Abstract
Mitochondrial homeostasis depends on mitophagy, the programmed degradation of mitochondria. Only a few proteins are known to participate in mitophagy. Here we develop a multidimensional CRISPR–Cas9 genetic screen, using multiple mitophagy reporter systems and pro-mitophagy triggers, and identify numerous components of parkin-dependent mitophagy1. Unexpectedly, we find that the adenine nucleotide translocator (ANT) complex is required for mitophagy in several cell types. Whereas pharmacological inhibition of ANT-mediated ADP/ATP exchange promotes mitophagy, genetic ablation of ANT paradoxically suppresses mitophagy. Notably, ANT promotes mitophagy independently of its nucleotide translocase catalytic activity. Instead, the ANT complex is required for inhibition of the presequence translocase TIM23, which leads to stabilization of PINK1, in response to bioenergetic collapse. ANT modulates TIM23 indirectly via interaction with TIM44, which regulates peptide import through TIM232. Mice that lack ANT1 show blunted mitophagy and consequent profound accumulation of aberrant mitochondria. Disease-causing human mutations in ANT1 abrogate binding to TIM44 and TIM23 and inhibit mitophagy. Together, our findings show that ANT is an essential and fundamental mediator of mitophagy in health and disease.
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Change history
20 November 2019
This article was amended to include three Supplementary Data files, which were missing originally from the Supplementary Information section.
References
Youle, R. J. & Narendra, D. P. Mechanisms of mitophagy. Nat. Rev. Mol. Cell Biol. 12, 9–14 (2011).
Ting, S. Y., Yan, N. L., Schilke, B. A. & Craig, E. A. Dual interaction of scaffold protein Tim44 of mitochondrial import motor with channel-forming translocase subunit Tim23. eLife 6, e23609 (2017).
Matsuda, N. et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J. Cell Biol. 189, 211–221 (2010).
Saito, T. & Sadoshima, J. Molecular mechanisms of mitochondrial autophagy/mitophagy in the heart. Circ. Res. 116, 1477–1490 (2015).
Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Cottet-Rousselle, C., Ronot, X., Leverve, X. & Mayol, J. F. Cytometric assessment of mitochondria using fluorescent probes. Cytometry A 79, 405–425 (2011).
Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T. & Miyawaki, A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem. Biol. 18, 1042–1052 (2011).
Hoshino, A. et al. Cytosolic p53 inhibits Parkin-mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart. Nat. Commun. 4, 2308 (2013).
Hasson, S. A. et al. High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy. Nature 504, 291–295 (2013).
McEwan, D. G. et al. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins. Mol. Cell 57, 39–54 (2015).
Lee, J. A., Beigneux, A., Ahmad, S. T., Young, S. G. & Gao, F. B. ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration. Curr. Biol. 17, 1561–1567 (2007).
Hammerling, B. C. et al. A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance. Nat. Commun. 8, 14050 (2017).
Takahashi, Y. et al. An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure. Nat. Commun. 9, 2855 (2018).
Yoshii, S. R., Kishi, C., Ishihara, N. & Mizushima, N. Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane. J. Biol. Chem. 286, 19630–19640 (2011).
Wong, Y. C. & Holzbaur, E. L. Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation. Proc. Natl Acad. Sci. USA 111, E4439–E4448 (2014).
Kawamata, H., Tiranti, V., Magrané, J., Chinopoulos, C. & Manfredi, G. adPEO mutations in ANT1 impair ADP-ATP translocation in muscle mitochondria. Hum. Mol. Genet. 20, 2964–2974 (2011).
Chevrollier, A., Loiseau, D., Reynier, P. & Stepien, G. Adenine nucleotide translocase 2 is a key mitochondrial protein in cancer metabolism. Biochim. Biophys. Acta 1807, 562–567 (2011).
Di Marino, D., Oteri, F., della Rocca, B. M., D’Annessa, I. & Falconi, M. Mapping multiple potential ATP binding sites on the matrix side of the bovine ADP/ATP carrier by the combined use of MD simulation and docking. J. Mol. Model. 18, 2377–2386 (2012).
Clémençon, B., Babot, M. & Trézéguet, V. The mitochondrial ADP/ATP carrier (SLC25 family): pathological implications of its dysfunction. Mol. Aspects Med. 34, 485–493 (2013).
