AbstractIn mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2伪 (eIF2伪) results in the induction of the transcription factor ATF41,2,3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2伪 kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2伪 kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2伪 phosphorylation. Blockade of the OMA1鈥揇ELE1鈥揌RI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1鈥揇ELE1鈥揌RI pathway therefore represents a potential聽therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction. Subscription info for Chinese customersWe have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal.Go to naturechina.comRent or Buy articleGet time limited or full article access on ReadCube.from$8.99Rent or BuyAll prices are NET prices. Source data for immunoblots are provided in Supplementary Fig. 1. Gating strategies for flow cytometry experiments are provided in Supplementary Fig. 2. RNA sequencing data described in this manuscript (associated with Extended Data Figs. 1, 7c) are deposited in the NCBI Gene Expression Omnibus (GEO) (GSE134986). There are no restrictions on data availability. Code availability Analysis of CRISPRi screen results was carried out using custom code (MAGeCK-iNC) developed in the Kampmann lab, which was previously described37 and is freely available at https://kampmannlab.ucsf.edu/mageck-inc. References1.Bao, X. R. et al. Mitochondrial dysfunction remodels one-carbon metabolism in human cells. eLife 5, e10575 (2016).PubMed聽 PubMed Central聽Google Scholar聽 2.Quir贸s, P. M. et al. Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals. J. Cell Biol. 216, 2027鈥?045 (2017).PubMed聽 PubMed Central聽Google Scholar聽 3.Viader, A. et al. Aberrant Schwann cell lipid metabolism linked to mitochondrial deficits leads to axon degeneration and neuropathy. Neuron 77, 886鈥?98 (2013).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 4.Mattson, M. P., Gleichmann, M. Cheng, A. Mitochondria in neuroplasticity and neurological disorders. Neuron 60, 748鈥?66 (2008).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 5.Sun, N., Youle, R. J. Finkel, T. The mitochondrial basis of aging. Mol. Cell 61, 654鈥?66 (2016).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 6.Suomalainen, A. Battersby, B. J. Mitochondrial diseases: the contribution of organelle stress responses to pathology. Nat. Rev. Mol. Cell Biol. 19, 77鈥?2 (2018).CAS聽 PubMed聽Google Scholar聽 7.Fiorese, C. J. et al. The transcription factor ATF5 mediates a mammalian mitochondrial UPR. Curr. Biol. 26, 2037鈥?043 (2016).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 8.M眉nch, C. Harper, J. W. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature 534, 710鈥?13 (2016).PubMed聽 PubMed Central聽 ADS聽Google Scholar聽 9.Papa, L. Germain, D. Estrogen receptor mediates a distinct mitochondrial unfolded protein response. J. Cell Sci. 124, 1396鈥?402 (2011).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 10.Papa, L. Germain, D. SirT3 regulates the mitochondrial unfolded protein response. Mol. Cell. Biol. 34, 699鈥?10 (2014).PubMed聽 PubMed Central聽Google Scholar聽 11.Zhao, Q. et al. A mitochondrial specific stress response in mammalian cells. EMBO J. 21, 4411鈥?419 (2002).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 12.Harding, H. P. et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol. Cell 11, 619鈥?33 (2003).CAS聽 PubMed聽Google Scholar聽 13.Wek, R. C., Jiang, H. Y. Anthony, T. G. Coping with stress: eIF2 kinases and translational control. Biochem. Soc. Trans. 34, 7鈥?1 (2006).CAS聽 PubMed聽Google Scholar聽 14.Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173鈥?183 (2013).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 15.Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647鈥?61 (2014).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 16.Chen, J. J. London, I. M. Regulation of protein synthesis by heme-regulated eIF-2伪 kinase. Trends Biochem. Sci. 20, 105鈥?08 (1995).CAS聽 PubMed聽Google Scholar聽 17.Kafina, M. D. Paw, B. H. Intracellular iron and heme trafficking and metabolism in developing erythroblasts. Metallomics 9, 1193鈥?203 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 18.Lu, L., Han, A. P. Chen, J. J. Translation initiation control by heme-regulated eukaryotic initiation factor 2伪 kinase in erythroid cells under cytoplasmic stresses. Mol. Cell. Biol. 21, 7971鈥?980 (2001).