The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.

Department of Cell Biology Faculty of Science Charles University 128 00 Prague Czech Republic

Laboratory of Bioenergetics Institute of Physiology Czech Academy of Sciences 142 00 Prague Czech Republic

MRC Mitochondrial Biology Unit University of Cambridge Cambridge CB2 0XY UK

Zobrazit více v PubMed

Hallberg B.M., Larsson N.G. Making proteins in the powerhouse. Cell Metab. 2014;20:226–240. doi: 10.1016/j.cmet.2014.07.001. PubMed DOI

Guerrero-Castillo S., Baertling F., Kownatzki D., Wessels H.J., Arnold S., Brandt U., Nijtmans L. The Assembly Pathway of Mitochondrial Respiratory Chain Complex I. Cell Metab. 2017;25:128–139. doi: 10.1016/j.cmet.2016.09.002. PubMed DOI

Sanchez-Caballero L., Guerrero-Castillo S., Nijtmans L. Unraveling the complexity of mitochondrial complex I assembly: A dynamic process. Biochim. Biophys Acta BBA Bioenerg. 2016;1857:980–990. doi: 10.1016/j.bbabio.2016.03.031. PubMed DOI

Signes A., Fernandez-Vizarra E. Assembly of mammalian oxidative phosphorylation complexes I–V and supercomplexes. Essays Biochem. 2018;62:255–270. doi: 10.1042/EBC20170098. PubMed DOI PMC

Stroud D.A., Surgenor E.E., Formosa L.E., Reljic B., Frazier A.E., Dibley M.G., Osellame L.D., Stait T., Beilharz T.H., Thorburn D.R., et al. Accessory subunits are integral for assembly and function of human mitochondrial complex I. Nature. 2016;538:123–126. doi: 10.1038/nature19754. PubMed DOI

Eisenberg-Bord M., Schuldiner M. Ground control to major TOM: Mitochondria-nucleus communication. FEBS J. 2017;284:196–210. doi: 10.1111/febs.13778. PubMed DOI

Fontanesi F., Soto I.C., Horn D., Barrientos A. Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly regulated cellular process. Am. J. Physiol. Cell Physiol. 2006;291:C1129–C1147. doi: 10.1152/ajpcell.00233.2006. PubMed DOI

Schagger H., Pfeiffer K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 2000;19:1777–1783. doi: 10.1093/emboj/19.8.1777. PubMed DOI PMC

Letts J.A., Fiedorczuk K., Sazanov L.A. The architecture of respiratory supercomplexes. Nature. 2016;537:644–648. doi: 10.1038/nature19774. PubMed DOI

Gu J., Wu M., Guo R., Yan K., Lei J., Gao N., Yang M. The architecture of the mammalian respirasome. Nature. 2016;537:639–643. doi: 10.1038/nature19359. PubMed DOI

Balsa E., Marco R., Perales-Clemente E., Szklarczyk R., Calvo E., Landazuri M.O., Enriquez J.A. NDUFA4 is a subunit of complex IV of the mammalian electron transport chain. Cell Metab. 2012;16:378–386. doi: 10.1016/j.cmet.2012.07.015. PubMed DOI

Zong S., Wu M., Gu J., Liu T., Guo R., Yang M. Structure of the intact 14-subunit human cytochrome c oxidase. Cell Res. 2018;28:1026–1034. doi: 10.1038/s41422-018-0071-1. PubMed DOI PMC

Cunatova K., Reguera D.P., Houstek J., Mracek T., Pecina P. Role of cytochrome c oxidase nuclear-encoded subunits in health and disease. Physiol. Res. 2020;69:947–965. doi: 10.33549/physiolres.934446. PubMed DOI PMC

Sommer N., Huttemann M., Pak O., Scheibe S., Knoepp F., Sinkler C., Malczyk M., Gierhardt M., Esfandiary A., Kraut S., et al. Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing. Circ. Res. 2017;121:424–438. doi: 10.1161/CIRCRESAHA.116.310482. PubMed DOI PMC

