Regulation of Respiratory Chain Supercomplex Formation and the Involvement of Rcf-proteins
by Bettina Homberg
Date of Examination:2021-05-19
Date of issue:2021-06-03
Advisor:Dr. Markus Deckers
Referee:Dr. Markus Deckers
Referee:Prof. Dr. Dörthe Katschinski
Persistent Address:
http://dx.doi.org/10.53846/goediss-8623
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Abstract
English
The mitochondrial oxidative phosphorylation machinery is comprised by five individual complexes and arranged in supramolecular structures, so-called supercomplexes, as it is shown for complex I, III, IV and V. In yeast S. cerevisiae complex I is substituted by three singular NADH dehydrogenases and therefore lacks multimeric complex I. Thus, it displays a suitable model for investigating the dynamics within supercomplex formation of complex III and IV. Although some players could be identified so far, it is mostly unknown which factors contribute to forming these complexes (III2IV(1-2)). Besides, the mechanism behind re-/arrangement of the supercomplexes to adapt to different conditions such as changing oxygen levels remains obscure. The yeast Rcf-proteins (respiratory supercomplex factors) Rcf1, Rcf2 and Rcf3 were identified as proteins with partly corresponding homologous domains and can interact independently with complex III and complex IV. This is a unique feature promoting an important role of the protein family for recruiting and/or association of the two complexes. Furthermore, they could serve as regulator of complex IV and prevent from undesired ROS production but the exact localization and functional domains remained unknown. A crosslinking approach on isolated yeast mitochondria could identify the interacting site of Rcf2 and Rcf3 at the periphery of complex IV contrary to first suggestions for the association at the interface of complex III and IV. During this investigation we discovered a putative novel subunit of complex IV – Min8 – and proved it as being involved in complex IV biogenesis. For determining the functionality of the different domains of Rcf-proteins, artificial fusion proteins out of Rcf1, Rcf2 and Rcf3 were constructed and expressed in various mutational strains. The effects of those proteins were assessed by various experiments addressing the respiration, complex biogenesis and ROS production. While Rcf1 operates on both, complex IV and supercomplex assembly suggesting an even higher dynamic than originally anticipated, Rcf2 and Rcf3 are acting predominantly at the site of supercomplexes. At the same time, we discovered that the functionality of transmembrane regions of Rcf2 depend on the overall topology of the protein. By further studying the involvement of the respective protein domains in supercomplex assembly, we found an interesting interaction of Rcf2 and its processed versions at the site of complex III2 under alleged hypoxic conditions in a COX5A mutant. This suggests a specific involvement within the adapting respiratory chain to altering oxygen levels.
Keywords: Mitochondria; Respiratory Chain; Cytochrome c Oxidase; Rcf-proteins; Supercomplexes