Volume 22, Issue 2, Pages 482-496 (January 2018) Defective Mitochondrial tRNA Taurine Modification Activates Global Proteostress and Leads to Mitochondrial Disease  Md Fakruddin, Fan-Yan Wei, Takeo Suzuki, Kana Asano, Takashi Kaieda, Akiko Omori, Ryoma Izumi, Atsushi Fujimura, Taku Kaitsuka, Keishi Miyata, Kimi Araki, Yuichi Oike, Luca Scorrano, Tsutomu Suzuki, Kazuhito Tomizawa  Cell Reports  Volume 22, Issue 2, Pages 482-496 (January 2018) DOI: 10.1016/j.celrep.2017.12.051 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2018 22, 482-496DOI: (10.1016/j.celrep.2017.12.051) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Generation of Mto1 KO Mice and ESCs and Analyses of the Taurine Modification in the ESCs (A) PECAM-1 and eosin staining of WT and Mto1 KO embryos at ∼E9.0. Arrows show the ventricles of the hearts. Scale bars, 0.5 mm (upper panel) and 0.2 mm (lower panel). (B) Alkaline phosphatase staining of WT and KO ESCs. Scale bars, 0.5 mm. (C) Growth rates of WT and KO ESC clones. n = 3 each. ∗∗∗∗p < 0.0001 by two-way ANOVA. Data are represented as the mean ± SEM. (D) Representative mass chromatograms of uridine (U), 5-taurinomethyluridine (τm5U), and 5-taurinomethyl 2-thiouridine (τm5s2U) in total RNA purified from WT and KO ESCs. (E) Secondary structure of murine mt-tRNATrp. (F) Representative mass chromatograms of tRNA fragments of mt-tRNATrp purified from WT and KO ESCs. Arrows indicate the peaks corresponding to fragments with or without τm5U. Red letters of U indicate 34U of mt-tRNATrp. MW, molecular weight. Cell Reports 2018 22, 482-496DOI: (10.1016/j.celrep.2017.12.051) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Mitochondrial Protein Translation and Metabolism in WT and Mto1 KO Cells (A) Metabolic labeling of mitochondrial translation in WT and KO cells. (B) BN-PAGE analysis of respiratory complex proteins in WT and KO cells. (C) Relative activities of complexes I–IV (CI–CIV) in WT and KO mitochondria. n = 3 for each. ∗∗∗∗p < 0.0001 vs. WT, by Student’s t test. (D) WT and KO cells were stained with JC-1 dye, and the relative membrane potential is shown. n = 7–8. ∗p < 0.05 vs. WT, by Student’s t test. (E–G) Lactate (E), relative NADH/NAD+ ratio (F), and relative ATP level (G) in WT and KO cells were analyzed using independent samples of WT and KO cells. n = 4 for each. ∗∗p < 0.01; ∗∗∗∗p < 0.0001 vs. WT, by Student’s t test. (H) Cytochrome c levels in cytosolic and mitochondrial fractions of WT and KO cells were examined by western blot. (I) Caspase 3/7 activities in WT and KO cells were examined. n = 3 for each. ∗∗p < 0.01 by Student’s t test. Data are represented as the mean ± SEM. Cell Reports 2018 22, 482-496DOI: (10.1016/j.celrep.2017.12.051) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Mitochondrial Protein Imbalance in Mto1 KO Cells (A) Representative transmission electron micrographs of mitochondria in WT and KO embryos at E9.0. Scale bars, 1 μm. Arrows show the swelling cristae in KO cells. (B) BN-PAGE analysis of Opa1 in WT and KO cells. (C) Western blot analysis of representative outer membrane proteins and inner membrane proteins. Coomassie brilliant blue (CBB) staining of the membrane was used as a loading control. (D) Representative images of confocal microscopic analysis of WT and KO cells expressing split GFP conjugated with outer membrane signal (Mtout-spGFP1–10) and spGFP11-mCherry. Scale bar, 10 μm. (E) Quantitative analysis of the data in (D). GFP fluorescence was normalized with mCherry fluorescence. ns = 19 and 32 for WT and KO, respectively. (F) Representative images of confocal microscopic analysis of WT and KO cells expressing split GFP conjugated with inner membrane signal (Mtin-spGFP1–10) and spGFP11-mCherry. Scale bar, 10 μm. (G) Quantitative analysis of GFP fluorescence in the data in (F). n = 30 for WT; n = 31 for KO. ∗∗∗p < 0.001, by Student’s t test. Data are represented as the mean ± SEM. Cell Reports 2018 22, 482-496DOI: (10.1016/j.celrep.2017.12.051) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Proteostatic Stress Responses in Mto1 KO Cells (A) Detection of protein aggregation in WT and KO cells. Scale bar, 10 μm. (B) Soluble and insoluble fractions of WT and KO cells were subjected to western blot analysis to detect Ndufb8, Sdha, and Opa1 as representative inner membrane proteins; Tomm 70, Tomm 40, and Tomm 34 as representative outer membrane proteins; and EF2 as a representative cytosolic protein. (C) Western blot analysis of the UPR-related proteins Atf4, Chop, phospho-EF2 (pEF2), and phospho-eIF2 (p-eIF2) in whole-cell lysates of WT and KO cells. (D) Global protein synthesis levels in WT and KO cells. CBB staining of the membrane was used as a loading control. (E) Relative expression levels of the UPR-related genes spliced Xbp1, Xbp1, and Chop in WT and KO cells. n = 3 for each. ∗∗∗p < 0.001 vs. WT, by Student’s t test. (F) Transmission electron micrographs of ER structure. Arrows indicate the dilated ER