Stress response silencing by an E3 ligase mutated in neurodegeneration

Abstract

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health^1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Fig. 1. The E3 ligase SIFI protects cells during mitochondrial import stress.

a, Outline of the synthetic lethality screen. sgRNA, singe guide RNA. b, ΔUBR4 cells are sensitive to the inhibition of mitochondrial import or ETC function. Darker grey dots represent the top 5% CasTLE score genes. c, Screen validation by depleting hits in mixtures of GFP-labelled WT and mCherry-labelled ΔUBR4 cells, reported as (ΔUBR4_sgRNA/WT_sgRNA)/(ΔUBR4_sgCNTRL/WT_sgCNTRL). sgCNTRL, control sgRNA. d, Chemical mitochondrial stress or growth in galactose-depleted conditions selectively depletes ΔUBR4 cells, reported as (ΔUBR4_treatment/WT_treatment)/(ΔUBR4_control/WT_control). e, Endogenous Flag–UBR4 and KCMF1–Flag were affinity purified, and binding partners were determined by mass spectrometry. TSC_norm, normalized total spectral counts. f, Cells lacking KCMF1 or the endogenous KCMF1-binding, calmodulin-binding (CALM) or UBR domains of UBR4 were depleted of TIMM8A and assessed by competition, reported as (UBR4(Δdomain)_sgTIMM8A/WT_sgTIMM8A)/(UBR4(Δdomain)_sgCNTRL/WT_sgCNTRL). UBR4 domain map visualizing location of endogenous domain deletions.

Fig. 2. SIFI targets DELE1 and HRI.

a, Top, outline of the import assay. Bottom, import assay using the model protein TRAP1 in WT or ΔUBR4 cells lacking TOMM40 (left) or the UBR4 genetic interactor TIMMDC1 (right). Similar results in n = 3 independent experiments. Mito., mitochondrial; si, small interfering RNA b, Stability reporter-based screen for UBR4 substrates identifies cleaved DELE1 and HRI. Upper schematic: map of reporter construct with GFP-tagged candidate substrate co-expressed with mCherry under control of an internal ribosome entry site (IRES). c, cDELE1 or HRI stability were monitored by flow cytometry (UBR4–ΔKCMF1: KCMF1-binding domain deleted in endogenous UBR4; same for the other domains). Similar results in n = 2 independent experiments. d, Endogenous HRI increases in cells lacking SIFI, as measured by western blotting. Similar results in n = 3 independent experiments. e, WT or ΔUBR4 cells that expressed endogenously tagged DELE1–HA were exposed to oligomycin (OM, 1 μM) before cycloheximide (CHX) and analysed by western blotting. Where indicated, carfilzomib (CFZ) was added. Similar results in n = 2 independent experiments. expo., exposure; FL, full length. f, ³⁵S-labelled cDELE1(142–515)–SUMO or HRI(1–138)–SUMO were ubiquitylated by SIFI, E1, UBE2A, UBE2D3 and ubiquitin (Ub). Similar results in n = 3 independent experiments. IP, immunoprecipitation. g, SIFI-dependent ubiquitylation requires UBE2A and UBE2D3. Similar results in n = 2 independent experiments. h, SIFI assembles predominantly K48-linked ubiquitin chains. (K0, all Lys mutated; K48only, only Lys48 present; K48R, only Lys48 mutated). Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.

