|
{"CAPTION EXTENDEDFIG1.png": "'\\n\\n**Extended Data Fig. 1** (**CRISPR library screening for PINK1- paraffin-mediated mitophagy. a, b,** Mitophagy was induced in paraffin- and mt-mKelima-expressing cells by treatment with the mitochondrial membrane potential uncoupler CCGP or a cocktail of suppressors of oxidative phosphorylation (OAK).\\n\\nMithophagy was analysed by flow cytometry for mt-mKelima (**a**) and western blotting for mitochondrial protein in the outer membrane (TOM20), inner membrane (ATFB), or matrix (PDH) (**b**).\\n\\n'", "CAPTION EXTENDEDFIG2.png": "\"\\n\\n**Extended Data Fig. 2 [Integration of seven mitophagy screens.****a**, GSEAof mitophagy accelerators. The top 1% of genes in aggregate _Z_-score were analysed using TopPGene Suite1n1. Representative functional categories and Bonferroni-corrected _P_values are shown. **b**, Proportion of genes encoding mitochondrial proteins annotated in MitoCart2.0*in the top 1% of mitophagy accelerators and decelerators (left), and percentage of MitoCart2.0 member genes identified as being either accelerators (green) or decelerators (red) of mitophagy (right). **c**, Five functional classes of proteins, based on MitoCart2.0 annotations, were present in the top 1% of mitophagy accelerators. The representation of each class within the top 1% was compared to its representation in MitoCart2.0*in vivo-tailed Fisher's exact test. **d**, Box-and-whisker plots of contrast significant mitophagy accelerator hits in each of the seven screens. Line, median; box; 75th-25th percentiles; whiskers (blue dots), 99th-18 percentiles. Genes involved in oxidative phosphorylation (OXPHOS) are indicated in yellow. Pathway enrichment was calculated using a Kolmogorov-Smirnov test. **e**, GSEA enrichment plot for OXPHOS (top) and ranked aggregate _Z_-scores of all genes (bottom). OXPHOS genes are indicated in yellow. **f**, GSEA of mitophagy accelerators analysed as in a. **g**-**1**, Genes in the KEGG endosomal sorting complexes required for transport (ESCRT) (**g**), homotypic fusion and vacuole protein sorting (HOPS) (**h**), and autophagosomes (**i**) pathways. Genes identified as mitophagy accelerators or decelerators are indicated in green and red, respectively. The diameter of each circle is proportional to the _Z_-score of the indicated gene. **j**, Principal component (PC) analysis isblot summarizing variation across the seven screens based on cumulative _Z_-scores of the top 100 genes, displayed as arrows. Autophagy-related genes are indicated. **k**, Mitochondrial membrane potential assessed by flow cytometry for TMRE is disrupted in CCCP treatment, but is increased after treatment with OAR cocktail. Similar results were obtained in two biological replicates.\\n\\n\"", "CAPTION EXTENDEDFIG3.png": "'\\n\\n**Extended Data Fig. 3 EssentialLC3 receptors for mitophagy in C2C12 mouse myoblasts.****a, b**, Validation as mitophagy decelerators of the indicated LC3 receptor geneRNAs, using one library gRNA and one non-library gRNA and using the m-mckemia assay (**a**) or western blotting of mitochondrial proteins in the outer membrane (TOM20 and TOM70), inner membrane (ATP8), or matrix (PPIH) (**b**): \\\\(n\\\\) = 3 biological replicates per gRNA, _P_values calculated by two-sided unpaired r-test relative to NTC_1. Mitophagic degradation of mitochondrial inner membrane and matrix proteins, but not outer membrane proteins, was blocked by gRNAtargeting _Tax1bp1_ for _Tbl1_, consistent with the notion that ubiquitinated outer membrane proteins can be degraded by the ubiquitin proteasome system. Similar results were obtained in two biological replicates. **c, d, LC3 receptor redundancy and TBK1 contribution.