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 19.Matts, R. L., Schatz, J. R., Hurst, R. Kagen, R. Toxic heavy metal ions activate the heme-regulated eukaryotic initiation factor-2伪 kinase by inhibiting the capacity of hemin-supplemented reticulocyte lysates to reduce disulfide bonds. J. Biol. Chem. 266, 12695鈥?2702 (1991).CAS聽 PubMed聽Google Scholar聽 20.Harada, T., Iwai, A. Miyazaki, T. Identification of DELE, a novel DAP3-binding protein which is crucial for death receptor-mediated apoptosis induction. Apoptosis 15, 1247鈥?255 (2010).CAS聽 PubMed聽Google Scholar聽 21.Quir贸s, P. M., Langer, T. L贸pez-Ot铆n, C. New roles for mitochondrial proteases in health, ageing and disease. Nat. Rev. Mol. Cell Biol. 16, 345鈥?59 (2015).PubMed聽Google Scholar聽 22.Baker, M. J. et al. Stress-induced OMA1 activation and autocatalytic turnover regulate OPA1-dependent mitochondrial dynamics. EMBO J. 33, 578鈥?93 (2014).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 23.Ehses, S. et al. Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1. J. Cell Biol. 187, 1023鈥?036 (2009).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 24.Kim, H., Botelho, S. C., Park, K. Kim, H. Use of carbonate extraction in analyzing moderately hydrophobic transmembrane proteins in the mitochondrial inner membrane. Protein Sci. 24, 2063鈥?069 (2015).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 25.Berlanga, J. J., Herrero, S. de Haro, C. Characterization of the hemin-sensitive eukaryotic initiation factor 2伪 kinase from mouse nonerythroid cells. J. Biol. Chem. 273, 32340鈥?2346 (1998).CAS聽 PubMed聽Google Scholar聽 26.Delaunay, J., Ranu, R. S., Levin, D. H., Ernst, V. London, I. M. Characterization of a rat liver factor that inhibits initiation of protein synthesis in rabbit reticulocyte lysates. Proc. Natl Acad. Sci. USA 74, 2264鈥?268 (1977).CAS聽 PubMed聽 ADS聽Google Scholar聽 27.Lee, H., Smith, S. B. Yoon, Y. The short variant of the mitochondrial dynamin OPA1 maintains mitochondrial energetics and cristae structure. J. Biol. Chem. 292, 7115鈥?130 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 28.Adjibade, P. et al. DDX3 regulates endoplasmic reticulum stress-induced ATF4 expression. Sci. Rep. 7, 13832 (2017).PubMed聽 PubMed Central聽 ADS聽Google Scholar聽 29.Walter, P. Ron, D. The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081鈥?086 (2011).CAS聽 PubMed聽 ADS聽Google Scholar聽 30.Wek, R. C. Role of eIF2伪 kinases in translational control and adaptation to cellular stress. Cold Spring Harb. Perspect. Biol. 10, a032870 (2018).PubMed聽Google Scholar聽 31.Zhang, S. et al. HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis. eLife 8, e46976 (2019).PubMed聽 PubMed Central聽Google Scholar聽 32.Chou, A. et al. Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury. Proc. Natl Acad. Sci. USA 114, E6420鈥揈6426 (2017).CAS聽 PubMed聽Google Scholar聽 33.Das, I. et al. Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit. Science 348, 239鈥?42 (2015).CAS聽 PubMed聽 PubMed Central聽 ADS聽Google Scholar聽 34.Acin-Perez, R. et al. Ablation of the stress protease OMA1 protects against heart failure in mice. Sci. Transl. Med. 10, eaan4935 (2018).PubMed聽Google Scholar聽 35.Sidrauski, C. et al. Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response. eLife 4, e07314 (2015).PubMed聽 PubMed Central聽Google Scholar聽 36.Chen, J. J. et al. Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation. J. Biol. Chem. 294, 18952鈥?8966 (2019).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 37.Tian, R. et al. CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons. Neuron 104, 239鈥?55 (2019).CAS聽 PubMed聽Google Scholar聽 38.Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867鈥?882 (2016).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 39.Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417鈥?19 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 40.Soneson, C., Love, M. I. Robinson, M. D. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000 Res. 4, 1521 (2015). Google Scholar聽 41.Love, M. I., Huber, W. Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).PubMed聽 PubMed Central聽Google Scholar聽 42.Kuleshov, M. V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 44, W90鈥揥97 (2016).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 43.Eisen, M. B., Spellman, P. T., Brown, P. O. Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 95, 14863鈥?4868 (1998).CAS聽 PubMed聽 ADS聽Google Scholar聽 44.Saldanha, A. J. Java Treeview鈥攅xtensible visualization of microarray data. Bioinformatics 20, 3246鈥?248 (2004).CAS聽 PubMed聽Google Scholar聽 45.Horlbeck, M. A. et al. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife 5, e19760 (2016).PubMed聽 PubMed Central聽Google Scholar聽 46.Nagy, T. Kampmann, M. CRISPulator: a discrete simulation tool for pooled genetic screens. BMC Bioinformatics 18, 347 (2017).PubMed聽 PubMed Central聽Google Scholar聽 47.Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676鈥?82 (2012).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 48.Rust, M. J., Bates, M. Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793鈥?96 (2006).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 49.Huang, B., Wang, W., Bates, M. Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810鈥?13 (2008).CAS聽 PubMed聽 PubMed Central聽 ADS聽Google Scholar聽 50.Wojcik, M., Hauser, M., Li, W., Moon, S. Xu, K. Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells. Nat. Commun. 6, 7384 (2015).CAS聽 PubMed聽 PubMed Central聽 ADS聽Google Scholar聽 51.Bossi, M. et al. Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species. Nano Lett. 8, 2463鈥?468 (2008).CAS聽 PubMed聽 ADS聽Google Scholar聽 52.Gorur, A. et al. COPII-coated membranes function as transport carriers of intracellular procollagen I. J. Cell Biol. 216, 1745鈥?759 (2017).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 53.Cox, J. Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367鈥?372 (2008).CAS聽Google Scholar聽 54.Liao, M. et al. Impaired dexamethasone-mediated induction of tryptophan 2,3-dioxygenase in heme-deficient rat hepatocytes: translational control by a hepatic eIF2伪 kinase, the heme-regulated inhibitor. J. Pharmacol. Exp. Ther. 323, 979鈥?89 (2007).CAS聽 PubMed聽Google Scholar聽 55.Dey, M. et al. PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2伪. Mol. Cell. Biol. 25, 3063鈥?075 (2005).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 56.Schmidt, E. K., Clavarino, G., Ceppi, M. Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6, 275鈥?77 (2009).CAS聽 PubMed聽Google Scholar聽 57.Han, H. et al. TRRUST v2: an expanded reference database of human and mouse transcriptional regulatory interactions. Nucleic Acids Res. 46, D380鈥揇386 (2018).CAS聽 PubMed聽Google Scholar聽 Download referencesAcknowledgementsWe thank G. Mohl, B. Herken and D. Swaney for contributions to preliminary experiments; J. Leong for support with FACS; C. Richards for contributions to data visualization; M. Boone, K. Shokat and members of the Kampmann lab for discussions; A. Frost, I. Jain, A. Samelson and E. Li for comments on the manuscript; E. Chow (UCSF Center for Advanced Technology) for support with next-generation sequencing; S. Elmes (UCSF Laboratory for Cell Analysis) for support with FACS; and D. Larson (UCSF Nikon Imaging Center) for support with fluorescence microscopy. This work was supported by the National Institutes of Health grants GM119139 (M.K.), DK26506 (M.A.C.), GM44037 (M.A.C.), OD022552 (A.P.W.), the Beckman Young Investigator Program (K.X.) and a Larry L. Hillblom Foundation Postdoctoral Fellowship (X.G.). M.K. and K.X. are Chan Zuckerberg Biohub Investigators. The DNA Technologies and Expression Analysis Core at the UC Davis Genome Center is supported by the NIH Shared Instrumentation Grant 1S10OD010786-01.Author informationAffiliationsInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA, USAXiaoyan Guo,聽Giovanni Aviles,聽Ruilin Tian聽 聽Martin KampmannChan Zuckerberg Biohub, San Francisco, CA, USAXiaoyan Guo,聽Giovanni Aviles,聽Ruilin Tian,聽Bret A. Unger,聽Ke Xu聽 聽Martin KampmannDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USAYi Liu聽 聽M. Almira CorreiaBiophysics Graduate Program, University of California, San Francisco, San Francisco, CA, USARuilin TianDepartment of Chemistry, University of California, Berkeley, Berkeley, CA, USABret A. Unger聽 聽Ke XuDepartment