Fig. 3. SIFI silences the mitochondrial stress response.

a, UBR4 deletion amplifies ISR signalling after arsenite (5 μM, 16 h) or CCCP (10 μM, 8 h) treatment, as detected by flow cytometry of uORF-ATF4-gated GFP translation. Upper schematic: map of ISR activation reporter, which measures uORF-gated translation of GFP controlled by IRES-mCherry. Similar results in n ≥ 2 independent experiments. b, WT and ΔUBR4 cells were treated with CCCP (16 h) and ATF4 was detected by western blotting. Similar results in n = 2 independent experiments. c, Western blot of WT and ΔUBR4 cells depleted of TIMM8A treated with arsenite (5 μM, 16 h). Similar results in n = 2 independent experiments. d, RNA-seq analysis of WT, ΔUBR4, WT sgTIMM8A, ΔUBR4 sgTIMM8A and arsenite-treated WT and ΔUBR4 cells. e, WT and ΔUBR4 cells were treated with arsenite (5 μM), and ATF4 was monitored by western blotting. Quantification of n = 4 independent experiments. Data shown as the mean ± s.e.m. f, WT and ΔUBR4 cells were depleted of CReP, treated with arsenite (5 μM) and analysed by western blotting. Similar results in n = 4 independent experiments. For gel source data, see Supplementary Fig. 1.

Fig. 4. SIFI targets mitochondrial precursors.

a, Stability of HRI variants in WT and ΔUBR4 cells analysed by flow cytometry. Similar results in n ≥ 3 independent experiments. b, AlphaFold2 model of the amino-terminal HRI domain. c, Deletion of two helices protects HRI against UBR4-dependent degradation. Similar results in n = 3 independent experiments. d, Autoradiography image of HRI variants analysed for SIFI-dependent ubiquitylation. Similar results in n = 2 independent experiments. e, Fluorescence image of TAMRA-labelled HRI helix 2 peptides analysed for SIFI-dependent ubiquitylation. Similar results in n = 2 independent experiments. f, cDELE1 stability reporters were analysed in WT and ΔUBR4 cells by flow cytometry. ΔN, amino-terminal deletion (152-end); ΔOQC, deletion of putative orphan QC motif; ΔiMTS, deletion of the region overlapping with the presequence-like helix. Similar results in n = 2 independent experiments. g, Ubiquitylation of a TAMRA-labelled presequence (MTS) peptide by SIFI, E1, UBE2A and UBE2D3 was monitored by fluorescence imaging. Similar results in n = 3 independent experiments. h, Fluorescence image of a TAMRA labelled presequence ubiquitylated by SIFI purified from WT and ΔKCMF1 cells. Experiment was performed once. i, Fluorescence image of modification of a TAMRA-labelled presequence with ubiquitin mutants. Similar results in n = 2 independent experiments. j–l, Flow cytometry results for MTS. j, Depletion of HSPA9 destabilizes a presequence reporter that is partially dependent on SIFI. Similar results in n = 2 independent experiments. k, Depletion of both TIMM8A and TIMM8B (siTIMM8A/8B) destabilizes a presequence–GFP fusion in a SIFI-dependent manner. Similar results in n = 2 independent experiments. l, Cells were treated with oligomycin (1 μM, 16 h), and the stability of a presequence–GFP fusion was determined by flow cytometry. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.

Fig. 5. Converging degrons couple stress resolution to stress response silencing and cell survival.

a, Degradation of WT-swap or degron/MTS-swap HRI reporters was monitored by flow cytometry. Similar results in n = 2 independent experiments b, The HRI helix 2 and the helical cDELE1 degron were fused in front of GFP, and localization was monitored by microscopy. Scale bar, 5 μm. Similar results in n = 3 independent experiments. c, The HRI helix 2 degron mediates import of citrate synthase (CS), as determined by flow cytometry. Experiment performed once and validated by microscopy. d, Autoradiography image of SIFI-dependent ubiquitylation of HRI(1–138)–SUMO in the presence of increasing concentration of presequence. Similar results in n = 2 independent experiments. e, Flow cytometry analysis of HRI stability in WT and ΔUBR4 cells depleted of TIMM8A and TIMM8B. Similar results in n = 2 independent experiments. f, Overexpression of mitochondrial precursors induces ISR signalling that depends on presequences and restricted by UBR4, as monitored through the uORF-ATF4 reporter. Similar results in n = 2 independent experiments. g, DELE1 or HRI depletion rescues synthetic lethality after loss of UBR4 and TIMM8A. h, ISRIB rescues the synthetic lethality after loss of UBR4 and mitochondrial import or ETC assembly factors. Some competitions were performed at the same time as experiments for Fig. 1c and some controls are therefore reshown. i, Model of regulated stress response silencing by the E3 ligase SIFI. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 1. UBR4 deletion sensitizes cells to mitochondrial protein import stress.