** **The indicated gRNAs were transduced singly or in combination by lentivirus infection, followed by analysis by flow cytometry for m-mckemia. Multiple gRNAs were superimposed on cells already targeted by _Tax1bp1_ gRNA (**d**), as indicated. \\\\(n\\\\) = 3 biological replicates per gRNA. _P_values calculated by one-way ANOVA with post hoc Tukey test, \"P_ < 0.05, \"P_ < 0.01, \"*P_ < 0.001 Data are mean +-x.d.\\n\\n'", "CAPTION EXTENDEDFIG4.png": "'\\n\\n**Extended DataFig. 4** **ANT is required in paraffin-mediated mitophagy.** A, Impaired mitophagy in the absence of ANT is confirmed by flux analysis using a lysosome inhibitor, bafilomycin A (\\\\(\\\\frac{1}{2}\\\\)M). Similar results were obtained in two biological replicates. **b**, **c**, Inhibition of mitophagy by CRISPR-mediated deletion of the indicated genes in mouse N2A (**b**) and human SH-SYSY (**c**) neuroblastoma cell lines. Representative flow tracings are shown on left, and quantification onright; \\\\(n\\\\) = 3biological replicates per gRNA, \\\\(P\\\\) values calculated by two-sided unpaired _t_-test relative to NTC, 1. Data are mean +- \\\\(s\\\\).d.\\n\\n'", "CAPTION EXTENDEDFIG5.png": "'\\n\\n**Extended Data Fig. 5**: **ANT is required for stabilization of PINK1.****a**, inhibition of ADP/ATP exchange worsens the loss of membrane potential in response to CCCP. TMR fluorescence intensity following treatment with CCCP was analysed by confocal laser scanning microscopy with the application of live time-series program. Cells were pretreated with control and boxykete acid (BA); \\\\(n\\\\) = 4 biological replicates per group. \\\\(P\\\\) values calculated by two-way repeated measures ANOVA. **b**, Mitophagy was induced by OXPHOS inhibitors (antimycin A and oligomycin) in the presence of the indicated concentration of the ADP/ATP transport inhibitor bongkeickacid, followed by flow cytometry for m-mecium. \\\\(n\\\\) = 3 biological replicates per group. \\\\(P\\\\) values calculated by one-way ANOVA with post hoc Tukey test; _\"P_ < 0.01. **c**, Genetic inhibition of ADP/ATP exchange worsens the loss of membrane potential in response to CCCP. _Ant_(_r_) cells were rescued by human wild-type (WT) or mutant ANTI; \\\\(n\\\\) = 3 biological replicates per group. \\\\(P\\\\) values calculated by two-way repeated measures ANOVA relative to NTC. **c**, ADP/ATP exchange rates is impaired in ADP/ATP-binding mutants (K330, K43E), but not in disease-causing mutants (A90D, V289M); \\\\(n\\\\) = 3 biological replicates per group. \\\\(P\\\\) values calculated by two-sided unpaired test relative to WT. **e**, Loss of ANT impairs PINK1-dependent mitophagy induced by oxidative stress, but does not impair PINK1-independent mitophagy caused by hypoxia or starvation; \\\\(n\\\\) = 3 biological replicates per gRNA. \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to NTC. \\\\(E\\\\), PINK1 accumulation in mitochondria is impaired in cells lacking ANT. Cells bearing gRNAs targeting the indicated genes were transduced with PINK1-GFP, followed by treatment with CCCP versus control, and then immunostained using anti-TOM20 antibody (red). GFP fluorescence is shown in green, and merged signal yellow. Scale bar, 20 \u03bcm. \\\\(g\\\\), PINK1 stabilization by CCCP treatment is preserved with wild-type ANT and ADP/ATP-binding double mutant (K43E/B24E), but not in known disease-causing mutants (A90D, A123D). **h**, Phosphorylation of PINK1 after CCCP treatment is preserved in the absence of ANT1 or