NDP 52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy
2018; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês
10.15252/embr.201846363
ISSN1469-3178
AutoresNorihiko Furuya, Soichiro Kakuta, Katsuhiko Sumiyoshi, Maya Ando, Risa Nonaka, Ayami Suzuki, Saiko Kazuno, Shinji Saiki, Nobutaka Hattori,
Tópico(s)MicroRNA in disease regulation
ResumoArticle11 October 2018free access Transparent processSource Data NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy Norihiko Furuya Corresponding Author [email protected] orcid.org/0000-0001-7695-4271 Division for Development of Autophagy Modulating Drugs, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neuroscience for Neurodegenerative Disorders, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Soichiro Kakuta Laboratory of Morphology and Image Analysis, Biomedical Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Cellular and Molecular Neuropathology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Katsuhiko Sumiyoshi Department of Health and Nutrition Collage of Human Science, Tokiwa University, Ibaraki, Japan Department of Cardiovascular Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Maya Ando Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Risa Nonaka Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Ayami Suzuki Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Saiko Kazuno Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Shinji Saiki Division for Development of Autophagy Modulating Drugs, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Nobutaka Hattori Corresponding Author [email protected] orcid.org/0000-0002-2034-2556 Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Norihiko Furuya Corresponding Author [email protected] orcid.org/0000-0001-7695-4271 Division for Development of Autophagy Modulating Drugs, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neuroscience for Neurodegenerative Disorders, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Soichiro Kakuta Laboratory of Morphology and Image Analysis, Biomedical Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Cellular and Molecular Neuropathology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Katsuhiko Sumiyoshi Department of Health and Nutrition Collage of Human Science, Tokiwa University, Ibaraki, Japan Department of Cardiovascular Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Maya Ando Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Risa Nonaka Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Ayami Suzuki Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Saiko Kazuno Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Shinji Saiki Division for Development of Autophagy Modulating Drugs, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Nobutaka Hattori Corresponding Author [email protected] orcid.org/0000-0002-2034-2556 Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Author Information Norihiko Furuya *,1,2,3, Soichiro Kakuta4,5, Katsuhiko Sumiyoshi6,7, Maya Ando3, Risa Nonaka8, Ayami Suzuki3, Saiko Kazuno9, Shinji Saiki1,3 and Nobutaka Hattori *,3 1Division for Development of Autophagy Modulating Drugs, Juntendo University Graduate School of Medicine, Tokyo, Japan 2Department of Neuroscience for Neurodegenerative Disorders, Juntendo University Graduate School of Medicine, Tokyo, Japan 3Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan 4Laboratory of Morphology and Image Analysis, Biomedical Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan 5Department of Cellular and Molecular Neuropathology, Juntendo University Graduate School of Medicine, Tokyo, Japan 6Department of Health and Nutrition Collage of Human Science, Tokiwa University, Ibaraki, Japan 7Department of Cardiovascular Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan 8Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan 9Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, Tokyo, Japan *Corresponding author. Tel: +81 3 3813 3111; Fax: +81 3 5813 7440; E-mail: [email protected] *Corresponding author. Tel: +81 3 3813 3111; Fax: +81 3 5800 0547; E-mail: [email protected] EMBO Rep (2018)19:e46363https://doi.org/10.15252/embr.201846363 PDFDownload PDF of article text and main figures.AM PDF 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 Parkin-mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N-terminal SKICH domain and