Evolution and diversification of the nuclear pore complex

The nuclear pore complex (NPC) is responsible for transport between the cytoplasm and nucleoplasm and one of the more intricate structures of eukaryotic cells. Typically composed of over 300 polypeptides, the NPC shares evolutionary origins with endo-membrane and intraflagellar transport system complexes. The modern NPC was fully established by the time of the last eukaryotic common ancestor and, hence, prior to eukaryote diversification. Despite the complexity, the NPC structure is surprisingly flexible with considerable variation between lineages. Here, we review diversification of the NPC in major taxa in view of recent advances in genomic and structural characterisation of plant, protist and nucleomorph NPCs and discuss the implications for NPC evolution. Furthermore, we highlight these changes in the context of mRNA export and consider how this process may have influenced NPC diversity. We reveal the NPC as a platform for continual evolution and adaptation

MidA is a putative methyltransferase that is required for mitochondrial complex I function

10 páginas, 6 figuras.-- et al.Dictyostelium and human MidA are homologous proteins that belong to a family of proteins of unknown function called DUF185. Using yeast two-hybrid screening and pull-down experiments, we showed that both proteins interact with the mitochondrial complex I subunit NDUFS2. Consistent with this, Dictyostelium cells lacking MidA showed a specific defect in complex I activity, and knockdown of human MidA in HEK293T cells resulted in reduced levels of assembled complex I. These results indicate a role for MidA in complex I assembly or stability. A structural bioinformatics analysis suggested the presence of a methyltransferase domain; this was further supported by site-directed mutagenesis of specific residues from the putative catalytic site. Interestingly, this complex I deficiency in a Dictyostelium midA- mutant causes a complex phenotypic outcome, which includes phototaxis and thermotaxis defects. We found that these aspects of the phenotype are mediated by a chronic activation of AMPK, revealing a possible role of AMPK signaling in complex I cytopathology.This work was supported by grants BMC2006-00394 and BMC2009-09050 to R.E. from the Spanish Ministerio de Ciencia e Innovación; to P.R.F. from the Thyne Reid Memorial Trusts and the Australian Research Council; to A.V. and O.G. from the Spanish National Bioinformatics Institute (www.inab.org), a platform of Genome Spain; to R.G. from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain (PI070167) and from the Comunidad de Madrid (GEN-0269/2006). S.C. is supported by a research contract from Consejería de Educación de la Comunidad de Madrid y del Fondo Social Europeo (FSE).Peer Reviewe

Dynamics and Architecture of the HOPS Tethering Complex in Yeast Vacuole Fusion

The evolvement of a complex endomembrane system, which is separating a variety of biochemical processes into distinct compartments, is a hallmark of eukaryotic cell development. Cellular homeostasis depends on the abilities of these lipid bilayer-enclosed organelles both to maintain distinct characteristics and to exchange materials. This is mainly achieved by a process called vesicular transport, which allows for a constant exchange of proteins, lipids and metabolites between different compartments. Lipid bilayer enclosed vesicles bud from the donor compartment, are transported to the target compartment and fuse with its surrounding membrane. The basic machineries involved in the process in budding and fusion have been intensely investigated in the last years. However, our knowledge about the processes, which confer target specificity and regulate intracellular membrane fusion, is still limited. Before fusion of two-compartments can occur, they have to specifically recognize and bind each other to allow for subsequent SNARE-induced fusion to take place. This early step in the fusion reaction is called tethering and involves the action of tethering factors and Rab GTPases. In my research, I focused on the HOPS protein complex that is implicated to function in the tethering process at the yeast vacuole, the fungal equivalent of lysosomes. To investigate the molecular properties that confer the functionality of this large hexameric complex, I established a method that allowed for the purification of substantial amounts of HOPS and investigated the interactions taking place between different subunits. This work paved the ground for electron microscopy analysis of the whole complex, which is currently performed and which yielded first, preliminary data. Furthermore, it allowed for the identification of the novel CORVET tethering complex at the endosome, which has several subunits in common with the HOPS complex. I was able to show that chimeric complexes exist, harboring both HOPS- and CORVET-specific subunits. This finding suggests that both complexes are dynamic and can interconvert. During my studies on the subunits’ interactions, I identified stable subcomplexes of the HOPS complex, for one of which I could show that it exists in vivo. The existence of such subcomplexes implies a much more dynamic functioning of the HOPS subunits than previously anticipated. This notion is further strengthened by my studies on the functionality of different subunits and subcomplexes. Intriguingly, my results show that the Rab Guanyl nucleotide exchange factor Vam6, which is needed to activate the vacuolar Rab Ypt7 for subsequent fusion and which is a component of the HOPS complex, loses its ability to interact with Ypt7 upon incorporation into a subcomplex or the fully assembled HOPS complex. In contrast to this, the subunit Vps41, which I could identify as a Rab effector, is active as a single protein and as part of the complex, suggesting that it might sequentially recruit the subcomplexes to assemble into the holo-complex at sites harboring active Ypt7. Another feature of Vps41 was addressed in my work. This protein was previously shown to be phosphorylated by the vacuolar casein kinase Yck3. I identified the phosphorylation sites in the Vps41 sequence, which allowed further studies on the effect of the phosphorylation on the functionality of the protein. In the phosphorylated state, the protein is displaced into the cytosol whereas it accumulates at endosomal-vacuolar fusion sites if phosphorylation is prevented. Intriguingly, we found that Ypt7 overexpression is able to partially rescue the loss of localization in the phosphomimetic mutant, indicating a cross-talk between these two layers of Vps41 regulation