of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USAYu-Hsiu T. Lin聽 聽Arun P. WiitaDepartment of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USAM. Almira CorreiaDepartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USAM. Almira CorreiaThe Liver Center, University of California, San Francisco, San Francisco, CA, USAM. Almira CorreiaDepartment of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USAMartin KampmannAuthorsXiaoyan GuoView author publicationsYou can also search for this author in PubMed聽Google ScholarGiovanni AvilesView author publicationsYou can also search for this author in PubMed聽Google ScholarYi LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarRuilin TianView author publicationsYou can also search for this author in PubMed聽Google ScholarBret A. UngerView author publicationsYou can also search for this author in PubMed聽Google ScholarYu-Hsiu T. LinView author publicationsYou can also search for this author in PubMed聽Google ScholarArun P. WiitaView author publicationsYou can also search for this author in PubMed聽Google ScholarKe XuView author publicationsYou can also search for this author in PubMed聽Google ScholarM. Almira CorreiaView author publicationsYou can also search for this author in PubMed聽Google ScholarMartin KampmannView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsX.G. and M.K. conceptualized and led the overall project, analysed results and wrote the manuscript, with input from all co-authors. X.G. and G.A. conducted and analysed the CRISPRi screen and follow-up experiments. Y.L. and M.A.C. conducted and analysed the experiments with purified proteins. R.T. and M.K. analysed and visualized RNA sequencing results. B.A.U. and K.X. conducted and analysed super-resolution microscopy experiments. Y.-H.T.L. and A.P.W. conducted and analysed mass spectrometry experiments.Corresponding authorCorrespondence to Martin Kampmann.Ethics declarations Competing interests The authors declare no competing interests. Additional informationPeer review information Nature thanks Cole Haynes and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Publisher鈥檚 note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended data figures and tablesExtended Data Fig. 1 Induction of ATF4 target genes under mitochondrial stress conditions and characterization of the ATF4 translational reporter.a, HEK293T cells were treated with 50 渭g聽ml鈭? doxycycline or 1.25 ng聽ml鈭? oligomycin for 16聽h, and transcript levels were compared to untreated cells using RNA sequencing for n聽=聽2 (doxycycline) or n聽=聽3 (oligomycin) independent experiments. Differentially expressed genes and P聽values were determined as described in the Methods (full datasets in Supplementary Tables 1, 2). Significantly induced genes (Padj聽 聽0.05), with at least a twofold increase in treated over untreated conditions, are included in the analysis. Enrichment analysis for targets of transcription factors that are annotated in the TRRUST database57 (statistical analysis described in the Methods) detected ATF4 as the only significant transcription factor (Padj聽 聽0.05) for both treatments. Genes induced by both treatments are listed, as well as genes annotated as ATF4 targets in TRRUST (dark green dots) or a previous study1 (light green dots). CARS is also known as CARS1. b, Quantification of the knockdown efficiency of sgRNAs that target the eIF2伪 kinases by qPCR (n聽=聽3 technical replicates). For HRI, two independent sgRNAs (1 and 2) were characterized. WT, wild type. c, d, Validation of the reporter cell line using the endoplasmic-reticulum stressor thapsigargin. Reporter cells expressing either individual (c) or triple (d) sgRNAs that target the indicated eIF2伪 kinases were exposed to 75聽nM thapsigargin for 8聽h before measuring reporter levels by flow cytometry. The induction of the ATF4 reporter by thapsigargin is blocked by knockdown of PERK. The fold change in reporter levels was quantified as in Fig. 1b (mean聽卤聽s.d., n聽=聽3 culture wells). e, Pharmacological inhibition of mitochondrial function using the mitochondrial ribosome inhibitor doxycycline, the electron transport chain inhibitors antimycin A and rotenone and the ATP synthase inhibitor oligomycin induces the ATF4 reporter. Reporter