a. Western blot analysis shows deletion of endogenous UBR4 from HEK293T cells as a prerequisite for a subsequent whole genome synthetic lethality CRISPR screen. Three different antibodies directed against UBR4 were used to establish successful deletion. b. GO analysis of genetic interactors of UBR4. Screen hits in the top 5% CasTLE score and with negative CasTLE effects were run through ClueGO to identify enriched pathways that are synthetic lethal with UBR4 deletion. P values were generated by ClueGO (Fisher’s exact test and Bonferroni correction). For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2. UBR4 stably interacts with KCMF1.

a. Endogenously FLAG-tagged UBR4 and KCMF1 were affinity-purified from 293T cells, and bound proteins were detected by Western blotting using specific antibodies. Similar results in n = 3 independent experiments. b. KCMF1 binds to a DOC domain in UBR4. FLAG-tagged fragments of UBR4 were immunoprecipitated from ΔUBR4 cells and co-precipitating endogenous KCMF1 was detected by Western blotting. Similar results in n = 2 independent experiments. c. Validation of the KCMF1 domain in endogenous UBR4. The DOC domain of UBR4 was excised from the endogenous UBR4 locus (UBR4 was already fused to a FLAG epitope) by CRISPR-Cas9 genome engineering. Similar approaches were used to eliminate the endogenous UBR- and calmodulin-binding regions in UBR4. Endogenous wildtype or mutant UBR4 was affinity-purified, and co-precipitating proteins were detected by Western blotting. Similar results in n = 3 independent experiments. d. Validation of mutant UBR4 by mass spectrometry. The KCMF1- or calmodulin-binding domains, or the UBR domain, were deleted in the endogenous locus of ^FLAGUBR4. Endogenous UBR4 complexes were affinity-purified and bound proteins were detected by mass spectrometry. Changes in interactions for mutant cell lines compared to wildtype UBR4 are depicted for select proteins. Spectral counts were normalized to bait (UBR4) spectral counts in each cell line. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 3. The SIFI complex targets cleaved DELE1 and HRI.

