Abstract
The low-density lipoprotein (LDL) is a major cholesterol carrier in circulation and is internalized into cells through LDL receptor (LDLR)-mediated endocytosis. The LDLR protein is highly expressed in the steroidogenic organs and LDL cholesterol is an important source for steroidogenesis. Cholesterol must be transported into the mitochondria, where steroid hormone biosynthesis initiates. However, how LDL cholesterol is conveyed to the mitochondria is poorly defined. Here, through genome-wide small hairpin RNA screening, we find that the outer mitochondrial membrane protein phospholipase D6 (PLD6), which hydrolyses cardiolipin to phosphatidic acid, accelerates LDLR degradation. PLD6 promotes the entrance of LDL and LDLR into the mitochondria, where LDLR is degraded by mitochondrial proteases and LDL-carried cholesterol is used for steroid hormone biosynthesis. Mechanistically, the outer mitochondrial membrane protein CISD2 binds to the cytosolic tail of LDLR and tethers LDLR+ vesicles to the mitochondria. The fusogenic lipid phosphatidic acid generated by PLD6 facilitates the membrane fusion of LDLR+ vesicles with the mitochondria. This intracellular transport pathway of LDL–LDLR bypasses the lysosomes and delivers cholesterol to the mitochondria for steroidogenesis.
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Data availability
The deep-sequencing data that support the findings of this study have been deposited in the National Center for Biotechnology Information Sequence Read Archive (PRJNA957231). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.
References
Hu, J., Zhang, Z., Shen, W.-J. & Azhar, S. Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones. Nutr. Metab. 7, 47 (2010).
Cole, T. J., Short, K. L. & Hooper, S. B. The science of steroids. Semin. Fetal Neonatal Med. 24, 170–175 (2019).
Ghayee, H. K. & Auchus, R. J. Basic concepts and recent developments in human steroid hormone biosynthesis. Rev. Endocr. Metab. Disord. 8, 289–300 (2007).
Tilokani, L., Nagashima, S., Paupe, V. & Prudent, J. Mitochondrial dynamics: overview of molecular mechanisms. Essays Biochem. 62, 341–360 (2018).
Wai, T. & Langer, T. Mitochondrial dynamics and metabolic regulation. Trends Endocrinol. Metab. 27, 105–117 (2016).
Otera, H., Miyata, N., Kuge, O. & Mihara, K. Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling. J. Cell Biol. 212, 531–544 (2016).
Santel, A. et al. Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells. J. Cell Sci. 116, 2763–2774 (2003).
Ishihara, N., Eura, Y. & Mihara, K. Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity. J. Cell Sci. 117, 6535–6546 (2004).
Cipolat, S., Martins de Brito, O., Dal Zilio, B. & Scorrano, L. OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc. Natl Acad. Sci. USA 101, 15927–15932 (2004).
Choi, S. Y. et al. A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis. Nat. Cell Biol. 8, 1255–1262 (2006).
Huang, H. & Frohman, M. A. Lipid signaling on the mitochondrial surface. Biochim. Biophys. Acta 1791, 839–844 (2009).
Zhukovsky, M. A., Filograna, A., Luini, A., Corda, D. & Valente, C. Phosphatidic acid in membrane rearrangements. FEBS Lett. 593, 2428–2451 (2019).
Brown, M. S. & Goldstein, J. L. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47 (1986).
Luo, J., Jiang, L., Yang, H. & Song, B. L. Routes and mechanisms of post-endosomal cholesterol trafficking: a story that never ends. Traffic 18, 209–217 (2017).
Luo, J., Yang, H. & Song, B. L. Mechanisms and regulation of cholesterol homeostasis. Nat. Rev. Mol. Cell Biol. 21, 225–245 (2020).
Chu, B. B. et al. Cholesterol transport through lysosome–peroxisome membrane contacts. Cell 161, 291–306 (2015).
Illingworth, D. R., Orwoll, E. S. & Connor, W. E. Impaired cortisol secretion in abetalipoproteinemia. J. Clin. Endocr. Metab. 50, 977–979 (1980).