Pajuelo Reguera D., Cunatova K., Vrbacky M., Pecinova A., Houstek J., Mracek T., Pecina P. Cytochrome c Oxidase Subunit 4 Isoform Exchange Results in Modulation of Oxygen Affinity. Cells. 2020;9:443. doi: 10.3390/cells9020443. PubMed DOI PMC

Nijtmans L.G., Taanman J.W., Muijsers A.O., Speijer D., Van den Bogert C. Assembly of cytochrome-c oxidase in cultured human cells. Eur. J. Biochem. 1998;254:389–394. doi: 10.1046/j.1432-1327.1998.2540389.x. PubMed DOI

Dennerlein S., Rehling P. Human mitochondrial COX1 assembly into cytochrome c oxidase at a glance. J. Cell Sci. 2015;128:833–837. doi: 10.1242/jcs.161729. PubMed DOI

Soto I.C., Fontanesi F., Liu J., Barrientos A. Biogenesis and assembly of eukaryotic cytochrome c oxidase catalytic core. Biochim. Biophys Acta BBA Bioenerg. 2012;1817:883–897. doi: 10.1016/j.bbabio.2011.09.005. PubMed DOI PMC

Vidoni S., Harbour M.E., Guerrero-Castillo S., Signes A., Ding S., Fearnley I.M., Taylor R.W., Tiranti V., Arnold S., Fernandez-Vizarra E., et al. MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase. Cell Rep. 2017;18:1727–1738. doi: 10.1016/j.celrep.2017.01.044. PubMed DOI

Mick D.U., Dennerlein S., Wiese H., Reinhold R., Pacheu-Grau D., Lorenzi I., Sasarman F., Weraarpachai W., Shoubridge E.A., Warscheid B., et al. MITRAC links mitochondrial protein translocation to respiratory-chain assembly and translational regulation. Cell. 2012;151:1528–1541. doi: 10.1016/j.cell.2012.11.053. 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

Aich A., Wang C., Chowdhury A., Ronsor C., Pacheu-Grau D., Richter-Dennerlein R., Dennerlein S., Rehling P. COX16 promotes COX2 metallation and assembly during respiratory complex IV biogenesis. Elife. 2018;7 doi: 10.7554/eLife.32572. PubMed DOI PMC

Maghool S., Cooray N.D.G., Stroud D.A., Aragao D., Ryan M.T., Maher M.J. Structural and functional characterization of the mitochondrial complex IV assembly factor Coa6. Life Sci. Alliance. 2019;2 doi: 10.26508/lsa.201900458. PubMed DOI PMC

Pacheu-Grau D., Wasilewski M., Oeljeklaus S., Gibhardt C.S., Aich A., Chudenkova M., Dennerlein S., Deckers M., Bogeski I., Warscheid B., et al. COA6 Facilitates Cytochrome c Oxidase Biogenesis as Thiol-reductase for Copper Metallochaperones in Mitochondria. J. Mol. Biol. 2020;432:2067–2079. doi: 10.1016/j.jmb.2020.01.036. PubMed DOI PMC

Soma S., Morgada M.N., Naik M.T., Boulet A., Roesler A.A., Dziuba N., Ghosh A., Yu Q., Lindahl P.A., Ames J.B., et al. COA6 Is Structurally Tuned to Function as a Thiol-Disulfide Oxidoreductase in Copper Delivery to Mitochondrial Cytochrome c Oxidase. Cell Rep. 2019;29:4114–4126. doi: 10.1016/j.celrep.2019.11.054. PubMed DOI PMC

Hock D.H., Reljic B., Ang C.S., Muellner-Wong L., Mountford H.S., Compton A.G., Ryan M.T., Thorburn D.R., Stroud D.A. HIGD2A is Required for Assembly of the COX3 Module of Human Mitochondrial Complex IV. Mol. Cell. Proteom. 2020;19:1145–1160. doi: 10.1074/mcp.RA120.002076. PubMed DOI PMC

