Cell lines and culture
HAP1 cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal calf serum (FCS) (BioSell) and 1% penicillin–streptomycin–glutamine (PSG) solution (Thermo Fisher Scientific). 293T (HEK293T), A549, HeLa, U2OS, BJEH and HCT116 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FCS and 1% PSG. SH-SY5Y cells were maintained in DMEM supplemented with 15% FCS, 1% PSG and 1 mM sodium pyruvate. K562 cells were maintained in Roswell Park Memorial Institute 1640 (RPMI) medium (Thermo Fisher) supplemented with 10% heat-inactivated FCS and 1% PSG. K562 differentiation was induced as previously described52. In brief, cells were pretreated with 200 nM imatinib (MedChemExpress, HY-15463) in IMDM at a density of 200,000 cells per ml for 24 h followed by treatment with 1 µM decatibine (MedChemExpress, HY-A0004R) in HEMA medium (IMDM, 20% FCS, 1% PSG and 2% BSA (Sigma Aldrich, A7030-50G)), 0.5 mg ml–1 holo-transferrin (Merck-Sigma, T0665), 2 U ml–1 erythropoietin (MedChemExpress, HY-P7164) and 20 ng ml–1 insulin (MedChemExpress, HY-P0035) for 3 days. HAP1 CHOPNeon WT and ∆HRI cells and clonal 293T ∆OMA1, ∆DELE1, ∆HRI and DELE1HA cells have been previously described21. All cell lines were tested initially for mycoplasma contamination.
Gene editing
To mutate EIF2S1 (which encodes eIF2α) endogenously into a non-phosphorylatable variant (EIF2S1S49A,S52A), 293T cells were transfected with a pX330 CRISPR plasmid (Addgene, 42230) containing a sgRNA targeting EIF2S1 exon 2, a donor vector encoding the serine 49 and 52 to alanine mutation and around 700 bp homology arms upstream and downstream of the sgRNA target site, and a puromycin-resistance vector. After 24 h of transfection, cells were selected with puromycin (1 μg ml–1), and single cell clones were derived and analysed for gene editing by PCR and Sanger sequencing.
Clonal and polyclonal knockout cell lines were also generated using the CRISPR–Cas9 system. Specifically, transient transfection of a pX330 containing the sgRNA of interest together with a puromycin-resistance or blasticidin-resistance vector was used to generate clonal 293T cells lacking OMA1, DELE1, HRI, HMOX1 and/or HMOX2. Lentiviral transduction of a pLentiCRISPR v.2 variant (derived from Addgene, 52961) containing the sgRNA of interest was used to delete OMA1, DELE1, HRI or NIX in A549, HeLa, U2OS, BJEH, HCT116, SH-SY5Y or K562 cells. After 24 h of transduction or transfection, cells were selected using puromycin (1 μg ml–1) or blasticidin (10 μg ml–1), and knockout efficiency was assessed by immunoblotting. Where indicated, clonal progeny of the polyclonal knockout populations were generated by single-cell cloning and verified by PCR, Sanger sequencing and immunoblotting. sgRNA sequences and primers for genotyping PCR used in this study are listed in Supplementary Table 1.
Haploid genetic screen for identification of CHOP regulators
Genome-wide mutagenesis of haploid HAP1 cells was carried out as previously described21. In brief, gene-trap virus particles were produced in 293T cells, concentrated by ultracentrifugation at 22,800 rpm for 2 h at 4 °C and stored at 4 °C overnight. To generate random genomic mutations via insertional mutagenesis by gene trapping, 1.5 × 107 haploid HAP1 CHOPNeon cells were transduced with concentrated retroviral particles 24 h after plating, followed by two additional transductions. The resulting library of mutants was expanded, plated at 20% confluence in a total of 20 T175 flasks (Sarstedt) and treated for 9 h with 5 μM DHA 48 h after plating. Cells were collected using trypsin–EDTA (0.25%, Gibco), passed through a 40 μm cell strainer (Greiner, 542040) and fixed with one volume of BD fix buffer I (BD Biosciences) for 10 min at 37 °C. The fixation was stopped with PBS (Gibco) containing 1% FCS, cells were passed through a 40 μm cell strainer, and approximately 1.5 × 109 cells were permeabilized with 1 pellet volume of cold BD Perm Buffer III (BD Biosciences) for 30 min on ice. Permeabilization was stopped with PBS containing 1% FCS, and cells were blocked in PBS with 1% FCS and 3% bovine serum albumin (BSA) for 30 min at room temperature. To increase fluorescence intensity of the CHOP(Neon) protein, cells were stained with anti-mNeonGreen antibodies (ProteinTech, 32F6) diluted 1:2,500 in PBS with 1% FCS and 1% BSA for 2.5 h on a rotor wheel at room temperature, followed by three 15-min washing steps with PBS and 1% FCS at room temperature. Primary antibodies were detected using AlexaFluor 488-conjugated secondary antibodies (anti-mouse-AF488, Life Technologies) diluted 1:500 in PBS supplemented with 1% FCS and 1% BSA for 1 h at room temperature on a rotor wheel protected from light. DAPI (Sigma-Aldrich, D9542) was added to the secondary antibody dilution at a final concentration of 2.5 μg ml–1 for a DNA counterstain. After three 15-min washing steps, cells were resuspended in PBS and 1% FCS, stored at 4 °C until sorting on a BD Fusion cell sorter (BD Biosciences, via FACSDiva v.8.0.2) using a 70 μm nozzle. Staining specificity was determined using a secondary antibody-only control. Haploid cells were identified on the basis of DNA content in the DAPI channel and of those, approximately 107 cells of the bottom 4% CHOP(Neon)-low and top 4% CHOP(Neon)-high cells were sorted into PBS and 10% FCS for isolation of gDNA.