C-terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly-ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52-mediated mitophagy, and the NDP52–MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria. Synopsis The autophagy receptor NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP), and the NDP52-MTPAP complex contributes to the ubiquitin-independent recognition of damaged mitochondria by autophagy. NDP52 irrupts into mitochondria proteasome-dependently, and interacts with MTPAP via its N-terminal SKICH domain upon mitophagy-inducing conditions. The NDP52-MTPAP complex attracts more LC3 to the damaged mitochondria than NDP52 alone. NDP52 interacts with MTPAP and irrupts into mitochondria ubiquitin-binding-independently. Introduction Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved dynamic membrane process in which cytoplasmic components including macromolecules and organelles are sequestered into double membrane structures called autophagosomes and delivered to lysosomes for degradation 12. Traditionally, autophagy has been considered to be a nonselective bulk process; however, accumulating evidences show that some of cytosolic components, including protein aggregates, dysfunctional organelles, and invading pathogens, are eliminated selectively by autophagy. Mitochondrial autophagy (mitophagy) is a mitochondrial quality control mechanism that selectively engulfs damaged mitochondria within autophagosomes (mitophagosomes) for degradation in lysosomes 13. Numerous clinical and animal studies indicate that mitochondrial dysfunction is involved in many processes and diseases, including aging, cancer, diabetes, and neurodegenerative diseases 45678. Mitochondrial quality control via mitophagy assumes an important role to maintain cellular homeostasis. Parkin-mediated mitophagy is one of the best-characterized mechanisms of mitophagy in mammalian cells 3. In damaged mitochondria, e.g., mitochondria with loss of membrane potential, under genetic or environmental stresses, or with unfolded protein accumulation, the protease activity of presenilin-associated rhomboid-like (PARL), which mediates cleavage of a mitochondrial Ser/Thr kinase, PTEN-induced putative kinase 1 (PINK1), is reduced. This stabilizes PINK1, which then phosphorylates the E3 ubiquitin ligase, Parkin, and ubiquitin at S65 9101112. Activated Parkin is recruited to damaged mitochondria and ubiquitylates outer mitochondrial membrane (OMM) proteins. Phosphorylated ubiquitin also activates Parkin E3 ligase activity 101112, and this feed-forward loop initiates Parkin-mediated mitophagy. Following ubiquitylation of OMM proteins by Parkin, damaged mitochondria are selectively engulfed by autophagosomes. Autophagy adaptor/receptor proteins containing both ubiquitin binding domains and microtubule-associated proteins 1A/1B light chain 3 (LC3)-interacting region (LIR) motifs, which bind to both ubiquitin and the autophagy-related protein 8 (Atg8) family (LC3 and GABARAP subfamily) proteins, contribute to recognition of ubiquitylated cargo in selective autophagy 13. Two autophagy receptors linked to xenophagy, optineurin (OPTN) and nuclear domain 10 protein 52 (NDP52, also known as CALCOCO2), are primary adapters for Parkin-mediated mitophagy 14. These proteins are involved in recruitment of autophagy factors and/or activation of TANK-binding kinase 1 (TBK1) 1415. Nuclear domain 10 protein 52 plays a role in the clearance of the ubiquitin-coated bacteria as a xenophagy receptor 16. NDP52 selectively binds with LC3C via a noncanonical LIR (CLIR) motif and has no or very weak affinity for other members of the Atg8 family 17. During Salmonella infection, NDP52 promotes activation of autophagy by binding with galectin 8, a protein that accumulates on vacuoles containing bacteria. NDP52 also promotes maturation of bacteria-containing autophagosome 1819. While NDP52 plays multiple roles in xenophagy, the role of NDP52 in mitophagy is not clear. Here, we report that NDP52 functions as a mitophagy receptor by interacting with mitochondrial RNA poly(A) polymerase (MTPAP; also known as PAPD1 or TUTase1) via its N-terminal SKICH domain and that this interaction is required for efficient autophagic engulfment of depolarized mitochondria. Our findings reveal a novel mechanism for recognition of damaged mitochondria by the autophagic membrane. Results