Timon-Gomez A., Garlich J., Stuart R.A., Ugalde C., Barrientos A. Distinct Roles of Mitochondrial HIGD1A and HIGD2A in Respiratory Complex and Supercomplex Biogenesis. Cell Rep. 2020;31:107607. doi: 10.1016/j.celrep.2020.107607. PubMed DOI

Acin-Perez R., Bayona-Bafaluy M.P., Fernandez-Silva P., Moreno-Loshuertos R., Perez-Martos A., Bruno C., Moraes C.T., Enriquez J.A. Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol. Cell. 2004;13:805–815. doi: 10.1016/S1097-2765(04)00124-8. PubMed DOI PMC

Barel O., Shorer Z., Flusser H., Ofir R., Narkis G., Finer G., Shalev H., Nasasra A., Saada A., Birk O.S. Mitochondrial complex III deficiency associated with a homozygous mutation in UQCRQ. Am. J. Hum. Genet. 2008;82:1211–1216. doi: 10.1016/j.ajhg.2008.03.020. PubMed DOI PMC

Carossa V., Ghelli A., Tropeano C.V., Valentino M.L., Iommarini L., Maresca A., Caporali L., La Morgia C., Liguori R., Barboni P., et al. A novel in-frame 18-bp microdeletion in MT-CYB causes a multisystem disorder with prominent exercise intolerance. Hum. Mutat. 2014;35:954–958. doi: 10.1002/humu.22596. PubMed DOI

Feichtinger R.G., Brunner-Krainz M., Alhaddad B., Wortmann S.B., Kovacs-Nagy R., Stojakovic T., Erwa W., Resch B., Windischhofer W., Verheyen S., et al. Combined Respiratory Chain Deficiency and UQCC2 Mutations in Neonatal Encephalomyopathy: Defective Supercomplex Assembly in Complex III Deficiencies. Oxid. Med. Cell. Longev. 2017;2017 doi: 10.1155/2017/7202589. PubMed DOI PMC

Lamantea E., Carrara F., Mariotti C., Morandi L., Tiranti V., Zeviani M. A novel nonsense mutation (Q352X) in the mitochondrial cytochrome b gene associated with a combined deficiency of complexes I and III. Neuromuscul. Disord. 2002;12:49–52. doi: 10.1016/S0960-8966(01)00244-9. PubMed DOI

Tucker E.J., Wanschers B.F., Szklarczyk R., Mountford H.S., Wijeyeratne X.W., van den Brand M.A., Leenders A.M., Rodenburg R.J., Reljic B., Compton A.G., et al. Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression. PLoS Genet. 2013;9:e1004034. doi: 10.1371/journal.pgen.1004034. PubMed DOI PMC

Moreno-Lastres D., Fontanesi F., Garcia-Consuegra I., Martin M.A., Arenas J., Barrientos A., Ugalde C. Mitochondrial complex I plays an essential role in human respirasome assembly. Cell Metab. 2012;15:324–335. doi: 10.1016/j.cmet.2012.01.015. PubMed DOI PMC

Protasoni M., Perez-Perez R., Lobo-Jarne T., Harbour M.E., Ding S., Penas A., Diaz F., Moraes C.T., Fearnley I.M., Zeviani M., et al. Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV. EMBO J. 2020;39:e102817. doi: 10.15252/embj.2019102817. PubMed DOI PMC

Diaz F., Fukui H., Garcia S., Moraes C.T. Cytochrome c oxidase is required for the assembly/stability of respiratory complex I in mouse fibroblasts. Mol. Cell. Biol. 2006;26:4872–4881. doi: 10.1128/MCB.01767-05. PubMed DOI PMC