Nguyen, T. N., Padman, B. S. & Lazarou, M. Deciphering the molecular signals of PINK1/Parkin mitophagy. Trends Cell Biol. 26, 733–744 (2016).
Okatsu, K. et al. PINK1 autophosphorylation upon membrane potential dissipation is essential for Parkin recruitment to damaged mitochondria. Nat. Commun. 3, 1016 (2012).
Jin, S. M. et al. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J. Cell Biol. 191, 933–942 (2010).
Sekine, S., et al. Reciprocal roles of Tom7 and OMA1 during mitochondrial import and activation of PINK1. Mol. Cell 73, 1028–1043 (2019).
Schweppe, D. K. et al. Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry. Proc. Natl Acad. Sci. USA 114, 1732–1737 (2017).
Graham, B. H. et al. A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator. Nat. Genet. 16, 226–234 (1997).
McWilliams, T. G. et al. mito-QC illuminates mitophagy and mitochondrial architecture in vivo. J. Cell Biol. 214, 333–345 (2016).
McWilliams, T.G. et al. Basal mitophagy occurs independently of PINK1 in mouse tissues of high metabolic demand. Cell Metab. 27, 439–449 (2018).
King, M. S. et al. Expanding the phenotype of de novo SLC25A4-linked mitochondrial disease to include mild myopathy. Neurol. Genet. 4, e256 (2018).
Morrow, R. M. et al. Mitochondrial energy deficiency leads to hyperproliferation of skeletal muscle mitochondria and enhanced insulin sensitivity. Proc. Natl Acad. Sci. USA 114, 2705–2710 (2017).
Esposito, L. A., Melov, S., Panov, A., Cottrell, B. A. & Wallace, D. C. Mitochondrial disease in mouse results in increased oxidative stress. Proc. Natl Acad. Sci. USA 96, 4820–4825 (1999).
Kaukonen, J. et al. Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science 289, 782–785 (2000).
McManus, M. J. et al. Mitochondrial DNA variation dictates expressivity and progression of nuclear DNA mutations causing cardiomyopathy. Cell Metab. 29, 78–90.e5 (2019).
Fontanesi, F. et al. Mutations in AAC2, equivalent to human adPEO-associated ANT1 mutations, lead to defective oxidative phosphorylation in Saccharomyces cerevisiae and affect mitochondrial DNA stability. Hum. Mol. Genet. 13, 923–934 (2004).
Thompson, K. et al. Recurrent de novo dominant mutations in SLC25A4 cause severe early-onset mitochondrial disease and loss of mitochondrial DNA copy number. Am. J. Hum. Genet. 99, 860–876 (2016).
Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Parnas, O. et al. A genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell 162, 675–686 (2015).
Hoshino, A. et al. Oxidative post-translational modifications develop LONP1 dysfunction in pressure overload heart failure. Circ. Heart Fail. 7, 500–509 (2014).
Kang, Y. et al. Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability. eLife 5, e17463 (2016).
Okatsu, K., Kimura, M., Oka, T., Tanaka, K. & Matsuda, N. Unconventional PINK1 localization to the outer membrane of depolarized mitochondria drives Parkin recruitment. J. Cell Sci. 128, 964–978 (2015).
Joshi, D. C. & Bakowska, J. C. Determination of mitochondrial membrane potential and reactive oxygen species in live rat cortical neurons. J. Vis. Exp. 51, 2704 (2011).
Kawamata, H., Starkov, A. A., Manfredi, G. & Chinopoulos, C. A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells. Anal. Biochem. 407, 52–57 (2010).
Chen, J., Xu, H., Aronow, B. J. & Jegga, A. G. Improved human disease candidate gene prioritization using mouse phenotype. BMC Bioinformatics 8, 392 (2007).
Calvo, S. E., Clauser, K. R. & Mootha, V. K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44 (D1), D1251–D1257 (2016).
