A mitochondrial iron-responsive pathway regulated by DELE1

Abstract

The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.

Keywords: DELE1; HRI; LONP1; erythroid cells; integrated stress response; iron; mitochondria; mitochondrial import; mitochondrial proteostasis.

Figure 1. DELE1 is a short-lived protein that is degraded by LONP1 after mitochondrial import.

(A) Endogenous DELE1-HA expressing HEK293T cells were treated with cycloheximide (CHX) for the indicated time periods. Cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. HSP90 is shown as a loading control. (B) IB for lysates of endogenous DELE1-HA HEK293T cells treated with MG132 or Bafilomycin A1 (Baf. A1) for 7 hours. (C-E) IB for lysates of endogenous DELE1-HA HEK293T cells transfected with the indicated siRNAs for 72 hours and treated with CHX for the last 2 hours before harvest. *; non-specific bands.

Figure 2. Iron deficiency stabilizes DELE1.

(A) Endogenous DELE1-HA HEK293T cells were treated with Deferoxamine (DFO), Deferiprone (DFP), CCCP, Hemin or succinyl acetone (SA) for 16 hours. The lysates were analyzed by immunoblotting (IB) with the indicated antibodies. (B) IB for lysates of tet-on DELE1-HA stable HeLa cells treated with the indicated concentration of DFO for 16 hours. Doxycycline was added 8 hours prior to DFO treatment to induce the expression of DELE1-HA. (C) IB for lysates of endogenous DELE1-HA HEK293T cells pre-treated with either DFO, DFP for 16 hours, or CCCP for 1.5 hours, and subsequently subjected to the CHX chase at the indicated time periods. (D) HEK293T DELE1 knockout (KO) cells (clone #24) were transiently infected with lentivirus expressing DELE1-HA. After 24 hours, cells were treated with DFO for 16 hours and were subsequently subjected to the CHX chase at the indicated time periods. Cell lysates were analyzed by IB. (E) Immunocytochemistry (ICC) for Tet-on DELE1 (wild-type, WT)-HA, DELE1 (delta 30, d30)-HA, DELE1 (delta 101, d101)-HA stable HeLa cells. Scale bars; 25 μm. A schematic representation of the domain structure of DELE1 is shown at the bottom. (F) IB for lysates of tet-on DELE1 WT-HA or DELE1 d101-HA stable HeLa cells were treated with DFP for 16 hours. Doxycycline was added 8 hours prior to DFO treatment to induce the expression of DELE1 WT-HA or DELE1 d101-HA. See also Figure S1.

Figure 3. DELE1 is stabilized on mitochondria by an iron deficiency-dependent mitochondrial import arrest.

(A) Tet-on DELE-HA stable HeLa cells were treated with DFO or CCCP for 16 hours. The subcellular localization of DELE1-HA was determined by immunocytochemistry (ICC). Scale bars; 10 μm. Line profiles for the indicated fluorescent intensities determined along the white lines are shown to the right. (B) Crude mitochondrial fractions were isolated from Endogenous DELE1-HA HEK293T cells that were treated with CHX for 30 min with or without the pre-treatment with DFO for 16 hours, and were subjected to in vitro proteinase K protection assay at the indicated proteinase K concentration in the indicated buffer conditions. Sensitivities of endogenous DELE1-HA and other mitochondrial marker proteins to proteinase K were determined by immunoblotting (IB). (C) Schematic representation for the TEV cleavage assay. Suppression of the proteases-mediated cleavage of DELE1 by import arrest (upper panel). Insertion of the TEV cleavage sequence after the predicted MPP cleavage site of DELE1 [DELE1(TEV30)] (lower panel). (D) DELE1 KO cells were transfected with the TEV sequence-inserted DELE1 (DELE1(TEV30)-HA) with or without the matrix-targeted TEV protease (Su9-TEV protease-HA). After 24 hours, cells were treated with DFO for the indicated time periods. Cell lysates were separated in an SDS-PAGE 7.5% polyacrylamide Midi gel and analyzed by IB. (E) IB for lysates of endogenous DELE1-HA HEK293T cells treated with CHX for 30 min with or without pre-treatment of DFO for 16 hours (lane 1–4), or transfected with the indicated siRNAs for 72 hours, and subsequently treated with CHX for 30 min (lane 5–10). (F) Knockdown efficiency of indicated proteins in (E) determined by IB. (G to I) HeLa cells transiently transfected with tag (−) PINK1 (G), Tet-on Su9-DHFR-3xFlag stable HeLa cells (H), or Tet-on DELE1-HA stable HeLa cells (I), were treated with DFO, DFP or CCCP for 16 hours. Doxycycline was added 8 hours prior to DFO treatment to induce the expression of Su9-DHFR-3xFlag (H) or DELE1-HA (I). Cell lysates were analyzed by IB. *; non-specific bands. See also Figure S2.

