The Acyl-CoA-binding domain-containing protein (ACBD3) plays multiple roles across the cell. Although generally associated with the Golgi apparatus, it operates also in mitochondria. In steroidogenic cells, ACBD3 is an important part of a multiprotein complex transporting cholesterol into mitochondria. Balance in mitochondrial cholesterol is essential for proper mitochondrial protein biosynthesis, among others. We generated ACBD3 knock-out (ACBD3-KO) HEK293 and HeLa cells and characterized the impact of protein absence on mitochondria, Golgi, and lipid profile. In ACBD3-KO cells, cholesterol level and mitochondrial structure and functions are not altered, demonstrating that an alternative pathway of cholesterol transport into mitochondria exists. However, ACBD3-KO cells exhibit enlarged Golgi area with absence of stacks and ribbon-like formation, confirming the importance of ACBD3 in Golgi stacking. The glycosylation of the LAMP2 glycoprotein was not affected by the altered Golgi structure. Moreover, decreased sphingomyelins together with normal ceramides and sphingomyelin synthase activity reveal the importance of ACBD3 in ceramide transport from ER to Golgi.

Department of Paediatrics and Inherited Metabolic Disorders Charles University 1st Faculty of Medicine and General University Hospital Prague 128 01 Prague Czech Republic

Institute of Physiology Academy of Sciences of the Czech Republic 142 00 Prague Czech Republic

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Uhlen M., Fagerberg L., Hallstrom B.M., Lindskog C., Oksvold P., Mardinoglu A., Sivertsson A., Kampf C., Sjostedt E., Asplund A., et al. Tissue-Based Map of the Human Proteome. Science. 2015;347:1260419. doi: 10.1126/science.1260419. PubMed DOI

The UniProt Consortium UniProt: A Worldwide Hub of Protein Knowledge. Nucleic Acids Res. 2019;47:D506–D515. doi: 10.1093/nar/gky1049. PubMed DOI PMC

Binder J.X., Pletscher-Frankild S., Tsafou K., Stolte C., O’Donoghue S.I., Schneider R., Jensen L.J. COMPARTMENTS: Unification and Visualization of Protein Subcellular Localization Evidence. Database J. Biol. Databases Curation. 2014;2014 doi: 10.1093/database/bau012. PubMed DOI PMC

Sohda M., Misumi Y., Yamamoto A., Yano A., Nakamura N., Ikehara Y. Identification and Characterization of a Novel Golgi Protein, GCP60, That Interacts with the Integral Membrane Protein Giantin. J. Biol. Chem. 2001;276:45298–45306. doi: 10.1074/jbc.M108961200. PubMed DOI

Li H., Degenhardt B., Tobin D., Yao Z.X., Tasken K., Papadopoulos V. Identification, Localization, and Function in Steroidogenesis of PAP7: A Peripheral-Type Benzodiazepine Receptor- and PKA (RIalpha)-Associated Protein. Mol. Endocrinol. Baltim. Md. 2001;15:2211–2228. doi: 10.1210/mend.15.12.0736. PubMed DOI

Liu J., Rone M.B., Papadopoulos V. Protein-Protein Interactions Mediate Mitochondrial Cholesterol Transport and Steroid Biosynthesis. J. Biol. Chem. 2006;281:38879–38893. doi: 10.1074/jbc.M608820200. PubMed DOI

Yue X., Bao M., Christiano R., Li S., Mei J., Zhu L., Mao F., Yue Q., Zhang P., Jing S., et al. ACBD3 Functions as a Scaffold to Organize the Golgi Stacking Proteins and a Rab33b-GAP. FEBS Lett. 2017;591:2793–2802. doi: 10.1002/1873-3468.12780. PubMed DOI

Liao J., Guan Y., Chen W., Shi C., Yao D., Wang F., Lam S.M., Shui G., Cao X. ACBD3 Is Required for FAPP2 Transferring Glucosylceramide through Maintaining the Golgi Integrity. J. Mol. Cell Biol. 2019;11:107–117. doi: 10.1093/jmcb/mjy030. PubMed DOI PMC