Li Y., D’Aurelio M., Deng J.H., Park J.S., Manfredi G., Hu P., Lu J., Bai Y. An assembled complex IV maintains the stability and activity of complex I in mammalian mitochondria. J. Biol. Chem. 2007;282:17557–17562. doi: 10.1074/jbc.M701056200. PubMed DOI

Hornig-Do H.T., Tatsuta T., Buckermann A., Bust M., Kollberg G., Rotig A., Hellmich M., Nijtmans L., Wiesner R.J. Nonsense mutations in the COX1 subunit impair the stability of respiratory chain complexes rather than their assembly. EMBO J. 2012;31:1293–1307. doi: 10.1038/emboj.2011.477. PubMed DOI PMC

Timon-Gomez A., Nyvltova E., Abriata L.A., Vila A.J., Hosler J., Barrientos A. Mitochondrial cytochrome c oxidase biogenesis: Recent developments. Semin. Cell Dev. Biol. 2018;76:163–178. doi: 10.1016/j.semcdb.2017.08.055. PubMed DOI PMC

Lobo-Jarne T., Perez-Perez R., Fontanesi F., Timon-Gomez A., Wittig I., Penas A., Serrano-Lorenzo P., Garcia-Consuegra I., Arenas J., Martin M.A., et al. Multiple pathways coordinate assembly of human mitochondrial complex IV and stabilization of respiratory supercomplexes. EMBO J. 2020;39:e103912. doi: 10.15252/embj.2019103912. PubMed DOI PMC

Ran F.A., Hsu P.D., Lin C.-Y., Gootenberg J.S., Konermann S., Trevino A., Scott D.A., Inoue A., Matoba S., Zhang Y., et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154:1380–1389. doi: 10.1016/j.cell.2013.08.021. PubMed DOI PMC

Schägger H. Tricine–SDS-PAGE. Nat. Protoc. 2006;1:16–22. doi: 10.1038/nprot.2006.4. PubMed DOI

Wittig I., Braun H.-P., Schägger H. Blue native PAGE. Nat. Protoc. 2006;1:418–428. doi: 10.1038/nprot.2006.62. PubMed DOI

Bentlage H.A.C.M., Wendel U., Schagger H., Ter Laak H.J., Janssen A.J.M., Trijbels J.M.F. Lethal infantile mitochondrial disease with isolated complex I deficiency in fibroblasts but with combined complex I and IV deficiencies in muscle. Neurology. 1996;47:243–248. doi: 10.1212/WNL.47.1.243. PubMed DOI

Hartmannova H., Piherova L., Tauchmannova K., Kidd K., Acott P.D., Crocker J.F., Oussedik Y., Mallet M., Hodanova K., Stranecky V., et al. Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6. Hum. Mol. Genet. 2016;25:4062–4079. doi: 10.1093/hmg/ddw245. PubMed DOI

Fernandez-Vizarra E., Ferrin G., Perez-Martos A., Fernandez-Silva P., Zeviani M., Enriquez J.A. Isolation of mitochondria for biogenetical studies: An update. Mitochondrion. 2010;10:253–262. doi: 10.1016/j.mito.2009.12.148. PubMed DOI

Heide H., Bleier L., Steger M., Ackermann J., Drose S., Schwamb B., Zornig M., Reichert A.S., Koch I., Wittig I., et al. Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex. Cell Metab. 2012;16:538–549. doi: 10.1016/j.cmet.2012.08.009. PubMed DOI

Van Strien J., Haupt A., Schulte U., Braun H.-P., Cabrera-Orefice A., Choudhary J.S., Evers F., Fernandez-Vizarra E., Guerrero-Castillo S., Kooij T.W.A., et al. CEDAR, an online resource for the reporting and exploration of complexome profiling data. bioRxiv. 2020 doi: 10.1101/2020.12.11.421172. PubMed DOI

