Investigation of early assembly of OXPHOS complexes during mitochondrial translation

Abstract

After a long time of coevolution between mitochondria and their eukaryotic host cells, the mitochondrial genome has diverged severely from it’s α- proteobacterial ancestors. Mitochondrial genes have mainly been transferred to the cytosolic genome and only a small number of highly hydrophobic core subunits of the oxidative phosphorylation system (OXPHOS) have been retained within the mitochondrial genome. Since the OXPHOS consist of subunits of both mitochondrial and cytosolic origins, the translation of mitochondrial proteins has to be coordinated with the translation and import of proteins translated on cytosolic ribosomes to provide efficient assembly of the OXPHOS subunits. One mechanism to regulate mitochondrial translation by cytosolic events is via regulators for mitochondrial translation. Such proteins are present during mitochondrial translation and can either interact with mitochondrial mRNAs, or associate with the mitochondrial ribosome. Even though many mitochondrial translation regulators are well characterized in yeast, little is known about them in human mitochondria. In this thesis, I was able to isolate the ribosome-associated early assembly intermediate of the cytochrome c oxidase, MITRAC, during the translation of COX1. Western blot and mass spectrometry analysis showed the presence of the insertase OXA1L within this complex and cryoEM analysis could localize the complex near the exit tunnel of the mitochondrial ribosome. In a second part, MITRAC15, which was characterized as a MITRAC subunit and a subunit of the PP-b early assembly intermediate, was functionally characterized. MITRAC15 could be shown to be a positive translation regulator of ND2 and is the first identified protein to possibly copurify with nascent chains of mitochondrial complex I proteins after puromycin treatment. Furthermore, it was shown that MITRAC15 is able to stall the assembly of the cytochrome c oxidase and therefore, links complex I and IV biogenesis.