Techn. Universiẗat, Diss., 2001--Dresden.

Universiẗat, Diss., 2001--Frankfurt (Main).

Univ., FB Chemie, Diss., 1998--Marburg

COX deficiency in human presents a plethora of phenotypes which is not surprising given the complexity of the enzyme structure and the multiple factors and many steps required for its assembly. A functional COX requires three mitochondrially encoded subunits (Cox1p, Cox2p and Cox3p), at least 10 nuclearly encoded subunits, some of which are tissue specific, and a yet unknown number of assembly factors. Mutations in four of these factors, hSco1p, hSco2p, hCox10p and hSurf1p, have been associated with lethal COX deficiency in patients. Sco proteins, conserved from prokaryotes to eukaryotes, are probably involved in the insertion of copper in COX. The role of hSco1p and hSco2p in this process was investigated in this work. Moreover the importance of some hSco mutations found in patients was analysed. Both in vitro and in vivo analyses show that the hSco proteins are localised in the mitochondria. Both proteins are per se unable to substitute for ySco1p. However, a chimeric construct consisting of the N-terminal portion, the TM and a part of the C-terminal portion of ySco1p and the remaining C-terminal part derived from hSco1p was able to complement a ysco1 null mutant strain. This construct was used to define the role of a point mutation (P174L) found in the hSCO1 gene of infants suffering from ketoacidotic coma. These mutation was shown to affect the COX activity and the levels of Cox1p and Cox2p. The fact that copper was able to suppress this mutation, strongly outlined the importance of Sco proteins in the copper insertion in COX. The C-terminal portions of recombinant hSco1p and hSco2p were purified from E. coli by affinity chromatography. The purified proteins were subjected to atomic emission and absorption analyses and were shown to specifically bind copper. A stoichiometry of 1:1 for hSco2p and of 0,6:1 for hSco1p was determined. To identify the Aa residues involved in copper binding, in vitro mutagenesis was performed. hSco1p and hSco2p, lacking the cysteines of the predicted metal binding site CxxxC, show a dramatic decrease in the ability to bind copper. A model for the structure of the metal binding site in hSco proteins is proposed. hSco proteins could bind copper with trigonal coordination, involving the two cysteines of the CxxxC motif and a conserved histidine. The purified recombinant proteins were also used in an enzymatic assay to test their ability to reduce disulfide bridges, similar to thioredoxin-like proteins involved in the assembly of bacterial COX. Both hSco proteins were not able to act as thioredoxins suggesting a role for the hSco proteins as copper chaperones. To define the pathway of the copper transfer to COX, hSco proteins were tested for their ability to interact with hCox17p, a mitochondrial copper chaperone, and with Cox2p, which contains two copper ions. An interaction between hSco1p and Cox2p was detected. Both hSco proteins were shown to homomerise and to form heterodimers one with each other. Two mutations found in hSCO2 patients suffering from hypertrophic cardiomyopathy, (E140K and S225F) were shown not to affect the copper binding properties, the intracellular localisation and the ability to form homomers. In accordance to these data, a model is proposed in which hSco2p dimers transfer copper to hSco1p dimers. hSco1p dimers interact with COX and insert copper in the binuclear centre of Cox2p.

COX deficiency in human presents a plethora of phenotypes which is not surprising given the complexity of the enzyme structure and the multiple factors and many steps required for its assembly. A functional COX requires three mitochondrially encoded subunits (Cox1p, Cox2p and Cox3p), at least 10 nuclearly encoded subunits, some of which are tissue specific, and a yet unknown number of assembly factors. Mutations in four of these factors, hSco1p, hSco2p, hCox10p and hSurf1p, have been associated with lethal COX deficiency in patients. Sco proteins, conserved from prokaryotes to eukaryotes, are probably involved in the insertion of copper in COX. The role of hSco1p and hSco2p in this process was investigated in this work. Moreover the importance of some hSco mutations found in patients was analysed. Both in vitro and in vivo analyses show that the hSco proteins are localised in the mitochondria. Both proteins are per se unable to substitute for ySco1p. However, a chimeric construct consisting of the N-terminal portion, the TM and a part of the C-terminal portion of ySco1p and the remaining C-terminal part derived from hSco1p was able to complement a ysco1 null mutant strain. This construct was used to define the role of a point mutation (P174L) found in the hSCO1 gene of infants suffering from ketoacidotic coma. These mutation was shown to affect the COX activity and the levels of Cox1p and Cox2p. The fact that copper was able to suppress this mutation, strongly outlined the importance of Sco proteins in the copper insertion in COX. The C-terminal portions of recombinant hSco1p and hSco2p were purified from E. coli by affinity chromatography. The purified proteins were subjected to atomic emission and absorption analyses and were shown to specifically bind copper. A stoichiometry of 1:1 for hSco2p and of 0,6:1 for hSco1p was determined. To identify the Aa residues involved in copper binding, in vitro mutagenesis was performed. hSco1p and hSco2p, lacking the cysteines of the predicted metal binding site CxxxC, show a dramatic decrease in the ability to bind copper. A model for the structure of the metal binding site in hSco proteins is proposed. hSco proteins could bind copper with trigonal coordination, involving the two cysteines of the CxxxC motif and a conserved histidine. The purified recombinant proteins were also used in an enzymatic assay to test their ability to reduce disulfide bridges, similar to thioredoxin-like proteins involved in the assembly of bacterial COX. Both hSco proteins were not able to act as thioredoxins suggesting a role for the hSco proteins as copper chaperones. To define the pathway of the copper transfer to COX, hSco proteins were tested for their ability to interact with hCox17p, a mitochondrial copper chaperone, and with Cox2p, which contains two copper ions. An interaction between hSco1p and Cox2p was detected. Both hSco proteins were shown to homomerise and to form heterodimers one with each other. Two mutations found in hSCO2 patients suffering from hypertrophic cardiomyopathy, (E140K and S225F) were shown not to affect the copper binding properties, the intracellular localisation and the ability to form homomers. In accordance to these data, a model is proposed in which hSco2p dimers transfer copper to hSco1p dimers. hSco1p dimers interact with COX and insert copper in the binuclear centre of Cox2p.