in KO cells. Scale bar, 1 μm. (G) Basal calcium levels in the cytoplasm of WT and KO cells were measured by Fluo-4. n = 86 each. ∗∗∗∗p < 0.0001, by Student’s t test. (H) Cells were stimulated with 10 μM thapsigargin, and calcium transients were measured using Fluo-4. n = 86 each. ∗∗∗∗p < 0.0001 between WT and KO, by two-way ANOVA. Data are represented as the mean ± SEM. Cell Reports 2018 22, 482-496DOI: (10.1016/j.celrep.2017.12.051) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Phenotypic Characterization of Heart- and Liver-Specific Mto1 KO Mice (A) Morphology of the heart-specific KO (HcKO) mouse and littermate control (Flox) at postnatal day 1 (upper panels). H&E staining of heart sections in the Flox and HcKO mice (lower panels). Scale bar, 2 mm. (B) Western blot analysis of respiratory subunits corresponding to CI–CV. CBB-stained membrane was used as a loading control. (C) Transmission electron micrographs of mitochondria and ER in the hearts of newborn Flox and HcKO mice. Scale bar, 1 μm. Arrows indicate ER structures. Arrowhead indicates aggregation-like structures. (D) Western blot analysis of Opa1 and Timm44 in mitochondria from the hearts of newborn mice. (E) Relative expression levels of the UPR-related genes spliced Xbp1, Xbp1, and Chop in the hearts of newborn mice. n = 3 for Flox, and n = 6 for HcKO. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 vs. Flox, by Student’s t test. Data are represented as the mean ± SEM. (F) Western blot analysis of UPR-related proteins in total heart lysates of newborn mice. (G) Appearance of Flox and liver-specific Mto1 KO (LcKO) mice at 8 weeks old. (H) Western blot analysis of mitochondrial proteins related to respiratory subunits (MTCOI and Ndufb8) and inner membrane proteins (Opa1 and Timm44). (I) Transmission electron micrographs of mitochondria in the liver of 8-week-old Flox and LcKO mice. Scale bar, 1 μm. Arrow indicates an aggregation of the cristae. Arrowhead indicates the dilation of ER. (J) Western blot analysis of the UPR-related proteins in the livers of Flox and LcKO mice. Cell Reports 2018 22, 482-496DOI: (10.1016/j.celrep.2017.12.051) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 TUDC Treatment Suppressed Proteostress via Opa1 in Mto1 KO Cells (A) Detection of protein aggregations in WT and KO cells with or without 50 μM TUDC treatment for 48 hr. Scale bars, 10 μm. (B) Western blot analysis of Opa1 in soluble and insoluble fractions from the cells. (C) Western blot analysis of the mitochondrial proteins Opa1, Timm44, and Tomm20 in mitochondrial fractions of cells. (D) Transmission electron micrographs of mitochondria in the cells. Scale bar, 1 μm. (E) Western blot analysis of Chop, pEF2, and actin in the total cell lysates of cells treated with TUDC. (F) Gene expression analysis of Chop in cells treated with TUDC. n = 3 each. ∗p < 0.05, by Student’s t test. (G) Global protein synthesis level in WT and KO cells. CBB staining of the membrane was used as a loading control. (H) Transmission electron micrographs of ER in cells. Arrow indicates the dilated ER. Arrowhead indicates the restored ER structure in KO cells treated with TUDC. Scale bar, 1 μm. (I) Basal calcium levels in cells. ns = 27–50. ∗∗∗∗p < 0.0001, by two-way ANOVA followed by Tukey’s comparison test. (J) Growth rate of cells with and without treatment of TUDC. n = 3 each. ∗∗∗∗p < 0.0001, by two-way ANOVA followed by Tukey’s comparison test. (K) Relative expression levels of Chop in cells with or without transfection of mouse Opa1. ∗∗∗∗p < 0.0001 by two-way ANOVA followed by Tukey’s comparison test. Data are represented as the mean ± SEM. Cell Reports 2018 22, 482-496DOI: (10.1016/j.celrep.2017.12.051) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 TUDC Treatment Improved Mitochondrial Functions in LcMto1 KO Mice (A) Western blot analysis of Opa1 in mitochondrial fractions purified from 8-week-old Flox and LcKO mice with or without the injection of TUDC for 1 week. (B) Transmission electron micrographs of mitochondria from Flox and LcKO mice with or without injection of TUDC. Scale bar, 1 μm. (C) Percentage of mitochondria with aberrant cristae aggregation was measured from transmission electron micrographs. ∗∗∗∗p < 0.0001, by two-way ANOVA followed by Tukey’s comparison test. (D) BN-PAGE analysis of complex III and IV proteins in WT and KO cells. (E) Relative activities of complex IV in the livers of Flox and LcKO mice injected with TUDC. n = 4 for each. ∗∗∗p < 0.001 vs. control. (F and G) Relative gene expression of Chop (F) and protein level of Chop (G) in livers of Flox and LcKO mice treated with TUDC or vehicle. n = 4 for each. ∗p < 0.05. (H) Relative gene expression levels of Fgf21 in the livers of Flox and LcKO mice treated with TUDC or vehicle. n = 4 for each. ∗∗∗∗p < 0.0001. Data are represented as the mean ± SEM. Cell Reports 2018 22, 482-496DOI: (10.1016/j.celrep.2017.12.051) Copyright © 2017 The Author(s) Terms and Conditions