Abreu, J. M. et al. Dyslipidemia’s influence on the secretion ovarian’s steroids in female mice. Res. Soc. Dev. 10, e298101321369 (2021).
Guo, T. et al. Low-density lipoprotein receptor affects the fertility of female mice. Reprod. Fertil. Dev. 27, 1222–1232 (2015).
Steinfeld, K. et al. Low testosterone in ApoE/LDL receptor double-knockout mice is associated with rarefied testicular capillaries together with fewer and smaller Leydig cells. Sci. Rep. 8, 5424 (2018).
Horton, J. D., Goldstein, J. L. & Brown, M. S. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J. Clin. Invest. 109, 1125–1131 (2002).
Abifadel, M. et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 34, 154–156 (2003).
Wang, Y., Huang, Y., Hobbs, H. H. & Cohen, J. C. Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR. J. Lipid Res. 53, 1932–1943 (2012).
Wang, J.-K. et al. Ablation of plasma prekallikrein decreases low-density lipoprotein cholesterol by stabilizing low-density lipoprotein receptor and protects against atherosclerosis. Circulation 145, 675–687 (2022).
Zelcer, N., Hong, C., Boyadjian, R. & Tontonoz, P. LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor. Science 325, 100–104 (2009).
Huang, H. et al. piRNA-associated germline nuage formation and spermatogenesis require MitoPLD profusogenic mitochondrial-surface lipid signaling. Dev. Cell 20, 376–387 (2011).
Baba, T. et al. Phosphatidic acid (PA)-preferring phospholipase A1 regulates mitochondrial dynamics. J. Biol. Chem. 289, 11497–11511 (2014).
Ciechanover, A. Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat. Rev. Mol. Cell Biol. 6, 79–87 (2005).
Quiros, P. M., Langer, T. & Lopez-Otin, C. New roles for mitochondrial proteases in health, ageing and disease. Nat. Rev. Mol. Cell Biol. 16, 345–359 (2015).
Wiedemann, N. & Pfanner, N. Mitochondrial machineries for protein import and assembly. Annu. Rev. Biochem. 86, 685–714 (2017).
Li, X. et al. Mitochondria-translocated PGK1 functions as a protein kinase to coordinate glycolysis and the TCA cycle in tumorigenesis. Mol. Cell 61, 705–719 (2016).
Wang, T. et al. SENP1–Sirt3 signaling controls mitochondrial protein acetylation and metabolism. Mol. Cell 75, 823–834.e5 (2019).
Ruan, L. et al. Cytosolic proteostasis through importing of misfolded proteins into mitochondria. Nature 543, 443–446 (2017).
Li, Y. et al. A mitochondrial FUNDC1/HSC70 interaction organizes the proteostatic stress response at the risk of cell morbidity. EMBO J. 38, e98786 (2019).
Wei, Z., Su, W., Lou, H., Duan, S. & Chen, G. Trafficking pathway between plasma membrane and mitochondria via clathrin-mediated endocytosis. J. Mol. Cell. Biol. 10, 539–548 (2018).
Itakura, E., Kishi-Itakura, C. & Mizushima, N. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151, 1256–1269 (2012).
Chen, Y. F. et al. Cisd2 deficiency drives premature aging and causes mitochondria-mediated defects in mice. Genes Dev. 23, 1183–1194 (2009).
Chen, P. L. et al. Vesicular transport mediates the uptake of cytoplasmic proteins into mitochondria in Drosophila melanogaster. Nat. Commun. 11, 2592 (2020).
Strappazzon, F. et al. AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1. Cell Death Differ. 22, 419–432 (2015).
Dudek, J. Role of cardiolipin in mitochondrial signaling pathways. Front. Cell Dev. Biol. 5, 90 (2017).
Paradies, G., Paradies, V., Ruggiero, F. M. & Petrosillo, G. Cardiolipin and mitochondrial function in health and disease. Antioxid. Redox Signal. 20, 1925–1953 (2014).
Wasilewski, M. et al. Optic atrophy 1-dependent mitochondrial remodeling controls steroidogenesis in trophoblasts. Curr. Biol. 22, 1228–1234 (2012).
Elustondo, P., Martin, L. A. & Karten, B. Mitochondrial cholesterol import. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1862, 90–101 (2017).
