Title: ATAD3B is a mitophagy receptor mediating clearance of oxidative stress‐induced damaged mitochondrial DNA
Abstract: Article5 March 2021free access Source DataTransparent process ATAD3B is a mitophagy receptor mediating clearance of oxidative stress-induced damaged mitochondrial DNA Li Shu Li Shu orcid.org/0000-0003-3230-9363 Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, ChinaThese authors contributed equally to this work Search for more papers by this author Chao Hu Chao Hu Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, ChinaThese authors contributed equally to this work Search for more papers by this author Meng Xu Meng Xu Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China Search for more papers by this author Jianglong Yu Jianglong Yu Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China Search for more papers by this author He He He He Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China Search for more papers by this author Jie Lin Jie Lin Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China Search for more papers by this author Hongying Sha Hongying Sha State Key Laboratory of Medical Neurobiology, Institute of Brain Science, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Bin Lu Bin Lu Attardi Institute of Mitochondrial Biomedicine, School of Life Sciences, Wenzhou Medical University, Wenzhou, China Search for more papers by this author Simone Engelender Simone Engelender Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Search for more papers by this author Minxin Guan Minxin Guan Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhiyin Song Corresponding Author Zhiyin Song [email protected] orcid.org/0000-0002-5003-081X Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China Search for more papers by this author Li Shu Li Shu orcid.org/0000-0003-3230-9363 Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, ChinaThese authors contributed equally to this work Search for more papers by this author Chao Hu Chao Hu Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, ChinaThese authors contributed equally to this work Search for more papers by this author Meng Xu Meng Xu Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China Search for more papers by this author Jianglong Yu Jianglong Yu Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China Search for more papers by this author He He He He Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China Search for more papers by this author Jie Lin Jie Lin Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China Search for more papers by this author Hongying Sha Hongying Sha State Key Laboratory of Medical Neurobiology, Institute of Brain Science, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Bin Lu Bin Lu Attardi Institute of Mitochondrial Biomedicine, School of Life Sciences, Wenzhou Medical University, Wenzhou, China Search for more papers by this author Simone Engelender Simone Engelender Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Search for more papers by this author Minxin Guan Minxin Guan Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, China Search for more papers by this author Zhiyin Song Corresponding Author Zhiyin Song [email protected] orcid.org/0000-0002-5003-081X Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China Search for more papers by this author Author Information Li Shu1, Chao Hu1, Meng Xu1, Jianglong Yu1, He He1, Jie Lin2, Hongying Sha3, Bin Lu4, Simone Engelender5, Minxin Guan6 and Zhiyin Song *,1 1Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China 2Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China 3State Key Laboratory of Medical Neurobiology, Institute of Brain Science, Shanghai Medical College, Fudan University, Shanghai, China 4Attardi Institute of Mitochondrial Biomedicine, School of Life Sciences, Wenzhou Medical University, Wenzhou, China 5Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel 6Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, China *Corresponding author. Tel: +86 027 68752235; E-mail: [email protected] The EMBO Journal (2021)40:e106283https://doi.org/10.15252/embj.2020106283 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondrial DNA (mtDNA) encodes several key components of respiratory chain complexes that produce cellular energy through oxidative phosphorylation. mtDNA is vulnerable to damage under various physiological stresses, especially oxidative