Assembly of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the correct insertion of subunits, accessory proteins, cofactors and prosthetic groups. Most of the fundamental biological knowledge concerning mitochondrial copper homeostasis and insertion of copper into COX derives from investigations in the yeast Saccharomyces cerevisiae. In this organism, Cox17 was the first identified factor involved in this pathway. It is a low molecular weight protein containing highly conserved twin Cx9C motifs and is localized in the cytoplasm as well as in the mitochondrial intermembrane space. It was shown that copper-binding is essential for its function.
So far, the role of Cox17 in the mammalian mitochondrial copper metabolism has not been well elucidated. Homozygous disruption of the mouse COX17 gene leads to COX deficiency followed by embryonic death, which implies an indispensable role for Cox17 in cell survival.
In this thesis, the role of COX17 in the biogenesis of the respiratory chain in HeLa cells was explored by use of siRNA. The knockdown of COX17 results in a reduced steady-state concentration of the copper-bearing subunits of COX and affects growth of HeLa cells accompagnied by an accumulation of ROS and apoptotic cells. Furthermore, in accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper center of COX, COX17 siRNA knockdown affects COX-activity and -assembly. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so called supercomplexes. While the abundance of COX dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. Accumulation of a novel ~150 kDa complex containing Cox1, but not Cox2 could be observed. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. Data presented here suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. Instead an interdependent assembly scenario for the formation of supercomplexes is proposed that requires the coordinated synthesis and association of individual complexes.:List of Figures and Tables
Abbreviations
Abstract
1 Indroduction
1.1 Mitochondria and the respriratory chain
1.2 The human mitochondrial genome
1.3 Homoplasmy and heteroplasmy
1.4 Mitochondrial disorders
1.4.1 Mutations in mitochondrial DNA
1.4.2 Mutations in nuclear DNA
1.5 Cytochrome c oxidase
1.6 Cytochrome c oxidase assembly
1.7 Copper and its trafficking in the cell
1.8 Mitochondrial copper metabolism
1.9 Cox17
1.10 Aims of the thesis
2 Materials and Methods
2.1 Materials
2.1.1 Chemicals and reagents
2.1.2 Antibodies
2.1.3 Plasmid
2.1.4 Kits
2.1.5 Marker
2.1.6 Enzymes
2.1.7 Primers
2.1.8 siRNAs
2.2 Methods
2.2.1 Cell culture
2.2.1.1 Cell culture: HeLa cells
2.2.1.2 Cell culture: HeLa cells transfected with pTurboRFP-mito
2.2.1.3 Subcultivation
2.2.1.4 Determination of cell number
2.2.1.5 Cell storage and thawing
2.2.2 Transient transfection of HeLa cells
2.2.3 Transfection of HeLa cells with pTurboRFP-mito
2.2.4 Immunocytochemistry
2.2.5 RNA extraction and quantitative real-time PCR
2.2.6 Isolation of mitochondria
2.2.6.1 Isolation of mitochondria for BN-PAGE Analysis
2.2.6.2 Isolation of mitochondria for localization studies
2.2.6.3 Isolation of bovine heart mitochondria
2.2.7 Proteinase K treatment of mitochondria and mitoplasts
2.2.8 Photometric activity assay
2.2.8.1 Citrate synthase activity
2.2.8.2 Cytochrome c oxidase activity
2.2.9 Blue native polyacrylamide gel electrophoresis (BN-PAGE)
2.2.9.1 In gel activity assay
2.2.9.2 2D-BN/SDS-PAGE
2.2.10 SDS-PAGE and Western blot analysis
2.2.11 Direct stochastic optical reconstruction microscopy (dSTORM)
2.2.12 Flow cytometric phenotyping
2.2.12.1 Determination of cell cyle phase
2.2.12.2 Identification of apoptotic cells
2.2.12.3 Detection of ROS
2.2.13 Oxygen measurement
2.2.14 Cu–His supplementation
3 Results
3.1 Subcellular localization of Cox17
3.2 Transient knockdown of COX17 in HeLa cells
3.2.1 Knockdown of COX17 mRNA
3.2.2 Knockdown of Cox17 protein
3.2.3 Effect of COX17 knockdown on the steady-state levels of OXPHOS
subunits
3.2.4 Effect of COX17 knockdown on the steady-state levels of copperbearing COX subunits
3.2.5 Subdiffraction-resolution fluorescence imaging
3.3 Phenotypical characterization
3.3.1 Growth analyis
3.3.2 Cell cycle analysis
3.3.3 Apoptosis assay
3.3.4 Detection of ROS
3.3.5 Oxygen measurement
3.4 Cytochrome c oxidase activity
3.5 Characterization of mt OXPHOS complexes
3.5.1 BN-PAGE/in gel activity assays
3.5.2 Supramolecular organization of COX
3.5.3 Molecular organization of Cox17
3.5.4 Molecular organisation of copper-bearing COX subunits Cox1 and
Cox2
3.5.5 Supramolecular organization of RC complexes
3.5.6 dSTORM of supercomplexes
3.6 Copper supplementation
4 Discussion
4.1 Dual localization of human Cox17
4.2 COX17 knockdown affects steady-state levels of copper-bearing
COX subunits Cox1 and Cox2
4.3 Supramolecular organization of RC is affected as an early response
to COX17 knockdown
4.4 Cox17 is primarily engaged in copper delivery to Sco1/Sco2
4.5 Copper supplementation alone cannot rescue the COX17
phenotype
4.6 Outlook
5 Appendix
6 PhD publication record
7 References
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:25394 |
| Date | 13 August 2010 |
| Creators | Oswald, Corina |
| Contributors | Rödel, Gerhard, Storch, Alexander, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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