Shinoda Y., Fujita K., Saito S., Matsui H., Kanto Y., Nagaura Y., Fukunaga K., Tamura S., Kobayashi T. Acyl-CoA Binding Domain Containing 3 (ACBD3) Recruits the Protein Phosphatase PPM1L to ER-Golgi Membrane Contact Sites. FEBS Lett. 2012;586:3024–3029. doi: 10.1016/j.febslet.2012.06.050. PubMed DOI

Islinger M., Costello J.L., Kors S., Soupene E., Levine T.P., Kuypers F.A., Schrader M. The Diversity of ACBD Proteins—From Lipid Binding to Protein Modulators and Organelle Tethers. Biochim. Biophys. Acta BBA Mol. Cell Res. 2020;1867:118675. doi: 10.1016/j.bbamcr.2020.118675. PubMed DOI PMC

Elustondo P., Martin L.A., Karten B. Mitochondrial Cholesterol Import. Biochim. Biophys. Acta BBA Mol. Cell Biol. Lipids. 2017;1862:90–101. doi: 10.1016/j.bbalip.2016.08.012. PubMed DOI

Hensen F., Cansiz S., Gerhold J.M., Spelbrink J.N. To Be or Not to Be a Nucleoid Protein: A Comparison of Mass-Spectrometry Based Approaches in the Identification of Potential MtDNA-Nucleoid Associated Proteins. Biochimie. 2014;100:219–226. doi: 10.1016/j.biochi.2013.09.017. PubMed DOI

Gerhold J.M., Cansiz-Arda Ş., Lõhmus M., Engberg O., Reyes A., van Rennes H., Sanz A., Holt I.J., Cooper H.M., Spelbrink J.N. Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure. Sci. Rep. 2015;5 doi: 10.1038/srep15292. PubMed DOI PMC

He J., Cooper H.M., Reyes A., Di Re M., Sembongi H., Litwin T.R., Gao J., Neuman K.C., Fearnley I.M., Spinazzola A., et al. Mitochondrial Nucleoid Interacting Proteins Support Mitochondrial Protein Synthesis. Nucleic Acids Res. 2012;40:6109–6121. doi: 10.1093/nar/gks266. PubMed DOI PMC

He J., Mao C.-C., Reyes A., Sembongi H., Di Re M., Granycome C., Clippingdale A.B., Fearnley I.M., Harbour M., Robinson A.J., et al. The AAA+ Protein ATAD3 Has Displacement Loop Binding Properties and Is Involved in Mitochondrial Nucleoid Organization. J. Cell Biol. 2007;176:141–146. doi: 10.1083/jcb.200609158. PubMed DOI PMC

Peralta S., Goffart S., Williams S.L., Diaz F., Garcia S., Nissanka N., Area-Gomez E., Pohjoismäki J., Moraes C.T. ATAD3 Controls Mitochondrial Cristae Structure in Mouse Muscle, Influencing MtDNA Replication and Cholesterol Levels. J. Cell Sci. 2018;131 doi: 10.1242/jcs.217075. PubMed DOI PMC

Mesmin B., Maxfield F.R. Intracellular Sterol Dynamics. Biochim. Biophys. Acta BBA Mol. Cell Biol. Lipids. 2009;1791:636–645. doi: 10.1016/j.bbalip.2009.03.002. PubMed DOI PMC

Rone M.B., Midzak A.S., Issop L., Rammouz G., Jagannathan S., Fan J., Ye X., Blonder J., Veenstra T., Papadopoulos V. Identification of a Dynamic Mitochondrial Protein Complex Driving Cholesterol Import, Trafficking, and Metabolism to Steroid Hormones. Mol. Endocrinol. 2012;26:1868–1882. doi: 10.1210/me.2012-1159. PubMed DOI PMC

Desai R., East D.A., Hardy L., Faccenda D., Rigon M., Crosby J., Alvarez M.S., Singh A., Mainenti M., Hussey L.K., et al. Mitochondria Form Contact Sites with the Nucleus to Couple Prosurvival Retrograde Response. Sci. Adv. 2020;6 doi: 10.1126/sciadv.abc9955. PubMed DOI PMC