cells were exposed to the indicated treatments for 16聽h before measuring reporter levels by flow cytometry. The fold change in reporter levels was quantified as in Fig. 1b (mean聽卤聽s.d., n聽=聽3 culture wells). f, The ATF4 reporter is induced in cells with CRISPRi knockdown of factors that are required for mitochondrial protein homeostasis (HSPD1 and LONP1) and mitochondrial ribosomal proteins (MRPL17 and MRPL22) (red), compared to wild-type cells (blue). Similar results in more than three independent experiments. g, DELE1 and HRI are not required to trigger the ISR in response to endoplasmic-reticulum stress. Reporter cells expressing non-targeting control sgRNAs or sgRNAs that target HRI or DELE1 were exposed to 75聽nM thapsigargin for 8聽h before measuring reporter levels by flow cytometry. The fold change in reporter levels (mean聽卤聽s.d., n聽=聽3 culture wells) is the ratio of median fluorescence values for thapsigargin-treated over untreated samples.Extended Data Fig. 2 Expression levels of DELE1 constructs and investigation of DELE1 cleavage and export mechanisms.a, Commercially available antibodies fail to detect DELE1. Lysates from either wild-type HEK293T cells or those expressing an sgRNA that targets DELE1 were probed with the indicated DELE1 antibodies and an antibody against 尾-actin. None of the bands detected by the DELE1 antibodies decreases in intensity in DELE1-knockdown cells. The asterisk indicates a non-specific band. Similar results in n聽 聽2 independent experiments. b, qPCR quantification of DELE1 mRNA levels in HEK293T cells in which DELE1 was knocked down by CRISPRi, and/or which express DELE1-mClover stably from the AAVS1 safe-harbour locus or via transient transfection (n聽=聽3 technical replicates). c鈥?b>e, Knockdown of PMPCB induces the ATF4 translational reporter in the absence of mitochondrial stressors in an HRI- and DELE1-dependent manner. c, Left, representative immunoblot of ATF4, DELE1-mClover and PMPCB in two PMPCB-knockdown cell lines and non-targeting control cells that were treated with 1.25聽ng聽ml鈭? oligomycin for 16聽h where indicated. Right, quantification (mean聽卤聽s.d., n聽=聽2 blots). d, e, HEK293T reporter cells were co-transfected with PMPCB-targeting sgRNAs 1 or 2 (with a BFP marker) and sgRNAs that target HRI (d) or DELE1 (e) (with a GFP maker) to generate a cell line with four populations as shown. The intensity of the ATF4 reporter was quantified using flow cytometry (mean聽卤聽s.d., n聽=聽3 culture wells). f, Quantification of the immunoblot shown in Fig. 3f (n聽=聽2 blots). g, Immunoblot of OPA1, DELE1-mClover and ATF4 in OPA1-knockdown and non-targeting control cells that were treated with 1.25聽ng聽ml鈭? oligomycin for 16聽h or untreated. Similar results in n聽 聽2 technical replicates. h, Measurement of mitochondrial potential by tetramethylrhodamine ethyl ester (TMRE) staining in cells that were treated with 1.25 ng聽ml鈭? oligomycin or 5聽渭M CCCP for 4聽h (mean聽卤聽s.d., n聽=聽3 culture wells, P聽values determined by two-tailed unpaired t test). i, Subcellular localization of DELE1L and DELE1S in OPA1-knockdown and non-targeting control cells that were treated with 1.25聽ng聽ml鈭? oligomycin for 16聽h or untreated. Similar results in n聽 聽2 technical replicates. For gel source data, see Supplementary Fig. 1.Extended Data Fig. 3 Characterization of DELE1 submitochondrial localization.a, b, Zoomed-out views of two-colour 3D-STORM super-resolution images of DELE1-mClover, TOM20 and HSP60. Two-colour DELE1-mClover (magenta) with TOM20 (a; green) or HSP60 (b; green), followed by the two separated colour channels. Scale bars, 1聽渭m. The boxed regions correspond to Fig. 3g, h. Similar results in n聽=聽3 independent experiments. c, Biochemical fractionation indicates that DELE1 associates with mitochondrial membranes. Cells stably expressing DELE1-mClover were fractionated into cytosol and mitochondria. Mitochondria were incubated in either isotonic buffer (10聽mM Tris HCl, pH聽6.7, 0.15聽mM MgCl2, 0.25聽mM sucrose, 1 mM DTT and protease inhibitor cocktail (Sigma, 5892970001)) or H2O (extreme hypotonic condition) for 5聽min, followed by centrifugation (10,000g for 10聽min) to separate the supernatant and pellet. The pellet was either dissolved with RIPA