ANTI. **l**, PINK1 transcription (b) and translation (j) are not changed by loss of ANT. **n** = 4 biological replicates per group. \\\\(P\\\\) values calculated by one-way ANOVA. **k**, The activities of the PINK1-cleaving proteases PARL and OMA1 are not changed by loss of ANT. **l**, General autophagy fluxis preserved in the absence of ANT. **n** = 3 biological replicates per group, \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to NTC. **m**, Suppression of TIM23-mediated protein translocation response to CCCP treatment is impaired in the absence of ANT1 or ANTI. **m**, as shown by import of SU9-GFP into intact cells; \\\\(n\\\\) = 3 biological replicates per gRNA, \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to NTC. Scale bar, 20 \u03bcm. Data are mean +- s.d. Similar results were obtained in two biological replicates (**f** - **h**, **j**, **k**). For gel source data, see Supplementary Fig. 1.\\n\\n'", "CAPTION EXTENDEDFIG6.png": "'\\n\\n**Extended Data Fig. 6**: **ANT mediates closure of TIM23 via TIM44.4**, Deletion of _Ant1_ or _Ant2_ does not affect expression of TIM or TOM proteins (right) or destabilize TIM and TOM complexes, as assessed by blue native PAGE (left). **b**, **c**, ANTI and ANTI2 bind to TIM23 and TIMM44, as assessed by corimmunoprecipitation (**b**) and blue native PAGE (**c**). The ANTI-TIM23 complex is marked with an asterisk asterisk. **d**, Wild-type ANTI and the ADP/ATP binding double mutant (K43/K2445) bind to the TIM23 complex component TIM23, whereas disease-causing mutants (A990D, A123D) do not. **c**, Closure of TIM23in response to CCCP treatment is impaired in the presence of disease-causing mutants (A90D, A123D), but is preserved in the presence of the ADP/ATP binding double mutant (K43E/R244E), as shown by import of SU9-GFP into mitochondria: \\\\(n\\\\) = 3 biological replicates per group. \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to empty. Scale bar, 40 um. **f**, ANTI binds to both TIMM23 and TIMM44. **g**, Mittophagys impaired in cells lacking TIMM44; \\\\(n\\\\) = 3 biological replicates per gRNA, \\\\(P\\\\) values calculated by two-sided unpaired \\\\(t\\\\) test relative to MC, **l**, **b**, PINI stabilization by CCCP treatment is abrogated in the absence of TIMM44. **l**, Rescue of mitophagy with wild-type ANT and ADP/ATP exchange mutants (K330, K43E/R244E), but not with known disease-causing mutants (A90D, A123D) and TIMM44-binding site mutant (G146E/K147D). Top left, schematic of ANT and sites of mutations. Bottom, western blotting demonstrating equivalent expression of ANT constructs. Right, quantification of mitophagy: \\\\(n\\\\) = 3 biological replicates per group, \\\\(P\\\\) values calculated by one-way ANOVA with post the Tukey test, \"_p_ < 0.01, \"_p_ < 0.001. **J**, Mutation of the predicted ANTI interactions site in TIMM44 abrogates binding to ANTI. **k**, Rescue of mitophagy with wild-type TIMM44, but not with binding site mutant (K43E/R244E); \\\\(n\\\\) = 3 biological replicates per group, \\\\(P\\\\) values calculated by one-way ANOVA with post hoc Tukey test, \"_p_ < 0.01, \"_p_ < 0.001. **I**, Mutation in TIMM44 of the ANTI interaction site does not abrogate TIMM44 binding to TIMM23. **Dataera** mean = s. d. Similar results were obtained in two biological replicates (**a**\u2013**d**, **f**, **b**\u2013**l**). For gel source data, see Supplementary Fig. 1.\\n\\n'", "CAPTION EXTENDEDFIG7.png": "'\\n\\n**Extended Data Fig. 7** **ANT is required for mitophagy in vivo, independently of transcriptional regulation.