1. Cell Biology

Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR

  1. Niko Amin-Wetzel
  2. Lisa Neidhardt
  3. Yahui Yan
  4. Matthias P Mayer
  5. David Ron  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. DKFZ-ZMBH Alliance, Germany
https://doi.org/10.7554/eLife.50793
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Cite this article
  1. Niko Amin-Wetzel
  2. Lisa Neidhardt
  3. Yahui Yan
  4. Matthias P Mayer
  5. David Ron
(2019)
Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR
eLife 8:e50793.
https://doi.org/10.7554/eLife.50793
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10 figures, 3 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Fusion of ERdj4’s J-domain to IRE1LD promotes efficient BiP association thereby repressing IRE1 activity in cells.

(A) Two dimensional plots of CHOP::GFP and XBP1s::Turquoise signals from CHO-K1 dual UPR reporter cells stably expressing the indicated IRE1 variants [IRE1 wild-type (wt), J-IRE1 or JQPD-IRE1 fusion;…

Figure 1—source data 1

Source data for Figure 1C.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig1-data1-v2.xlsx
Figure 1—source data 2

Source data for Figure 1E .

https://cdn.elifesciences.org/articles/50793/elife-50793-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
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Modification of the endogenous Ern1 locus to introduce IRE1LD variants.

(A) Schematic description of the homologous recombination platform for creating IRE1-encoding alleles at the endogenous Ern1 locus (ΔIRE1, Kono et al., 2017). Cas9-CRISPR guides generated a large …

Figure 2 with 2 supplements
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Binding of MPZ-N peptide to IRE1LD does not promote IRE1LD dimerisation.

(A) Size-exclusion chromatography (SEC) elution profiles of TAMRA (TMR)-labelled wild-type IRE1LD at the indicated concentrations in presence and absence of MPZ-N peptide. TMR fluorescence is …

Figure 2—source data 1

Source data for Figure 2A and B.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig2-data1-v2.xlsx
Figure 2—source data 2

Source data for Figure 2D.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
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Biochemical properties of disulphide-linked dimeric IRE1LD Q105C SS and monomeric variants used to study MPZ-N’s interaction with IRE1LD.

(A) Size-exclusion chromatography (SEC) elution profiles of wild-type (wt) and monomeric IRE1LD W125A and IRE1LD P108A proteins at the indicated concentrations. Protein absorbance at 280 nm (A280) …

Figure 2—figure supplement 1—source data 1

Source data for Figure 2—figure supplement 1A.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig2-figsupp1-data1-v2.xlsx
Figure 2—figure supplement 1—source data 2

Source data for Figure 2—figure supplement 1D.

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Figure 2—figure supplement 2
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Implications of the distance constraint arising from the paramagnetic relaxation enhancement (PRE) experiments with IRE1LD and an MPZ-proxyl-labelled peptide (Karagöz et al., 2017) to the possible modes of peptide binding.

(A) Cartoon representation of the IRE1LD dimer with protomers in light and dark grey (PDB: 2HZ6). The two helices flanking the MHC-like groove are coloured in blue. The isoleucines (Ile) whose peaks …

Figure 3 with 1 supplement
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Identification of flexible regions in IRE1LD that are important for the regulation of IRE1 activity in cells.

(A) Left panel shows a bar diagram of the percentage of amide hydrogen exchange (%ex) of the indicated by IRE1LD segments after 30 and 300 s incubation in D2O. The amino acids (aa) covered by the …

Figure 3—source data 1

Source data for Figure 3A.

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Figure 3—figure supplement 1
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IRE1’s tail region is involved in maintaining the repressed state of IRE1 in vivo.

(A) Schematic representation of IRE1. The signal peptide (SP), luminal domain (LD), comprised of a structured core (CLD) and an unstructured tail, the transmembrane (TM) domain and the cytosolic …

Figure 4 with 1 supplement
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Cells expressing IRE1LD deletion variants exhibit a de-repressed IRE1 phenotype that correlates with less BiP bound to IRE1.

(A) Bar diagram of median XBP1::Turquoise and CHOP::GFP signals from untreated and tunicamycin (Tm)-treated CHO-K1 dual UPR reporter cells with Ern1 alleles encoding wild-type (wt) or the indicated …

Figure 4—source data 1

Source data for Figure 4A.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig4-data1-v2.xlsx
Figure 4—source data 2

Source data for Figure 4B.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
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Single or double deletion of a flexible loop and the tail within IRE1LD de-repressed IRE1 basal activity.