NDP52 localizes to damaged mitochondria and is degraded by autophagy To assess the involvement of NDP52 in mitophagy, we incubated HeLa cells stably expressing GFP-Parkin (HeLa-GFP-Parkin cells) with valinomycin, a mitochondrial uncoupler. In HeLa-GFP-Parkin cells, Parkin activity can be monitored by ubiquitylation of an N-terminal GFP-tag as a pseudo-substrate 20. In untreated cells, endogenous NDP52 predominantly localized to the perinuclear region (Fig 1A). In cells incubated with valinomycin for 90 min, a portion of NDP52 colocalized with GFP-Parkin localized at mitochondria. However, colocalization of NDP52 with Parkin localized at mitochondria was reduced in cells treated with valinomycin for 180 min. The majority of NDP52 was degraded after incubation with valinomycin for 3 h while minimal degradation occurred to the remaining NDP52 after incubation with valinomycin for 6–24 h (Fig EV1A). In valinomycin-treated cells, degradation of NDP52 was inhibited by bafilomycin A1, a specific vacuolar ATPase inhibitor, and not by epoxomicin, a specific proteasome inhibitor (Fig EV1B). These results indicate that NDP52 is degraded soon (within 3 h) after valinomycin treatment. In addition, degradation of a large proportion of mitochondrial proteins was strongly activated in the same period. Therefore, to clarify the role of NDP52 in Parkin-mediated mitophagy, we focused on the contribution of NDP52 in the early mitophagy period. Figure 1. Mitophagosome formation is reduced by NDP52 knockdown A. HeLa cells stably expressing GFP-Parkin (HeLa-GFP-Parkin cells) were treated with 1 μM valinomycin and immunostained with anti-NDP52 and anti-Tom20 antibodies. Boxed areas in the images are shown in the next panels on the right. Colocalization of GFP-Parkin, NDP52, and Tom20 was determined using Line Scan. Fluorescence intensities of each channel were measured along the dotted white arrow. Scale bars, 10 μm. Images are representative of three independent experiments. B. HeLa-GFP-Parkin cells were transfected with siNDP52 or scrambled siRNA. Seventy-two hours after transfection, cells were incubated with 1 μM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments. C. Mitophagy flux assay using Mtphagy dye. Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment. Results are a summary of three independent experiments. Values are the means ± SEM. *P < 0.05, **P < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t-test. D. Fluorescence images of NDP52 KD and control cells expressing mCherry-LC3 and MTS-TagBFP. Arrows indicate mCherry-LC3 puncta colocalized with MTS-TagBFP. Scale bars, 10 μm. Boxed areas in images are shown in the next panels on the right. Images are representative of four independent experiments. E. The number of mCherry-LC3 puncta colocalized with MTS-TagBFP (representing mitophagosomes) per cell was counted. Results were from at least six microscopic fields of three independent experiments. Values are the means ± SEM. **P < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t-test. F, G. Electron micrographs of NDP52 KD and control cells. Asterisks indicate mitochondria sequestered by autophagosomes (mitophagosomes, yellow dotted line). The ratios of mitophagosomes per mitochondrial area are shown in (G). Ten cells from three independent experiments were counted. Values are the means ± SD. *P < 0.05 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t-test. Source data are available online for this figure. Source Data for Figure 1 [embr201846363-sup-0012-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Effect of valinomycin treatment on the levels of NDP52 and mitochondrial proteins HeLa-GFP-Parkin cells incubated with valinomycin for 0–24 h. Cell lysates were subjected to Western blotting using the indicated antibodies. HeLa-GFP-Parkin cells incubated with valinomycin for 3 h in the presence or absence of 100 nM bafilomycin A1 or epoxomicin. Cell lysates were subjected to Western blotting using the indicated antibodies. HeLa-GFP-Parkin cells expressing mCherry-NDP52 were incubated with valinomycin. Cells were immunostained with anti-Tom20 antibody. Colocalization of GFP-Parkin, NDP52, and Tom20 was determined using Line Scan. Fluorescence intensities of each channel were measured along the white arrow. Scale bars, 10 μm. Mitophagy flux assay in FIP200 KD cells. Cells transfected with