Abstract
In this work we studied molecular and functional effects of the loss of the smallest nuclear encoded subunit of cytochrome c oxidase COX8A in fibroblasts from a patient with a homozygous splice site mutation and in CRISPR/Cas9 genome-edited HEK293T cells. In both cellular model systems, between 20 to 30% of the residual enzymatic activity of cytochrome c oxidase (COX) was detectable. In immunoblots of BN-PAGE separated mitochondria from both cellular models almost no monomers and dimers of the fully assembled COX could be visualized. Interestingly, supercomplexes of COX formed with complex III and also with complexes I and III retained considerable immunoreactivity, while nearly no immunoreactivity attributable to subassemblies was found. That indicates that COX lacking subunit 8A is stabilized in supercomplexes, while monomers and dimers are rapidly degraded. With transcriptome analysis by 3′-RNA sequencing we failed to detect in our cellular models of COX8A deficiency transcriptional changes of genes involved in the mitochondrial unfolded protein response (mtUPR) and the integrated stress response (ISR). Thus, our data strongly suggest that the smallest subunit of cytochrome c oxidase COX8A is required for maintenance of the structural stability of COX monomers and dimers.
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Abbreviations
- COX:
-
cytochrome c oxidase
- IMM:
-
inner mitochondrial membrane
- OXPHOS:
-
oxidative phosphorylation
- RC:
-
respiratory chain
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Acknowledgements
This work is dedicated to Dr. Alexander A. Konstantinov who passed away in May 2020. The authors wish to thank Prof. Mike Ryan and Dr. David Stroud for providing the HEK293T cell line.
Funding
This work was financially supported by the DFG – Deutsche Forschungsgemeinschaft (grants KU 911/21-2 and KU 911/22-1 to W. S. K.; ZS 99/3-2 and ZS 99/4-1 to GZ), by the Polish National Science Center (grants No. 2019/34/A/NZ1/00352 to AS and 2015/18/E/NZ1/00737 to BK), and the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska–Curie grant agreement No. 665735 (Bio4Med).
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The authors declare no conflicts of interest in financial or any other sphere. All the procedures carried out in the research with participation of humans were in compliance with the ethical standards of the National Research Ethics Committee and with the Helsinki Declaration of 1964 and its subsequent changes or with comparable ethics standards. Informed voluntary consent was obtained from the participants of the study.
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Rotko, D., Kudin, A.P., Zsurka, G. et al. Molecular and Functional Effects of Loss of Cytochrome c Oxidase Subunit 8A. Biochemistry Moscow 86, 33–43 (2021). https://doi.org/10.1134/S0006297921010041
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DOI: https://doi.org/10.1134/S0006297921010041