a. Genetic interactors of UBR4 control mitochondrial protein import. Mitochondrial import of GFP11-tagged HMT2 was monitored in WT cells stably expressing mitochondrially targeted GFP(1–10). Genetic interactors of UBR4 or known protein import regulators were depleted with specific siRNAs. Similar results in n = 2 independent experiments. b. UBR4 does not regulate mitochondrial protein import. Import of GFP11-tagged TRAP1 was analyzed as above. When indicated, ΔUBR4 cells were used or the genetic interactors of UBR4, TIMM13 and HIGD2A, were depleted using sgRNAs. Similar results in n = 3 independent experiments. c. Chemical stressors that deplete ΔUBR4 cells in competition assays, compromised mitochondrial protein import. Import of GFP11-tagged HMT2 was analyzed in the presence of indicated drugs CCCP(5 μM), arsenite (10 μM), OM (2.5 μM), Antimycin A (10 μM), BTdCPU (10 μM) for 16 h by flow cytometry, as described above. Similar results in n = 2 independent experiments. d. Depletion of eIF2α, the eIF2B subunit EIF2B4, or the eIF2α phosphatase PPP1R15B causes synthetic lethality with loss of UBR4, as seen in our synthetic lethality screen described earlier. e. Validation of synthetic lethality between EIF2B4 and PPP1R15B (CReP) by cell competition assays. The second eIF2α phosphatase PPP1R15A (GADD34) also shows weak synthetic lethality with UBR4 deletion. f. HRI and cleaved DELE1 are degraded through UBR4 and KCMF1, while the quality control E3 ligases UBR5 or RNF126 are not required. E3 ligases were deleted from 293 T cells by CRISPR/Cas9-mediated genome engineering and the stability of HRI or cDELE1 was monitored as GFP-tagged proteins by flow cytometry. Experiment performed once, similar results obtained with siRNA depletions. g. Degradation of orphan cDELE1, which is not bound to HRI, requires a central domain in cDELE1. A stability reporter expressing either cDELE1 or an internal deletion resistant to HRI depletion (cDELE1^Δ228–276) were monitored by flow cytometry in either wildtype or ΔUBR4 cells. When indicated, HRI was depleted by specific sgRNAs. Similar results in n = 2 independent experiments. h. Expression of HRI or DELE1 is not induced by mitochondrial stress, as seen by RNAseq in cells depleted of TIMM8A or treated with arsenite. Data was taken from RNAseq experiments described in Fig. 3d. i. Expression of HRI or DELE1 is not induced by deletion of UBR4 or KCMF1, as seen by qRT-PCR. Graph shows mean ± SD of 3 independent experiments. j. Degradation of an overexpressed wildtype HRI reporter does not require DELE1. Stability of the HRI reporter was monitored in cells treated with control siRNAs or siRNAs targeting DELE1, by flow cytometry. Experiment performed once. k. Mutation of K196 in HRI, which is required for autophosphorylation and activation, prevents UBR4-dependent degradation, as seen by flow cytometry. Similar results in n = 3 independent experiments.

Extended Data Fig. 4. The SIFI complex targets HRI and cDELE1 for proteasomal degradation.

a. HRI is ubiquitylated by the SIFI complex. The SIFI complex was purified from 293 T cells expressing endogenously FLAG-tagged UBR4. It was incubated with ³⁵S-labeled HRI^1–138-SUMO or MBP-SUMO as a control, respectively, E1, UBE2D3 and UBE2A as E2 enzymes, and ubiquitin. Reaction products were visualized by autoradiography. Experiment performed once. b. All SIFI subunits are required for HRI ubiquitylation. SIFI complexes were purified from cells expressing endogenously FLAG-tagged UBR4. When indicated, cells with internal deletions of the KCMF1-binding domain, the calmodulin-binding domain, or the UBR domain in endogenous UBR4 were used. ³⁵S-labeled HRI^1–138-SUMO, E1, UBE2D3, UBE2A and ubiquitin were added, and reaction products were visualized by autoradiography. Similar results in n = 2 independent experiments. c. The SIFI complex mediates HRI ubiquitylation in cells. Ubiquitin conjugates were purified under denaturing conditions from cells expressing HRI^FLAG and ^HISubiquitin, and modified HRI was detected by αFLAG Western blotting. Cells were treated with proteasome inhibitor (CFZ, 2 μM) for 6 h prior to harvesting. Similar results in n = 2 independent experiments. d. The SIFI complex modifies HRI with ubiquitin chains predominantly linked to K48 of ubiquitin. Ubiquitylation of ³⁵S-labeled HRI^1–138-SUMO was analyzed as described above, but in the presence of indicated ubiquitin mutants (K0: all Lys residues mutated to Arg; K6only: all Lys residues except for K6 mutated to Arg). Experiment performed once. e. HRI and cDELE1 are degraded through the proteasome. Cells were analyzed for levels of stability reporters encoding HRI-GFP or cDELE1-GFP by flow cytometry. The proteasome inhibitor carfilzomib (2 μM) or the lysosome inhibitor bafilomycin A (700 nM) were added for 6 h as indicated. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 5. The SIFI complex silences the integrated stress response.

