecipitates with Ire1. A. The UPR activated by inducible CPY/CPY*-HA was measured with a 4×UPRE-GFP reporter. Fold induction is the ratio of median FITC intensity of cells in 2% galactose to cells in 2% raffinose after 4 hours. Error bars are SD; N≥4. B. FLAG-tagged Ire1 was immunoprecipitated from cells expressing CPY-HA or CPY*-HA (2 hours, 2% galactose); CPY/CPY*-HA was detected with anti-HA. The asterisk indicates unglycosylated CPY/CPY*.
Fig 2
Fig 2
Ire1 binds specific regions in CPY* containing basic and hydrophobic residues. A. Binding of 500 nM GST-cLD to a peptide array tiling along the sequence of CPY* was detected with anti-GST. B. The contribution of each amino acid in CPY*'s sequence to Ire1-cLD binding (WT and W426A) was calculated by averaging the intensity of spots containing that amino acid (see Methods). Values were plotted along the sequence of CPY*. SP: signal peptide. C. An array composed of peptides with single amino acid substitutions in peptide F17 from the CPY* tiling array (AQLAPYQRTGRNVYD) was probed with GST-cLDW426A and GST-cLD (Fig. S3). The binding intensity of the mutated peptides was normalized to the intensity of the wild-type peptide. Large arrows highlight the basic residues in F17. Blue: introduced basic residues. Red: introduced acidic residues. D. A peptide array of chaperone substrates shows that GST-cLDW426A and Kar2 bind a different subset of peptides.
Fig 3
Fig 3
A. Fluorescence anisotropy of ΔEspP-FAM (50 nM) and FAM-F17 (100 nM) binding to Ire1-cLDW426A. K1/2 for ΔEspP-FAM is 0.75±0.03 μM with a Hill coefficient of 1.2 and K1/2 for FAM-F17 is 172±4 μM with a Hill coefficient of 1.4. B. Induction of a HAC1 mRNA splicing reporter after a 3 hour incubation with 0 or 5 mM DTT in CRY1 Δire1∷KAN strains expressing no Ire1, WT Ire1, or Ire1-ΔMFY. N=5; error bars are SD; p value calculated by Student T-test between WT and ΔMFY. C. ΔMFY increases the K1/2 for Ire1-cLDW426A binding to ΔEspP-FAM to 42±2 μM, while the Hill coefficient decreases to 0.9. For A and C, N≥3; error bars are SD.
Fig 4
Fig 4
The addition of peptide causes Ire1 oligomerization. A. C(s) analysis of velocity sedimentation of 7 μM Ire1-cLD WT (blue) and with 50 μM ΔEspP (red). Without peptide: RMSD: 0.0105, f/f0: 1.65. With peptide: RMSD: 0.0080, f/f0: 2.75. B. C(s) analysis of the velocity sedimentation of 9 μM Ire1-cLDW426A (blue) and with 20 μM of ΔEspP (red). Without peptide: RMSD=0.0104, f/f0: 1.46. With peptide: 0.0086, f/f0: 1.52. C. Comparison of the sedimentation equilibrium (20,000×g, 12 hrs) of Ire1-cLDW426A (blue) and with a 2:1 molar ratio of ΔEspP (red) with simulated Ire1 monomer (dotted) and dimer (solid). The full data set and fit to a monomer-dimer model is presented in Fig. S8.
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Comment in

  • Cell biology. Sensing ER stress.
    Kawaguchi S, Ng DT. Kawaguchi S, et al. Science. 2011 Sep 30;333(6051):1830-1. doi: 10.1126/science.1212840. Science. 2011. PMID: 21960615 No abstract available.

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