Mitochondria-associated membranes (MAMs) are central microdomains that fine-tune bioenergetics by the local transfer of calcium from the endoplasmic reticulum to the mitochondrial matrix. Here, we report an unexpected function of the endoplasmic reticulum stress transducer IRE1α as a structural determinant of MAMs that controls mitochondrial calcium uptake. IRE1α deficiency resulted in marked alterations in mitochondrial physiology and energy metabolism under resting conditions. IRE1α determined the distribution of inositol-1,4,5-trisphosphate receptors at MAMs by operating as a scaffold. Using mutagenesis analysis, we separated the housekeeping activity of IRE1α at MAMs from its canonical role in the unfolded protein response. These observations were validated in vivo in the liver of IRE1α conditional knockout mice, revealing broad implications for cellular metabolism. Our results support an alternative function of IRE1α in orchestrating the communication between the endoplasmic reticulum and mitochondria to sustain bioenergetics.
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Mass spectrometry data have been deposited in ProteomeXchange (PXD013313). Metabolomics data have been deposited as a Mendeley dataset at https://data.mendeley.com/ (https://doi.org/10.17632/dtdf7wk3mb.1). Source data for Figs. 1–7 and for Supplementary Figs. 1–4 and 7 have been provided in Supplementary Table 6. Source data for Fig. 6 and Supplementary Fig. 6 have been provided in Supplementary Tables 3 and 4. Source data for Fig. 7 and Supplementary Fig. 7 can be found in Supplementary Table 5. All data that support the findings of this study are available from the corresponding author on reasonable request.
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We thank S. Lavandero, D. Rojas-Rivera and M. Chiong for initial feedback in this project and for sharing reagents, T. Iwawaki for providing IRE1α null animals and L. Qi for providing IRE1α mutant constructs, G. Hajnoczky for MAM linker constructs, T. Calì and M. Brini for the SPLICS constructs, D. Ron for providing IRE1α null MEFs, J. Ponce for animal care, and N. Bossut, F. Aprahamian, A. Florizoone, M. Crabbe, K. Welkenhuyzen, C. Villablanca, T. Vervliet and T. Luyten for their support. This work was funded by FONDECYT 1140549, FONDAP program 15150012, the Millennium Institute P09-015-F and the European Commission R&D MSCA-RISE 734749 (to C.H.); the Michael J. Fox Foundation for Parkinson’s Research target validation grant number 9277, FONDEF ID16I10223, FONDEF D11E1007, US Office of Naval Research-Global N62909-16-1-2003, US Air Force Office of Scientific Research FA9550-16-1-0384, ALSRP Therapeutic Idea Award AL150111, Muscular Dystrophy Association 382453, Seed grant Leading House for the Latin American Region, Switzerland and CONICYT-Brazil 441921/2016-7 (to C.H.), FONDECYT 1160332 and FONDAP15150012 (to J.C.C.); the Spanish Ministry of Economy and Competitiveness SAF2014-52228-R, Unidad de Excelencia María de Maeztu, funded by the MINECO (ref: MDM-2014-0370) and Fundació la Marató de TV3 20134030 (to R.V.); NIH NS095892 (to R.L.W.); FONDECYT 11180825 (to H.U.); FONDECYT 3150113 and EMBO ASTF 385-2016 (to A.C.-S.); FONDECYT 3140355 (to E.R.-F.); FONDECYT 3140458 and 11170291 (to F.J.); FONDECYT 3180427 (to Y.H.); FONDECYT 3190738 (to A.S.-C.); FONDECYT 11170546 and CONICYT PAI 77170091 (to C.T.-R.); R01DK113171, R01CA198103 and R01DK103185 (to R.J.K.); FONDECYT 1150766 (to F.A.C.); the Research Council KU Leuven grant OT14/101 (to G.B.); the Research Foundation – Flanders (FWO) G.0C91.14N, G.0A34.16N and the FWO Scientific Research Community “Ca2+ signaling in health, disease and therapy” W0.019.17 (to G.B.); FWO (G049817N, G076617N) and KU Leuven (C16/15/073) (to P.A.); a FWO doctorate fellowship (to M.K.); the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant 675448 (to M.L.S.); CONICYT fellowship PCHA/Doctorado Nacional/2016-21160232 (to M.G.-Q.); the George E. Hewitt Foundation for a postdoctoral fellowship (to D.E.M.); Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche – Projets blancs; under the frame of E-Rare-2, the ERA-Net for Research on Rare Diseases; Association pour la recherche sur le cancer; Cancéropôle Ile-de-France; Chancelerie des universités de Paris (Legs Poix), Fondation pour la Recherche Médicale; a donation from Elior; the European Research Area Network on Cardiovascular Diseases (MINOTAUR), Gustave Roussy Odyssea, the European Union Horizon 2020 Project Oncobiome; Fondation Carrefour; High-end Foreign Expert Program in China (GDW20171100085); Institut National du Cancer; Inserm (HTE); Institut Universitaire de France; LeDucq Foundation; the LabEx Immuno-Oncology; the RHU Torino Lumière; the Seerave Foundation; the SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination; and the SIRIC Cancer Research and Personalized Medicine (to G.K.).