Insertion site mapping and analysis
To extract gDNA from the sorted cell populations de-crosslinking was performed at 56 °C overnight followed by DNA isolation using a QIAamp DNA Mini kit (Qiagen, 51306) according to the manufacturer’s instructions. Gene-trap insertion sites of CHOP(Neon)-high and CHOP(Neon)-low populations were recovered as previously described21. The amplified libraries were sequenced on a NextSeq1000 (Illumina) with a read length of 60 nucleotides. Demultiplexing of indexed sequencing reactions was performed, allowing one mismatch. Reads were aligned to the human reference genome (hg19) and analysed as previously described21. Bowtie53 (v.1.0.1) was used to align reads to the human genome, allowing one mismatch, followed by mapping to the coordinates of RefSeq protein-coding genes with intersectBED54 (v.2.26.0). Only integrations in the sense orientation were considered disruptive and used for downstream analyses. To identify CHOP(Neon) regulators in DHA-treated cells per gene, the number of unique gene-trap insertion sites in the query gene versus the whole sample was compared between the CHOP(Neon)-high and CHOP(Neon)-low cell populations using a two-sided Fisher’s exact test and Benjamini–Hochberg FDR correction. Data were plotted as the combined number of unique mutations identified in the CHOP(Neon)-high and CHOP(Neon)-low population (x axis) versus their mutation ratio (high versus low) normalized to the respective sizes of the datasets (y axis). Fishtail plots were created using GraphPad Prism 10.
Treatments, transfections and transductions
Unless otherwise stated, cells were treated with 20 μM CCCP (Sigma-Aldrich, C2759), 1 μM oligomycin A (Sigma-Aldrich, 75351), 10 μM antimycin A (Sigma-Aldrich, A8674), 10 μM tunicamycin (MedChemExpress, HY-A0098), 5 μM DHA (MedChemExpress, HY-N0176), 100 μM SA (Sigma-Aldrich D1415), 20 μM NMPP (CaymanChemical, Cay20846-5), 250 nM ISRIB (Sigma-Aldrich SML0843), 20 μM haemin (Sigma-Aldrich, 51280), 5 mM GSH (Merck-Sigma, G4251), 10 mM NAC (Sigma-Aldrich, A9165), 100 μM FeCl2 (Sigma-Aldrich 372870) or 100 μM DFO (Sigma-Aldrich, D9533) for the indicated times. To induce robust haem starvation with SA, NMPP or the combination, media were supplemented with 5% haem-depleted FCS instead of normal FCS. To this end, heat-inactivated FCS was incubated with 10 mM ascorbic acid for 8 h at 37 °C and dialysed 3 times against PBS. Haemin stock solutions were prepared according to a previously described method55, with the exception that ethylene glycol was used as a solvent. To prepare a 5 mM haemin stock solution, 32.6 mg haemin chloride was dissolved in 500 μl 1 M NaOH, 500 μl 0.5 M Tris and 8.4 ml ethylene glycol. Finally, the pH was adjusted to pH 7.4 by the addition of 600 µl 1 M HCl and the solution was stored at −20 °C. A solution lacking haemin chloride was used as the control. For all haem rescue experiments, haemin was added 2 h (for SA or NMPP) or 12 h (for DHA) before haem-starvation induction. For induction of shRNAs from the pLKO vector, cells were treated with 500 ng ml–1 doxycycline hyclate (Biomol, Cay14422-1) for 3 days. shRNA sequences used in this study are listed in Supplementary Table 1.
Where indicated, cells were transfected using polyethylenimine (PEI 25000, Polysciences) or Turbofectin (OriGene Technologies). PEI was used to transfect 293T cells at a PEI-to-DNA ratio of 3:1. Turbofectin was used to transfect HAP1 cells at a Turbofectin-to-DNA ratio of 2.5:1. Transfection reagents and DNA dilutions were prepared separately in OptiMEM (Gibco), incubated for 10 min at room temperature, mixed by pipetting and added to 50% confluent cells after an additional 20 min of incubation. For activity analysis of ectopically expressed HRI in presence or absence of haemin, 293T DELE1 and HRI double-knockout cells were grown in 24-well plates and transfected with 100 ng HRI and 400 ng DELE1 or empty-vector plasmid DNA. After 4 h of transfection, cells were treated with the indicated haemin concentration and cells were collected 24 h after transfection. For HRI and/or DELE1 purifications, 293T EIF2S1S49A,S52A WT or HRI knockout cells were grown on 15 cm cell culture dishes, transfected with 30 μg plasmid DNA, treated with haemin 24 h after transfection and collected after an additional 24 h of culture.
The generation of lentiviral and retroviral particles was performed as previously described21. In brief, 293T cells were transfected with lentiviral (pCMVd8.2dVPR, pCMV-VSV-G, pAdVAntage) or retroviral (pCMV-Gag-Pol, pCMV-VSV-G, pAdVAntage) packaging plasmids and the indicated transfer vector encoding the cDNA or Cas9 and sgRNA of interest. After 48 h of transfection, virus particles were collected, filtered through a 0.45 μm syringe filter (Sarstedt), and the indicated cell line was transduced with 1:2 diluted virus supernatant containing protamine sulfate. After 24 h of transduction, cells were selected for at least 48 h with puromycin (1 μg ml–1), blasticidin (10 μg ml–1) or hygromycin (300 μg ml–1), followed by a 24-h recovery period in antibiotic-free medium.
DNA cloning
The coding sequences of genes expressed in this study were amplified from human cDNA or synthesized as codon-optimized gene blocks (IDT) listed in Supplementary Table 2. Point mutations were introduced by overlap-extension PCR or by gene synthesis. All oligonucleotide sequences are listed in Supplementary Table 1. Amplified DNA was subjected to restriction digest and ligation using standard cloning procedures. All cloned constructs were sequence-verified by Sanger sequencing.