Cytochrome c oxidase (COX) is one of two known Cu-containing enzymes in mitochondria. Delivery and insertion of copper into COX are very complex processes that require multiple steps and involve a large number of assisting factors. One of the involved components is Cox11p, a copper binding protein in the inner mitochondrial membrane that is conserved from prokaryotes to eukaryotes. Cox11p is essential for respiratory growth and implicated in the assembly of the CuB site located in subunit Cox1p of COX. In the thesis the topology of Cox11p was determined and evidence for its association with the mitochondrial translation machinery is provided. The interaction of Cox11p with mitoribosomes is mediated by its single evolutionary conserved transmembrane segment and appears to be indirect and mediated by another conserved membrane protein(s). A model is proposed in which the CuB site is co-translationally formed by a transient interaction between Cox11p and the nascent Cox1p in the mitochondrial intermembrane space. In addition the genetic and biochemical characterization of S. pombe Cox11p homologue was performed. Two versions of cox11+ gene are detected in a haploid S. pombe genome. Cells lacking either of the cox11+ copies remain respiratory competent, whereas deletion of both S. pombe cox11+ alleles appears to result in either spore lethality or in severe decrease of spores viability. Thus, both versions of SpCox11p are functional and important. In S. pombe Cox11p exists as a tandem with the mitoribosomal protein Rsm22p. This precursor protein is cleaved during mitochondrial import into two mature protein species corresponding to Rsm22p- and Cox11p-like moieties.

Diese Arbeit beschäftigt sich mit dem mitochondrialen Sco1-Protein der Hefe Saccharomyces cerevisiae sowie mit weiteren Vertretern der Sco-Proteinfamilie. Sco1p ist essenziell für die Assemblierung der Cytochrom c Oxidase (COX), dem terminalen Komplex der Atmungskette. Aufgrund von genetischen Daten wurde angenommen, dass es an der Insertion von Cu-Ionen in den COX-Komplex beteiligt ist. Dabei existieren zwei unterschiedliche Vorstellungen über seine Wirkweise: Einerseits könnte Sco1p als Cu-Chaperon selbst Cu-Ionen binden und anschließend auf die Cu-tragenden COX-Untereinheiten Cox1p und/oder Cox2p übertragen. Andererseits könnte es als Disulfidreduktase die in die Cu-Bindung involvierten Cysteinreste von Cox2p reduzieren und somit die Voraussetzung für eine Cu-Anheftung an Cox2p schaffen. In beiden Fällen wird den unter den Sco-Proteinen konservierten Aminosäuren Cystein(148), Cystein(152) und Histidin(239) eine Schlüsselrolle zugedacht. Es wurde gezeigt, dass diese Aminosäuren tatsächlich essenziell für die Funktion von Sco1p sind. Die Daten dieser Arbeit sprechen dafür, dass Sco1p als Cu-Chaperon fungiert: Sco1p zeigt keine Aktivität als Disulfidreduktase. Außerdem interagiert Sco1p mit Cox17p - dem Protein, das Cu-Ionen in die Mitochondrien importiert - und geht mit Cox2p eine Wechselwirkung ein. Im Rahmen der Interaktionsanalysen wurde weiterhin gezeigt, dass Sco1p homomere Komplexe ausbildet. Ein weiterer Schwerpunkt dieser Arbeit lag in Untersuchungen zum homologen Sco2p aus Saccharomyces cerevisiae, das im Gegensatz zu Sco1p nicht essenziell für eine funkionsfähige COX ist. Trotz seiner großen Ähnlichkeit ist Sco2p nicht in der Lage, die Funktion von Sco1p zu erfüllen. Im Rahmen dieser Arbeit konnt aber demonstriert werden, dass Sco2p zumindest teilweise Sco1p ersetzen kann. Somit kann für beide Proteine angenommen werden, dass sie überlappende Funktionen besitzen. Übereinstimmend wurde nachgewiesen, dass Sco2p - wie Sco1p - in der Lage ist, mit Cox17p und mit Cox2p zu interagieren und außerdem heteromere Komplexe mit Sco1p formiert. Es wurde ein Modell zur Wirkweise von Sco1p und Sco2p entwickelt.

