Abstract
In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) 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 eIF2α 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 eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1–DELE1–HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1–DELE1–HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.
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Data availability
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.
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Acknowledgements
We 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.
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X.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.
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Extended data figures and tables
Extended 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 μg ml−1 doxycycline or 1.25 ng ml−1 oligomycin for 16 h, and transcript levels were compared to untreated cells using RNA sequencing for n = 2 (doxycycline) or n = 3 (oligomycin) independent experiments. Differentially expressed genes and P values were determined as described in the Methods (full datasets in Supplementary Tables 1, 2). Significantly induced genes (Padj < 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 < 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 eIF2α kinases by qPCR (n = 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 eIF2α kinases were exposed to 75 nM thapsigargin for 8 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 ± s.d., n = 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 h before measuring reporter levels by flow cytometry. The fold change in reporter levels was quantified as in Fig. 1b (mean ± s.d., n = 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 nM thapsigargin for 8 h before measuring reporter levels by flow cytometry. The fold change in reporter levels (mean ± s.d., n = 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 β-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 > 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 = 3 technical replicates). c–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 ng ml−1 oligomycin for 16 h where indicated. Right, quantification (mean ± s.d., n = 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 ± s.d., n = 3 culture wells). f, Quantification of the immunoblot shown in Fig. 3f (n = 2 blots). g, Immunoblot of OPA1, DELE1-mClover and ATF4 in OPA1-knockdown and non-targeting control cells that were treated with 1.25 ng ml−1 oligomycin for 16 h or untreated. Similar results in n > 2 technical replicates. h, Measurement of mitochondrial potential by tetramethylrhodamine ethyl ester (TMRE) staining in cells that were treated with 1.25 ng ml−1 oligomycin or 5 μM CCCP for 4 h (mean ± s.d., n = 3 culture wells, P 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 ng ml−1 oligomycin for 16 h or untreated. Similar results in n > 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 μm. The boxed regions correspond to Fig. 3g, h. Similar results in n = 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 mM Tris HCl, pH 6.7, 0.15 mM MgCl2, 0.25 mM sucrose, 1 mM DTT and protease inhibitor cocktail (Sigma, 5892970001)) or H2O (extreme hypotonic condition) for 5 min, followed by centrifugation (10,000g for 10 min) to separate the supernatant and pellet. The pellet was either dissolved with RIPA buffer or incubated with 0.1 M NaCO3 (pH 11.4) for 30 min at 4 °C. 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 = 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 ± s.d., n = 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(ΔN100)-mClover and DELE1(ΔN148)-mClover (green) with the mitochondrial stain MitoTracker (red). Scale bars, 7 μm. Similar results in n = 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 dimension) in untreated cells (left) and cells treated with 1.25 ng ml−1 oligomycin for 16 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 = 3 independent experiments. g, Colocalization of transiently expressed HRI-mClover and DELE1-mClover with the mitochondrially targeted plasmid mRuby (mito7-mRuby). Scale bars, 7 μm (n = 1 culture well).
Extended Data Fig. 4 Protein kinase assay of purified HRI.
a, Purified recombinant HRI, DELE1 and eIF2α. A total of 800 ng of each recombinant protein was subjected to SDS–PAGE and stained with Coomassie blue (n = 1 gel). b, c, HRI kinase reactions were performed with 1 μM recombinant yeast eIF2α and various amounts of purified recombinant HRI protein. Reactions were stopped at the time points indicated by removing 5-μl aliquots of the kinase reaction mixture and mixing with an equal volume of 2× SDS loading buffer. The SDS samples were then dotted on nitrocellulose blots and subjected to immunoblotting analysis with antibodies against phosphorylated eIF2α. b, Representative dot blot. c, Densitometric quantification of dot blots expressed as the average value from n = 2 individual experiments. To enable a reaction in a linear range, 25 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 ± s.e.m., n = 3 individual reactions, fit for data shown in Fig. 4f). Kinetic constants were determined by fitting to the Michaelis–Menten equation using a least-squares fit in Prism v.6.07. The constants calculated from HRI + 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 eIF2α 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 ng ml−1 oligomycin for 16 h. Similar results in n = 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 ng ml−1 puromycin for 1 or 2 h or left untreated, followed by a 10-min incubation with puromycin (10 μg ml−1). 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 = 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 nM antimycin A (AA), 40 nM rotenone (rot), 50 μg ml−1 doxycycline (dox) or 5 μM CCCP for 2 and 4 h. Left, representative blots. Right, ATF4 levels were quantified and normalized to β-actin (mean ± s.d., n = 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 h (see 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 ± s.d., n = 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 h or left untreated. β-actin and LONP1 were probed as markers for cytosol and mitochondria, respectively. Similar results in n = 2 independent experiments. d, Examination of OPA1 cleavage in cells treated with a broad range of mitochondrial toxins. Similar results in n = 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 ng ml−1 oligomycin for 4 h. Similar results in n = 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 ng ml−1 oligomycin for 2 and 4 h. Similar results in n = 2 technical replicates. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 The DELE1–HRI 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 ng ml−1 oligomycin for 16 h, and cells were counted (mean ± s.d., n = 3 culture wells, P 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 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 ± s.d., n = 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 ng ml−1 oligomycin for 16 h, and transcriptomes were analysed by RNA sequencing for n = 3 independent experiments. Differentially expressed genes and P values were determined as described in the Methods (full datasets in Supplementary Tables 2, 5–7). The heat map only includes genes that changed significantly in expression upon oligomycin treatment (Padj < 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 value calculated by Fisher’s 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).
Supplementary 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.
Supplementary 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).
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Guo, X., Aviles, G., Liu, Y. et al. Mitochondrial stress is relayed to the cytosol by an OMA1–DELE1–HRI pathway. Nature 579, 427–432 (2020). https://doi.org/10.1038/s41586-020-2078-2
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DOI: https://doi.org/10.1038/s41586-020-2078-2
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