siFIP200 oligo were incubated with valinomycin for 6 h in the presence or absence bafilomycin A1. Results are a summary of three independent experiments. Values are the means ± SEM. **P < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t-test. mCherry-NDP52 was expressed in HeLa-GFP-Parkin cells and immunostained with anti-Lamp2 antibody. Scale bars, 10 μm. Source data are available online for this figure. Download figure Download PowerPoint As was the case with endogenous NDP52, mCherry-NDP52 predominantly localized to the perinuclear region without valinomycin treatment and partially colocalized with Parkin localized at mitochondria in cells incubated with valinomycin for 90 min (Fig EV1C). Colocalization of mCherry-NDP52 with Parkin localized at mitochondria was also reduced in cells treated with valinomycin for 180 min. NDP52 knockdown induces a delay in mitophagy To clarify the role of NDP52 in Parkin-mediated mitophagy, we knocked down NDP52 expression in HeLa-GFP-Parkin cells using siRNA (Fig 1B–G). The NDP52 siRNA oligo completely blocked endogenous NDP52 expression 72 h after transfection. NDP52 knockdown (KD) did not influence proteasomal degradation of mitofusin 1 (MFN1), a Parkin substrate 21; degradation of the OMM protein, Tom20; or degradation of the inner mitochondrial membrane (IMM) proteins, Tim23 and OPA1 (Fig 1B). To precisely assess the effect of NDP52 KD on mitophagy, we quantified mitophagic flux using Mtphagy dye (Fig 1C). Mtphagy dye stains mitochondria, and its fluorescence intensity depends on the pH conditions. When mitochondria are transported to lysosomes by mitophagy, Mtphagy dye exhibits higher fluorescence intensity. We confirmed that the fluorescence enhancement of Mtphagy dye during valinomycin treatment is autophagy dependent and lysosomal pH dependent (Fig EV1D). Mitophagy in control cells was precipitously induced by valinomycin treatment and reached a plateau after treatment for approximately 6 h. NDP52 KD significantly reduced mitophagic flux 2–6 h after valinomycin treatment. Thereafter, mitophagic flux in NDP52 KD cells caught up with that in control cells. This indicates that NDP52 KD delays mitophagy. To assess the effect of NDP52 KD on mitophagosome formation, we expressed mCherry-LC3 and MTS-TagBFP, which fluorescently label autophagosomes and mitochondria, respectively, and investigated colocalization of the mCherry and TagBFP signals (reflecting mitophagosomes) using IN Cell Analyzer (Fig 1D and E). In control cells, the number of mCherry-LC3 puncta with MTS-TagBFP signal was increased 1–3 h after valinomycin treatment. However, the number of mCherry-LC3 puncta colocalized with MTS-TagBFP was significantly reduced by NDP52 KD at 1, 2, and 3 h after valinomycin treatment. Nevertheless, the number of mitophagosomes was comparable in NDP52 KD and control cells at 6–24 h after valinomycin treatment. Furthermore, the majority of NDP52 was degraded and its level reached its minimum at 3 h after valinomycin treatment (Fig 1B). Therefore, we assumed that NDP52 was not involved in mitophagosome formation after 3 h. To more precisely clarify the effect of NDP52 KD on mitophagy, we performed electron microscopy analysis to compare the number and morphology of mitophagosomes in NDP52 KD and control cells (Fig 1F and G). To quantify mitophagosomes, cells were incubated with both valinomycin and bafilomycin A1 for 2 h, which caused accumulation of mitophagosomes by inhibition of fusion between mitophagosomes and lysosomes. Mitochondria sequestered by autophagosomes were observed in both NDP52 KD and control cells. However, the ratio of mitophagosomes to total mitochondrial area was significantly lower in NDP52 KD cells compared with controls (6.1 and 14.1%, respectively). These results indicate that NDP52 is involved in mitophagy in the early period (within 3 h) of valinomycin treatment and may be involved in efficient engulfment of damaged mitochondria by autophagosomes. Both N-terminal SKICH and C-terminal LIM-L domains of NDP52 are required for mitophagy To investigate the precise behavior of NDP52 in valinomycin-treated cells, we performed time-lapse imaging of mCherry-NDP52 and GFP-Parkin (Fig 2A and C, Movies EV1 and EV2). GFP-Parkin formed ring-like structures surrounding mitochondria from 20 min after valinomycin treatment (Movie EV1). Almost at the same