a. Deletion of UBR4 increases ISR signaling in response to cells being treated with oligomycin (0.2 μM) for 16 h or BTdCPU (7.5 μM) for 8 h. ISR activation was monitored by flow cytometry using the uORF-ATF4 reporter described above. Similar results in n = 2 independent experiments. b. UBR4 deletion increases ISR signaling. Wildtype or ΔUBR4 cells were treated for 16 h with increasing concentrations of arsenite and analyzed for ATF4 levels by Western blotting. Similar results in n = 2 independent experiments. c. Deletion of UBR4 increases ISR signaling in cells treated for 16 h with increasing concentrations of BTdCPU, as monitored by Western blots detecting ATF4. Similar results in n = 2 independent experiments. d. UBR4 deletion increases ISR signaling in cells treated for 16 h with increasing concentrations of antimycin A, as detected by ATF4 expression. Similar results in n = 2 independent experiments. e. Deletion of TIMM8A induces ATF4 accumulation more strongly in ΔUBR4 cells. WT or ΔUBR4 cells depleted of TIMM8A were treated with antimycin A (0.6 μM) for 16 h. Similar results in n = 2 independent experiments. f. Deletion of KCMF1 increases ISR signaling to a similar extent as UBR4 deletion, as detected using the uORF-dependent ISR reporter in flow cytometry. Cells were treated with OM (0.2 μM) for 8 h. Similar results in n = 2 independent experiments g. Deletion of KCMF1- and calmodulin-binding domains in the endogenous UBR4 locus increases ISR signaling in response to 5 μM sodium arsenite for 16 h, as determined by flow cytometry using the uORF-dependent ISR reporter. Similar results in n = 2 independent experiments h. UBR4 does not restrict ISR signaling in response to endoplasmic reticulum stress. Wildtype or ΔUBR4 cells were treated with thapsigargin or tunicamycin for 8 h and analyzed for ATF4 levels by Western blotting. Experiment performed once. i. ER stress activation by thapsigargin does not induce DELE1 cleavage. Cells were treated with thapsigargin (1 μM) or oligomycin (1 μM) for the indicated times. Experiment performed once. j. UBR4 deletion increases ISR signaling, as read out by ATF4 activation. Wildtype or ΔUBR4 cells were either treated with 5 μM sodium arsenite (left panel) or depleted of TIMM8A (right panel) and expression of established ATF4 target genes was determined by qPCR. Graph shows mean ± SD of 3 independent experiments. k. UBR4 depletion increases ISR signaling in neurons derived from induced pluripotent stem cells by NGN2 activation. Differentiation was ensured by qRT-PCR against OCT4 and β3-tubulin and ISR target gene expression was measured by qRT-PCR. As indicated, either 5 μM sodium arsenite or ISRIB were added. Graph shows mean ± SD of 3 independent experiments. Statistical significance was determined using a two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Exact p-Values: OCT4: sgCNTRL p < 0.0001; sgUBR4 p < 0.0001. β3-tubulin: sgCNTRL p = 0.0046; sgUBR4 p = 0.0014. VEGFA: sgUBR4 arsenite vs. unt. p = 0.0011; sgUBR4 arsenite vs. sgCNTRL arsenite p = 0.0052; sgUBR4 arsenite/ISRIB vs. sgUBR4 arsenite p = 0.0242. DDIT4: sgUBR4 arsenite vs. unt. p < 0.0001; sgUBR4 arsenite vs. sgCNTRL arsenite p = 0.0143; sgUBR4 arsenite/ISRIB vs. sgUBR4 arsenite p < 0.0001. ASNS: sgUBR4 arsenite vs. unt. p = 0.0004; sgUBR4 arsenite vs. sgCNTRL arsenite p = 0.0089; sgUBR4 arsenite/ISRIB vs. sgUBR4 arsenite p = 0.0236. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 6. The SIFI complex silences the cellular response to mitochondrial import stress.