Courtney, K. C. et al. C24 sphingolipids govern the transbilayer asymmetry of cholesterol and lateral organization of model and live-cell plasma membranes. Cell Rep. 24, 1037–1049 (2018).
Venugopal, S. et al. Plasma membrane origin of the steroidogenic pool of cholesterol used in hormone-induced acute steroid formation in Leydig cells. J. Biol. Chem. 291, 26109–26125 (2016).
Sandhu, J. et al. Aster proteins facilitate nonvesicular plasma membrane to ER cholesterol transport in mammalian cells. Cell 175, 514–529.e20 (2018).
Rigotti, A., Miettinen, H. E. & Krieger, M. The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues. Endocr. Rev. 24, 357–387 (2003).
Chen, M. et al. Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs. Cell Death Differ. 25, 1905–1920 (2018).
Ipsaro, J. J., Haase, A. D., Knott, S. R., Joshua-Tor, L. & Hannon, G. J. The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis. Nature 491, 279–283 (2012).
Watanabe, T. et al. MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline. Dev. Cell 20, 364–375 (2011).
Curley, M. et al. A young testicular microenvironment protects Leydig cells against age-related dysfunction in a mouse model of premature aging. FASEB J. 33, 978–995 (2019).
Shen, Z. Q. et al. CISD2 maintains cellular homeostasis. Biochim. Biophys. Acta Mol. Cell Res. 1868, 118954 (2021).
Kameoka, S., Adachi, Y., Okamoto, K., Iijima, M. & Sesaki, H. Phosphatidic acid and cardiolipin coordinate mitochondrial dynamics. Trends Cell Biol. 28, 67–76 (2018).
Barneda, D. et al. The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix. eLife 4, e07485 (2015).
Fei, W. et al. A role for phosphatidic acid in the formation of ‘supersized’ lipid droplets. PLoS Genet. 7, e1002201 (2011).
Hoppins, S. et al. The soluble form of Bax regulates mitochondrial fusion via MFN2 homotypic complexes. Mol. Cell 41, 150–160 (2011).
Meeusen, S. et al. Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1. Cell 127, 383–395 (2006).
Zhao, H. et al. AMPK-mediated activation of MCU stimulates mitochondrial Ca2+ entry to promote mitotic progression. Nat. Cell Biol. 21, 476–486 (2019).
Acknowledgements
This work was supported by grants from the National Natural Science Foundation (32293203, 91954203 and 32021003) and Ministry of Science and Technology (2018YFA0800703). B.-L.S. acknowledges support from the Tencent Foundation through the XPLORER PRIZE.
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Contributions
B.-L.S. conceived of the project. B.-L.S. and J.L. directed the research. Y.-X.Z. and B.-L.S. designed the experiments. Y.-X.Z. and J.W. performed the genome-wide shRNA screen. Y.-X.Z. and P.-Y.S. performed the cell-free mitochondria recruitment assay. Y.-X.Z. and G.D. performed the PLD6 protein purification assay. Y.-X.Z. performed all of the other experiments and analysed the data with B.-L.S. Y.-X.Z., A.H., J.L. and B.-L.S. wrote the paper with input from all authors.
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Extended data
Extended Data Fig. 1 A genome-wide screen identifies the genes modulating LDLR level.
a, Schematic representation of the screening strategy. Huh7 cells were infected with lentivirus expressing a genome-wide shRNA library and then treated with 4 µg/ml puromycin for 4 days. Surviving cells were incubated with the anti-LDLR antibody and secondary antibody (2nd Ab) at 4 °C and subjected to fluorescence-activated cell sorting (FACS). After 5 rounds of sorting, genomic DNA was isolated from the populations with top 0.3% fluorescence and unsorted cells, and shRNA cassettes were PCR-amplified and subjected to deep sequencing. b, The procedure of the screening based on fluorescence-activating cell sorter. c, Bioinformatics classification of the hits based on biological processes and molecular functions. d, e, Validation of the hits from screening. Huh7 cells were transfected with siRNAs targeting the indicated genes for 48 h and harvested for real-time quantitative PCR analysis (d) and immunoblotting analysis (e). Data are normalized to control cells and presented as mean ± SEM (n = 3 biological repeats). Unpaired two-tailed Student’s t-test. The experiment was repeated twice with similar results obtained. f, Topology of human PLD6 protein. The transmembrane domain spans 5–27 amino acids, the 156th histidine residue (H156) is the catalytic site. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 2 PLD6 is involved in LDLR degradation.