McKenzie M., Lazarou M., Ryan M.T. Chapter 18 Analysis of respiratory chain complex assembly with radiolabeled nuclear- and mitochondrial-encoded subunits. Methods Enzymol. 2009;456:321–339. doi: 10.1016/S0076-6879(08)04418-2. PubMed DOI

Arroyo J.D., Jourdain A.A., Calvo S.E., Ballarano C.A., Doench J.G., Root D.E., Mootha V.K. A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation. Cell Metab. 2016;24:875–885. doi: 10.1016/j.cmet.2016.08.017. PubMed DOI PMC

Stiburek L., Hansikova H., Tesarova M., Cerna L., Zeman J. Biogenesis of eukaryotic cytochrome c oxidase. Physiol. Res. 2006;55(Suppl. 2):S27–S41. PubMed

Huttemann M., Klewer S., Lee I., Pecinova A., Pecina P., Liu J., Lee M., Doan J.W., Larson D., Slack E., et al. Mice deleted for heart-type cytochrome c oxidase subunit 7a1 develop dilated cardiomyopathy. Mitochondrion. 2012;12:294–304. doi: 10.1016/j.mito.2011.11.002. PubMed DOI PMC

Fukuda R., Zhang H., Kim J.W., Shimoda L., Dang C.V., Semenza G.L. HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell. 2007;129:111–122. doi: 10.1016/j.cell.2007.01.047. PubMed DOI

Huttemann M., Lee I., Liu J., Grossman L.I. Transcription of mammalian cytochrome c oxidase subunit IV-2 is controlled by a novel conserved oxygen responsive element. FEBS J. 2007;274:5737–5748. doi: 10.1111/j.1742-4658.2007.06093.x. PubMed DOI

Kaila V.R., Verkhovsky M.I., Wikstrom M. Proton-coupled electron transfer in cytochrome oxidase. Chem. Rev. 2010;110:7062–7081. doi: 10.1021/cr1002003. PubMed DOI

Stoldt S., Wenzel D., Kehrein K., Riedel D., Ott M., Jakobs S. Spatial orchestration of mitochondrial translation and OXPHOS complex assembly. Nat. Cell Biol. 2018;20:528–534. doi: 10.1038/s41556-018-0090-7. PubMed DOI

Richter-Dennerlein R., Oeljeklaus S., Lorenzi I., Ronsor C., Bareth B., Schendzielorz A.B., Wang C., Warscheid B., Rehling P., Dennerlein S. Mitochondrial Protein Synthesis Adapts to Influx of Nuclear-Encoded Protein. Cell. 2016;167:471–483. doi: 10.1016/j.cell.2016.09.003. PubMed DOI PMC

Wang C., Richter-Dennerlein R., Pacheu-Grau D., Liu F., Zhu Y., Dennerlein S., Rehling P. MITRAC15/COA1 promotes mitochondrial translation in a ND2 ribosome-nascent chain complex. EMBO Rep. 2020;21:e48833. doi: 10.15252/embr.201948833. PubMed DOI PMC

Quiros P.M., Prado M.A., Zamboni N., D’Amico D., Williams R.W., Finley D., Gygi S.P., Auwerx J. Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals. J. Cell Biol. 2017;216:2027–2045. doi: 10.1083/jcb.201702058. PubMed DOI PMC

Fornuskova D., Stiburek L., Wenchich L., Vinsova K., Hansikova H., Zeman J. Novel insights into the assembly and function of human nuclear-encoded cytochrome c oxidase subunits 4, 5a, 6a, 7a and 7b. Biochem. J. 2010;428:363–374. doi: 10.1042/BJ20091714. PubMed DOI

Haplotype variability in mitochondrial rRNA predisposes to metabolic syndrome

Mitochondrial translation is the primary determinant of secondary mitochondrial complex I deficiencies

Generation and characterization of human U-2 OS cell lines with the CRISPR/Cas9-edited protoporphyrinogen oxidase IX gene