Mammalian CcO (cytochrome c oxidase) is a hetero-oligomeric protein complex composed of 13 structural subunits encoded by both the mitochondrial and nuclear genomes. To study the role of nuclear-encoded CcO subunits in the assembly and function of the human complex, we used stable RNA interference of COX4, COX5A and COX6A1, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)-293 cells. Knockdown of Cox4, Cox5a and Cox6a resulted in reduced CcO activity, diminished affinity of the residual enzyme for oxygen, decreased holoCcO and CcO dimer levels, increased accumulation of CcO subcomplexes and gave rise to an altered pattern of respiratory supercomplexes. An analysis of the patterns of CcO subcomplexes found in both knockdown and overexpressing cells identified a novel CcO assembly intermediate, identified the entry points of three late-assembled subunits and demonstrated directly the essential character as well as the interdependence of the assembly of Cox4 and Cox5a. The ectopic expression of the heart/muscle-specific isoform of the Cox6 subunit (COX6A2) resulted in restoration of both CcO holoenzyme and activity in COX6A1-knockdown cells. This was in sharp contrast with the unaltered levels of COX6A2 mRNA in these cells, suggesting the existence of a fixed expression programme. The normal amount and function of respiratory complex I in all of our CcO-deficient knockdown cell lines suggest that, unlike non-human CcO-deficient models, even relatively small amounts of CcO can maintain the normal biogenesis of this respiratory complex in cultured human cells.
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Research Article|
May 27 2010
Novel insights into the assembly and function of human nuclear-encoded cytochrome c oxidase subunits 4, 5a, 6a, 7a and 7b
Daniela Fornuskova;
Daniela Fornuskova
1Department of Pediatrics, First Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, Prague 2, 128 08, Czech Republic
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Lukas Stiburek;
Lukas Stiburek
1Department of Pediatrics, First Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, Prague 2, 128 08, Czech Republic
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Laszlo Wenchich;
Laszlo Wenchich
1Department of Pediatrics, First Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, Prague 2, 128 08, Czech Republic
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Kamila Vinsova;
Kamila Vinsova
1Department of Pediatrics, First Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, Prague 2, 128 08, Czech Republic
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Hana Hansikova;
Hana Hansikova
1Department of Pediatrics, First Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, Prague 2, 128 08, Czech Republic
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Jiri Zeman
Jiri Zeman
1
1Department of Pediatrics, First Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, Prague 2, 128 08, Czech Republic
1To whom correspondence should be addressed (email jzem@lf1.cuni.cz).
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Publisher: Portland Press Ltd
Received:
November 05 2009
Revision Received:
March 12 2010
Accepted:
March 22 2010
Accepted Manuscript online:
March 22 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 428 (3): 363–374.
Article history
Received:
November 05 2009
Revision Received:
March 12 2010
Accepted:
March 22 2010
Accepted Manuscript online:
March 22 2010
Citation
Daniela Fornuskova, Lukas Stiburek, Laszlo Wenchich, Kamila Vinsova, Hana Hansikova, Jiri Zeman; Novel insights into the assembly and function of human nuclear-encoded cytochrome c oxidase subunits 4, 5a, 6a, 7a and 7b. Biochem J 15 June 2010; 428 (3): 363–374. doi: https://doi.org/10.1042/BJ20091714
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