time, mCherry-NDP52 began to colocalize with part of the Parkin-ring-like structures. At first, mCherry-NDP52 was recruited to the edges of the Parkin-positive ring structures as dot-shaped structures, which then elongated along the ring structures and overlapped with Parkin-positive mitochondria. Some mCherry-NDP52 signals were also observed inside Parkin-ring-like structures after elongation (Fig 2B and D, yellow arrowheads). While mCherry-tagged OPTN, another essential mitophagy receptor, was recruited and accumulated to most Parkin-ring structures as previously described 22 (Fig 2C, Movie EV3), mCherry-NDP52 tended to be recruited to smaller ring structures and rarely localized to larger mitochondrial rings. mCherry-NDP52-positive mitochondria immediately disappeared or translocated to the perinuclear region. Furthermore, after valinomycin treatment, much of the mCherry signal coalesced and formed an aggregation at the perinuclear area. Immunofluorescence staining showed that a portion of mCherry-NDP52 colocalized with the lysosomal membrane protein, lysosomal-associated membrane protein 2 (Lamp2; Fig EV1E). mCherry is a pH-stable fluorescent protein; therefore, this mCherry region indicates accumulation of sequestrated mCherry-NDP52 in lysosomes via the autophagic/mitophagic pathway. Thus, disappearance or perinuclear translocation of NDP52-localized mitochondria likely represents mitophagy. These results indicate that NDP52 plays a role as a mitophagy receptor in the early period of valinomycin-induced Parkin-mediated mitophagy. Figure 2. SKICH and LIM-L domains of NDP52 are both required for mitophagy A, C. Images of HeLa-GFP-Parkin cells expressing mCherry-NDP52 (A) or mCherry-OPTN (C) under valinomycin treatment. The images are from Movies EV2 and EV3, respectively. Cyan, GFP-Parkin; magenta, mCherry-NDP52 or OPTN. Scale bars, 10 μm. Images are representative of five independent experiments. B. Time-lapse images of HeLa-GFP-Parkin cells expressing mCherry-NDP52 under valinomycin treatment. The images are representative of five independent experiments and correspond to the boxed regions of Fig 2A and Movie EV2. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin-ring structures. D. mCherry-NDP52 or OPTN signals colocalized with Parkin-ring structures and existing inside of Parkin-ring structures (irrupted) were quantified. Data were from five independent experiments in cells incubated with valinomycin for 2 h. Values are the means ± SEM. **P < 0.01 compared with mCherry-OPTN-expressing cells, determined with one-way ANOVA followed by the Student's t-test. E. Mitophagic flux in NDP52 KD cells expressing siRNA-resistant NDP52 or NDP52 mutants. Cells were incubated with valinomycin for 3 h. Results are a summary of three independent experiments. Values are the means ± SEM. *P < 0.05, **P < 0.01 vs. scrambled oligo-transfected cells; †P < 0.05, ††P < 0.01 vs. empty vector-transfected cells, determined with one-way ANOVA followed by the Tukey–Kramer post hoc test. F. HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 μM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by Western blotting. Data are representative of three independent experiments. G. Images of HeLa-GFP-Parkin cells expressing mCherry-NDP52 ∆LIM-L or mCherry-NDP52 ΔSKICH treated with valinomycin for 2 h. The images are from Movie EV4. Cyan, GFP-Parkin; magenta, mCherry-NDP52 mutant. Scale bars, 10 μm. Images are representative of three independent experiments. Source data are available online for this figure. Source Data for Figure 2 [embr201846363-sup-0013-SDataFig2.pdf] Download figure Download PowerPoint Human NDP52 contains an N-terminal skeletal muscle and kidney-enriched inositol phosphatase carboxy homology (SKICH) domain (which is required for TBK1 interaction via Nap1 or Sintbad), a noncanonical LC3-interacting region motif for LC3C interaction (CLIR), a coiled-coil domain for homodimerization, and a C-terminal Lin1, Isl-1 and Mec-3 (LIM)-like (LIM-L) domain 16172324. The region containing the LIM-L domain includes two zinc finger motifs associated with poly-ubiquitin chain interaction 1625. To determine which NDP52 region is important for mitophagosome formation, we constructed NDP52 deletion mutants lacking the SKICH domain, CLIR motif and upstream acidic amino acid cluster, or LIM-L domain (Fig EV2A). To assess the