(A) Two dimensional contour plots of untreated and tunicamycin (Tm)-treated CHO-K1 CHOP::GFP and XBP1s::Turquoise dual UPR reporter cells expressing the indicated alleles (as in Figure 4A) analysed …

Figure 5 with 1 supplement
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Impaired BiP binding and monomerisation of IRE1LD ΔΔ in vitro.

(A) Left panel shows Bio-Layer Interferometry (BLI)-derived association (assoc.) and dissociation (dissoc.) traces of streptavidin sensors loaded with the indicated biotinylated ligands [a fusion of …

Figure 5—source data 1

Source data for Figure 5A.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig5-data1-v2.xlsx
Figure 5—source data 2

Source data for Figure 5C.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
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The ∆∆ deletion does not affect the stability of the IRE1LD dimer.

(A) Bio-Layer Interferometry (BLI)-derived association (assoc.) and dissociation (dissoc.) traces of streptavidin sensors loaded with the indicated biotinylated ligands [wild-type (wt) or IRE1LD ∆∆] …

Figure 5—figure supplement 1—source data 1

Source data for Figure 5—figure supplement 1B.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig5-figsupp1-data1-v2.xlsx
Figure 5—figure supplement 1—source data 2

Source data for Figure 5—figure supplement 1C.

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Figure 6 with 1 supplement
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BiP-mediated monomerisation of IRE1LD ∆∆ assessed by hydrogen exchange mass spectrometry (HX-MS).

(A) Difference plot of deuteron incorporation comparing wild-type (wt) IRE1LD with the monomeric mutants IRE1LD W125A (orange trace) or IRE1LD P108A (pink trace) after 30 s incubation in D2O [see Tab…

Figure 6—source data 1

Source data for Figure 6A and Figure 6—figure supplement 1A.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig6-data1-v2.xlsx
Figure 6—source data 2

Source data for Figure 6C and Figure 6—figure supplement 1C.

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Figure 6—figure supplement 1
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HX-MS evidence for impaired BiP- and ERdj4-driven monomerisation of IRE1LD ΔΔ.

(A) Difference plot of deuteron incorporation into the indicated peptic peptides of wild-type (wt) versus IRE1LD W125A or wt versus IRE1LD P108A after 300 s incubation in D2O (as in Figure 6A) (see F…

Figure 6—figure supplement 1—source data 1

Source data for Figure 6—figure supplement 1E.

https://cdn.elifesciences.org/articles/50793/elife-50793-fig6-figsupp1-data1-v2.xlsx
Figure 7 with 1 supplement
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Analysis of bimodally-distributed isotope clusters of IRE1LD peptic peptides reveals active destabilisation of the IRE1LD dimer by BiP.

(A) Intensity distributions of the isotope clusters of peptide 655.273+ (residues 297–302) from IRE1LD, untreated or exposed to BiP, ERDj4 and ATP (30 min at 30°C) following different incubation …

Figure 7—source data 1

Source data for Figure 7A and B and Figure 7—figure supplement 1A, B, C.

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Figure 7—figure supplement 1
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Analysis of the bimodal distributions of the isotope clusters detected by HX-MS.

(A) Shown are original spectra of peptic peptide 655.273+ (residues 297–302) from monomeric IRE1LD P108A (left panel) or wild-type IRE1LD (right panel) at the indicated time point of incubation in D2…

Figure 8
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Cartoon depicting features of BiP-mediated regulation of IRE1 activity.

In stressed cells unfolded proteins compete for BiP, exposing IRE1LD to a default dimeric active state, specified by the kinetics of the monomer-dimer equilibrium (left panel). In compensated cells …

Author response image 1
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BiP binding score based on Blond-Elguindi et al. (1993) of hepta-peptides within the entire human IRE1LD’s sequence.

Hepta-peptides having a score of >10 are predicted to have an extremely high probability of binding (black dashed line), peptides with scores between +6 and +10 are predicted to have odds of three …

Author response image 2
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Bar diagram of median XBP1::Turquoise and CHOP::GFP signals from untreated and tunicamycin (Tm)-treated CHO-K1 dual UPR reporter cells with Ern1 alleles encoding wild type (wt) or the indicated mutant variants of IRE1 (∆loop, missing residues 313-338).

Data from two independent experiments is shown (means ± range)..