Structure and functional architecture of the Mediator middle module from budding yeast

Mediator is a central coactivator complex required for regulated transcription by RNA polymerase (Pol) II in all eukaryotes. Budding yeast Mediator has a size of 1.4 MDa and consists of 25 subunits arranged in the head, middle, tail, and kinase modules. It is thought that Mediator forms an interface between the general RNA polymerase (RNA Pol) II machinery and transcriptional activators leading to promotion of pre-initiation complex (PIC) assembly. Mediator middle module from budding yeast consists of seven subunits Med1, 4, 7, 9, 10, 21, and 31 and was investigated during this thesis both structurally and functionally. Previously, the structure of a subcomplex comprising the C-terminal region of Med7 (Med7C) and Med21 was solved by X-ray crystallography and protocols for obtaining larger recombinant complexes were established in the laboratory. As structural and functional studies of Mediator are limited by the availability of protocols for the preparation of modules, I pursued these studies and established protocols for obtaining pure endogenous and recombinant complete Mediator middle module. Another subcomplex of the middle module, comprising the N-terminal part of subunit Med7 (Med7N) and the highly conserved subunit Med31 (Soh1) was successfully crystallized and its structure solved during this work. It is found, that it contains a unique structure and acts also as a functional entity (termed submodule). The Med7N/31 submodule shows a novel fold, with two conserved proline-rich stretches in Med7N wrapping around the righthanded four-helix bundle of Med31. In vitro, Med7N/31 is required for activated transcription and can act in trans when added exogenously. In vivo, Med7N/31 has a predominantly positive function on the expression of a specific subset of genes, including genes involved in methionine metabolism and iron transport. Comparative phenotyping and transcriptome profiling identified specific and overlapping functions of different Mediator submodules. Crystallization screening of larger middle module (sub-)complexes did not result in crystal formation, even after removal of some flexible regions. Thus alternative methods were applied to characterize the middle module topology. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion mobility mass spectrometry, limited proteolysis, light scattering, and small angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module, giving a potential explanation of why crystallization of larger complexes was unsuccessful. Moreover, based on systematic protein-protein interaction analysis, a new model for the subunit-subunit interaction network within the middle module of the Mediator is proposed. In this model, the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes Med7N/21, Med7C/31, and Med4/9. The subunits Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4. Furthermore, first steps in establishing an in vitro assay to test endogenous and recombinant middle module functionality have been initiated and will provide the basis for future studies

Funkční charakterizace nových komponent savčího mitochondriálního proteomu.

1 Abstrakt Mitochondriální proteom savců je tvořen ~1500 různými proteiny, z nichž není stále přibližně jedna čtvrtina plně charakterizována. Jedním z těchto proteinů je TMEM70 podílející se na biogenezi eukaryotické F1Fo-ATP syntázy. Mutace v TMEM70 způsobují izolovaný nedostatek ATP syntázy, což často vede u pacientů k letálním neonatálním mitochondriálním encefalokardiomyopatiím. Abychom porozuměli molekulárnímu mechanismu působení TMEM70, vytvořili jsme konstitutivní Tmem70 knockout myší model, který byl embryonálně letální s narušenou biogenesí ATP syntázy. Následně vytvořený myší indukovatelný Tmem70 knockout model byl letální v 8. týdnu po indukci. Především vykazoval funkční poruchu jater, což je v kontrastu k převážně kardiologickému fenotypu u lidí v počátku onemocnění. Analýza jaterních mitochondrií odhalila tvorbu labilních subkomplexů ATP syntázy postrádajících podjednotku c. V případě deficitu TMEM70 tedy nebyl inkorporován c-oligomer do ATP syntázy, což vedlo ke kritickému poškození produkce energie mitochondriemi, analogickému k dysfunkci TMEM70 u lidí. V modelech s deficitem TMEM70 dosáhl nedostatek ATP syntázy limitu pro jeho patologický projev, který jsme stanovili na 30 %. Pozorovali jsme také kompenzační zvýšení obsahu většiny komplexů OXPHOS, ale neočekávaně také ANT a PiC, komponent...1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...1. lékařská fakultaFirst Faculty of Medicin