biological activities of these mutants in NDP52 KD cells, we designed them to be siRNA-resistant (siR). Expression of full-length siR-NDP52 and siR-NDP52∆CLIR, but not siR-NDP52∆SKICH or siR-NDP52∆LIM-L, restored the mitophagic flux inhibited by NDP52 KD (Fig 2E). However, siR-NDP52∆LIM-L expression partially rescued the mitophagic flux inhibited by NDP52 KD. Intriguingly, co-expression of siR-NDP52∆SKICH and siR-∆LIM-L restored the mitophagic flux to levels comparable with those of full-length siR-NDP52-expressing cells and control cells. As expected, LC3, TBK1, and ubiquitin co-immunoprecipitated with mCherry-tagged full-length NDP52, while the ∆SKICH and ∆LIM-L mutants had no interaction with TBK1 and ubiquitin, respectively (Fig 2F). The LC3 interaction was diminished by SKICH domain deletion, but was not influenced by CLIR or LIM-L domain deletion. In HeLa-GFP-Parkin cells, endogenous LC3C expression and the NDP52-LC3C interaction were not detected (Fig EV2C). These data can explain the activity of NDP52∆CLIR and indicate that both N-terminal SKICH and C-terminal LIM-L domains are essential for the function of NDP52. Click here to expand this figure. Figure EV2. Ubiquitin-binding-deficient mutants of NDP52 fail to localize at damaged mitochondria Schematic structures of NDP52 truncated mutants used in this study. HeLa-GFP-Parkin cells expressing mCherry-NDP52 ΔSKICH, ∆CLIR, or ∆LIM-L were immunostained with anti-Tom20 antibody. Scale bars, 10 μm. Colocalization of GFP-Parkin, mCherry-NDP52 mutant, and Tom20 was determined using Line Scan. Fluorescence intensities of each channel were measured along the white arrow. HeLa-GFP-Parkin cells expressing mCherry-NDP52 or ∆CLIR were incubated with valinomycin and bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using an anti-RFP antibody. Immunoprecipitates were analyzed by Western blotting. HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with valinomycin and bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using an anti-RFP antibody. Immunoprecipitates were analyzed by Western blotting. HeLa-GFP-Parkin cells expressing mCherry-NDP52∆ZF2 or D439R/C443K were incubated with valinomycin for 2 h. Immunostaining was performed with anti-Tom20 antibody. Scale bar, 10 μm. Colocalization of GFP-Parkin, mCherry-NDP52 mutant, and Tom20 was assessed using Line Scan. Mitophagic flux in NDP52 KD cells transfected with siRNA-resistant NDP52 or NDP52 mutants. Cells were incubated with valinomycin for 3 h. Results are a summary of three independent experiments. Values are the means ± SEM. **P < 0.01 vs. scrambled oligo-transfected cells; †P < 0.05, ††P < 0.01 vs. empty vector-transfected cells, determined with one-way ANOVA followed by the Tukey–Kramer post hoc test. mCherry-NDP52 was expressed in TBK1 KD cells. Cells were incubated with 1 μM valinomycin for 2 h and immunostained with anti-Tom20 antibody. Scale bars, 10 μm. Colocalization of GFP-Parkin, mCherry-NDP52 mutant, and Tom20 was determined using Line Scan. Source data are available online for this figure. Download figure Download PowerPoint Without valinomycin treatment, ∆LIM-L was predominantly localized to the perinuclear region, similar to full-length NDP52, whereas most mCherry-NDP52 ΔSKICH showed diffuse localization in the cytosol (Fig 2G, Movie EV4). NDP52∆SKICH, but not NDP52∆LIM-L, colocalized with Parkin-localized ring-shaped mitochondria after valinomycin treatment (Figs 2G and EV2B, Movie EV4). While some mCherry-∆SKICH-positive mitochondria immediately disappeared, most NDP52∆SKICH accumulated on depolarized mitochondria. Unlike full-length NDP52, mCherry-∆SKICH colocalized with smaller GFP-Parkin rings and with larger mitochondria. Indeed, deletion of the SKICH domain increased colocalization of NDP52 with Parkin at mitochondria. These results indicate that the SKICH domain of NDP52 is required for the localization of NDP52 in normal conditions and for the migration of damaged mitochondria to lysosomes. The LIM-L domain of NDP52 contains an unconventional zinc finger (ZF1) and a C2H2-type zinc finger (ZF2), and only ZF2 interacts with mono-ubiquitin or poly-ubiquitin chains 25. As previously reported, interaction between NDP52 and ubiquitin was abolished by deletion of the ZF2 domain and point mutations of the D439 and C443 resid
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