Figure 4. DELE1 activates an HRI-mediated ISR following iron deficiency.

(A, B) HEK293T cells were treated with DFO, DFP or CCCP for the indicated time periods (A) or were treated with the indicated concentrations of DFO for 16 hours (B). Cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. (C) ATF4 reporter (ATF4 uORF mApple)-expressing HeLa cells were treated with the indicated concentrations of DFO for 16 hours and were subjected to flow cytometry analysis. Data are shown as mean ± S.D. (N=4). ***P = 0.0001 (One-way ANOVA followed by Dunnett’s multiple comparison’s test). (D) IB for lysates of endogenous DELE1-HA HEK293T cells treated with DFO with or without ferric ammonium citrate (FAC) for 16 hours. (E) IB for lysates of HeLa cells treated with the indicated compounds for 16 hours. (F) IB for lysates of endogenous DELE1-HA HEK293T cells treated with DFO for 16 hours in the presence or absence of hemin. (G) IB for lysates of HeLa cells transfected with indicated siRNAs for 72 hours, and treated with DFO, DFP, or CCCP for the last 16 hours. (H) IB for lysates of HEK293T WT or DELE1 KO cell lines (clone #24 and #51) treated with DFO, 1 mM DFP, or CCCP for 16 hours. The lysates were analyzed by IB. (I) Quantitative PCR for ISR target gene expression in HEK293T WT or DELE1 KO (clone #24) cells treated with DFO for 16 hours. Shown are mean ± S.D. (N=3). **** P<0.0001, *** P<0.001, and ** P<0.01 (One-way ANOVA followed by Turkey’s multiple comparison). (J) DELE1(WT)-HA or DELE1(d246–272)-HA was expressed in DELE1 KO HEK293T cells (clone #24) through lentivirus infection for 40 hours. Cells were treated with DFO for the last 16 hours before harvest. Cell lysates were analyzed by IB. See also Figure S3.

Figure 5. DELE1 on mitochondrial surface activates HRI.

(A) Flow cytometry analysis to determine mitochondrial membrane potential in HeLa cells treated with either DFO, DFP or CCCP for 16 hours, and with TMRM for the last 15 min. (B) HeLa cells were treated with either DFO, DFP or CCCP for 16 hours. Cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. (C, D) IB for lysates of endogenous DELE1-HA HEK293T cells (C) or HeLa cells (D) transfected with indicated siRNAs for 72 hours, and treated with DFO or DFP for the last 16 hours before harvest. *; non-specific bands. (E) Endogenous DELE1-HA was immunoprecipitated with anti-HA antibody from the indicated HEK293T cells with or without 16 hours of DFO treatment. The samples were subjected to IB. IP; immunoprecipitation. (F) Proximity Ligation Assay (PLA) between overexpressed DELE1-HA and endogenous HRI. Tet-on DELE1-HA stable HeLa cells that stably express Mito-BFP were treated with DFO or DFP for 20 hours, or CCCP for 4 hours, under Doxycycline-added conditions, and were subjected to PLA. White arrow heads indicate representative PLA signals that were observed exactly on or close proximity to mitochondria. Scale bars; 5 μm. Magnified merge images of the squared area are shown to the right. Low magnification images are shown in Figure S4A. (G) DELE1-HA or the OMM-tethered DELE1 [TOM20(SS)-DELE1(d101)-HA] or TOM20(SS)-DELE1(d200)-HA] was expressed in DELE1 KO HEK293T cells (clone #24) through lentivirus infection for 44 hours. Cells were treated with DFO for the last 20 hours or CCCP for the last 4 hours before harvest. Cell lysates were analyzed by IB (upper panel). Schematic representation of TOM20(SS)-DELE1(d101)-HA and TOM20(SS)-DELE1 (d200)-HA (lower panel). The OMM-targeting signal of TOM20 is shown as a blue box. (H) IB for lysates of DELE1 KO HEK293T cells transfected with indicated siRNAs for 48 hours, and infected with the OMM-tethered DELE1-HA-expressing lentiviruses for the last 24 hours before harvest. See also Figure S4.