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Integrated supplementary information
(A) IRE1α-KO cells reconstituted with IRE1α-HA or empty vector (Mock), processed for (WB) for the indicated proteins. Bottom: RT-PCR for Xbp1 mRNA splicing after 8 h of 0.5 µg/ml tunicamycin (Tm) treatment (n = 3 independent experiments). (B) CRISPR control and IRE1α KO cells, or an additional set of cells (C) were analysed as described in A (n =3 independent experiments). (D-E) CRISPR control and IRE1α KO (clone 2) cells imaged for calcium levels in the cytosol and mitochondria (arrow = 100 µM ATP). Right: maximum peak for normalized Fura2 were quantified (total cells analysed: Control 2: n = 45; IRE1α 2: n = 75). (F) WB analysis for the indicated proteins of cells described in 1G normalized to GAPDH (IP3R1: n = 6; IP3R3: n = 7: SERCA2b: n = 4; MCU: n = 7; VDAC1: n = 6; all n are biologically independent). (G) CRISPR control and IRE1α KO cells were processed as indicated in F (IP3R1: n = 5; IP3R3: n = 5; SERCA2b: n = 4; MCU: n = 3; VDAC1: n = 3; all n are biologically independent samples). (H-I) Same cells as in F were simultaneously imaged for calcium levels as in D-E (arrow = 50 µM 3M3FBS) (cells analysed: Mock: n = 93; IRE1α-HA: n = 97) (J) Maximum peak from Fura2/Rhod2 measurements from same samples as in Fig. 1e, f calculated with non-linear regression analyses to obtain correlation constant (K). (K-L) Cells were loaded with 45Ca2+ to determine ER 45Ca2+ loading at steady-state levels (K, n = 8 independent experiments) or after 5-10 minutes of 45Ca2+ loading (L, n = 4 independent experiments). (M) Unidirectional 45Ca2+ efflux from the ER loaded to 45Ca2+ steady-state levels. Graph displays normalized ER 45Ca2+ content (%) over time (n = 8 independent experiments). (N) Resting Fura2-AM ratio (340/405) measurements for the indicated cell lines (cells analysed: Mock: n = 99; IRE1α-HA: n = 109). All plots represent mean and SEM. Statistical differences were detected with unpaired two-tail Student´s t-test or Two-way ANOVA (L). Source data have been provided in Supplementary Table 6.
(A) ATP measurement in the indicated cell lines with a luminescence assay (n = 18 biologically independent samples). (B) Indicated cell lines imaged using AT01 cytosolic FRET probe. Scale bar = 20 µm (total cells analysed: Mock: n = 91 cells; IRE1α-HA: n = 62 cells). (C) Indicated cells were processed for WB analysis to measure pAMPK followed by quantification using total AMPK (dashed line indicated spliced gel to avoid irrelevant lanes) (n = 6 biologically independent samples). (D) Indicated cells were imaged for LC3-II immunofluorescence and confocal microscopy after 6 h treatment with or without 2 h pre-exposure to starvation (CLQ = 1 µM chloroquine, BAF = 0.1 µM bafilomycin A1). Right panel: LC3-II dot count per cell were quantified (n = 3 independent experiments; total cells analysed from left to right: 67; 89; 65; 99; 70; 91; 80; 91; 51; 103; 69; 96). Scale bar = 20 µm). (E) ATP determination of cells described in B with the indicated treatments after 24 h of 1 µM CLQ (n = 17 biologically independent samples). (F) Same cells as in E imaged with TEM. Scale bar = 4 µm. Graph bars representing the area and circularity for the indicated conditions (control: n = 55 cells; IRE1α: n = 60 cells). (G) Live imaging of CRISPR IRE1α KO cells transiently expressing KDEL-RFP with IRE1α-HA or Mock. Graph bars represent the mean for the indicated parameter (n = 3 independent experiments, Scale bar = 10 µm). (H-I) Indicated cells were imaged with TEM. Scale bar = 200 nm. Distance between cristae was determined (total mitochondria analysed: Mock: n = 36; IRE1α-HA: n = 35; Control: n = 32; IRE1α: n = 32). (J) Summary of two different approximations to quantify MAM length. (K) ER longitudinal contact distance (red arrows) was measured by the 2 approaches described in J (n = 3 independent experiments). Scale bar = 500 nm. (Total events analysed: CRISPR control: n = 17 contacts; CRISPR IRE1α: n = 28 contacts). All plots represent mean and SEM. Unpaired or paired (K) Student´s t-test was used. Source data have been provided in Supplementary Table 6.