Tables

Table 1
Data collection and refinement statistics of IRE1LDQ105C SS.
Data collection
Synchrotron stationsDls i04-1
Space groupP6522
a,b,c; Å182.77, 182.77, 68.45
α, β, γ; ⁰90.00, 90.00, 120.00
Resolution, Å91.39–3.55 (3.89–3.55)*
Rmerge0.180 (2.242)*
I/σ(I)11.7 (1.5)*
CC1/21.000 (0.797)*
No. of unique reflections8590 (1996)*
Completeness, %100.0 (100.0)*
Redundancy19.3 (19.9)*
Refinement
Rwork/Rfree0.323/0.332
No. of atoms (non H)1784
Average B-factors127
RMS Bond lengths Å0.003
RMS Bond angles,⁰0.606
Ramachandran favoured region, %95.85
Ramachandran outliers, %0
MolProbity score†1.51 (100th)
PDB code6SHC

  1. * Values in parentheses are for highest-resolution shell.

    † 100† 100th percentile is the best among structures of comparable resolutions. 0th percentile is the worst.

Table 2
List of IRE1LD peptic peptides analysed by hydrogen-1H/2H-exchange mass spectrometry (HX-MS) containing the respective m/z values, charge (z) and sequence of each peptide.

Note that the N-terminal amide hydrogen of each peptic fragment exchanges too fast to be detectable with this method. Hence, the N-terminal residue was excluded from the data analysis.