** Equivalent amounts of PINK1 and higher parkins transcription in _Antf_20 heart and skeletal muscle (SM); \\\\(n\\\\) = 4 per group, _P_values calculated by two-sided unpaired _k_-test. Data are mean +s.d.\\n\\n'", "CAPTION FIG1.png": "'relative to non-targeting control;NTC) or by western blotting of mitochondrial proteins in the outer membrane (OMM-TOM20), inner membrane (IMM-ATPB), or matrix (PDH) (**e**). Similar results were obtained in two biological replicates. For gel source data, see Supplementary Fig. 1.**f**, Suppression of mitophagy in primary ratneurons. Left, visualization of neuronal mitochondria with tetramethylrhodamine ethylester (TMRF) dye. Middle, representative image showing coating of mitochondria (labelled with Mirc-Snap) with the mitophagy receptor OPTN, indicating active mitophagy. Right, quantification of cells undergoing active mitophagy: \\\\(n\\\\) = 6 (untreated control), 6 (treated control), 4 (_Ant1_), and 5biological replicates (_Ant2_), \\\\(P\\\\) values calculated by one-way ANOVA with post hoc Dunnett\\'s multiple comparison test; \\\\(P\\\\) < 0.05, \"_P_ < 0.01. Scale bars, 5 \u03bcm, 0.8 \u03bcm (inset). Data are mean +-s.d.\\n\\nFig. 1: **Multidimensional mitophagy screen reveals that ANT is required for mitophagy.****a**. Outline of CRISPR\u2013Cas9 genome-wide genetic screen, using four reporter assays and two modes of mitophagy induction. **b**, Most significant hits in each of the seven screens. MT, MitoTracker; OM-G, outer membrane GFP; Mat-G, matrix GFP. Representative previously known genes shown by open symbols, previously unknown genes by coloured symbols; line, median; box, 75th\u201325th percentiles; \u2018whiskering\u2019, 99th\u20131st percentiles; duplicate experiments. **c**, Ranked aggregate _z_\u2019scores of all genes. Representative previously known genes labelled in grey, previously unknown in black. **d**, **e**, Validation as mitophagy decelerators of the indicated genes, using both a gRNA chosen from the screening library, and an independent non-library gRNA, followed by flow cytometry for matrix-targeted mkcima (**d**, \\\\(n\\\\) = 3 biological replicates per gRNA, \\\\(P\\\\) values calculated by two-sided unpaired _d_-test'", "CAPTION FIG2.png": "'Fig. 2 **ANT mediates suppression of TIM23 via TIM44.****a**, Inhibition of ADP/ATP transport with the indicated inhibitors accelerates mitophagy, in sharp contrast to genetic deletion of ANTI (bottom right); \\\\(n\\\\) = 3 per group., Mitochondrial admi is elevated in cells lacking ANT, and reduced in response to CCCP, compared to control cells, consistent with reverse ADP/ATP exchange in low admi: \\\\(n\\\\) = 3 per gRNA. \\\\(P\\\\) values calculated by two-way repeated measures ANOVA relative to NTC. **c**, Rescue of mitophagy with wild type (WT) ANT and ADP/ATP-binding mutant (K43k/R244K), but not with disease-causing mutants (A90D, A123D). Top left, schematic of ANT mutations. Bottom, equivalent expression of constructs. Right, quantification of mitophagy; \\\\(n\\\\) = 3 per group. **d**, PINK1 stabilization by CCCP treatment is abrogated in the absence of ANTI or 2. **e**, Suppression of TIM23-mediated import of [**15**S]Sup-DHFR into isolated mitochondria in response to CCCP is impaired in the absence of ANTI (p., precursor; m, mature). **f**, Interaction between ANTI and TIM44 identified in the mitochondrial interactome database XlinkDB. **g**, Deletion of TIMM44 impairs binding of ANTI to TIMM23. **h**, Mutation of the predicted TIMM44 interaction site in ANTI or disease-causing ANTI mutation (A90D) abrogates binding of ANTI to TIMM44, while ADP/ATP exchange mutants (K43K/R244E and K330) do not. **i**, Model of ANTI-mediated suppression of TIM23 in response to compromise of mitochondrial bioenergetics. Data are mean +- \\\\(x\\\\) < .d_.