Gene regulation during stress response transcription in Saccharomyces Cerevisiae

DYNAMIC TRANSCRIPTOME ANALYSIS MEASURES RATES OF MRNA SYNTHESIS AND DECAY IN YEAST To obtain rates of mRNA synthesis and decay in yeast, we established dynamic transcriptome analysis (DTA). DTA combines non-perturbing metabolic RNA labeling with dynamic kinetic modeling. DTA reveals that most mRNA synthesis rates are around several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. During the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. During the recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems.MEDIATOR PHOSPHORYLATION PREVENTS STRESS RESPONSE TRANSCRIPTION DURING NON STRESS CONDITIONS The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an endpoint of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast S. cerevisiae is phosphorylated at multiple sites a 17 out of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells

Development of native electrophoretic techniques for the isolation and characterization of mitochondrial complexes

In the first part of this work, the development of a novel two-dimensional native gel electrophoretic system (2-D BN/hrCNE) is described. This new system simplifies proteomics and biochemical analysis of mega protein complexes that are dissociated into the constituent complexes during 2-D electrophoresis, thereby reducing the complexity of the system considerably. This technique is exceptionally well suited for the in-gel detection of fluorescence-labeled proteins and the identification of individual enzymes and protein complexes by specific in-gel assays on native gels. In the second part, a new technique for the native immunoblotting of blue native gels (NIBN) was developed. This new technique allows for the identification of conformation-specific antibodies and the discrimination of antibodies recognizing linear epitopes of denatured proteins. Identification of conformation-specific antibodies is becoming increasingly important not only for the electron microscopic identification of native proteins but also for structural investigations in general. For this purpose, a commonly used protocol for Western blotting of blue native gels was modified in such a way that the native state of proteins and protein complexes was retained throughout the complete protocol. Instead of using the denaturing methanol in Western blotting protocols, mild detergents such as Tween 20, digitonin and Brij 35 were used for the obligatory removal of protein bound Coomassie-dye. The detection of respiratory complex I by activity staining on the blot membrane demonstrated that all three non-ionic detergents preserved the native state of complex I. The native state of the enzyme on the blot membrane was also monitored and confirmed with the help of a set of conformation-specific antibodies. NIBN can be used as a simple alternative method to the demanding native ELISA to screen for conformation-specific antibodies for structural studies. Unlike the time consuming native ELISA, NIBN does not require introduction of appropriate affinity tags and purification of the target protein by chromatography. Thus, the NIBN technique is especially useful for microscale projects and for proteins not easily accessible to genetic manipulation. The third part aimed at identification of the immediate protein interaction partners of Cox26, a hydrophobic protein that has been identified by our group as a novel component of yeast respiratory supercomplex. Multi-dimensional electrophoretic techniques were applied to identify non-covalent and covalent protein-protein interactions of Cox26. Three-dimensional electrophoresis (BNE/BNE/SDS-PAGE) gave both qualitative and quantitative information on covalent and non-covalent interactions of Cox26 and subunits of cytochrome c oxidase (complex IV), and showed that most of the Cox26 protein was non-covalently bound to the complex IV moiety of the respirasomes. Four-dimensional electrophoresis (BNE/BNE/SDS/SDS-PAGE) applying reducing and non-reducing conditions revealed that a minor fraction of Cox26 used a single cysteine residue in the center of a predicted transmembrane helix to form a disulfide bond with the Cox2 subunit of complex IV. A structural role of Cox26 protein in the assembly/stability of respiratory strings or patches has been suggested. The last part of this work focused on the isolation and characterization of native and morphologically intact nucleoids from bovine heart mitochondria, since only a few studies on nucleoid organization and composition have been carried out on mammalian tissues. The nucleoids appeared as distinct bands (apparent mass around 30-36 MDa) in blue native-PAGE on large pore gels. The moderate variation in particle size seems to reflect variations in the binding of loosely nucleoid-associated components like respiratory chain complexes. The estimated 30-36 MDa mass of nucleoids on native gels suggested that each nucleoid contains one mtDNA molecule provided that nucleoids contains equal amounts of