ResiduesM/zZSequence
24–36631.3452STSTVTLPETLL
37–45938.4781FVSTLDGSL
46–59396.7304HAVSKRTGSIKWTL
77–85927.4351LPDPNDGSL
86–106779.0873YTLGSKNNEGLTKLPFTIPEL
96–106636.3802LTKLPFTIPEL
107–1191316.6801VQASPSRSSDGIL
120–128390.1993YMGKKQDIW
130–134735.4241YVIDLL
134–145631.8322LTGEKQQTLSSA
147–1571090.5631ADSLSPSTSLL
157–168730.8742LYLGRTEYTITM
168–175522.2422MYDTKTRE
176–183535.7722LRWNATYF
186–1951031.4471AASLPEDDVD
196–208727.8372YKMSHFVSNGDGL
209–221703.3432VVTVDSESGDVLW
221–232697.8562WIQNYASPVVAF
233–2401050.5371YVWQREGL
241–248332.8643RKVMHINV
253–258406.7352LRYLTF
280–287444.282KSKLTPTL
288–2961017.5251YVGKYSTSL
297–302655.2731YASPSM
303–316474.2683VHEGVAVVPRGSTL
317–335956.9782PLLEGPQTDGVTIGDKGES
343–360534.3074VKFDPGLKSKNKLNYLRN
365–378503.5883IGHHETPLSASTKM
379–404516.6066LERFPNNLPKHRENVIPADSEKKSFE
410–424810.3762VDQTSENAPTTVSRD
410–443727.1495VDQTSENAPTTVSRDVEEKPAHAPARPEAPVDSM
Key resources table
Reagent type
(species) or resource		DesignationSource or
reference		IdentifiersAdditional
information
Strain, strain background (Escherichia coli)BL21 C3013 E. coliNEBCat no: C3013I
Strain, strain background (Escherichia coli)Origami B(DE3) E. coliNovagen/MERCKCat no: 70837
AntibodyAnti-mouse IRE1α serum (rabbit polyclonal)Bertolotti et al., 2000NY200used at 1/1000
Antibodyanti-hamster BiP (chicken polyclonal)Avezov et al., 2013Anti-BiPused at 1/1000
AntibodyAnti-GST (polyclonal rabbit)Ron and Habener, 1992Anti-CHOPused at 1/1000
Cell line, (Cricetulus griseus)Clone S21 a derivative of RRID: CVCL_0214Sekine et al., 2016CHO-K1 S21CHO CHOP::GFP, XBP1s::Turquoise dual UPR reporter cell line
Cell line, (Cricetulus griseus)CHO-K1 S21 CHOP::GFP, XBP1s::Turquoise ∆LD 15Kono et al., 2017∆IRE1CHO CHOP::GFP, XBP1s::Turquoise dual UPR reporter, Ern1 null cell line
Cell line, (Cricetulus griseus)CHO-K1 S21 CHOP::GFP, XBP1s::Turquoise IRE1 wild-typeThis paperIRE1 wild-typeCHO CHOP::GFP, XBP1s::Turquoise dual UPR reporter, Ern1 null cell line reconstituted with IRE1 wild-type
Cell line, (Cricetulus griseus)CHO-K1 S21 CHOP::GFP, XBP1s::Turquoise IRE1 ∆∆This paperIRE1 ∆∆CHO CHOP::GFP, XBP1s::Turquoise dual UPR reporter, Ern1 null cell line reconstituted with IRE1 ∆∆ (missing residues 313–338 and 391–444)
Peptide, recombinant proteinMPZ-NKaragöz et al., 2017MPZ-N12-mer peptide (MPZ-N) derived from myelin protein zero
Peptide, recombinant proteinFAM-MPZ-NKaragöz et al., 2017FAM-MPZ-NFAM labelled 12-mer peptide (MPZ-N) derived from myelin protein zero
Software, algorithmPrismGraphPad
Software, algorithmFlowJo,LLC,
Software, algorithmData Analysis 4.1Bruker
Chemical compound, drugTunicamycinMelfordCat no: T2250
Chemical compound, drug2-DeoxyglucoseSigmaCat no: D6134
Chemical compound, drug4μ8cTocris BioscienceCat no: 4479
Chemical compound, drugDigitoninCalbiochemCat no: 300410
Chemical compound, drugBiotin-NHS esterSigmaCat no: H1759
Chemical compound, drugProtease inhibitorsSigma Aldrich (MERCK)S8830
Chemical compound, drugOregon Green-iodoacetic acidThermoFisherCat no: O6010
Chemical compound, drugTAMRA-maleimideSigmaCat no: 94506
Chemical compound, drugPhosphocreatineSigmaCat no: 10621714001
Chemical compound, drugCreatine kinaseSigmaCat no: C3755
Recombinant DNA reagenthaBiP_27–654_pQE10 (plasmid)Petrova et al., 2008UK173N-terminally His6-tagged hamster BiP
Recombinant DNA reagenthaBiP_27–654_V461F_pQE10 (plasmid)Petrova et al., 2008UK182N-terminally His6-tagged hamster BiP V461F
Recombinant DNA reagenthaBiP_27–654_ADDA_pQE10 (plasmid)Preissler et al., 2015aUK984N-terminally His6-tagged hamster BiP ADDA
Recombinant DNA reagentH6_Ulp1_pET28b (plasmid)This studyUK1249H6-tagged Ulp1
Recombinant DNA reagentpCEFL_mCherry_3XFLAG_C (plasmid)Sekine et al., 2016UK1314pCEFL with 3XFLAG_C tagged from mCherry-tagged plasmid
Recombinant DNA reagentBPPTSP_SubA_22–347_3XFLAG_KDEL_pUC57_Acc65I_based_pCEFL_mCherry (plasmid)This studyUK14523xFLAG-tagged SubA with KDEL on mCherry-tagged plasmid
Recombinant DNA reagentBPPTSP_SubA_22–347_S272A_3XFLAG_KDEL_pUC57_Acc65I_based_pCEFL_mCherry (plasmid)This studyUK14593xFLAG-tagged SubAS272Awith KDEL mCherry-tagged plasmid
Recombinant DNA reagenthIRE1_19–486_dC_GST_del3UTR _pCDNA3 (plasmid)Amin-Wetzel et al., 2017UK1703C-GST-tagged cysteine-free human IRE1
Recombinant DNA reagentCHO_IRE1_guideC15.1_pSpCas9(BB)−2A-mCherry (plasmid)Kono et al., 2017UK1903Cas9 and guide targeting IRE1 in CHO-K1 ∆LD clone 15 (mCherry-tagged)
Recombinant DNA reagentCHO_IRE1_hIRE1-LD_reptemp4_pCR-Blunt2-TOPO (plasmid)Kono et al., 2017UK1968Repair template for wild-type IRE1 reconstitution in CHO-K1 cells