stress. mtDNA damage leads to mitochondrial dysfunction, and dysfunctional mitochondria can be removed by mitophagy, an essential process in cellular homeostasis. However, how damaged mtDNA is selectively cleared from the cell, and how damaged mtDNA triggers mitophagy, remain mostly unknown. Here, we identified a novel mitophagy receptor, ATAD3B, which is specifically expressed in primates. ATAD3B contains a LIR motif that binds to LC3 and promotes oxidative stress-induced mitophagy in a PINK1-independent manner, thus promoting the clearance of damaged mtDNA induced by oxidative stress. Under normal conditions, ATAD3B hetero-oligomerizes with ATAD3A, thus promoting the targeting of the C-terminal region of ATAD3B to the mitochondrial intermembrane space. Oxidative stress-induced mtDNA damage or mtDNA depletion reduces ATAD3B-ATAD3A hetero-oligomerization and leads to exposure of the ATAD3B C-terminus at the mitochondrial outer membrane and subsequent recruitment of LC3 for initiating mitophagy. Furthermore, ATAD3B is little expressed in m.3243A > G mutated cells and MELAS patient fibroblasts showing endogenous oxidative stress, and ATAD3B re-expression promotes the clearance of m.3243A > G mutated mtDNA. Our findings uncover a new pathway to selectively remove damaged mtDNA and reveal that increasing ATAD3B activity is a potential therapeutic approach for mitochondrial diseases. SYNOPSIS The quality control machinery ensuring removal of damaged mitochondrial DNA (mtDNA) upon oxidative stress remains unclear. This work identifies mitochondrial protein ATAD3B as recruitment factor for LC3 to initiate PINK1/Parkin-independent mitophagy and promote the clearance of damaged mtDNA in mammalian cells. ATAD3B is a novel regulator of oxidative stress-induced mitophagy. Oxidative stress reduces ATAD3B-ATAD3A hetero-oligomerization and induces ATAD3B localization at the mitochondrial outer membrane. ATAD3B contains a LIR motif that binds to LC3 and initiates mitophagy in a PINK1-independent manner. ATAD3B promotes the clearance of damaged mtDNA by mitophagy. Introduction Mitochondria are ubiquitous and highly dynamic eukaryotic organelles, which produce about 90% of the cellular ATP through oxidative phosphorylation (OXPHOS) in most mammalian cells. Also, mitochondria play an essential role in a series of cellular processes, including tricarboxylic acid cycle (TCA), β-oxidation of fatty acids, calcium homeostasis, apoptosis, and cellular signaling (Suen et al, 2008). Unlike other organelles, mitochondria contain circular double-stranded DNA, called mitochondrial DNA (mtDNA), which is usually located at the mitochondrial matrix and encodes a series of critical subunits of the oxidative phosphorylation as well as tRNAs and rRNAs necessary for their synthesis (Yan et al, 2019). In addition, mtDNA plays an important role in innate immune responses and inflammatory pathology (West & Shadel, 2017). Each cell contains hundreds to thousands of mtDNA copies, and each mitochondrion contains one or more copies of mtDNA, which is packaged by a series of proteins, including TFAM, POLG, prohibitins, and ATAD3, to form an mtDNA-protein complex called nucleoid (Lee & Han, 2017). In contrast to the nuclear DNA (nDNA), mtDNA is exclusively transmitted through maternal inheritance (Schon et al, 2012; Yan et al, 2019). Damage of the mtDNA occurs in response to a series of physiological stresses due to the lack of protective histones in the structure and effective repair mechanisms (Kujoth et al, 2005). mtDNA is susceptible to damage by oxygen reactive species, which lead to the formation of deoxyribose rings, apurinic/apyrimidinic (AP) sites, strand breaks, and other damages (Shokolenko et al, 2009; Kazak et al, 2012). Moreover, damaged mtDNA can result in the dysfunction of the mitochondrial respiratory chain, leading to increased production of reactive oxygen species (ROS), which will damage the mtDNA even further (Hiona & Leeuwenburgh, 2008). These mechanisms contribute to the very high mutation rate of the mtDNA, which is about 10- to 17-fold higher than that in the nDNA (Tuppen et al, 2010; Schon et al, 2012). Furthermore, the inability to eliminate mutated or damaged mtDNA causes the accumulation of mitochondria with mtDNA heteroplasmy, where increased damaged mtDNA coexists with wild-type (WT) mtDNA (Bacman et al, 2013; Yan et al, 2019). mtDNA mutations and mitochondrial dysfunction