Gel electrophoresis and immunoblotting
For analysis of protein expression by denaturing gel-electrophoresis, cells were treated as described in the figure legends, washed with PBS and lysed with SDS-sample buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue and 4% β-mercaptoethanol). For phosphatase treatment of protein lysates, cells were lysed in DISC buffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol) supplemented with protease inhibitor (complete protease inhibitor cocktail, Roche, 11697498001) and 1% NP-40 for 15 min on ice, and lysates were cleared twice by centrifugation at 20,000g for 10 min at 4 °C. Supernatants were transferred to a microfuge tube, and FastAP buffer and FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific, EF0654) were added. Samples were incubated at 37 °C for 1 h, and the reaction was stopped by the addition of SDS sample buffer. All samples were denatured for 10 min at 95 °C. Subsequently, equal amounts of protein were subjected to denaturing SDS–PAGE and transferred to PVDF membrane (Millipore) by semi-dry transfer using a Bolt gel electrophoresis and transfer system (Thermo Fisher Scientific) according to the manufacturer’s instructions and homemade transfer buffer (190 mM glycine, 25 mM Tris and 20% ethanol). Bolt gradient gels (4–12%; Thermo Fisher) and a MOPS or MES buffer system (Thermo Fisher) were used for routine immunoblot analyses. For analysis of HRI phosphorylation, proteins were separated using 7.5% Tris–glycine gels and a Tris buffer system (25 mM Tris, 192 mM glycine and 0.1% SDS). After transfer, membranes were blocked by incubation with Tris-buffered saline (25 mM Tris-HCl, pH 7.6, and 150 mM NaCl), containing 0.02% Tween 20 (TBST) and 5% dry milk (Sigma Aldrich) for 1 h at room temperature. After incubation with primary antibodies for 2 h at room temperature or at 4 °C overnight, membranes were washed three times with TBST and incubated with secondary antibodies (goat-anti-rabbit/mouse HRP-conjugated, diluted in TBST + 5% dry milk, Bio-Rad) for 1 h at room temperature. Membranes were washed three times with TBST, enhanced chemiluminescence solution56 was applied and the signal was detected using a Bio-Rad Chemidoc MP system. The antibodies used for immunoblotting are listed in Supplementary Table 3.
To analyse DELE1-containing and HRI-containing protein assemblies in cytosolic extracts by native gel electrophoresis, cells were seeded and treated as described in the figure legends, washed with PBS and lysed for 20 min on ice with NativePAGE sample buffer (Thermo Fisher) supplemented with protease inhibitors and 0.02% digitonin (Sigma-Aldrich, D141). Subsequently, cytosolic extracts were cleared twice by centrifugation at 20,000g for 10 min at 4 °C, NativePAGE G-250 sample additive was added to a final concentration of 0.025% (v/v) and samples were separated on 3–12% Bis-Tris Mini protein gels (Invitrogen) according to the manufacturer’s protocol using NativePAGE cathode and anode buffers (Thermo Fisher). Native mark (Fisher scientific) was used to estimate the molecular weight of protein assemblies. Subsequently, proteins were transferred to PVDF membrane by semi-dry transfer using NuPAGE transfer buffer (Invitrogen) and a Bolt gel electrophoresis and transfer system (Thermo Fischer) according to the manufacturer’s protocol. Protein assemblies were detected by immunoblotting as described above.
Isolation and fractionation of mitochondrial membranes
To determine haem levels in the cytosol and different mitochondrial subfractions, 293T WT cells were treated with 5 μM DHA for 6 h. Afterwards, cells were collected by scraping, washed with PBS and cell pellets were collected by centrifugation at 450g for 5 min. Cells were resuspended with homogenization buffer (20 mM HEPES-KOH pH 7, 220 mM mannitol, 70 mM sucrose and 20 μM EGTA) and lysed on ice by passing the cell suspension through a 25 G needle (0.50 × 16 mm, Sterican, B. Braun) attached to a 1 ml syringe (Omnifix-F Luer, B. Braun) in 3 cycles of 10 passes each, with cooling intervals between cycles. Cell debris was removed by centrifugation twice at 700g for 5 min at 4 °C, and supernatant was transferred into new tubes and centrifuged at 8,000g for 15 min at 4 °C to pellet the crude mitochondrial fraction. Cytosolic fractions were transferred into new tubes and centrifuged again at 20,000g for 5 min at 4 °C to remove residual mitochondria. Mitochondrial pellets were resuspended in EM buffer (10 mM HEPES-KOH, pH 7.4, and 1 mM EDTA–KOH pH 8.0) followed by lysis with SEM buffer (250 mM sucrose, 10 mM HEPES-KOH, pH 7.4, and 1 mM EDTA–KOH pH 8.0) containing 0.1% digitonin for 20 min on ice to release outer mitochondrial membrane and intermembrane space fractions into the solution. Subsequently, the inner mitochondrial membrane and matrix were re-isolated by centrifugation at 10,000g for 10 min at 4 °C. The pellets were resuspended with SEM buffer containing 1% Triton X-100, incubated on ice for 20 min and labelled as inner mitochondrial membrane together with mitochondrial matrix fractions.
Haem measurements
Commercial apoHRP, APO/HRP4C-peroxidase (BBI Solutions, APO/HRP4C), was used in all enzyme-based haem detection assays. To deplete residual haem from apoHRP present in the preparation, apoHRP was dissolved to 4 mg ml–1 in PBS and haem was extracted by the addition of 5 ml acetone and 125 μl concentrated hydrochloric acid per mg apoHRP. Acetone-extracted apoHRP was recovered by centrifugation at 2,000g for 2 min at room temperature, dissolved to a final concentration of 50 μM in PBS and stored at −20 °C.