Recombinant DNA reagentSmt3_cgERdj4_24–222_pET-21a (plasmid)Amin-Wetzel et al., 2017UK2012N-Smt3-tagged Chinese amster ERdj4 24–222
Recombinant DNA reagentSmt3_J4_domain_24–90_pET-21a (plasmid)Amin-Wetzel et al., 2017UK2041N-Smt3-tagged Chinese hamster ERdj4 24–90
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_24_444 (plasmid)This studyUK2042N-His6-Smt3-tagged wild-type human IRE1LD24–444
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_24_444 Q105C (plasmid)Amin-Wetzel et al., 2017UK2045N-His6-Smt3-tagged cysteine-free human IRE1LD Q105C24–444
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_24_444 R234C (plasmid)Amin-Wetzel et al., 2017UK2048N-His6-Smt3-tagged cysteine-free human IRE1LD24–444, R234C (FRET probe)
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_24_444 S112C (plasmid)Amin-Wetzel et al., 2017UK2076N-His6-Smt3-tagged cysteine-free human IRE1LD24–444, S112C (FRET probe)
Recombinant DNA reagentSmt3_cgERdj4_24–222_GS6_MalE_pET21a (plasmid)Amin-Wetzel et al., 2017UK2108N-Smt3-ERdj4-MBP Chinese hamster 24–222
Recombinant DNA reagentSmt3_cgERdj4_24–222_QPD_GS6_MalE_pET21a (plasmid)Amin-Wetzel et al., 2017UK2119N-Smt3-ERdj4-MBP Chinese hamster residues 24–222 H54Q
Recombinant DNA reagentIRE1a_LD_∆C_24–443_AviTag_H6_pET30a (plasmid)This studyUK2246C-Avi-His6-tagged cysteine-free human IRE1LD24–444
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD∆C_Q105C_24_390 (plasmid)This studyUK2304N-His6-Smt3-tagged cysteine-free human IRE1LD Q105C24–390
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD_dC_24_390_∆313–338_S112C (plasmid)This studyUK2370N-His6-Smt3-tagged cysteine-free human IRE1LD ∆∆ (313-338, 391-444) S112C, FRET probe
Recombinant DNA reagentCHO_IRE1_hIRE1-LD_d313-338_reptemp4_pCR-Blunt2-TOPO (plasmid)This studyUK2384Repair template for IRE1 ∆loop (d313-338) reconstitution in CHO-K1 cells
Recombinant DNA reagentCHO_IRE1_hIRE1-LD_d391-444_reptemp4_pCR-Blunt2-TOPO (plasmid)This studyUK2385Repair template for IRE1 ∆tail (d391-444) reconstitution in CHO-K1 cells
Recombinant DNA reagentCHO_IRE1_hIRE1-LD_d313-338_d391-440_reptemp4_pCR-Blunt2-TOPO (plasmid)This studyUK2386Repair template for IRE1 ∆∆ (d313-338, 391–444) reconstitution in CHO-K1 cells
Recombinant DNA reagenthIRE1α_19–486_dC_ d313-338_d391-440_GST_del3UTR _pCDNA3 (plasmid)This studyUK2401C-GST-tagged cysteine-free human IRE1 ∆∆ (missing residues 313–338 and 391–444)
Recombinant DNA reagenthIRE1α_19–486_dC_ d313-338_GST_del3UTR _pCDNA3 (plasmid)This studyUK2404C-GST-tagged cysteine-free human IRE1 ∆loop (missing residues 313–338)
Recombinant DNA reagenthIRE1α_19–486_dC_ d391-440_GST_del3UTR _pCDNA3 (plasmid)This studyUK2406C-GST-tagged cysteine-free human IRE1 ∆∆ (missing residues 391–444)
Recombinant DNA reagentMet_ERdj4_24–120_Ire1a_LD∆C_24–443_AviTag_H6_pET30a (plasmid)This studyUK2408C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD24–444 protein
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD_dC_24_444_P108A (plasmid)This studyUK2410N-His6-Smt3-tagged cysteine-free human IRE1LD P108Amonomeric mutant 24–444
Recombinant DNA reagentpET22b_H7_Smt3_Ire1a_LD_dC_24_444_W125A (plasmid)This studyUK2411N-His6-Smt3-tagged cysteine-free human IRE1LD W125Amonomeric mutant 24–444
Recombinant DNA reagentMet_ERdj4_24–120_Ire1a_LD∆C_24–443_S112C_AviTag_H6_pET30a (plasmid)This studyUK2412C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD24–444 protein, S112C (FRET probe)
Recombinant DNA reagentJ4_WT_IRE1_LD_CHORepairTemplate (plasmid)This studyUK2425Repair template for chimeric J-IRE1 reconstitution in CHO-K1 cells
Recombinant DNA reagentJ4_QPD_IRE1_LD_CHORepairTemplate_V1 (plasmid)This studyUK2426Repair template for chimeric JQPD-IRE1 reconstitution in CHO-K1 cells
Recombinant DNA reagentMet_ERdJ4_24–120_Ire1a_LD∆C_24–443_P108A_AviTag_H6_pET30a (plasmid)This studyUK2428C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD24–444 protein containing monomerising mutation P108A
Recombinant DNA reagentMet_ErdJ4_24–120_IRE1a_LD∆C_24–390_∆313–338_AviTag_H6_pET30a (plasmid)This studyUK2458C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD ∆∆protein (313-338, 391-444)
Recombinant DNA reagentIRE1a_LD∆C_24–390_∆313–338_AviTag_H6_pET30a (plasmid)This studyUK2459C-Avi-His6-tagged cysteine-free human IRE1LD ∆∆(d313-338, 391–444)
Recombinant DNA reagentMet_ERdJ4_24–120_Ire1a_LD∆C_24–443_Q105C_AviTag_H6_pET30a (plasmid)This studyUK2558C-Avi-His6-tagged cysteine-free chimeric J-ERdj4 human IRE1LD24–444 protein containing mutation Q105C

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