a, Expressional profiling of PLD6 and LDLR in the indicated mouse tissues. The same amount of total protein was loaded. The experiment was repeated three times with similar results obtained. b, Representative confocal images showing mouse adrenal gland and testis stained with anti-PLD6 and anti-LDLR antibodies. Nuclei were counterstained with Hoechst. Scale bars, 100 µm (main), 50 µm (inset). The experiment was repeated three times with similar results obtained. c, PLD6 mRNA levels in different cell types. Data are normalized to NIH3T3 cells and presented as mean ± SEM (n = 3 biological repeats). Unpaired two-tailed Student’s t-test. d, Immunoblotting analysis of PLD6 protein in the cells transfected with the indicated amounts of PLD6-expressing plasmids and mouse tissues. CHC, clathrin heavy chain. The experiment was repeated twice with similar results obtained. e, Mitochondrial morphology revealed by the anti-TOM20 antibody in the indicated cell types. Nuclei were counterstained with DAPI. Scale bars, 10 µm (main), 2.5 µm (inset). The experiment was repeated twice with similar results obtained. f, Representative confocal images showing mitochondrial morphology revealed by the anti-TOM20 antibody in HeLa cells infected with lentivirus expressing negative control (NC) or the indicated PLD6 variants. Scale bars, 10 µm (main), 2.5 µm (inset). g, Quantification of mitochondrial morphology shown in (f). Data are presented as mean ± SEM (n = 306, 272, 245, 317 cells from 3 independent experiments). h, Representative confocal images showing the localization of Raf1-PABD in HeLa cells infected with lentivirus expressing negative control (NC) or the indicated PLD6 variants. Scale bars, 10 µm (main), 2.5 µm (inset). i, Percentages of cells with Raf1-PABD recruitment to mitochondria over total cells shown in (h). Data are presented as mean ± SEM (n = 200 cells from 3 independent experiments). Unpaired two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 3 The PA levels on the outer mitochondrial membrane regulate LDLR level.
a, Diagram showing PLD6-catalyzed conversion of cardiolipin to phosphatidic acid, which is then converted to diacylglycerol and lysophosphatidic acid by LPIN1 and PA-PLA1, respectively. b, Sanger sequencing analysis of two lines of LPIN1 KO HeLa cells (LPIN1-KO1 and LPIN1-KO2) and PA-PLA1 KO HeLa cells. WT, wild-type HeLa cells. c, Immunoblotting analysis of LDLR expression in WT and two lines of LPIN1 knockout HeLa cells (LPIN1-KO1 and LPIN1-KO2). CHC, clathrin heavy chain. The experiment was repeated twice with similar results obtained. d, Immunoblotting analysis of LDLR expression in WT and PA-PLA1 knockout HeLa cells (PA-PLA1 KO). The experiment was repeated twice with similar results obtained. e, f, Immunoblotting analysis of LDLR expression in LPIN1 KO cells and PA-PLA1 KO cells transfected with the indicated plasmids (0.3 µg of pBA-EGFP-LDLR, 0.3/1 µg of pCMV-LPIN1-3×Flag or pCMV-PA-PLA1-3×Flag) for 48 h. LPIN1(Δ678–682) and PA-PLA1 (Δ611–886) are catalytic domain-deleted forms. The experiment was repeated three times with similar results obtained. g, HeLa cells were infected with the indicated lentivirus for 48 h, fixed and immunostained with anti-Flag and anti-MYC antibodies. Scale bars, 10 µm. h, Percentages of cells with Raf1-PABD recruitment to the mitochondria over total cells in (g). Data are represented as mean ± SEM (n = 150 cells from 3 independent experiments). Unpaired two-tailed Student’s t-test. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 4 Identification of mitochondrial proteases responsible for LDLR degradation.