Acknowledgements
We thank the staff of the Penn Medicine Biobank including J. Weaver, D. Birtwell, H. Williams, P. Baumann, and M. Risman, as well as the Regeneron Genetics Center (RGC). The Penn Medicine Biobank was funded by a gift from the Smilow family and by the Penn Cardiovascular Institute and the Perelman School of Medicine. A.H. was supported by the Uehara Memorial Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research and JSPS Overseas Research Fellowships. S.W. was supported by a fellowship from the ADA (1-16-PDF-117) and Toyobo Biotechnology Foundation. C.S.E. was supported by a Hanna H. Gray Fellowship from the Howard Hughes Medical Institute. E.L.F.H. was supported by the NIH NINDS (R37 NS060698). D.C.W. was supported by the NIH (NS021328, OD010944, MH108592, MH110185) and DOD (W81XWH-16-1-0401) Z.A. was supported by the NIH (HL094499, DK107667) and the AHA (Established Investigator Award).
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Contributions
A.H. and W.W. contributed equally to this work. A.H., W.W. and Z.A. conceived the project and designed experiments. A.H. performed CRISPR library screen and C.M.-R., M.P.M. and K.S.R. analysed NGS data. C.S.E. and E.L.F.H. performed rat primary neuron imaging. D.C.W. provided Ant1 knockout mice, and W.W., S.W., J.L., K.L., M.J.M. and D.C.W. performed mouse experiments. A.H., W.W. and B.G. conducted flow cytometry and W.W., C.B. and P.P. performed blue native PAGE. S.Y., M.L. and S.D. collected ANT1 mutant patient data. A.H., W.W. and Z.A. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 CRISPR library screening for PINK1–parkin-mediated mitophagy.
a, b, Mitophagy was induced in parkin- and mt-mKeima-expressing cells by treatment with the mitochondrial membrane potential uncoupler CCCP or a cocktail of suppressors of oxidative phosphorylation (OAR). Mitophagy was analysed by flow cytometry for mt-mKeima (a) and western blotting for mitochondrial protein in the outer membrane (TOM20), inner membrane (ATPB), or matrix (PDH) (b). c, Representative gate setting of cell sorting for each of the four indicated mitophagy assays: 1. loss of MitoTracker labelling of mitochondrial membrane; 2. loss of ectopically expressed outer membrane-targeted GFP (GFP–OMP25); 3. loss of ectopically expressed matrix GFP protein (COX8–GFP); and 4. altered fluorescence of matrix-targeted mKeima. d, Genes in the KEGG mitophagy pathway. Genes identified as mitophagy accelerators or decelerators in CRISPR knockout, defined as Z-score >1.5 in at least one screen, are indicated in green and red, respectively. The diameter of each circle is proportional to the Z-score of the indicated gene. Similar results were obtained in two biological replicates (a–c). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 Integration of seven mitophagy screens.
a, GSEA of mitophagy accelerators. The top 1% of genes in aggregate Z-score were analysed using ToppGene Suite31,43. Representative functional categories and Bonferroni-corrected P values are shown. b, Proportion of genes encoding mitochondrial proteins annotated in MitoCarta2.044 in the top 1% of mitophagy accelerators and decelerators (left), and percentage of MitoCarta2.0 member genes identified as being either accelerators (green) or decelerators (red) of mitophagy (right). c, Five functional classes of proteins, based on MitoCarta2.0 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 MitoCarta2.0 via a two-tailed Fisher’s exact test. d, Box-and-whisker plots of most significant mitophagy accelerator hits in each of the seven screens. Line, median; box, 75th–25th percentiles; whiskers (blue dots), 99th–1st 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 decelerators analysed as in a. g–i, Genes in the KEGG endosomal sorting complexes required for transport (ESCRT) (g), homotypic fusion and vacuole protein sorting (HOPS) (h), and autophagosome (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 biplot 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.
Extended Data Fig. 3 Essential LC3 receptors for mitophagy in C2C12 mouse myoblasts.
a, b, Validation as mitophagy decelerators of the indicated LC3 receptor gene gRNAs, using one library gRNA and one non-library gRNA and using the mt-mKeima assay (a) or western blotting of mitochondrial proteins in the outer membrane (TOM20 and TOM70), inner membrane (ATPB), or matrix (PDH) (b); n = 3 biological replicates per gRNA, P values calculated by two-sided unpaired t-test relative to NTC_1. Mitophagic degradation of mitochondrial inner membrane and matrix proteins, but not outer membrane proteins, was blocked by gRNA targeting Tax1bp1 or Tbk1, 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 mt-mKeima. Multiple gRNAs were superimposed on cells already targeted by Tax1bp1 gRNA (c) or Tbk1 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 ± s.d.