(A) ATP measurements in cells treated with 100 ng/ml tunicamycin (Tm) for the indicated times (NT: n = 18; 4h: n = 12; 24h: n = 8; all n are biologically independent experiments). (B) IRE1α KO cells reconstituted with IRE1α-HA or Mock or CRISPR cells (C) were processed for western blot analyses to monitor levels of indicated proteins. Cells were treated with 100 ng/ml Tm for the indicated times (n = 3 independent experiments). (D) IRE1α KO cells were reconstituted with IRE1α-HA and processed to obtain purified MAM fractions after 4 h treatment with 1 µg/ml Tm. Fractions were analysed by western blot (WB) for indicated UPR and MAM markers (Cr: crude mitochondria; H: homogenate; M: MAMs; P: pure mitochondria; C: cytosol; E: ER) (n = 2 independent experiments). (E) Same cells described in D were treated with 0.1 µg/ml Tm for indicated time points and stained for ERp72, TOM20 and HA using indirect immunofluorescence. Scale bar = 20 µm. Confocal microscopy analysis and co-localization coefficient was calculated (n = 3) (total cells analysed for t = 0 and 16; n = 40; t = 8; n = 43). (F) IRE1α KO cells reconstituted with IRE1α-HA or empty vector (Mock) were processed to obtain subcellular fractions and analysed by WB to monitor the levels of indicated proteins. Right panel: quantification in the expression of IP3R3 normalized to calnexin (CNX) in the ER fraction (n = 6 independent experiments). (G) IRE1α KO cells reconstituted with IRE1α-HA or empty vector (Mock) transiently expressing control or 9x MAMs linker were imaged for TEM to measure MAMs width. Graph represents the frequency distribution of MAMs width. All plots represent mean and SEM. Statistical differences were detected with unpaired Student´s t-test; two-way ANOVA (A). Source data have been provided in Supplementary Table 6.
(A) Left: scheme representing IRE1α structure and the different mutants used, including D123P, a deletion of the entire cytosolic region and a deletion of the entire N-terminal region. Right panel: cells stably expressing designated constructs described in were processed for western blot analysis to measure levels of indicated proteins (n = 3 independent experiments). (B) Left panels: IRE1α KO cells reconstituted with the indicated constructs were imaged for calcium levels in the cytosol (Fura2-AM) in a calcium free media (n = 4). Arrow = stimulation with 100 µM ATP. Right panel: The maximum peak for normalized Fura2-AM ratio is presented (total cells analysed: Mock: n = 99 cells; IRE1α-HA: n = 109 cells; IRE1α-D123P-HA: n = 78 cells; IRE1α-ΔN-HA: n = 92 cells and IRE1α-ΔC-HA: n = 91 cells). (C) IRE1α KO cells reconstituted with empty vector (Mock), IRE1α-HA, IRE1α-P830L-HA, or IRE1α-ΔC-HA were processed for western blot (WB) analysis to monitor the levels of indicated proteins. Dashed lines indicate cropped gels to eliminate irrelevant lanes from the same image (n = 2 independent experiments). (D) and (E) HEK 293T cells were transiently transfected with indicated constructs and immunoprecipitated (IP) with an anti-HA antibody. WB analysis was performed for the indicated proteins in IPs and total input (n = 2 independent experiments). (F) IRE1α KO cells reconstituted with IRE1α-HA or Mock were stained with PLA using antibodies to detect IP3R3 and VDAC1 proteins. Right panel: mean number of dot counts per cell were quantified (n = 2 independent experiments). All plots represent mean and SEM. Statistical differences were detected with ANOVA and Dunnett’s multiple comparison test. Source data have been provided in Supplementary Table 6.