Hornig-Do H.-T., Günther G., Bust M., Lehnartz P., Bosio A., Wiesner R.J. Isolation of Functional Pure Mitochondria by Superparamagnetic Microbeads. Anal. Biochem. 2009;389:1–5. doi: 10.1016/j.ab.2009.02.040. PubMed DOI

Brantová O., Tesařová M., Hansíková H., Elleder M., Zeman J., Sládková J. Ultrastructural Changes of Mitochondria in the Cultivated Skin Fibroblasts of Patients with Point Mutations in Mitochondrial DNA. Ultrastruct. Pathol. 2006;30:239–245. doi: 10.1080/01913120600820112. PubMed DOI

Cesnekova J., Rodinova M., Hansikova H., Zeman J., Stiburek L. Loss of Mitochondrial AAA Proteases AFG3L2 and YME1L Impairs Mitochondrial Structure and Respiratory Chain Biogenesis. Int. J. Mol. Sci. 2018;19:3930. doi: 10.3390/ijms19123930. PubMed DOI PMC

Bilal F., Pérès M., Andrieu-Abadie N., Levade T., Badran B., Daher A., Ségui B. CD95. Volume 1557. Humana Press; New York, NY, USA: 2017. Method to Measure Sphingomyelin Synthase Activity Changes in Response to CD95L; pp. 207–212. PubMed DOI

Sbodio J.I., Hicks S.W., Simon D., Machamer C.E. GCP60 Preferentially Interacts with a Caspase-Generated Golgin-160 Fragment. J. Biol. Chem. 2006;281:27924–27931. doi: 10.1074/jbc.M603276200. PubMed DOI

Klima M., Tóth D.J., Hexnerova R., Baumlova A., Chalupska D., Tykvart J., Rezabkova L., Sengupta N., Man P., Dubankova A., et al. Structural Insights and in Vitro Reconstitution of Membrane Targeting and Activation of Human PI4KB by the ACBD3 Protein. Sci. Rep. 2016;6 doi: 10.1038/srep23641. PubMed DOI PMC

Chen Y., Patel V., Bang S., Cohen N., Millar J., Kim S.F. Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3. PLoS ONE. 2012;7:e49906. doi: 10.1371/journal.pone.0049906. PubMed DOI PMC

Bekier M.E., Wang L., Li J., Huang H., Tang D., Zhang X., Wang Y. Knockout of the Golgi Stacking Proteins GRASP55 and GRASP65 Impairs Golgi Structure and Function. Mol. Biol. Cell. 2017;28:2833–2842. doi: 10.1091/mbc.e17-02-0112. PubMed DOI PMC

Miller W.L. Steroid Hormone Synthesis in Mitochondria. Mol. Cell. Endocrinol. 2013;379:62–73. doi: 10.1016/j.mce.2013.04.014. PubMed DOI

Midzak A., Rone M., Aghazadeh Y., Culty M., Papadopoulos V. Mitochondrial Protein Import and the Genesis of Steroidogenic Mitochondria. Mol. Cell. Endocrinol. 2011;336:70–79. doi: 10.1016/j.mce.2010.12.007. PubMed DOI PMC

Desai R., Campanella M. Exploring Mitochondrial Cholesterol Signalling for Therapeutic Intervention in Neurological Conditions. Br. J. Pharmacol. 2019;176:4284–4292. doi: 10.1111/bph.14697. PubMed DOI PMC

Midzak A., Papadopoulos V. Adrenal Mitochondria and Steroidogenesis: From Individual Proteins to Functional Protein Assemblies. Front. Endocrinol. 2016;7 doi: 10.3389/fendo.2016.00106. PubMed DOI PMC

Papadopoulos V., Guarneri P., Kreuger K.E., Guidotti A., Costa E. Pregnenolone Biosynthesis in C6-2B Glioma Cell Mitochondria: Regulation by a Mitochondrial Diazepam Binding Inhibitor Receptor. Proc. Natl. Acad. Sci. USA. 1992;89:5113–5117. doi: 10.1073/pnas.89.11.5113. PubMed DOI PMC