are associated with various human diseases, ranging from severe inherited disorders to common late-onset diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), Kearns–Sayre syndrome (KSS), and Leber’s hereditary optic neuropathy (LHON) (Tuppen et al, 2010; El-Hattab et al, 2015). MELAS is due to the mutation of mtDNA transfer RNA Leu (UUR) at nucleotide 3243A > G, which is the most common human pathogenic mtDNA point mutation (El-Hattab et al, 2015). In general, clinical manifestations occur when the ratio of mutants to WT mtDNA exceeds about 4:1 (Bacman et al, 2013; Wallace & Chalkia, 2013). However, there is currently no effective treatment for halting the progression of mtDNA mutation-related human diseases. In most cells, mitochondrial autophagy (mitophagy) can clear WT or mutant mtDNA, which is a selective pathway to eliminate the damaged or dysfunctional mitochondria (de Vries et al, 2012; Yan et al, 2019). However, mtDNA mutation alone is not sufficient to initiate selective mitophagy (de Vries et al, 2012). The well-known pathway of mitophagy is mediated by the PINK1-Parkin pathway (Youle & Narendra, 2011). In this pathway, PINK1 accumulates on depolarized mitochondria, recruiting, and activating Parkin. Activated Parkin then ubiquitinates numerous mitochondrial outer membrane proteins to mediate the clearance of damaged mitochondria via mitophagy (Youle & Narendra, 2011). Besides, under certain physiological conditions, such as hypoxia, mitophagy can also be initiated via mitophagy receptors, including NIX, BNIP3, FUNDC1, Bcl2L13, Prohibitin 2, and FKBP8 (Zhang & Ney, 2009; Novak et al, 2010; Liu et al, 2012; Murakawa et al, 2015; Bhujabal et al, 2017; Wei et al, 2017). Mitophagy receptors contain an LC3-interacting region (LIR) that physically binds to LC3 (a key autophagosomal membrane protein), connecting the mitochondria and autophagosomes. Once LC3 is recruited to the mitochondria, autophagosomes engulf the mitochondria and deliver it to the lysosome for degradation (Liu et al, 2014). LIR is composed of a [W/F/Y]xx[L/I/V] core motif that interacts with two hydrophobic pockets in the LC3 (or other ATG8 family proteins) anchored in the phagophore membrane (Birgisdottir et al, 2013). The LIR motif of mitophagy receptors is widely recognized as being critical for the selective sequestration of dysfunctional or damaged mitochondria. ROS are highly reactive molecules, consisting of hydrogen peroxide (H2O2), hydroxyl radical (·OH), and superoxide anion (O2−). The electron transport chain of mitochondria is the primary producer of ROS, which readily attack nDNA and mtDNA, and cause a variety of DNA lesions, including DNA strand breaks, oxidized DNA bases, and abasic sites (Alexeyev et al, 2013). 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), also known as 8-hydroxy-2′-deoxyguanosine (8-OHdG), is among the most common and well-characterized ROS-induced DNA lesions and has been used as a biomarker for oxidative stress (Ock et al, 2012). During oxidative stress, ROS act as an initiator and mediator of autophagy, which in turn contributes to the removal of the irreversibly oxidized biomolecules, including DNA, proteins, and lipids in cells (Filomeni et al, 2015; Shefa et al, 2019). Although mtDNA is vulnerable to oxidative stress, whether and how mitophagy removes oxidative stress-induced damaged mtDNA remains poorly understood. Here, we report that mitophagy is critical for the clearance of damaged mtDNA during oxidative stress, and identify a novel mitophagy receptor ATAD3B, which regulates the removal of human damaged mtDNA via mitophagy. Upon oxidative stress or mtDNA damage, ATAD3B binds to LC3B to initiate mitophagy by its LIR motif. Moreover, we find that re-expression of ATAD3B decreases the level of mutated mtDNA (3243A > G) in MELAS patient-derived cells suggesting a novel therapeutic strategy for mitochondrial diseases. Results Mitophagy regulates the clearance of oxidative stress-induced damaged mtDNA Mitochondria contain their genome, mitochondrial DNA (mtDNA), which usually exists in hundreds of copies in a mammalian cell. Due to the insufficient mtDNA repair systems and lack of protective histones, physiological and pathological stresses, especially oxidative stress, easily damage mtDNAs (Yakes & Van Houten, 1997; Alexeyev et al, 2013; Yan et al, 2019). Damaged mitochondria can be eliminated by mitophagy (Youle & Narendra, 