buffer or incubated with 0.1聽M NaCO3 (pH聽11.4) for 30聽min at 4鈥壜癈. The supernatant and pellet from NaCO3-treated samples were collected for western blotting. Unlike the soluble matrix proteins LONP1 and HSPD1, which can be extracted with H2O incubation, only a small proportion of DELE1 is present in the supernatant. NaCO3 can extract the majority of the DELE1S, but not DELE1L. This is similar to the pattern of the mitochondrial membrane protein VDAC, suggesting that DELE1L is probably a membrane associated protein (n聽=聽2 independent experiments). For gel source data, see Supplementary Fig. 1. d, The indicated DELE1-mClover constructs were transiently overexpressed in reporter cells, and reporter induction was quantified by flow cytometry (mean聽卤聽s.d., n聽=聽3 culture wells). Subcellular localization was evaluated by microscopy in cells that also express the mitochondrially targeted plasmid mRuby. e, Lack of colocalization of transiently expressed DELE1(螖N100)-mClover and DELE1(螖N148)-mClover (green) with the mitochondrial stain MitoTracker (red). Scale bars, 7聽渭m. Similar results in n聽=聽2 culture wells. f, Increased detection of DELE1-mClover outside the mitochondria after treatment with oligomycin. 3D-STORM super-resolution images of stably expressed DELE1-mClover (colours indicate depth in the z聽dimension) in untreated cells (left) and cells treated with 1.25聽ng聽ml鈭? oligomycin for 16聽h (right). The areas in red boxes in the top panels are shown at a higher magnification in the bottom panels. Similar results in n聽=聽3 independent experiments. g, Colocalization of transiently expressed HRI-mClover and DELE1-mClover with the mitochondrially targeted plasmid mRuby (mito7-mRuby). Scale bars, 7聽渭m (n聽=聽1 culture well).Extended Data Fig. 4 Protein kinase assay of purified HRI.a, Purified recombinant HRI, DELE1 and eIF2伪. A total of 800聽ng of each recombinant protein was subjected to SDS鈥揚AGE and stained with Coomassie blue (n聽=聽1 gel). b, c, HRI kinase reactions were performed with 1聽渭M recombinant yeast eIF2伪 and various amounts of purified recombinant HRI protein. Reactions were stopped at the time points indicated by removing 5-渭l aliquots of the kinase reaction mixture and mixing with an equal volume of 2脳 SDS loading buffer. The SDS samples were then dotted on nitrocellulose blots and subjected to immunoblotting analysis with antibodies against phosphorylated eIF2伪. b, Representative dot blot. c, Densitometric quantification of dot blots expressed as the average value from n聽=聽2 individual experiments. To enable a reaction in a linear range, 25聽nM HRI and a 5-min incubation time were used for all the subsequent experiments. d, Enzyme kinetic constants for HRI activity with or without DELE1 in the presence or absence of haemin (mean聽卤聽s.e.m., n聽=聽3 individual reactions, fit for data shown in Fig. 4f). Kinetic constants were determined by fitting to the Michaelis鈥揗enten equation using a least-squares fit in Prism v.6.07. The constants calculated from HRI聽+聽haemin (marked with asterisks) are not accurate because at this range of substrate concentrations, the enzymatic reaction is first order and never reaches Vmax. But higher substrate concentrations cannot be used to obtain Vmax conditions, as purified eIF2伪 protein will precipitate at higher concentrations.Extended Data Fig. 5 Measurement of protein synthesis under mitochondrial stress.a, Immunoblot of HRI and ATF4 in wild-type and HRI-knockout cells. Cells were untreated or treated with 1.25聽ng聽ml鈭? oligomycin for 16聽h. Similar results in n聽=聽2 independent experiments. For gel source data, see Supplementary Fig. 1. b, Newly synthesized protein was labelled with puromycin. Wild-type or HRI-knockout cells were treated with 1.25聽ng聽ml鈭? puromycin for 1 or 2聽h or left untreated, followed by a 10-min incubation with puromycin (10聽渭g聽ml鈭?). Protein from each sample was quantified by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, 23225) and equally loaded. Two immunoblots are shown from separate experiments probed with anti-puromycin (top) and Ponceau S staining (middle). Bottom, quantification of newly synthesized protein (ratio of anti-puromycin signal to Ponceau S signal) for these blots from n聽=聽2 experiments.Extended