Boujrad N., Hudson J.R., Papadopoulos V. Inhibition of Hormone-Stimulated Steroidogenesis in Cultured Leydig Tumor Cells by a Cholesterol-Linked Phosphorothioate Oligodeoxynucleotide Antisense to Diazepam-Binding Inhibitor. Proc. Natl. Acad. Sci. USA. 1993;90:5728–5731. doi: 10.1073/pnas.90.12.5728. PubMed DOI PMC

Garnier M., Boujrad N., Ogwuegbu S.O., Hudson J.R., Papadopoulos V. The Polypeptide Diazepam-Binding Inhibitor and a Higher Affinity Mitochondrial Peripheral-Type Benzodiazepine Receptor Sustain Constitutive Steroidogenesis in the R2C Leydig Tumor Cell Line. J. Biol. Chem. 1994;269:22105–22112. doi: 10.1016/S0021-9258(17)31762-3. PubMed DOI

Papadopoulos V., Brown A.S. Role of the Peripheral-Type Benzodiazepine Receptor and the Polypeptide Diazepam Binding Inhibitor in Steroidogenesis. J. Steroid Biochem. Mol. Biol. 1995;53:103–110. doi: 10.1016/0960-0760(95)00027-W. PubMed DOI

Fan J., Li X., Issop L., Culty M., Papadopoulos V. ACBD2/ECI2-Mediated Peroxisome-Mitochondria Interactions in Leydig Cell Steroid Biosynthesis. Mol. Endocrinol. 2016;30:763–782. doi: 10.1210/me.2016-1008. PubMed DOI PMC

Andersen J.-P., Zhang J., Sun H., Liu X., Liu J., Nie J., Shi Y. Aster-B Coordinates with Arf1 to Regulate Mitochondrial Cholesterol Transport. Mol. Metab. 2020;42:101055. doi: 10.1016/j.molmet.2020.101055. PubMed DOI PMC

Saiki R., Nagata A., Kainou T., Matsuda H., Kawamukai M. Characterization of Solanesyl and Decaprenyl Diphosphate Synthases in Mice and Humans. FEBS J. 2005;272:5606–5622. doi: 10.1111/j.1742-4658.2005.04956.x. PubMed DOI

Huttlin E.L., Bruckner R.J., Paulo J.A., Cannon J.R., Ting L., Baltier K., Colby G., Gebreab F., Gygi M.P., Parzen H., et al. Architecture of the Human Interactome Defines Protein Communities and Disease Networks. Nature. 2017;545:505–509. doi: 10.1038/nature22366. PubMed DOI PMC

Greninger A.L., Knudsen G.M., Betegon M., Burlingame A.L., DeRisi J.L. The 3A Protein from Multiple Picornaviruses Utilizes the Golgi Adaptor Protein ACBD3 To Recruit PI4KIIIβ. J. Virol. 2012;86:3605–3616. doi: 10.1128/JVI.06778-11. PubMed DOI PMC

Kumagai K., Hanada K. Structure, Functions and Regulation of CERT, a Lipid-transfer Protein for the Delivery of Ceramide at the ER–Golgi Membrane Contact Sites. FEBS Lett. 2019;593:2366–2377. doi: 10.1002/1873-3468.13511. PubMed DOI

Yamaji T., Hanada K. Establishment of HeLa Cell Mutants Deficient in Sphingolipid-Related Genes Using TALENs. PLoS ONE. 2014;9:e88124. doi: 10.1371/journal.pone.0088124. PubMed DOI PMC

Housden B.E., Muhar M., Gemberling M., Gersbach C.A., Stainier D.Y.R., Seydoux G., Mohr S.E., Zuber J., Perrimon N. Loss-of-Function Genetic Tools for Animal Models: Cross-Species and Cross-Platform Differences. Nat. Rev. Genet. 2017;18:24–40. doi: 10.1038/nrg.2016.118. PubMed DOI PMC

Zimmer A.M., Pan Y.K., Chandrapalan T., Kwong R.W.M., Perry S.F. Loss-of-Function Approaches in Comparative Physiology: Is There a Future for Knockdown Experiments in the Era of Genome Editing? J. Exp. Biol. 2019;222:jeb175737. doi: 10.1242/jeb.175737. PubMed DOI