To measure cellular haem levels, cells were treated as indicated and lysed with PBS or DISC buffer containing protease inhibitor and 1% Triton-X100 as described above. Protein concentrations were determined by Bradford assay and adjusted to equalize protein concentrations across all samples. Subsequently, apoHRP solution was added to a final concentration of 12.5 μM followed by incubation for 10 min on ice. To detect HRP-bound haem, TMB ELISA substrate (Serva, 37068.01) was added to the reaction, and absorbance at 562 nm was measured on a microplate reader (Tecan Spark, via SparkControl, v.3.1) after incubation at room temperature in the dark for 10 min. Haem binding to purified, endogenous, stably or transiently expressed HRI was assessed analogously, with the exception that biotin-eluted or pH-shift-eluted HRI was denatured for 15 min at 98 °C before incubation with 12.5 μM apoHRP. For analysis of haem release from HRI after treatment of cells with CCCP, oligomycin A or DHA, Strep-tagged HRI, stably expressed in 293T EIF2S1S49A,S52A WT or DELE1 knockout cells in presence or absence of stable L-DELE1 co-expression, was used as a handle for affinity capture.
Soret-band measurements
For analysis of haem binding to HRI by UV–vis spectroscopy, the absorbance of HRI purified in the presence or absence of S-DELE1 was recorded from 250 nm to 600 nm using a V630 Bio spectrophotometer (Jasco) equipped with a quartz cuvette (Hellma Analytics, 10 mm path length, 105-202-15-40) and Jasco Spectra Manager II software. The UV–vis spectrum was blanked against elution buffer and curves were normalized to the protein concentration calculated based on the absorbance at 280 nm using extinction coefficients of 116,180 M−1 cm−1 for the HRI dimer and 190,340 M−1 cm−1 for the HRI–DELE1 tetramer. Equal protein loading was subsequently confirmed by Coomassie gel analysis.
Immunoprecipitation and affinity purifications
For purification of HRI from cytosolic extracts, cells were treated as indicated, collected by scraping in ice-cold PBS, washed and lysed in DISC buffer supplemented with protease inhibitor, phosphatase inhibitor (Thermo Fisher, A32957) and 0.02% digitonin for 20 min on ice. Cytosolic extracts were cleared by centrifugation twice at 20,000g for 10 min at 4 °C. Subsequently, cleared cytosolic extracts were incubated with either Strep-Tactin Sepharose resin (IBA, 2-1201-010), anti-Flag M2 magnetic beads (Sigma, M8823) or GFP-Trap magnetic agarose (Chromotek, gtma-20) for precipitation of StrepTagII-containing, Flag-containing or GFP-containing protein complexes, respectively. Immunoprecipitations of endogenous HRI were performed with anti-eIF2AK1 antibodies (Proteintech, 20499-1-AP) coupled to Pierce protein A beads (Thermo Fisher, 20333) at an antibody-to-cell lysate ratio of 1:1,000. All beads were washed five times with detergent free DISC buffer before addition of supernatants. After incubation at 4 °C on a rotor wheel for 1.5 to 3 h, beads were washed six times with DISC buffer containing 0.1% Triton-X100. After the last wash step, all washing buffer was removed, and bead bound proteins were eluted with SDS sample buffer for 10 min at 95 °C before analysis by gel electrophoresis. For sequential immunoprecipitations or haem measurements, Strep-tagged proteins were eluted with 50 mM biotin in elution buffer (50 mM NaCl, 20 mM HEPES pH 8.0, and 10% glycerol), GFP-containing protein complexes were eluted with 3C protease (Sigma-Aldrich, GE27-0843-01) in the experiments relating to sequential affinity purification and elution, and anti-Flag or anti-EIF2AK1 antibody-bound proteins were eluted with 200 mM glycine pH 2.5 followed by neutralization with Tris-HCl pH 10.5. Unless otherwise indicated in the figure, 1–1.5% of input material was loaded for immunopurification control immunoblotting.
Medium-scale HRI and DELE1 affinity purification from mammalian cells
To purify large quantities of Strep-tagged HRI, Flag-tagged S-DELE1 or StHA-tagged S-DELE1 for in vitro analyses, 293T EIF2S1S49A,S52A cells were transfected with the respective construct and grown in the presence or absence of 20 μM haemin as described above. After 48 h of transfection, cells were collected, washed with PBS and lysed for 30 min on ice with modified DISC buffer (20 mM HEPES, pH 7.5, 150 mM NaCl and 10% glycerol) supplemented with protease inhibitor, phosphatase inhibitor and 0.02% digitonin. Cell debris was removed by centrifugation twice at 21,000g for 10 min at 4 °C, and cytosolic extracts were incubated with Strep-Tactin Sepharose resin (IBA, 2-1201-010) or anti-Flag M2 magnetic beads (Sigma, M8823) for 1.5 h at 4 °C. Beads were washed six times with lysis buffer and Strep-tagged or StHA-tagged proteins were eluted with 50 mM biotin in elution buffer for haem binding, mass photometry or crosslinking experiments. For in vitro haem release assays, Strep-tagged HRI was not eluted after washing but instead incubated with pH-eluted Flag-tagged S-DELE1 or elution buffer for an additional 4 h. Subsequently, supernatant was collected, beads were washed three times with lysis buffer and HRI was eluted with biotin. For phosphorylation analysis of exogenously expressed HRI in presence or absence of S-DELE1 by mass spectrometry (MS), Strep-tagged HRI was eluted with SDS sample buffer for 10 min at 95 °C. Flag-tagged DELE1 was eluted with 200 mM glycine pH 2.5 followed by neutralization with Tris-HCl, pH 10.5.