Data Fig. 6 Examination of the mitochondrial stress response with a broad range of mitochondrial toxins and in non-HEK293T cells.a, Immunoblot of ATF4 in wild-type HEK293T cells and those treated with sgRNAs targeting DELE1, HRI or OMA1 under different mitochondrial stress conditions. Cells were left untreated or treated with 40聽nM antimycin A (AA), 40聽nM rotenone (rot), 50聽渭g聽ml鈭? doxycycline (dox) or 5聽渭M CCCP for 2 and 4聽h. Left, representative blots. Right, ATF4 levels were quantified and normalized to 尾-actin (mean聽卤聽s.d., n聽=聽2 blots). b, A broad range of mitochondrial toxins stimulates the accumulation of DELE1S. Cells stably expressing DELE1-mClover were untreated or treated with a panel of mitochondrial toxins for 16聽h (see聽Methods for details) and subjected to western blotting with antibodies detecting DELE1-mClover, ATF4 and actin. Top, representative blot. Middle, bottom, ATF4, DELE1L-mClover and DELE1S-mClover levels were quantified (mean聽卤聽s.d., n聽=聽2 blots). c, Subcellular localization of DELE1L and DELE1S in cells that were treated with a broad range of mitochondrial toxins. Biochemical fractionation of cells stably expressing DELE1-mClover that were either treated with different mitochondrial toxins as indicated for 16聽h or left untreated. 尾-actin and LONP1 were probed as markers for cytosol and mitochondria, respectively. Similar results in n聽=聽2 independent experiments. d, Examination of OPA1 cleavage in cells treated with a broad range of mitochondrial toxins. Similar results in n聽=聽2 independent experiments. e, Immunoblot of ATF4 in wild-type cells and those treated with sgRNAs targeting DELE1, HRI or OMA1 in the WTC11 human iPSC line. Cells were left untreated or treated with 1.25聽ng聽ml鈭? oligomycin for 4聽h. Similar results in n聽=聽2 independent experiments. f, Immunoblot of ATF4 in wild-type cells and those treated with sgRNAs targeting DELE1, HRI or OMA1 in the human HeLa cell line. Cells were left untreated or treated with 1.25聽ng聽ml鈭? oligomycin for 2 and 4聽h. Similar results in n聽=聽2 technical replicates. For gel source data, see Supplementary Fig. 1.Extended Data Fig. 7 The DELE1鈥揌RI pathway can be maladaptive and its blockade induces an alternative program.a, Knockdown of OMA1, DELE1 or HRI is protective during oligomycin treatment. HEK293T cells expressing non-targeting control sgRNA or sgRNAs that target HRI, DELE1 or OMA1 were left untreated or treated with 2.5聽ng聽ml鈭? oligomycin for 16聽h, and cells were counted (mean聽卤聽s.d., n聽=聽3 culture wells, P聽values determined by two-tailed unpaired t test). b, Knockdown of HRI is protective for cells in which the mitochondrial ribosomal protein MRPL17 is depleted, but sensitizes cells in which the mitochondrial protease LONP1 is depleted. HEK293T cells were co-transduced with a lentiviral construct expressing GFP and an sgRNA that targets HRI, and with a lentiviral construct expressing BFP and a non-targeting control sgRNA or sgRNAs that target MRPL17, MRPL22 or LONP1. Cells were cultured for 9聽d and the proportions of cells expressing GFP and BFP were quantified by flow cytometry on days 3, 6, 8 and 9 after infection (top). Thus, the effect of knockdown of HRI on proliferation in different genetic backgrounds could be evaluated in an internally controlled experiment. In parallel, the ATF4 reporter was quantified (bottom) (mean聽卤聽s.d., n聽=聽3 culture wells). c, HEK293T cells that were either infected with a non-targeting control sgRNA or sgRNAs that target OMA1, DELE1 or HRI were untreated or treated with 1.25聽ng聽ml鈭? oligomycin for 16聽h, and transcriptomes were analysed by RNA sequencing for n聽=聽3 independent experiments. Differentially expressed genes and P聽values were determined as described in the Methods (full datasets in Supplementary Tables 2, 5鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"supplementary material anchor\" href=\"/articles/s41586-020-2078-2#MOESM11\">7). The heat map only includes genes that changed significantly in expression upon oligomycin treatment (Padj聽 聽0.05), by at least twofold in at least one genetic background. Hierarchical clustering reveals four major gene clusters. Gene groups are indicated by dots in different colours: ATF4 target