a, Knockdown efficiency of the siRNAs in Fig. 2d. HeLa cells transfected with scrambled siRNA (siNC, negative control) were used as control. Data are presented as mean ± SEM (n = 3 biological repeats). Unpaired two-tailed Student’s t-test. b-d, HTRA2, PMPCB and PITRM1 can cleave LDLR protein in vitro. Purified LDLR-His protein (10 μg) and HTRA2-His (1 μg), PMPCB-MYC (1 μg) or PITRM1-MYC (1 μg) proteins were incubated at 37 °C for 16 h. The reaction samples were resolved by 12% SDS-PAGE, visualized by silver stain and analyzed by immunoblotting with the indicated antibodies. The experiment was repeated twice with similar results obtained. e, Knockdown efficiency of the indicated siRNAs. HeLa cells transfected with scrambled siRNA (siNC, negative control) were used as control. Data are presented as mean ± SEM (n = 3 biological repeats). Unpaired two-tailed Student’s t-test. f, Immunoblotting analysis determining the involvement of TOM and TIM complexes in regulating endogenous LDLR levels. HeLa cells were transfected the indicated siRNA for 48 h and harvested for immunoblotting analysis. NC, negative control. The experiment was repeated three times with similar results obtained. g, Immunoblotting analysis showing the involvement of TIM complex in PLD6-induced LDLR degradation. HeLa cells (WT) and HeLa cells stably expressing doxycycline (DOX)-inducible PLD6 (Tet-on-PLD6-3×Flag) were transfected with scrambled siRNA (siNC, negative control) and siRNAs targeting TIM22 for 48 h, treated with 2 μg/ml DOX for 24 h, and then harvested for immunoblotting analysis. The experiment was repeated twice with similar results obtained. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 5 PLD6 promotes LDLR translocation into the mitochondria.
a, Representative confocal images showing the effect of PLD6 on mitochondrial localization of LDLR in Fig. 3e. H295R cells were transfected with scrambled siRNA (siNC, negative control) or siRNA targeting PLD6 (siPLD6). Mitochondria were isolated, treated without (-) or with (+) 10 µg/ml proteinase K (ProK) for 30 min and immunostained with the indicated antibodies. Scale bars, 2 µm. The experiment was repeated twice with similar results obtained. b, Representative confocal images showing the effect of PLD6 on mitochondrial localization of EGFR. H295R cells were transfected with scrambled siRNA (siNC, negative control) or siRNA targeting PLD6 (siPLD6). Mitochondria were isolated, treated without (-) or with (+) 10 µg/ml proteinase K (ProK) for 30 min and immunostained with the indicated antibodies. Scale bars, 2 µm. c, Percentages of EGFR+ mitochondria over total mitochondria shown in (b). Data are presented as mean ± SEM (n = 12, 9, 9, 9 images for each treatment). Unpaired two-tailed Student’s t-test. ProK, proteinase K. Source numerical data are available in source data.
Extended Data Fig. 6 PLD6 facilitates LDL translocation into the mitochondria.
a, Representative confocal images showing the co-localization between Dil-LDL and TOM20+ mitochondria in the indicated cells incubated in cholesterol-depleting medium for 16 h and then 10 µg/ml Dil-LDL for 5 h. Scale bars, 10 µm (main), 2 µm (inset). The experiment was repeated twice with similar results obtained. b, Representative confocal images showing the effect of PLD6 on mitochondrial localization of Dil-LDL shown in Fig. 4e. H295R cells were transfected with scrambled siRNA (siNC, negative control) or siRNA target PLD6 (siPLD6), and mitochondria were isolated and treated as in Fig. 4a. Scale bars, 2 µm. The experiment was repeated twice with similar results obtained. c, Representative confocal images showing the effect of overexpressed PLD6 protein on mitochondrial localization of Dil-LDL. H295R cells were transfected with the plasmid expressing negative control (NC) or the indicated amounts (μg) of PLD6-expressing plasmid, treated as in Fig. 4a, and immunostained with the anti-Flag and anti-TOM20 antibodies. Scale bars, 15 µm (main), 2.5 µm (inset). d, Quantification of the relative fluorescence intensity of Dil-LDL in the mitochondria shown in (c). Data are normalized to control cells and presented as mean median (interquartile range) (n = 30 cells for each treatment). Non-parametric two-sided Mann-Whitney test. e, Representative confocal images showing the antagonistic effect of LPIN1 and PA-PLA1 on PLD6-induced mitochondrial localization of Dil-LDL. H295R cells were infected with the indicated lentivirus and treated as in Fig. 4a. Scale bars, 15 µm (main), 2.5 µm (inset). f, Quantification of the relative fluorescence intensity of Dil-LDL in the mitochondria shown in (e). Data are normalized to control cells and presented as median (interquartile range) (n = 24 cells for each treatment). Non-parametric two-sided Mann-Whitney test. Source numerical data are available in source data.