GST pull-down assay
GST-fused HRI or HRI mutants were expressed in Escherichia coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) in LB medium. When the optical density (OD600) of bacterial cultures reached 0.2, protein expression was induced using 1 mM IPTG (Thermo Scientific, R0393) at 8 °C, 120 rpm for 48 h. Bacteria were pelleted by centrifugation (4 °C, 6,000g, 10 min). Bacteria were lysed by sonication on ice in lysis buffer (30 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% glycerol and 0.5 mM DTT) supplemented with protease inhibitor and phosphatase inhibitor. Lysates was cleared by centrifugation (4 °C, 18,000g, 30 min) and the cleared lysate was incubated with glutathione sepharose 4B beads (Cytiva, GE17-0756-01) at 4 °C for 1 h with rolling. The beads were pelleted by (4 °C, 700g, 20 s) and washed 3 times with washing buffer (30 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) supplemented with 0.25 mM DTT. Beads immobilized with GST-fusion were ready to incubate with cell lysates. 293T cells were transfected with a plasmid encoding triple HA-tagged S-DELE1. Cells were pelleted by centrifugation (4 °C, 500g, 5 min) 2 days after transfection. Cells were lysed in DISC buffer supplemented with protease inhibitor, phosphatase inhibitor and 0.02% digitonin. Cell lysate was cleared by centrifugation (4 °C, 20,000g, 12 min) and the cleared lysate was incubated with beads immobilized with GST-fusion at 4 °C for 1 h. Beads were pelleted by centrifugation (4 °C, 700g, 20 s) and washed 5 times with washing buffer. Proteins immobilized on the beads were eluted in SDS sample buffer, heated at 95 °C for 10 min and then subjected to SDS–PAGE and immunoblotting analyses. For the dephosphorylation reaction coupled with GST pull-down assays, GST-fusion-coupled beads were incubated with lambda protein phosphatase (New England Biolabs) according to the manufacturer’s instructions. In these experiments, phosphatase inhibitor was omitted.
Protein purification from bacteria
6×His-TEV-tagged HRI, HRI(K196R) or HRI along with S-DELE1 were expressed in the same manner for GST-fusion proteins but at 8 °C and 120 rpm for 48 h. Bacteria were pelleted by centrifugation (4 °C, 6,000g, 10 min). Bacteria were lysed by sonication on ice in lysis buffer (30 mM Tris-HCl pH 8.0, 500 mM NaCl, 25 mM imidazole, 10% glycerol and 0.5 mM DTT) supplemented with protease inhibitor and phosphatase inhibitor. Lysate was cleared by centrifugation (4 °C, 18,000g, 30 min) and then incubated with Ni-NTA resin (HisPur, 88221) at 4 °C for 1 h with rolling. The solution was loaded into gravity-flow columns. Resin was washed 3 times with washing buffer (30 mM Tris-HCl pH 8.0, 150 mM NaCl, 50 mM imidazole, 5% glycerol and 0.25 mM DTT). Immobilized proteins were eluted in elution buffer (30 mM Tris-HCl pH 8.0, 150 mM NaCl, 500 mM imidazole, 0.25 mM DTT) and concentrated using ultracentrifuge filters (Amicon). Concentrated proteins were loaded onto a superose 6 increase 10/300 GL column (Cytiva) connected to a chromatography system (ÄKTA pure) and eluted with SEC buffer (30 mM HEPES pH 7.5, 100 mM NaCl and 0.25 mM DTT). Fractions eluted in the main peak were collected and checked by SDS–PAGE for purity. Fractions of high purity (>95% judged by Coomassie brilliant blue staining) were further concentrated using ultracentrifuge filters (Amicon), then directly used for subsequent experiments or snap-frozen in liquid N2 and then stored at −80 °C.
6×His-MBP-GST-TEV-tagged eIF2α or eIF2α(S49A,S52A) were similarly purified. Protein was expressed at 16 °C, 120 rpm overnight. After elution from Ni-NTA resin, proteins were incubated with TEV protease (a gift from K.-P. Hopfner) to remove the tag. For size exclusion chromatography, a superdex 75 10/300 GL column (Cytiva) was used.
In vitro kinase assay
To determine the linearity range of HRI and eIF2α kinase reactions, 0–200 nM HRI, 20 μM eIF2α and 40 μM ATP were reacted in PK buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mM EDTA, 2 mM DTT and 0.01% Brij 35) at 37 °C for 0–12 min. To determine the initial velocity and maximal velocity of the HRI and eIF2α kinase reaction, 50 nM HRI, 1.25–40 μM eIF2α and 40 μM ATP were reacted in PK buffer at 37 °C for 0–10 min. To determine the IC50 of haemin for HRI alone or HRI with S-DELE1, 50 nM HRI or HRI–S-DELE1 was incubated with 0–20 μM haemin at 25 °C for 10 min and then reacted with 5 μM eIF2α and 40 μM ATP in PK buffer at 37 °C for 10 min. Reactions were terminated by the addition of SDS sample buffer. Next, 3 μl of the reaction was subjected to dot blot analysis using nitrocellulose membranes and a phospho-eIF2α(Ser52) antibody.
Mass photometry
The molecular mass of HRI or DELE1 was determined by mass photometry. Measurements were performed using a TwoMP mass photometer (Refeyn). Before each measurement, the focus was adjusted by applying 10 μl mass photometry buffer (30 mM HEPES pH 7.5, and 100 mM NaCl) to a new flow chamber. Next, 10 μl HRI or DELE1 was added to the mass photometry buffer to a final concentration of 50 nM immediately before mass photometry measurements. Videos were recorded for 60 s and data were collected and analysed using Refeyn AcquireMP 2.3 and Refeyn DiscoverMP 2.3, respectively.
Sample preparation for determination of HRI phosphorylation sites by MS
To assess phosphorylation levels of overexpressed HRI after haem release and kinase activation by S-DELE1, Strep-tagged HRI purified from human cells was subjected to denaturing gel electrophoresis as described above. Gels were stained with 0.1% Coomassie brilliant blue R in 10% acetic acid and 20% ethanol, destained with 10% acetic acid and 30% ethanol, and HRI gel bands were cut into small pieces and incubated in 100% acetonitrile for 15 min at room temperature. To reduce proteins, samples were incubated in 10 mM DTT at 60 °C for 1 h, followed by a wash in acetonitrile for 15 min at room temperature. To alkylate proteins, samples were incubated in 55 mM chloroacetamide for 30 min at room temperature in the dark. Samples were destained by alternating washes in acetonitrile and 50 mM ammonium bicarbonate for 15 min at room temperature. Protein material was digested overnight at 37 °C using trypsin (10 ng µl–1, Promega). After overnight incubation, supernatants were acidified with formic acid (1% final concentration) and dried using a SpeedVac centrifuge (Eppendorf, Concentrator Plus). The samples were resuspended in 0.1% formic acid before liquid chromatography–tandem MS (LC–MS/MS) analysis.