Extended Data Fig. 4 ANT is required in parkin-mediated mitophagy.
a, Impaired mitophagy in the absence of ANT is confirmed by flux analysis using a lysosome inhibitor, bafilomycin A (1 μM). 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-SY5Y (c) neuroblastoma cell lines. Representative flow tracings are shown on left, and quantification on right; n = 3 biological replicates per gRNA, P values calculated by two-sided unpaired t-test relative to NTC_1. Data are mean ± s.d.
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. TMRE 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 bongkrekic 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 bongkrekic acid, followed by flow cytometry for mt-mKeima; 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. Ant1KO cells were rescued by human wild-type (WT) or mutant ANT1; n = 3 biological replicates per group, P values calculated by two-way repeated measures ANOVA relative to NTC. d, ADP/ATP exchange rate is impaired in ADP/ATP-binding mutants (K33Q, K43E), but not in disease-causing mutants (A90D, V289M); n = 3 biological replicates per group, P values calculated by two-sided unpaired t 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. f, 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 in yellow. Scale bar, 20 μm. g, PINK1 stabilization by CCCP treatment is preserved in wild-type ANT and ADP/ATP-binding double mutant (K43E/R244E), but not in known disease-causing mutants (A90D, A123D). h, Phosphorylation of PINK1 after CCCP treatment is preserved in the absence of ANT1 or ANT2. i, j, PINK1 transcription (i) 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 flux is 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 in response to CCCP treatment is impaired in the absence of ANT1 or ANT2, 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 μm. 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.
Extended Data Fig. 6 ANT mediates closure of TIM23 via TIM44.
a, 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, ANT1 and ANT2 bind to TIMM23 and TIMM44, as assessed by co-immunoprecipitation (b) and blue native PAGE (c). The ANT–TIM23 complex is marked with an asterisk. d, Wild-type ANT1 and the ADP/ATP binding double mutant (K43E/R244E) bind to the TIM23 complex component TIMM23, whereas disease-causing mutants (A90D, A123D) do not. e, Closure of TIM23 in 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 μm. f, ANT1 binds to both TIMM23 and TIMM44. g, Mitophagy is impaired in cells lacking TIMM44; n = 3 biological replicates per gRNA, P values calculated by two-sided unpaired t-test relative to NTC_1. h, PINK1 stabilization by CCCP treatment is abrogated in the absence of TIMM44. i, Rescue of mitophagy with wild-type ANT and ADP/ATP exchange mutants (K33Q, 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 hoc Tukey test, **P < 0.01, ***P < 0.001. j, Mutation of the predicted ANT1 interaction site in TIMM44 abrogates binding to ANT1. k, Rescue of mitophagy with wild-type TIMM44, but not with binding site mutant (K282D); n = 3 biological replicates per group, P values calculated by one-way ANOVA with post hoc Tukey test, **P < 0.01, ***P < 0.001. l, Mutation in TIMM44 of the ANT1 interaction site does not abrogate TIMM44 binding to TIMM23. Data are mean ± s.d. Similar results were obtained in two biological replicates (a–d, f, h–j, l). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 ANT is required for mitophagy in vivo, independently of transcriptional regulation.
Equivalent amounts of PINK1 and higher parkin transcription in Ant1KO heart and skeletal muscle (SM); n = 4 per group, P values calculated by two-sided unpaired t-test. Data are mean ± s.d.
Supplementary information
Supplementary Figure 1
This file contains uncropped scans with size marker indications
Supplementary Data 1
Raw counts of screens.
Supplementary Data 2
Z-scores of accelerators.
Supplementary Data 3
Z-scores of decelerators.
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Hoshino, A., Wang, Wj., Wada, S. et al. The ADP/ATP translocase drives mitophagy independent of nucleotide exchange. Nature 575, 375–379 (2019). https://doi.org/10.1038/s41586-019-1667-4
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DOI: https://doi.org/10.1038/s41586-019-1667-4
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