2011). However, how to remove the damaged mtDNA remains largely unknown. To investigate mtDNA damage, we used quantitative PCR (Q-RCR) to amplify mtDNA and quantify mtDNA lesions in several cell lines, including HEK293, HeLa, and MEFs. This assay is based on the principle that many kinds of DNA lesions can slow down or block the progression of DNA polymerase. Therefore, if equal amounts of DNA from differently treated samples are amplified under identical conditions, DNA with fewer lesions will amplify to a greater extent than more damaged DNA (Van Houten et al, 2000). In response to hydrogen peroxide (H2O2), a universal intracellular mediator of oxidative stress, we observed significant mtDNA damage, reflected by the reduced mtDNA amplification and increased mtDNA lesions in HEK293 cells (Appendix Fig S1A–C). The extent of damaged mtDNAs was remarkably reduced 1 h after washout (Appendix Fig S1B), indicating that the damaged mitochondria were removed or repaired. H2O2 treatment also damaged nDNA but quickly recovered due to harboring efficient DNA repair systems (Appendix Fig S1D–F). However, because mtDNA repair systems in mitochondria are insufficient (Kazak et al, 2012; Alexeyev et al, 2013), we hypothesize that autophagy might significantly contribute to the elimination of H2O2-induced damaged mtDNA. Therefore, we depleted ATG5, a key regulator of autophagy, to further assess the elimination of damaged mtDNA. Upon ATG5 knockdown (KD), most H2O2 -induced mtDNA damage remained 1 h after washout (Appendix Fig S1B and C), suggesting that autophagy machinery is required for the removal of damaged mtDNAs. To further investigate the mtDNA damage and its clearance, we next used 3-nitropropionic acid (3-NPA), a naturally potent mtDNA damage inducer that increases cellular mitochondrial ROS production (Acevedo-Torres et al, 2009). Both H2O2 and 3-NPA treatment resulted in markedly increased mitochondrial ROS production (Appendix Fig S1G), which is well known to cause mtDNA lesions. As expected, 3-NPA treatment also resulted in significant mtDNA lesions, which were removed 1 h after washout (Fig 1A and B). Similar to H2O2 treatment, ATG5 KD significantly inhibited the removal of 3-NPA-induced damaged mtDNA (Fig 1A and B). Figure 1. Mitophagy is required for the clearance of oxidative stress-induced damaged mtDNA Assessment of mtDNA damage by quantitative PCR. HEK293 cells infected with control (empty vector) or shATG5 for 5 days. Cells were then incubated with DMSO or 4 mM 3-NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h. Cells with or without washout were used for the extraction of total DNA. All DNA samples were used for amplification of 8.9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment. PCR products were quantitated by PicoGreen staining using Micro Plate Reader. Data are presented as mean ± SD (n = 3 independent experiments), and statistical significance was assessed by two-tailed Student’s t-test, N.S., not significant, *P < 0.05, **P < 0.01. The data in (A) were further calculated for the frequency of mtDNA damage. The equation was seen in “Materials and Methods”. Data are presented as mean ± SD (n = 3 independent experiments), and statistical significance was assessed by a two-tailed Student’s t-test, N.S., not significant, **P < 0.01. Representative images show 8-oxo-dG staining. Control or shATG5 HeLa cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and then either fixed immediately or washed with fresh medium and incubated for another 1 h. Cells were immunostained with DAPI and anti-8-oxo-dG antibody and analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in (C). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), and statistical significance was assessed by a two-tailed Student’s t-test, N.S., not significant, *P < 0.05, **P < 0.01. Control or shATG5 HEK293 cells stably expressing mito-Keima. Control cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and shATG5 cells were treated with 200 µM H2O2 as a negative control. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Right panels show the pixel intensity of red (mitochondria within lysosomes) and green (mitochondria in the cytoplasm) from a line. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (E). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), and statistical significance was assessed by a two-way ANOVA, N.S., not significant, **P < 0.01. HeLa cells expressing GFP-LC3 were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were stained with anti-Tom20 and anti-DNA antibodies and then analyzed by confocal microscopy. The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA/Tom20. Scale bar, 10 µm. Quantification of the LC3 punta colocalizing or contacting with mtDNA/Tom20 in a cell. n = 30 cells from 3 coverslips, data are presented as mean ± SD (n = 30), and statistical significance was assessed by a one-way ANOVA with Tukey’s multiple comparisons test, ***P < 0.001. Download figure Download PowerPoint To directly visualize mtDNA damage, 8-oxo-dG (or 8-OHdG), which is one of the major ROS-induced base-modified DNA products and widely accepted as a marker of oxidative DNA lesions (Ock et al, 2012), was detected by immunostaining. After exposure to H2O2 or 3-NPA, HeLa cells displayed a remarkable increase in the intensity of cytoplasmic 8-oxo-dG fluorescence (excluding the fluorescence in the nucleus), indicating the prominent increase of mtDNA lesions. Incubation with a standard medium for 1 h after washout resulted in a significant decrease of cytoplasmic 8-oxo-dG fluorescence intensity (Fig 1C and D) while there was no significant decrease in cytoplasmic 8-oxo-dG fluorescence intensity 1 h after washout in ATG5 KD cells (Fig 1C and D), indicating that ATG5 KD blocks the clearance of damaged mtDNA. Overall, these findings suggest that autophagy is responsible for the clearance of H2O2 and 3-NPA-induced damaged mtDNA. To investigate how damaged mtDNA is eliminated, mitochondrial autophagy (mitophagy) was then measured. Mito-Keima, a useful tool in the assessment of single mitophagic events (Katayama et al, 2011), was used to evaluate mitophagy. Upon treatment with DMSO, no mitophagy was detected based on the absence of red fluorescence in HEK293 cells expressing mito-keima (Fig 1E and F). However, in response to 3-NPA or H2O2, a remarkable increase in red fluorescence was detected in WT cells but not in ATG5 KD cells (Fig 1E and F), suggesting that mitophagy can eliminate 3-NPA or H2O2-induced damaged mitochondria. In addition, transmission electron microscope analysis revealed that H2O2 treatment led to some mitochondria engulfed by autophagosome (Appendix Fig S1H), further confirming that H2O2 treatment induces mitophagy. Our data are consistent with the previous finding that the direct generation of mitochondrial ROS using a mitochondrial-targeted photosensitizer can induce mitophagy (Wang et al, 2012). Furthermore, 3-NPA or H2O2 treatment increased the number of GFP-LC3 puncta, part of which colocalized with mitochondria and mtDNA (Fig 1G and H), further demonstrating that mitophagy contributes to the clearance of oxidative stress-induced damaged mtDNA. Identification of ATAD3B as a strong novel regulator of oxidative stress-induced mitophagy To investigate how oxidative stress-induced damaged mtDNA initiates mitophagy signal, we screened for an mtDNA-related mitophagy regulator by using a mito-Keima assay. mtDNA and a set of mtDNA binding or associated proteins are packaged into nucleoprotein complexes referred to as an “mtDNA nucleoid.” We carried out a screen where we knocked down over 20 nucleoid-associated proteins (including mtDNA binding protein LONP1, SSBP1, PEO1, POLG2, POLRMT, TFAM, and ATAD3) by short hairpin RNAs in mito-Keima expressing HEK293 cells and analyzed the effect of knockdown by Western blotting or quantitative PCR (Appendix Fig S2A–C). Upon H2O2 treatment, we then assessed control and nucleoid-associated protein knockdown cells by confocal microscopy and flow cytometry analysis. As illustrated in Fig 2A, a set of mtDNA binding proteins are associated with H2O2-induced mitophagy. Among them, the effect of ATAD3 KD is similar to that of ATG5 KD on H2O2-induced mitophagy (Fig 2A). Several recent studies have reported that ATAD3 gene cluster deletions are associated with the fatal congenital pontocerebellar hypoplasia and aberrant mtDNA organization (Desai et al, 2017), leading us to focus on the role of ATAD3 on the clearance of damaged mtDNA. Figure 2. ATAD3B is a novel and robust regulator of oxidative stress-induced mitophagy Screening of mitophagy regulators for removing damaged mtDNA. HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors. Five days later, cells were treated with 200 µM H2O2 for 2 h. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima. ShATG5 was used as a negative control. Right panels for each image show the FACS-based mito-Keima dot plots. The y-axis represents the fluorescence emission of mito-Keima at pH 4.0 (lysosome), while the x-axis indicates mito-Keima at pH 7.0 (mitochondria). The percentages of cells within the different regions are indicated. Scale bar, 10 µm. Control or ATAD3B KO HeLa cells stably expressing mito-Keima were treated with 4 mM 3-NPA for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (B). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), and statistical significance was assessed by two-tailed Student’s t-test, N.S., not significant, **P < 0.01. Control or KO ATAD3B HeLa cells were treated with 4 mM 3-NPA for 2 h. Cells were washed with fresh medium and incubated for another 1 h. Cells were then fixed and immunostained with DAPI and anti-8-oxo-dG antibodies and were analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in cells described in (D). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), and statistical significance was assessed by two-tailed Student’s t-test, N.S., not significant, **P < 0.01. Download figure Download PowerPoint There are three ATAD3 protein isoforms, and they belong to the family of ATPase AAA domain-containing proteins. Among the ATAD3 family, ATAD3A and ATAD3B are the most prominent members and share high levels of similarity at the protein level (Appendix Fig S3A). While ATAD3A is conserved among all multicellular organisms, ATAD3B is specifically expressed in primates, including Pan troglodytes and Homo sapiens (Appendix Fig S3B). Human ATAD3B is highly expressed in embryonic stem cells and various cancer cells (Merle et al, 2012). Besides, according to the Human Proteome Map database, ATAD3B is also moderately expressed in various adult tissues, including the frontal cortex, retina, liver, ovary, testis, pancreas, and B cells (Appendix Fig S3C). Since the targeting sequences used to knockdown ATAD3 (Table S1) are common for both ATAD3A and ATAD3B, we next investigated which one is responsible for regulating mtDNA-related mitophagy. For this, we used CRISPR/Cas9 technology to create ATAD3A and ATAD3B double knockout (ATAD3 DKO) HeLa cell lines (Fig EV1A) and re-expressed ATAD3 isoforms to determine which one may rescue mtDNA-related mitophagy. mito-Keima assay revealed that in response to 3-NPA or H2O2, the expression of ATAD3A-Flag in ATAD3 DKO cells promoted no increment of mitophagy when compared to control (ATAD3 DKO) cells (Fig EV1B–F). On the other hand, the expression of ATAD3B-Flag in ATAD3 DKO cells dramatically increased the level of mitophagy compared with control (ATAD3 DKO) cells upon 3-NPA or H2O2 treatment (Fig EV1B–F), suggesting that ATAD3B, but not ATAD3A, promotes 3NPA or H2O2-induced mitophagy. In addition, ATAD3B-Flag expression markedly increased H2O2-induced mitophagy in MEFs that naturally lack ATAD3B (Fig EV1G and H). Moreover, ATAD3B knockout (KO) significantly decreased 3-NPA-induced mitophagy that is measured by mito-Keima assay (Fig 2B and C). These findings suggest that ATAD3B promotes oxidative stress-induced mitophagy independent of ATAD3A. Click here to expand this figure. Figure EV1. ATAD3B promotes oxidative stress-induced mitophagy A. Control and ATAD3 DKO (ATAD3A and ATAD3B double knockout) HeLa cell lines were lysed and analyzed by Western blotting using anti-ATAD3 or anti-tubulin antibodies. B. ATAD3 DKO HeLa cells were infected with lentiviral particles containing control, ATAD3A-Flag, or ATAD3B-Flag. Cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-tubulin antibodies. C–F. WT or ATAD3 DKO HeLa cells stably expressing mito-Keima were infected with lentiviral particles containing control, ATAD3A-Flag, or ATAD3B-Flag. Five days later, cells were treated with DMSO, 200 µM H2O2 (C), or 4 mM 3-NPA (E) for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. The relative ratio of red to green fluorescence intensity (561 nm/458 nm) of DMSO, H2O2-treated (D), or 3-NPA-treated (F) cells were then quantified respectively by ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), and statistical significance was assessed by a two-way ANOVA, *P < 0.05, **P < 0.01. G. MEFs stably expressing mito-Keima were infected with lentiviral particles containing control or ATAD3B-Flag and further cultured for 5 days. Cells were then incubated with DMSO or 200 µM H2O2 for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and