Cunatova 2017 MiP2017
The major part of ATP in mammalian organisms is generated by the oxidative phosphorylation apparatus (OXPHOS), located in the inner mitochondrial membrane. Regulation of this process is partly accomplished by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain. The largest of these subunits is COX4, which is incorporated into the enzyme complex at early assembly stage, before the COX2 subunit is associated with COX1 to complete the functional chain of the electron-transferring cofactors.
Employing novel CRISPR CAS9-10A paired nickase technology, we created a unique HEK293-based cellular model with complete absence of subunit COX4. Double knock-out of both isoforms 1 and 2 of COX4 (COX4-1/4-2 KO) showed absence of the majority of COX subunits resulting in total COX deficiency. Electrophoretic analyses revealed that the residual content of the COX1 subunit was accumulated as the S1 assembly intermediate. Moreover, the levels of complex I subunits as well as the content of assembled complex were decreased in COX4-1/4-2 KO. On the other hand, levels of complexes II, III, and V were not significantly changed. Pulse-chase metabolic labelling of 13 proteins encoded by mtDNA and synthesized in mitochondria uncovered severely decreased translation of COX and complex I subunits, while complex III and V components were less affected. Partial impairment of mitochondrial proteosynthesis correlated with a decreased content of mitochondrial ribosomal proteins. As expected, mitochondrial respiration was undetected in the COX4-1/4-2 KO cells. The lack of mitochondrial ATP production was compensated by increased glycolytic capacity.
In summary, the HEK293-based cellular model of COX4-1/2 KO presented with the phenotype of complete COX absence and full reliance on glycolytic ATP production. We hypothesise that the decrease of mitochondrial protein synthesis represents a secondary effect of respiratory chain dysfunction.
Labels: MiParea: mtDNA;mt-genetics
Organism: Human Tissue;cell: HEK
Enzyme: Complex I, Complex II;succinate dehydrogenase, Complex III, Complex V;ATP synthase Regulation: ATP production
Affiliations and support
- Dept Bioenergetics, Inst Physiology CAS, Vídeňská 1083, Prague, Czech Republic. – [email protected]
- The project is supported by Grant Agency of the Czech Republic (16-13671S).