Extended Data Fig. 7 Vesicular transport along microtube mediates mitochondrial entry of the LDL/LDLR complex.
a, WT and LDLR knockout (LDLR-KO) H295R cells were infected with the indicated lentivirus expressing PLD6, full-length (1–860 a.a.) or C-terminal truncated (1–810 a.a.) LDLR as indicated and treated as in Fig. 4a. Scale bars, 15 µm (main), 2.5 µm (inset). b, Quantification of the relative fluorescence intensity of Dil-LDL in the mitochondria shown in (a). Data are normalized to control cells and presented as median (Interquartile range) (n = 37, 33, 32, 31, 34, 35, 40, 35 cells from 3 independent experiments). Non-parametric two-sided Mann-Whitney test. c, Topology of human LDLR. The cytoplasmic tail spans amino acids 811–860 and NPXY is the endocytic motif. d, Requirement of dynamin-dependent endocytosis and microtubule machinery for LDL entering the mitochondria. H295R cells were infected with control or PLD6-expressing lentivirus and treated as in Fig. 4a. Cells were pretreated with DMSO, 10 µM Nocodazole or 30 µM Dyngo-4a for 30 min prior to Dil-LDL exposure. Cells were then fixed and immunostained with anti-TOM20 and anti-Flag antibodies. Scale bars, 15 µm (main), 2.5 µm (inset). e, Quantification of the relative fluorescence intensity of Dil-LDL in the mitochondria shown in (d). Data are normalized to control cells and presented as median (Interquartile range) (n = 32, 30, 30, 30, 28, 32 cells from 2 independent experiments). Non-parametric two-sided Mann-Whitney test. Source numerical data are available in source data.
Extended Data Fig. 8 CISD2 is required for LDL translocation into the mitochondria.
a, H295R cells were transfected with the indicated siRNA and harvested for real-time quantitative PCR analysis. Data are normalized to cells transfected with scrambled siRNA (NC, negative control) and presented as mean ± SEM (n = 3 biological repeats). Unpaired two-tailed Student’s t-test. b, Representative confocal images showing H295R cells transfected with indicated siRNA, infected with PLD6-expressing lentivirus and treated as in Fig. 4a. NC, negative control. Scale bars, 15 µm (main), 2.5 µm (inset). c, Quantification of the relative fluorescence intensity of Dil-LDL in the mitochondria shown in (b). Data are normalized to control cells and presented as median (Interquartile range) (n = 21, 23, 23, 23, 23, 22, 23, 21, 23, 23, 23 cells from 2 independent experiments). Non-parametric two-sided Mann-Whitney test. NC, negative control. Source numerical data are available in source data.
Extended Data Fig. 9 CISD2 interacts with LDLR and mediates its mitochondrial localization.