To assess DELE1-dependent phosphorylation of endogenous HRI in response to haem deficiency or repression of HRI phosphorylation by haemin, 293T EIF2S1S49A,S52A WT or DELE1 KO cells were treated as indicated and endogenous HRI was purified using protein-A-coupled EIF2AK1 antibodies as described above, with the exception that the last three washes were performed using detergent-free DISC buffer. After the last wash step, all washing buffer was removed, beads were snap-frozen in liquid nitrogen and stored at −80 °C until further processing. Bead pellets were resuspended in sodium deoxycholate buffer (4% SDC in 100 mM Tris-HCl pH 8.5) and denatured for 5 min at 95 °C with shaking (1,000 rpm). Samples were reduced and alkylated with 10 mM TCEP and 40 mM 2-chloroacetamide for 5 min at 45 °C with shaking (1,000 rpm) and digested with 0.5 µg LysC (Wako Chemicals) and 0.5 µg trypsin (Promega, sequencing grade) for 16 h at 30 °C. After digestion, peptides were acidified with trifluoroacetic acid and desalted using SDB-RPS solid-phase extraction discs (3M). The samples were resuspended in 0.1% formic acid before LC–MS/MS analyses.
LC–MS/MS data acquisition for determination of phosphorylation sites
Samples were measured on an Eclipse mass spectrometer (Thermo Fisher Scientific) coupled online to a Dionex Ultimate 3000 RSLCnano system (Thermo Fisher Scientific). The liquid chromatography setup consisted of a 75 μm × 2 cm trap column and a 75 μm × 40 cm analytical column, packed in-house with Reprosil Pur ODS-3 1.9 μm particles (Dr Maisch). Peptides were loaded onto the trap column using 0.1% formic acid in water at a flow rate of 5 μl min–1 and separated using a 50 min linear gradient from 4% to 32% of solvent B (0.1% (v/v) formic acid and 5% (v/v) DMSO in acetonitrile) at a 300 nl min–1 flow rate. nanoLC solvent A was 0.1% (v/v) formic acid and 5% (v/v) DMSO in HPLC-grade water. The Eclipse mass spectrometer was operated in data-dependent-acquisition (DDA) mode and positive ionization mode. Full-scan MS1 spectra were recorded in the Orbitrap from 360 to 1,300 m/z at 60,000 resolution using an automatic gain control target value of 100% and a maximum injection time of 50 ms. The cycle time was set to 2 s. Orbitrap readout MS2 scans were performed using higher energy collision-induced dissociation and a normalized collision energy of 30%. For full proteome analysis, the precursor isolation window was set to 1.3 m/z with 15,000 MS2 resolution, an automatic gain control target value of 200% and maximum injection time of 22 ms. Only precursors with a charge state of 2–6 were selected and dynamic exclusion was set to 30 s. Endogenous HRI (phospho)peptide levels were monitored using a parallel reaction monitoring assay with the same gradient settings used in the DDA experiment. PROCAL57 retention time peptides were added to each sample before LC–MS measurements.
Processing of raw MS data for determination of phosphorylation sites
DDA raw MS data files were processed using MaxQuant58 (v.2.4.0.0) with default settings and intensity based absolute quantification (iBAQ) enabled (protein FDR = 0.01, PSM FDR = 0.01, site FDR = 0.01, maximum missed trypsin site = 2) enabled. Phosphorylation of Ser, Thr and Tyr residues (phospho (STY)) was included as a variable modification, and ‘match between runs’ was activated. Spectra were searched against forward and reverse sequences of the human reference proteome, including isoforms (UniProt UP000005640, taxon ID 9606).
The MaxQuant output was further analysed using Perseus59 (v.1.6.15.0). For analysis of differentially phosphorylated sites, phosphosite intensities from the phospho(STY)Sites.txt file were log2-transformed and filtered for a minimum number of three valid values in at least one group. Missing values were imputed from the normal distribution, and differentially phosphorylated sites on HRI between HRI + haemin and HRI + haemin + DELE1 samples were identified by two-sided Student’s t-test (S0 = 0.5; permutation-based FDR < 0.05, 250 randomizations). To ensure equal HRI protein expression between samples, iBAQ values from the proteinGroups.txt file for HRI were compared across samples. For the lollipop plot depicting the phosphorylation sites, summed intensities of phosphosites were normalized to the relative expression levels of HRI (derived from log2-transformed iBAQ values) in the respective sample.
The recorded parallel reaction monitoring raw MS data files were imported into Skyline-daily60 (v.25.1) for data filtering and analysis. A spectral library was constructed with the HRI (phospho)peptide MS/MS spectra identified in the DDA experiment. Peaks were integrated using automatic settings followed by manual curation. The summed area under the fragment ion traces was exported for quantitative comparison. Phosphopeptide intensities were normalized to the summed intensity of unmodified HRI peptides in the respective sample.
