Ubiquinol-cytochrome c reductase (complex III) is a central component of the respiratory chain of the inner mitochondrial membrane. It transfers electrons from reduced ubiquinone to ferricytochrome c. Correctly assembled and functional complex III is an essential prerequisite for oxidative energy metabolism. Complex III deficiency has been reported to be associated with several neurodegenerative diseases. Formation and assembly of complex III requires a multitude of specific nuclearly encoded proteins. For example, gene specific translational activators for cytochrome b synthesis as well as three non-subunit proteins, which are important for assembly and/or stability have been detected. The role of Bcs1p in assembly of Rieske FeS protein and Qcr10p into complex III has been clasified recently. The role of the two putative chaperones, Cbp3p and Cbp4p, is not known. In spite of the similar phenotype of cbp3D and cbp4D strains, that suggests the role of both proteins in the same step of complex III assembly, we were able for the first time to demonstrate differences on the molecular level between both deletion mutants. We show by BN-PAGE that cbp3D and cbp4D mutants are disturbed in complex III assembly and accumulate intermediate-sized forms of the complex. Moreover deletion of CBP3 interferes with the formation of complex III/IV supracomplexes. Our studies show that Cbp3p and Cbp4p interact and are present in high molecular weight complexes, some of which might represent intermediates of complex III assembly. Overexpression of Cbp4p cannot substitute for the function of Cbp3p, but high level expression of Cbp3p can partially compensate for the lack of Cbp4p. Because lipids play an important role for complex III assembly and stability, we analysed the mitochondrial lipid composition of cbp3D and cbp4D mutants. Our data show that mitochondria of both mutants exhibit a wild type-like lipid composition, that favors the idea that Cbp3p and Cbp4p are specific assembly factors for complex III rather than components of the mitochondrial lipid metabolism. By complementation studies we have shown that Cbp3 proteins of S. cerevisiae, S. pombe and human are (partially) functional homologues. A yeast model based on chimeric constructs of S. cerevisiae and human proteins was constructed, which allows to test the pathogenicity of human mutations. To define the role/s of Cbp3p and Cbp4p in the assembly pathway of complex III, interactions of selected subunits with both assembly factors were analysed by TAP- or co-immunoprecipitation. Based on the results of Cbp3p and Cbp4p topologies, BN-PAGE analysis of null mutant strains and interaction studies a model for complex III assembly and the roles of Cbp3p and Cbp4p in this process are proposed. I present a hypothesis, according to which Cbp3p and Cbp4p form a ?scaffold? for the assembly of all three putative sub-complexes, may act independently in the first steps of bc1 complex assembly (e. g. the formation of sub-complexes) and interact together to assist the final assembly of sub-complexes into a mature enzyme. / Der Ubiquinol-Cytochrom c Reductase (Komplex III) ist eine zentrale Komponente der Atmungskette der inneren Mitochondrienmembran. Er transferiert Elektronen von reduziertem Ubiquinon auf Ferricytochrom c. Der korrekt assemblierte und funktionale Komplex III ist eine essenzielle Voraussetzung für den oxidativen Energiemetabolismus. Komplex III Defizienz ist assoziiert mit verschiedenen neurodegenerativen Krankheiten...
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:swb:14-1122027648324-54732 |
| Date | 22 June 2005 |
| Creators | Kronekova, Zuzana |
| Contributors | Technische Universität Dresden, Mathematik und Naturwissenschaften, Biologie, Institut für Genetik, Prof. Dr. Gerhard Rödel, Prof. Dr. Michael Göttfert, Dr. Leo Nijtmans, Prof. Dr. Gerhard Rödel |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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