genes annotated by the TRRUST database (dark green, Padj value calculated by Enrichr) or a previous study1 (light green, P聽value calculated by Fisher鈥檚 exact test), genes co-expressed with HRI in the ARCHS4 database (orange, Padj value calculated by Enrichr), mitochondrially encoded genes (gold) and genes encoding heat-shock proteins (brown).Extended Data Table 1 Cloning strategies and oligonucleotide sequences used for generation of plasmidsFull size tableExtended Data Table 2 Protospacer sequences of individually cloned sgRNAsFull size tableExtended Data Table 3 qPCR primer sequencesFull size tableSupplementary information Supplementary Discussion: This file contains discussion of Mapping the site of stress-induced cleavage in DELE1, Michaelis-Menten kinetics of DELE1 stimulation of HRI, Differences between mitochondrial stressors, and Open questions.Reporting SummarySupplementary Figure 1: Source Data (Immunoblots). The original source images for all data obtained by electrophoretic separation. The full scanned images show the uncropped form with molecular weight markers and loading controls. Blots are labeled according to the corresponding Figure panel within the main or Extended Data Figures.Supplementary Figure 2: Gating Strategies (Flow cytometry). Representative flow cytometry data with gating strategies. Similar results were obtained in n 2 independent experiments.Supplementary Table 1: Differentially expressed genes upon doxycycline treatment in WT HEK293T cells, determined using DESeq2 (1.20.0) based on n = 2 experimental replicates for each the treated and untreated condition (see Methods for details of statistical analysis).Supplementary Table 2: Differentially expressed genes upon oligomycin treatment in WT HEK293T cells, determined using DESeq2 (1.20.0) based on n = 3 experimental replicates for each the treated and untreated condition (see Methods for details of statistical analysis).Supplementary Table 3: Results from the CRISPRi screen. Phenotypes for genes targeted in the CRISPRi screens for untreated and oligomycin-treated cells were analyzed using MAGeCK-iNC (see Methods for details).Supplementary Table 4: sgRNA counts from the CRISPRi screen. Raw counts for all sgRNAs, provided for each sample of the CRISPRi screen and separated by sublibrary (H1-H7) of the genome-wide library are provided in individual tabs.Supplementary Table 5: Differentially expressed genes upon oligomycin treatment in OMA1 KD HEK293T cells, determined using DESeq2 (1.20.0) based on n = 3 experimental replicates for each the treated and untreated condition (see Methods for details of statistical analysis).Supplementary Table 6: Differentially expressed genes upon oligomycin treatment in DELE1 KD HEK293T cells, determined using DESeq2 (1.20.0) based on n = 3 experimental replicates for each the treated and untreated condition (see Methods for details of statistical analysis).Supplementary Table 7: Differentially expressed genes upon oligomycin treatment in HRI KD HEK293T cells, determined using DESeq2 (1.20.0) based on n = 3 experimental replicates for each the treated and untreated condition (see Methods for details of statistical analysis).Rights and permissionsReprints and PermissionsAbout this articleCite this articleGuo, X., Aviles, G., Liu, Y. et al. Mitochondrial stress is relayed to the cytosol by an OMA1鈥揇ELE1鈥揌RI pathway. Nature 579, 427鈥?32 (2020). https://doi.org/10.1038/s41586-020-2078-2Download citationReceived: 25 July 2019Accepted: 03 February 2020Published: 04 March 2020Issue Date: 19 March 2020DOI: https://doi.org/10.1038/s41586-020-2078-2 Gustavo Della-Flora Nunes, Emma R. Wilson, Leandro N. Marziali, Edward Hurley, Nicholas Silvestri, Bin He, Bert W. O鈥橫alley, Bogdan Beirowski, Yannick Poitelon, Lawrence Wrabetz M. Laura Feltri Nature Communications (2021) Cheryl Q. E. Lee, Baptiste Kerouanton, Sonia Chothani, Shan Zhang, Ying Chen, Chinmay Kumar Mantri, Daniella Helena Hock, Radiance Lim, Rhea Nadkarni, Vinh Thang Huynh, Daryl Lim, Wei Leong Chew, Franklin L. Zhong, David Arthur Stroud, Sebastian Schafer, Vinay Tergaonkar, Ashley L. St John, Owen J. L. Rackham Lena Ho Nature Communications (2021) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.