a. The SIFI complex limits signal duration, not amplitude. Wildtype or ΔUBR4 cells were treated with 5 μM sodium arsenite and cell lysates were analyzed for ATF4 levels by Western blotting over time. Quantification of 4 independent experiments shown in Fig. 3e. b. The SIFI complex also limits signal duration after ISR activation with 5 μM antimycin A (AM). Cell lysates were analyzed as described above. Similar results in n = 3 independent experiments. c. The SIFI complex, not the GADD34 phosphatase, mediates stress response silencing in response to arsenite. WT or ΔUBR4 cells were depleted of GADD34, as indicated, and treated with 5 μM arsenite. At different times, samples were analyzed for ATF4 expression by Western blotting. Similar results in n = 2 independent experiments. d. The SIFI complex, not the CReP phosphatase, mediates stress response silencing after antimycin A treatment. WT or ΔUBR4 cells were depleted of CReP, as indicated, and treated with 0.6 μM antimycin A. At different times, samples were analyzed for ATF4 expression by Western blotting. Similar results in n = 2 independent experiments. e. The SIFI complex, not the GADD34 phosphatase, mediates stress response silencing in response to antimycin A. WT or ΔUBR4 cells were depleted of GADD34, as indicated, and treated with 0.6 μM antimycin A. At different times, samples were analyzed for ATF4 expression by Western blotting. Similar results in n = 2 independent experiments. f. The SIFI complex does not mediate degradation of GADD34, as shown by a GADD34 stability reporter in flow cytometry. Similar results in n = 2 independent experiments. g. The SIFI complex does not mediate degradation of CReP, as shown by a CReP stability reporter in flow cytometry. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 7. The SIFI complex detects helical degrons in HRI and DELE1.

a. Deletion or mutation of two helices in HRI at the same time, but not manipulation of a single helix, protects HRI from UBR4-dependent degradation. The stability of indicated mutants was analyzed in wildtype or ΔUBR4 cells by flow cytometry using the GFP/mCherry-based degradation reporter. Similar results in n ≥ 2 independent experiments. b. The SIFI complex ubiquitylates a single HRI peptide irrespectively of whether the SIFI complex was purified from control cells or cells treated with arsenite (40 μM for 4 h). Experiment performed once. c. Peptides encompassing a single HRI helix compete for ubiquitylation of the entire amino-terminal HRI domain (residues 1–138). ³⁵S-labeled HRI^1–138-SUMO was incubated with affinity-purified SIFI complexes, E1, UBE2A and UBE2D3, and ubiquitin. 200 μM of purified peptides encompassing the helices comprising degron 1 or degron 2, respectively, were added, and reaction products were analyzed by autoradiography. Similar results in n = 2 independent experiments d. Changing the amino-terminus of cleaved DELE1 does not affect its stability, as seen by flow cytometry. Similar results in n = 2 independent experiments e. Capping of the amino-terminus of cleaved DELE1 with threonine, an amino acid not recognized by the N-end rule, does not change its stability, as seen by flow cytometry. Similar results in n = 2 independent experiments. f. A helical DELE1 degron similar to HRI helices is ubiquitylated by the SIFI complex as a TAMRA-labeled peptide, while a distinct DELE1 peptide was not modified. Similar results in n = 2 independent experiments. g. Other top SIFI substrates are mostly composed of α-helices. The stability of top SIFI substrates identified in our screen was analyzed in wildtype or ΔUBR4 cells by flow cytometry, using our degradation reporters. Similar results in n ≥ 2 independent experiments. AlphaFold2 models of each substrate are shown below. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 8. The SIFI complex recognizes mitochondrial presequences.