Host specificity of tropical bark and ambrosia beetles was surveyed by rearing the beetles from 13 host trees in a lowland rainforest in Papua New Guinea. Ploeophagous bark beetles show narrow host specificity (usually family-level) typical for herbivorous insects, fungus-growing ambrosia beetles display almost no host fidelity. In both groups of species, the local diversity of plants is unlikely to have played a role in the clade diversification. The ambrosia symbiosis (scolytine beetles and fungi) is shown to be less specific than previously assumed, based on a discovery of new association between Scolytodes unipunctatus (genus of phloem feeders) and three unrelated groups of ambrosia fungi. The hypothesis that apparent polyphagy may conceal specialized populations within a species of a herbivore is tested for Homona mermerodes (Lepidoptera, Tortricidae). The haplotype diversity of the species show no congruence with host plants or geographic origin, confirming polyphagy of the species.

Diese Arbeit beschäftigt sich mit dem mitochondrialen Sco1-Protein der Hefe Saccharomyces cerevisiae sowie mit weiteren Vertretern der Sco-Proteinfamilie. Sco1p ist essenziell für die Assemblierung der Cytochrom c Oxidase (COX), dem terminalen Komplex der Atmungskette. Aufgrund von genetischen Daten wurde angenommen, dass es an der Insertion von Cu-Ionen in den COX-Komplex beteiligt ist. Dabei existieren zwei unterschiedliche Vorstellungen über seine Wirkweise: Einerseits könnte Sco1p als Cu-Chaperon selbst Cu-Ionen binden und anschließend auf die Cu-tragenden COX-Untereinheiten Cox1p und/oder Cox2p übertragen. Andererseits könnte es als Disulfidreduktase die in die Cu-Bindung involvierten Cysteinreste von Cox2p reduzieren und somit die Voraussetzung für eine Cu-Anheftung an Cox2p schaffen. In beiden Fällen wird den unter den Sco-Proteinen konservierten Aminosäuren Cystein(148), Cystein(152) und Histidin(239) eine Schlüsselrolle zugedacht. Es wurde gezeigt, dass diese Aminosäuren tatsächlich essenziell für die Funktion von Sco1p sind. Die Daten dieser Arbeit sprechen dafür, dass Sco1p als Cu-Chaperon fungiert: Sco1p zeigt keine Aktivität als Disulfidreduktase. Außerdem interagiert Sco1p mit Cox17p - dem Protein, das Cu-Ionen in die Mitochondrien importiert - und geht mit Cox2p eine Wechselwirkung ein. Im Rahmen der Interaktionsanalysen wurde weiterhin gezeigt, dass Sco1p homomere Komplexe ausbildet. Ein weiterer Schwerpunkt dieser Arbeit lag in Untersuchungen zum homologen Sco2p aus Saccharomyces cerevisiae, das im Gegensatz zu Sco1p nicht essenziell für eine funkionsfähige COX ist. Trotz seiner großen Ähnlichkeit ist Sco2p nicht in der Lage, die Funktion von Sco1p zu erfüllen. Im Rahmen dieser Arbeit konnt aber demonstriert werden, dass Sco2p zumindest teilweise Sco1p ersetzen kann. Somit kann für beide Proteine angenommen werden, dass sie überlappende Funktionen besitzen. Übereinstimmend wurde nachgewiesen, dass Sco2p - wie Sco1p - in der Lage ist, mit Cox17p und mit Cox2p zu interagieren und außerdem heteromere Komplexe mit Sco1p formiert. Es wurde ein Modell zur Wirkweise von Sco1p und Sco2p entwickelt.

Cytochrome c oxidase (COX) is one of two known Cu-containing enzymes in mitochondria. Delivery and insertion of copper into COX are very complex processes that require multiple steps and involve a large number of assisting factors. One of the involved components is Cox11p, a copper binding protein in the inner mitochondrial membrane that is conserved from prokaryotes to eukaryotes. Cox11p is essential for respiratory growth and implicated in the assembly of the CuB site located in subunit Cox1p of COX. In the thesis the topology of Cox11p was determined and evidence for its association with the mitochondrial translation machinery is provided. The interaction of Cox11p with mitoribosomes is mediated by its single evolutionary conserved transmembrane segment and appears to be indirect and mediated by another conserved membrane protein(s). A model is proposed in which the CuB site is co-translationally formed by a transient interaction between Cox11p and the nascent Cox1p in the mitochondrial intermembrane space. In addition the genetic and biochemical characterization of S. pombe Cox11p homologue was performed. Two versions of cox11+ gene are detected in a haploid S. pombe genome. Cells lacking either of the cox11+ copies remain respiratory competent, whereas deletion of both S. pombe cox11+ alleles appears to result in either spore lethality or in severe decrease of spores viability. Thus, both versions of SpCox11p are functional and important. In S. pombe Cox11p exists as a tandem with the mitoribosomal protein Rsm22p. This precursor protein is cleaved during mitochondrial import into two mature protein species corresponding to Rsm22p- and Cox11p-like moieties.