Sample preparation for determination of protein–protein crosslinks by MS
To assess confirmational changes occurring in HRI after haem release and kinase activation by S-DELE1, Strep-tagged HRI purified from human cells in the presence or absence of S-DELE1 was crosslinked for 30 min at room temperature with 125 μM DSS. The crosslinking reaction was terminated by the addition of 25 mM Tris-HCl pH 7.5 for 10 min on ice. Crosslinked proteins were denatured by the addition of 4 M urea in 50 mM Tris buffer. To reduce and alkylate disulfides, 10 mM tris(2-carboxyethyl)phosphine (TCEP, Thermo Fisher Scientific) and 40 mM 2-chloroacetamide (Sigma-Aldrich) were added. Samples were incubated at 37 °C for 20 min. Following incubation, samples were diluted 1:3 with MS-grade water (VWR). Proteins were enzymatically digested overnight at 37 °C using 1 µg LysC and 2 µg trypsin (Promega). After digestion, the reaction was acidified to a final concentration of 1% trifluoroacetic acid (Merck), and peptides were desalted using Sep-Pak C18 1cc vacuum cartridges (Waters). A total of 200 ng peptides was loaded onto Evotips Pure (Evosep).
LC–MS/MS data acquisition for determination of protein–protein crosslinks
Peptides were eluted from Evotips onto a 15-cm PepSep C18 column (15 cm × 150 µm, 1.5 µm particle size, Bruker Daltonics) using an Evosep One HPLC system, using the 30 samples per day method. MS analysis was carried out on an Orbitrap Exploris 480 (Thermo Fisher), operated in DDA mode. Full MS scans were collected from m/z 300 to 1,650 Th at a resolution of 60,000 (at m/z 200 Th). The top 15 most intense precursor ions were selected for fragmentation using stepped higher-energy C-trap dissociation at normalized collision energies of 19, 27 and 35. MS2 spectra were acquired with a resolution of 30,000 (at m/z 200 Th) across a dynamic m/z range. Normalized automatic gain control targets were set to 300% for MS1 and 100% for MS2, with a maximum injection time of 25 ms for MS1 and auto for MS2. Ions with a charge state of +2 were excluded to prioritize crosslinked precursors.
Processing of raw MS data for determination of protein–protein crosslinks
Raw data were analysed using Proteome Discoverer (v.2.5.0.400), incorporating XlinkX/PD nodes61. Crosslinked peptides were identified via database search against a FASTA file containing the relevant protein sequences. DSS/BS3 was selected as the crosslinking reagent. Carbamidomethylation of cysteines was set as a static modification, whereas oxidation of methionines and N-terminal acetylation were defined as variable modifications. Trypsin/P was used as the specified protease, allowing for up to two missed cleavages. Peptide identifications were accepted with a minimum score of 40 and a delta score of at least 4. A 1% FDR at the peptide level was applied for filtering.
Analytical flow cytometry
For analysis of CHOP(Neon) fluorescence in HAP1 ∆HRI cells transiently expressing cDNAs of interest, cells were treated as indicated 24 h after transfection, detached using trypsin–EDTA (0.25%, Gibco) and measured on a BD LSRFortessa flow cytometer (BD Biosciences). Co-transfection of mCherry was used to identify transfected cells.
The mitochondrial membrane potential was measured using the ratiometric probe JC-1 (MedChemExpress, HY-15534), mitochondrial ROS production was determined using the fluorescent ROS probe MitoSOX (Thermo Fisher, M36007) and free cellular iron was assessed using FerroOrange (CST, 36104). Cells were treated as indicated, collected by trypsinization, washed with PBS and incubated with 1 µM MitoSOX, 1 µM JC-1 or 1 µM FerroOrange in serum-free medium for 30 min at 37 °C. Subsequently, cells were washed with serum-free medium and fluorescence was recorded on a BD LSRFortessa flow cytometer. JC-1 was excited at 488 nM and fluorescence was recorded using 585/42 nm (JC-1 monomers) and 530/30 nm (JC-1 aggregates) emission filters. MitoSOX was excited at 405 nM, and emission was recorded using a 610/20 nm filter. FerroOrange was excited at 561 nm and emission was recorded using a 582/15 nm filter.
To assess protein aggregation by flow cytometry, 1 million K562 cells were fixed with 4% formaldehyde (Sigma Aldrich) in PBS for 30 min at room temperature. Subsequently, cells were washed with PBS, permeabilized with PBS supplemented with 0.5% Triton-X100 (Sigma Aldrich) for 30 min at room temperature and stained for 30 min at room temperature with Proteostat (Enzo, ENZ-51023, 1:1,250 in PBS). Before detection, cells were washed once with PBS, and proteostat fluorescence was recorded on a BD LSRFortessa flow cytometer using 488 nm laser excitation and 585/42 emission filters.
All data were acquired using FACSDiva (BD, v.8.0.1) and analysed using FlowJo software (BD, v.10.4). Cells were identified by forward scatter area versus sideward scatter area gating, and doubles were excluded on the basis of sideward scatter height versus area gating. For analysis of HRI domain deletion and truncation mutant activity, the top 10% of mCherry-positive cells were gated and their mean mNeon intensity was divided by the mean mNeon intensity of untransfected (mCherry-negative) cells. For analysis of CHOP(Neon) fluorescence induction by invertebrate DELE1 and HRI constructs in response to DHA, background fluorescence in the green channel (induced by DHA treatment) of untransfected, DMSO-treated or DHA-treated cells was subtracted from the mean mNeon fluorescence of all mCherry-positive cells. MitoSOX, FerroOrange and proteostat data represent unprocessed mean fluorescence intensity values from single cells normalized to the respective control. JC-1 data represent the mean fluorescence intensity of red, JC-1 monomers divided by the mean fluorescence intensity of green JC-1 aggregates and normalized to the DMSO control.
Microscopy
For analysis of mitochondrial morphology following haem starvation, BJEH cells were plated onto live-cell imaging slides (Ibidi, 80807), treated as indicated, and stained for 30 min with 100 nM TMRM (Thermo Fisher, T668) at 37 °C. Subsequently, cells were washed with normal medium, and images of mitochondria were acquired with an DMi8 (Leica) scanning confocal microscope equipped with a ×40 water-immersion objective (Leica, Plan Apochromat NA 1.1), a white light laser excitation and HyD detector unit (Leica, TCS SP8 X), and a humidified environmental chamber (Okolab, 5% CO2, 37 °C). All images were acquired using Leica Application Suite X (Leica, v.3.5.7) and mitochondrial morphology was scored in Fiji (v.2.16.0/1.54p) and ImageJ (v.1.54f). Cells with short, rounded mitochondria were scored as ‘fragmented’, whereas cells containing unusually long and highly connected mitochondria were scored as ‘elongated’.