a. Helical degrons in HRI and cDELE1 resemble mitochondrial presequences in amino acid composition (left panel) and structure (right panels). Presequences were aligned with COBALT (https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi). The structures of the HRI degron and the presequences of citrate synthase (CS) or COQ9 are AlphaFold2 models. The DELE1 helix is from its cryo-EM structure and the ALDH2 presequence is its actual structure when bound to TOMM20. b. A prediction algorithm for mitochondrial presequences identifies the helical HRI and cDELE1 degrons. Internal MTS sequences were predicted using iMLP: iMTS-L predictor service (https://csb-imlp.bio.rptu.de/). A score above 0 is predictive of an internal MTS. Orange shaded boxes correspond to identified degrons in HRI and DELE1. c. The second HRI degron (helix 2) efficiently competes with mitochondrial presequences for access to the SIFI complex. A TAMRA-labeled COX8A presequence peptide (10 μM) was incubated with affinity-purified SIFI complex, E1, UBE2A and UBE2D3, and ubiquitin. As indicated, 100 μM of purified peptides encompassing the helices comprising degron 1 or degron 2 were added, and reaction products were analyzed after gel electrophoresis by fluorescence. Similar results in n = 2 independent experiments d. The SIFI complex, but not the quality control E3 ligase UBR5, ubiquitylates a presequence peptide. Ubiquitylation was analyzed as described above. Experiment performed once. e. The entire SIFI complex is required for presequence ubiquitylation. A TAMRA-labeled COX8A presequence peptide was incubated with affinity-purified SIFI complex purified from WT cells or cells carrying deletions of the endogenous KCMF1 binding-, calmodulin-, or UBR-domains in UBR4. E1, UBE2A and UBE2D3, and ubiquitin were added and reaction products were analyzed after gel electrophoresis by fluorescence. Similar results in n = 2 independent experiments. f. The SIFI complex ubiquitylates a TAMRA-labeled presequence peptide irrespectively of whether the E3 ligase had been purified from control cells or cells treated with arsenite (40 μM for 4 h). g. The SIFI complex modifies presequences with ubiquitin chains predominantly composed of K48-linkages. A TAMRA labeled COX8A presequence peptide was incubated with SIFI complex, E1, UBE2A and UBE2D3 and the indicated ubiquitin mutants (ubi-K0: all Lys residues mutated to Arg; ubi-K6only: all Lys residue except for K6 mutated to Arg), and reaction products were analyzed as above. Experiment performed once. h. The COX8A presequence is a SIFI-dependent degradation signal. The presequence was cloned as a fusion to GFP into the degradation reporter and assessed for its effects on protein stability by flow cytometry. As indicated, the proteasome inhibitor carfilzomib (CFZ) or the lysosome inhibitor bafilomycin A (BafA) were added. Note that only the cytoplasmic fraction of this fusion protein can be targeted via SIFI and the proteasome. Similar results in n = 2 independent experiments. i. A fusion between a COX8A presequence peptide carrying mutations in four Leu residues and GFP is not degraded through UBR4, the proteasome or the lysosome, as determined by flow cytometry. Similar results in n = 2 independent experiments. j. The mitochondrial import receptor TOMM20 competes with the SIFI complex for recognition of mitochondrial presequences. A TAMRA-labeled COX8A presequence was incubated with increasing concentrations of the cytoplasmic domain of TOMM20 or TOMM20^I74SV109S, which is incapable of binding presequences. The SIFI complex, E1, E2s, and ubiquitin were added, and ubiquitylation was monitored by gel electrophoresis and fluorescence imaging. Experiment performed once. k. Mutation of presequence residues required for TOMM20 binding also ablates ubiquitylation by the SIFI complex. Similar results in n = 2 independent experiments. l. Import inhibition leads to accumulation of mitochondrial precursor proteins that still contain their presequence. UBR4 deletion further increases precursor abundance, as seen by Western blotting after expressing of HA-tagged mitochondrial proteins in either WT or ΔUBR4 cells treated with mitochondrial import blocker oligomycin (1 μM, 16 h) and ISRIB. Similar results in n = 2 independent experiments. m. Inhibition of mitochondrial protein import upon depletion of TIMM16 stabilizes HRI. Similar results in n = 2 independent experiments. n. Inhibition of mitochondrial protein import upon depletion of TIMM16 stabilizes cDELE1, as determined by flow cytometry. Similar results in n = 2 independent experiments. o. Deletion of UBR4 partially stabilizes a presequence reporter if import was prevented by TIMM16 depletion, as seen by flow cytometry. Similar results in n = 2 independent experiments. p. Activation of ISR signaling in ΔUBR4 cells upon overexpression of the mitochondrial protein NIPSNAP1 is dependent on HRI and DELE1. HRI and DELE1 were depleted by specific siRNAs and ISR activation was monitored using the uORF-ATF4 reporter using flow cytometry. When indicated, NIPSNAP1 was overexpressed. Similar results in n = 2 independent experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 9. HRI and DELE1 mediate stress response signaling without affecting mitochondrial protein import.