a, Topology of the human CISD2 protein. Amino acids 61–135 are the cytoplasmic region. b, Subcellular localization of endogenous CISD2 in H295R cells infected with lentivirus expressing EGFP-Sec61β or TOM20-EGFP. Scale bars, 10 µm. The experiment was repeated twice with similar results obtained. c, Subcellular localization of overexpressed CISD2 in H295R cells transfected with the plasmids expressing MYC-CISD2 and EGFP-Sec61β or TOM20-EGFP. Scale bars, 10 µm. The experiment was repeated three times with similar results obtained. d, Immunoblotting analysis showing LDLR and CISD2 expression in H295R cells and two lines of CISD2 knockout H295R cells (CISD2-KO1 and CISD2-KO2). The experiment was repeated twice with similar results obtained. e, Representative confocal images showing the effect of CISD2 on mitochondrial localization of LDLR. H295R cells were transfected with scrambled siRNA (siNC, negative control) or siRNA targeting CISD2 (siCISD2) and treated as in Fig. 3a. Mitochondria were isolated, treated without (-) or with (+) 10 µg/ml proteinase K (ProK) and immunostained with the indicated antibodies. Scale bars, 2 µm. f, Percentages of LDLR+ mitochondria over total mitochondria in H295R cells in (e). Data are presented as mean ± SEM (n = 11 images for each treatment). Unpaired two-tailed Student’s t-test. ProK, proteinase K. g, Schematic diagrams showing CISD2 and its various forms. The mutant (Mut) protein has C99S, C101S, C110S, H114Q mutations. h-j, The interaction of CISD2 and LDLR analyzed by co- immunoprecipitation. HeLa cells were transfected with the plasmids expressing MYC-tagged CISD2 variants and LDLR-3×Flag for 48 h. Cell lysates were immunoprecipitated with anti-Flag antibody coupled agarose and subjected to immunoblotting. The experiment was repeated twice with similar results obtained. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 10 PLD6 promotes mitochondrial fusion in vitro.
a, Coomassie blue staining of the indicated proteins. b, Diagram showing the in vitro mitochondrial fusion assay. c, d, Recombinant PLD6 protein was recruited to the mitochondrial membrane in vitro. Purified PLD6 protein and mitochondria were mixed with S2 fusion buffer in a 50 µl reaction system at 37 °C for 60 min. The mixture was washed using the indicated MIB buffer, and the pellet was analyzed. e, f, Representative confocal (e) and transmission electron microscope images (f) showing mitochondrial fusion in vitro. Scale bars, 0.5 µm (e), 200 nm (f). g, PLD6 promotes mitochondrial fusion in vitro. Fusion efficiency is presented as the number of fused (mCherry+ and EGFP+) mitochondria over those of EGFP+ mitochondria. Data are presented as mean ± SEM (n = 406, 391, 380 randomly selected EGFP+ mitochondria for each treatment). One-way analysis of variance followed by Dunnett’s test. h, i, Knockdown of TAZ increases LDLR level. HeLa cells were transfected with the indicated siRNA and harvested for qPCR (h) and immunoblotting analysis (i). QPCR results are presented as mean ± SEM (n = 3 biological repeats). Unpaired two-tailed Student’s t-test. j, Representative confocal images showing NBD-cholesterol labeled-LDL in WT but not LDLR knockout HeLa cells. Scale bars, 10 µm. k, Diagram showing NBD-cholesterol loading onto the inner mitochondrial membrane. l, Immunoblotting analysis showing LDLR degradation in the isolated mitochondria in the absence (-) or presence (+) of PLD6. m, The amount of NBD-cholesterol in the indicated mitoplasts in the absence (-) or presence (+) of PLD6. Data are presented as mean ± SEM (n = 3 biological repeats). Unpaired two-tailed Student’s t-test. The experiment was repeated twice (a, c, d, e, i, j) or three time (l, f) with similar results obtained. Source numerical data and unprocessed blots are available in source data.
Supplementary information
Supplementary Information
Supplementary Figs. 1–7 and associated figure captions.
Supplementary Data
Supplementary Table 1. A list of candidate genes from screening. Supplementary Table 2. Sequences of siRNA, shRNA, single guide RNA and quantitative PCR primers. Source data Supplementary Fig. 1. Statistical source data. Source data Supplementary Fig. 2. Statistical source data. Source data Supplementary Fig. 5. Statistical source data.
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Zhou, YX., Wei, J., Deng, G. et al. Delivery of low-density lipoprotein from endocytic carriers to mitochondria supports steroidogenesis. Nat Cell Biol 25, 937–949 (2023). https://doi.org/10.1038/s41556-023-01160-6
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DOI: https://doi.org/10.1038/s41556-023-01160-6
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