Stiburek L., Vesela K., Hansikova H., Pecina P., Tesarova M., Cerna L., Houstek J., Zeman J. Tissue-Specific Cytochrome c Oxidase Assembly Defects Due to Mutations in SCO2 and SURF1. Biochem. J. 2005;392:625–632. doi: 10.1042/BJ20050807. PubMed DOI PMC

Schägger H., von Jagow G. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 KDa. Anal. Biochem. 1987;166:368–379. doi: 10.1016/0003-2697(87)90587-2. PubMed DOI

Schägger H., von Jagow G. Blue Native Electrophoresis for Isolation of Membrane Protein Complexes in Enzymatically Active Form. Anal. Biochem. 1991;199:223–231. doi: 10.1016/0003-2697(91)90094-A. PubMed DOI

Gnaiger E. Mitochondr Physiol Network 19.12. 5th ed. Oroboros MiPNet Publications; Innsbruck, Austria: 2020. Mitochondrial Pathways and Respiratory Control: An Introduction to OXPHOS Analysis. DOI

Pejznochova M., Tesarova M., Hansikova H., Magner M., Honzik T., Vinsova K., Hajkova Z., Havlickova V., Zeman J. Mitochondrial DNA Content and Expression of Genes Involved in MtDNA Transcription, Regulation and Maintenance during Human Fetal Development. Mitochondrion. 2010;10:321–329. doi: 10.1016/j.mito.2010.01.006. PubMed DOI

Ondruskova N., Honzik T., Vondrackova A., Stranecky V., Tesarova M., Zeman J., Hansikova H. Severe Phenotype of ATP6AP1-CDG in Two Siblings with a Novel Mutation Leading to a Differential Tissue-Specific ATP6AP1 Protein Pattern, Cellular Oxidative Stress and Hepatic Copper Accumulation. J. Inherit. Metab. Dis. 2020;43:694–700. doi: 10.1002/jimd.12237. PubMed DOI PMC

Luft J.H. Permanganate; a New Fixative for Electron Microscopy. J. Biophys. Biochem. Cytol. 1956;2:799–802. doi: 10.1083/jcb.2.6.799. PubMed DOI PMC

Janovska P., Melenovsky V., Svobodova M., Havlenova T., Kratochvilova H., Haluzik M., Hoskova E., Pelikanova T., Kautzner J., Monzo L., et al. Dysregulation of Epicardial Adipose Tissue in Cachexia Due to Heart Failure: The Role of Natriuretic Peptides and Cardiolipin. J. Cachexia Sarcopenia Muscle. 2020;11:1614–1627. doi: 10.1002/jcsm.12631. PubMed DOI PMC

Paluchova V., Oseeva M., Brezinova M., Cajka T., Bardova K., Adamcova K., Zacek P., Brejchova K., Balas L., Chodounska H., et al. Lipokine 5-PAHSA Is Regulated by Adipose Triglyceride Lipase and Primes Adipocytes for De Novo Lipogenesis in Mice. Diabetes. 2020;69:300–312. doi: 10.2337/db19-0494. PubMed DOI PMC

Paluchova V., Vik A., Cajka T., Brezinova M., Brejchova K., Bugajev V., Draberova L., Draber P., Buresova J., Kroupova P., et al. Triacylglycerol-Rich Oils of Marine Origin Are Optimal Nutrients for Induction of Polyunsaturated Docosahexaenoic Acid Ester of Hydroxy Linoleic Acid (13-DHAHLA) with Anti-Inflammatory Properties in Mice. Mol. Nutr. Food Res. 2020;64:e1901238. doi: 10.1002/mnfr.201901238. PubMed DOI

Sistilli G., Kalendova V., Cajka T., Irodenko I., Bardova K., Oseeva M., Zacek P., Kroupova P., Horakova O., Lackner K., et al. Krill Oil Supplementation Reduces Exacerbated Hepatic Steatosis Induced by Thermoneutral Housing in Mice with Diet-Induced Obesity. Nutrients. 2021;13:437. doi: 10.3390/nu13020437. PubMed DOI PMC

Case report: A rare variant m.4135T>C in the MT-ND1 gene leads to Leber hereditary optic neuropathy and altered respiratory chain supercomplexes