Supplementary Figure 5 Enhanced expression of IP3R1 reverts the defects in calcium signalling observed in IRE1α null cells.
(A) IRE1α KO cells stably expressing CRa-IP3R1 or CRa-Control were imaged for calcium in the cytosol and mitochondria simultaneously with Fura2-AM and Rhodamine2N-AM (Rhod2). Integration of the maximum peak of individual cells from Fura2-AM and Rhod2-AM signals to calculate the non-linear regression k value and SEM (n = 5 independent experiments; total cells analysed: CRa-IP3R1: n = 87 cells; CRa-Control: n = 65 cells). (B) CRa-IP3R3 or CRa-Control were stained for proximity ligation assay (PLA) with anti-IP3R3 and anti-VDAC1 antibodies and visualized by confocal microscopy. Scale bar = 10 µm. Right panel: mean dots per cell were quantified (n = 2 independent experiments). Source data have been provided in Supplementary Table 6.
(A) Upper panel: scheme representing Ern1 structure and deletion strategy (Ern1ΔR). Down, left: livers from Ern1 and Ern1ΔR processed for WB for the indicated proteins. Western blot (WB) is representative of a minimum of three independent experiments. Bottom panel, right: Ern1 and Ern1ΔR mice were intraperitoneally injected with 1 mg/Kg tunicamycin (Tm) or vehicle for 6 h and then Xbp1 mRNA splicing was evaluated by RT-PCR (n = 2 independent experiments). (B) Illustration of the flow chart of the MAMs proteomics. Left: representative WB of wild-type liver subcellular fractionation highlighting the MAMs fraction. Middle: silver staining of representative MAMs fractions. Right: Venn diagram representing the intersections between the hits obtained in the MAMs proteomics of this study, with two other studies, (related to Supplementary Tables 3 and 4). (C-D) Indicated liver samples were processed to obtain subcellular fractions and analysed by WB against the indicated antibodies. (C) Total extracts (Ern1 n = 6 animals; Ern1ΔK n = 5 animals). (D) ER fraction (Ern1 n = 5 animals; Ern1ΔK n = 4 animals). (E) Indicated liver samples were processed to obtain subcellular fractions and analysed by WB for the indicated markers (Cr: crude mitochondria; H: homogenate; M: MAMs; P: pure mitochondria; C: cytosol; E: ER) Representative WB of n = 3 animals. All plots represent mean and SEM. Source data have been provided in Supplementary Tables 3 and 4.
(A) Venn diagram for metabolomics data showing the overlap in metabolites altered in Ern1ΔK and Ern1ΔR. (B-C) Pathway impact probability and significant alterations in metabolites calculated with MetaboanalystR from Supplementary Table 5. (B) Altered pathways only in Ern1ΔR (n = 3 animals per group). (C) Altered pathways only in Ern1ΔR and Ern1ΔK intersection (n = 3 and 4 animals per group, respectively). (D) Complement to Fig. 7a–d: Whiskers and dot plots of indicated metabolites comparing Ern1 and Ern1ΔK liver samples (4 animals per group). (E-F) Ern1 control and Ern1ΔK liver samples were processed to obtain total tissue homogenates (E) and isolated mitochondria (F) and analysed for mitochondrial respiration (ATP levels) and ROS production (n = 3 animals per group). Statistical differences were detected with one (D) or two –tailed (B-C) Student´s t-test. Source data have been provided in Supplementary Table 5.
All gels and blots presented in this work are displayed unprocessed and labeled. The name of the main figure and panel corresponds to the processed images presented in the final manuscript. Specific lanes are indicated with a blue dashed line.
Supplementary Figures 1–7, titles and legends for Supplementary Tables 1–6.
ER morphological parameters.
MAMs morphometric values.
MAM total protein list.
MAMs consensus proteins.
Statistics source data.
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Carreras-Sureda, A., Jaña, F., Urra, H. et al. Non-canonical function of IRE1α determines mitochondria-associated endoplasmic reticulum composition to control calcium transfer and bioenergetics. Nat Cell Biol 21, 755–767 (2019). https://doi.org/10.1038/s41556-019-0329-y
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