a. Depletion of HRI suppresses increased ISR activation in ΔUBR4 cells treated with 5 μΜ sodium arsenite for 16 h, as monitored by Western blotting using antibodies against ATF4. Similar results in n = 2 independent experiments. b. Depletion of HRI or DELE1 suppresses increased ISR activation in ΔUBR4 cells treated with 25 μΜ oligomycin for 8 h, as monitored by Western blotting using antibodies against ATF4. Similar results in n = 2 independent experiments. c. Depletion of HRI or DELE1 suppresses increased ISR activation in ΔUBR4 cells treated with 0.6 μΜ antimycin A for 16 h, as monitored by Western blotting using antibodies against ATF4. Similar results in n = 2 independent experiments. d. Depletion of HRI or DELE1 suppresses increased ISR activation in ΔUBR4 cells treated with 5 μΜ BTdCPU for 8 h, as monitored by Western blotting using antibodies against ATF4. Similar results in n = 2 independent experiments. e. Depletion of HRI and DELE1 by siRNA does not restore mitochondrial protein import in cells lacking TIMMDC1. Wildtype or ΔUBR4 cells were depleted of TIMMDC1 using specific sgRNAs, as indicated. Mitochondrial protein import was monitored by reconstitution of GFP upon expression of TRAP1-GFP11 co-expressed with BFP in cells stably expressing GFP(1^–10) in the mitochondrial matrix. GFP formation upon successful import was monitored by flow cytometry. Experiment was validated using the alternative mitochondrial import substrate HMT2-GFP11. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 10. Stress response silencing restores cell survival.

a. Pharmacological stress response silencing in cells lacking UBR4 or KCMF1 through ISRIB. Wildtype, ΔUBR4, or ΔKCMF1 cells were treated with 5 μΜ sodium arsenite for 16 h and, as indicated, ISRIB. ATF4 levels were monitored by Western blotting. Similar results in n = 2 independent experiments. b. ISRIB inhibits stress response activation in cells that were lacking UBR4 or KCMF1 and were treated with antimycin A (0.6 μΜ) for 16 h. Similar results in n = 2 independent experiments. c. ISRIB inhibits stress response activation in cells that were lacking UBR4 or KCMF1 and were treated with BtdCPU (5 μΜ) for 8 h. Similar results in n = 2 independent experiments. d. ISRIB does not restore mitochondrial protein import in cells depleted of TIMM13. Import was measured upon GFP reconstitution by flow cytometry, as described above. Experiment was validated using the alternative mitochondrial import substrate HMT2-GFP11. e. ISRIB rescues ISR activation in human embryonic stem cells. As indicated, UBR4 was depleted by sgRNAs. Sodium arsenite (1.25 μΜ) and/or ISRIB were added for 8 h and ATF4 activation was monitored by Western blotting. Similar results in n = 2 independent experiments. f. Pharmacological silencing of the ISR with ISRIB rescues the synthetic lethality between UBR4 deletion and chemical mitochondrial stressors. Cell competition assays were performed as described above. Some competitions were performed at the same time as for Fig. 1d and are therefore re-reshown from Fig. 1d. g. Pharmacological silencing of the ISR with ISRIB rescues the cells depleted of the disease gene TIMM8A. Cell competition assays were performed as described above. For gel source data, see Supplementary Fig. 1.