Structure predictions
Computational protein structure predictions were obtained using AlphaFold23 (v.3) and visualized using UCSF ChimeraX62 (v.1.8). The DELE1–HRI interaction was modelled using two copies of HRI(1–143) and two copies of DELE1(238–436) as input. Overlays between human and H. vulgaris proteins are based on the following AlphaFold models from UniProt: Q96E52 (192–524), T2M7Q7 (110–391), Q14154 (229–436), T2MDI1 (135–420), Q9BQI3 (63–238; 376–630); and XP047128012.1 (45–220; 303–562).
Seahorse assays
Mitochondrial function was assessed using an Agilent Seahorse XF Cell Mito Stress Test kit and XFe96/XF Pro FluxPak Mini (Agilent Technologies, 103015-100 and 103793-100, respectively), and the oxygen consumption rate was monitored using a Seahorse XFe96 Analyzer (Agilent Technologies) according to the manufacturer’s instructions. 293T cells (2 × 104 per well for DHA, 1 × 104 per well for SA and SA + NMPP) were seeded in a XFe96/XF 96-well plate coated with poly-l-lysine (Sigma-Aldrich) 1 day before treatment. Cells were then treated for 4 h with 5 μM DHA and for 24 h with 100 μM SA or 100 μM SA + 20 μM NMPP. One hour before the assay, cells were washed twice with Seahorse XF DMEM medium (Agilent Technologies, 103575-100) supplemented with 1 mM pyruvate, 2 mM glutamine and 10 mM glucose (Agilent Technologies, 103578-100, 103579-100 and 103577-100, respectively). Subsequently, cells were placed in a CO2-free BioTek Cytation 1 imaging reader, in which bright-field images of each well were taken. The mito-stress test was performed following a standard protocol. For oligomycin, FCCP and rotenone–antimycin A, concentrations of 1.5 μM, 1 μM and 0.5 μM, respectively, were used. To determine cell numbers for normalization, Hoechst 33342 (Thermo Fisher Scientific, 62249) was added to the rotenone–antimycin A solution to reach a final concentration of 10 μM. After completion of the mito-stress test, cells were returned to the BioTek Cytation 1 imaging reader, in which fluorescence images of the Hoechst 33342 signal were taken. The resulting cell numbers were imported into Wave software (v.2.6.1) and normalization was applied. Seahorse Analytics (v.1.0.0.796) was used for analyses.
Statistics and reproducibility
For the genome-wide genetic screen, 2.19 × 107 single cells were interrogated phenotypically according to their CHOP(Neon) signal and genetically by deep sequencing of gene-trap integration sites. This process produced a total of 2,743,720 unique mutations in the sense orientation of the affected genes. A two-sided Fisher’s exact test was used to calculate enrichment of mutations in the high or low channel, and P values were FDR-corrected using the Benjamini–Hochberg method, as previously described21.
For haem measurements, data show the mean ± s.d. of at least three independent biological replicates, with each experiment containing technical duplicates or triplicates. The exact number of independent biological replicates (n) is stated in the figure legends. HRI haem-binding data were normalized to the average signal intensity of the respective experiment to account for differences in the background signal between biologically independent experiments.
For the phosphorylation analysis by MS of overexpressed HRI (Fig. 4b and Extended Data Fig. 8i), n = 4 independent biological replicates were measured in the same run. For the MS analysis of endogenous HRI phosphorylation after DHA or SA treatment (Extended Data Fig. 8j,k), n = 3 (WT – DMSO) or n = 4 (all other samples) independent biological replicates were measured in the same run. For MS analysis of endogenous HRI phosphorylation after haemin treatment (Extended Data Fig. 8l), n = 4 independent biological replicates were measured in the same run. For cross-linking MS experiments (Fig. 4c,d and Extended Data Fig. 8p), n = 4 independent biological replicates were analysed in the same run, and crosslinks identified in at least three independent biological replicates are depicted.
Immunoblots and protein gels are representative of at least three independent biological replicates with similar results obtained. Quantifications of immunoblotting data and the corresponding statistical analyses show the mean ± s.d., with the sample size (n) stated in the figure legends. Immunoblotting data were acquired using Image Lab (Bio-Rad, v.5.2) or FusionCapt Advance FX7 (Vilber, v.17.04a). Chemiluminescence was quantified using Image Lab (Bio-Rad, v.5.2) and normalized to the average signal intensity of the respective experiment and to a loading control to account for differences in the background signal between biologically independent experiments and protein loading, respectively63. Uncropped blots and protein gels are presented in Supplementary Fig. 1.
Mitochondrial morphology data show the mean ± s.d. of n = 3 independent biological replicates, with each experiment containing at least 30 cells per condition.
Analytical flow cytometry and Seahorse data show the mean ± s.d. of n = 3 independent biological replicates, with each experiment containing a minimum of technical triplicates.
No statistical methods were used to predetermine sample sizes. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessments. Statistical significance was determined by t-tests (comparison of two groups) or one-way or two-way ANOVA (comparison of multiple groups) tests followed by the appropriate multiple comparison test using GraphPad Prism 10. All statistical tests are two-sided. The specific test used in each experiment is indicated in the figure legends. All numerical values are presented in Supplementary Data 5.
Inclusion and ethics statement
This study was conducted using commercially available human cell lines. No human participants or animals were involved, and no personally identifiable information was used. All experimental procedures followed institutional biosafety and ethical guidelines. The research team collaborated across different career stages and institutions, with all contributors meeting authorship criteria and being appropriately credited.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