\\\\(P\\\\) values by one-way ANOVA with post hoc Tukey test (**a**, **c**); \\\\(p\\\\) < 0.05, \"_p_ < 0.01, \"_p_ < 0.001. Similar results were obtained in two biological replicates. For gel source data, see Supplementary Fig. 1(**c**\u2013**e**, **g**, **h**).\\n\\n'", "CAPTION FIG3.png": "'_Ant_20 mice. Scale bars, 2 \u03bcm. Similar results were obtained from three mice in each group. B, Dilated cardiomyopathy in _Ant_20 mice: heart weights (top left), sample images (top right) and quantification (bottom) of echocardiography; \\\\(n\\\\) = 7 per group. LVID, left ventricle internal diameter systolic (s) and diastolic (d); LVDW, left ventricle posterior wall; EF, ejection fraction. **1**. Echocardiography of patient bearing homozygous loss-of-function. _ANTI_ mutations. Left, systolic; right, diastole, **3**. Electron micrographs of endomyocardial biopsy from same patient as in L. Localized distension of outer membrane (blue arrows) with apparent release of mitochondrial matrix content (green) into the cytoplasm. Scale bars, 1 \u03bcm. Data are mean +-s.d. (**a**, **e**). _P_values by two-sided unpaired \\\\(t\\\\) test (**a**, **d**, **h**), one-way ANOVA with post hoc Tukey test (**b**); \\\\(P\\\\) < 0.05, \"_P_ < 0.01, \"_P_ < 0.001. For gel source data, see Supplementary Fig. 1.\\n\\nFig. 3: **ANTI is required for mitophagy in vivo.****a**, Blunted mitophagy in heart and brain of _Ant_20 mice, illustrate at by reduced coating of mitochondria by p62, PINK1 and parin. **b**, Blunted mitophagy in skeletal muscle of _Ant_20 mice, shown by intramuscular transfection of mitoQC plasmid. Line, mean; \\\\(n\\\\) = 4 mice, 47 fibres (WT) and 4 mice, 50 fibres (_Ant_20). Scale bars, 10 \u03bcm. **c**, Rescue or mitophagy with wild type (WT) ANTI and mutant lacking mCherry exchange activity (K3IQ), but not with disease-causing mutant (A900). Line, mean; \\\\(n\\\\) = 3 mice, 24 fibres (WT), 3 mice, 16 fibres (Luc), 3 mice, 23 fibres (k33Q) and 3 mice, 21 fibres (A90D). Scale bars, 10 \u03bcm. **d**, Accumulation of mitochondrial DNA (right) in heart (top) and muscle (bottom) of _Ant_20 mice despite absence of nuclear-encoded biogenesis (left): \\\\(n\\\\) = 4 per group. **e\u2013g**. Accumulation of mitochondrial proteins (**e**) and of disorganized and aberrant mitochondria (**f**) in heart, and deep red colouring of mitochondria in skeletal muscle (**g**) of _Ant_20 mice. Scale bars, 2 \u03bcm. Similar results were obtained from three mice in each group. **b**, Dilated cardiomyopathy in _Ant_20 mice: heart weights (top left), sample images (top right) and quantification (bottom) of echocardiography; \\\\(n\\\\) = 7 per group. LVID, left ventricle internal diameter systolic (s) and diastolic (d); LVDW, left ventricle posterior wall; EF, ejection fraction. **1**. Echocardiography of patient bearing homozygous loss-of-function. _ANTI_ mutations. Left, systolic; right, diastole, **3**. Electron micrographs of endomyocardial biopsy from same patient as in L. Localized distension of outer membrane (blue arrows) with apparent release of mitochondrial matrix content (green) into the cytoplasm. Scale bars, 1 \u03bcm. Data are mean +-s.d. (**a**, **e**). _P_values by two-sided unpaired \\\\(t\\\\) test (**a**, **d**, **h**), one-way ANOVA with post hoc Tukey test (**b**); \\\\(P\\\\) < 0.05, \"_P_ < 0.01, \"_P_ < 0.001. For gel source data, see Supplementary Fig. 1.\\n\\n'"} |