Figure 6. Determination of critical factors that are required for the iron deficiency-induced ISR activation.

(A) A schematic representation of DELE1 domain structures. See text for details. (B) DELE1 (WT) or DELE1 deletion mutants that lacked indicated regions were expressed in DELE1 KO HEK293T cells through lentivirus infection for 40 hours. Cells were treated with DFO for the last 16 hours before harvest. Cell lysates were analyzed by immunoblotting (IB). (C) The indicated DELE1 deletion mutants were transiently transfected in DELE1 KO HEK293T cells. After 24 hours, cells were lysed and each DELE1 was immunoprecipitated with anti-HA antibody. The samples were subjected to IB. IP; immunoprecipitation. (D) DELE1 WT or d102-200 were transiently infected in DELE1 KO HEK293T cells (clone #24). Cells were subsequently treated with DFO for the last 20 hours before harvest. Each DELE1 was immunoprecipitated with anti-HA antibody. Samples were subjected to IB. (E, F) DELE1 WT or DELE1 deletion mutants that lacked indicated regions (E) or linker-inserted DELE1(d102-200) mutants (F) were expressed in DELE1 KO HEK293T cells through lentivirus infection for 40 hours. Cells were treated with DFO for the last 16 hours before harvest. Cell lysates were analyzed by IB. (G) A model for the role of 102–200 a.a. region of DELE1 in HRI activation during iron deficiency. See text for details. (H) IB for lysates of HeLa cells transfected with indicated siRNAs for 72 hours, and treated with DFO for the last 16 hours. (I) IB for lysates of HeLa cells transfected with indicated siRNAs for 72 hours, and treated with DFO, DFP, or CCCP for the last 16 hours. (J) Knockdown efficiency of ABCB7 in (I) determined by quantitative PCR (qPCR). (K) Endogenous DELE1-HA HEK293T cells were transfected with indicated siRNAs for 72 hours. Cells were treated with DFO for 16 hours, followed by the CHX chase for the last 30 min before harvest. Cell lysates were analyzed by IB. *; non-specific bands. (L) Knockdown efficiency of ABCB7 in (K) determined by qPCR. See also Figure S5 and Figure S6.

Figure 7. DELE1-HRI-ISR pathway protects the erythropoietic cell lineage against ferroptosis.

(A, B) MEL wildtype (WT), two independent DELE1 KO (clone #3 and clone #8), and HRI KO (clone #1) cells were cultured with 2% DMSO-containing medium in the presence or absence of DFO for 24 hours. Cell lysates were analyzed by immunoblotting (IB) with the indicated antibodies. HSP90 in (A) and NCL in (B) are shown as loading controls. (C) IB for lysates of WT, DELE1 KO, or DELE1(WT)-HA-transfected DELE1 KO MEL cells. Cells were treated with DFO for the last 24 hours before harvest. (D) MEL WT, DELE1 KO, and HRI KO cells were cultured with 2% DMSO-containing medium in the presence or absence of DFO for 24 hours. Cell lysates were subjected to RNA sequencing analysis. Differences between WT and HRI KO (X axis) or WT and DELE1 KO (Y axis) in log₂ fold changes (FC) of each gene expression before and after iron chelation (DFO/mock) are plotted. Representative ISR target genes are shown in magenta. (E) Relative mRNA expression of representative ISR target genes selected from the RNA sequencing data. (F) MEL WT, DELE1 KO (clone #3 and #8), and HRI KO (clone #1) cells were cultured with 2% DMSO-containing medium in the presence or absence of DFO for 48 hours. Dead cells were determined by flow cytometry for Propidium iodide (PI) positive cells. Data are shown as mean ± S.D. (N=5–6). ***P<0.001 and **P<0.01 (One-way ANOVA followed by Turkey’s multiple comparison). (G) A proposed model of the iron-deficiency-induced mitochondrial import regulation of DELE1 and the subsequent activation of the HRI-ISR pathway. See text for details. See also Figure S7.