DNA, protein and RNA (Miyakawa et al., 1987). Electron microscopic analysis of native nucleoids, which was performed by Dr. Karen Davies from the Max-Planck-Institute of Biophysics, Department of Structural Biology, Frankfurt, showed homogenous pool of particles with dimensions in 85x100 nm (in negative stain) and 100x150 nm (in cryo-tomography). Some of the nucleoids showed dumbbell-shape indicating dimerization of nucleoids. Recent EM and high-resolution light microscopy analysis of mammalian nucleoids have reported that nucleoids have a size of 70 nm in average. We also observed the same size of 70 nm in cryo-tomogramms when we applied harsher treatment of the native nucleoid particles with dimensions 100x150 nm. This observation is in agreement with published nucleoid sizes from both EM and high-resolution light microscopy, if we assume that native nucleoids have been dissociated under harsher treatment. The protein composition of bovine heart mt-nucleoids was analyzed by a number of complementary approaches to identify low and highly abundant, easily dissociating and tightly bound proteins, and to rank the 90 most abundant mt-nucleoid proteins. Native and denaturing gel electrophoresis techniques were coupled to LC-MS/MS to achieve a comprehensive protein component analysis. Qualitative MS analysis of highly purified nucleoids identified more than 400 proteins, including well known nucleoid proteins such as mitochondrial transcription factor and mtDNA-binding protein (TFAM), mitochondrial single-stranded DNA-binding protein (mtSSB), mitochondrial DNA polymerase subunit gamma-2 (POLG2) and mitochondrial helicase C26H10ORF2 protein (Twinkle). These proteins were ranked according to Mascot scores, and sorted according to presumed functional properties. A large group of proteins involved in protein synthesis comprised an almost complete set of subunits of mitochondrial ribosomes suggesting that the nucleoids contained significant amounts of mitochondrial ribosomes. Identification of sixty six proteins from the oxidative phosphorylation (OXPHOS) system comprising around 100 proteins in total suggested that OXPHOS proteins are also associated with mt-nucleoids. Interestingly, TFAM, described as a main mtDNA packaging factor in human and other mammalian cells, was not confirmed here as a major nucleoid component from bovine heart mitochondria. Fluorescence staining of protein spots on 2-D IEF/SDS gels clearly identified TFAM, but according to the stain intensity, this protein did not rank in the list of the 90 most abundant nucleoid proteins. Western blot analysis of sucrose gradient fractions revealed an enrichment of putative TFAM isoform in nucleoid fractions. Unexpectedly, the uncharacterized mitochondrial protein Es1 was identified as the most abundant nucleoid protein in bovine heart nucleoids instead. This implicates that nucleoid organization may differ between species and tissues. A functional characterization of Es1 is required to clarify its role in mammalian nucleoids.Das erste Ziel dieser Arbeit war die Entwicklung eines neuen zwei-dimensionalen Elektrophoresesystems, bei dem der native Zustand von Proteinen und Proteinkomplexen in beiden aufeinanderfolgenden Laufrichtungen erhalten bleiben sollte. Damit sollte die proteinchemische und biochemische Untersuchung riesiger Proteinkomplexe, wie der Superkomplexe der mitochondrialen Atmungskette, wesentlich erleichtert werden. Gleichzeitig sollten die gravierenden Nachteile der bisher bekannten zwei-dimensionalen Blau-Nativ Elektrophorese (2-D BN/BN Elektrophorese) vermieden werden. Dieses System hat nämlich den Nachteil, dass der verwendete Coomassie-Farbstoff in der zweiten Dimension der Elektrophorese die In-Gel-Farbreaktionen und die Detektierbarkeit von Fluoreszenz-markierten Proteinen beträchtlich stört. Deswegen wurde im neuen 2-D Elektrophoresesystem (2-D BN/hrCNE) die sogenannte “high-resolution clear native electrophoresis“ (hrCNE) für die zweite Laufrichtung verwendet, die eine ungestörte Proteinidentifizierung im Gel über Fluoreszenzmarkierung und enzymatische Tests erlaubt. Diese neue Kombination zweier Nativ-Elektrophoresen (BNE und hrCNE) vereinfacht proteomische und biochemische Analysen von Superkomplexen, weil BNE zur Isolierung von Superkomplexen geeignet ist und die nachfolgende hrCNE, Einzelkomplexe aus den Superkomplexen abspaltet und voneinander trennt. Damit wird die Komplexität des Systems beträchtlich reduziert. Das System eignet sich besonders für die Identifizierung von Membranproteinkomplexen anhand spezifischer Fluoreszenzmarkierungen oder anhand ihrer enzymatischen Eigenschaften, die direkt im Nativ-Gel analysiert werden können. Verwendung fand dieser Ansatz vor allem bei der Identifizierung und Charakterisierung von Superkomplexen der mitochondrialen Atmungskette aus unterschiedlichen Spezies und Geweben. Die Analyse der obligat aeroben Hefe Yarrowia lipolytica führte zur Identifizierung bisher nicht bekannter Typen von Superkomplexen. Ein zweites Ziel der Arbeit war die Entwicklung einer Methode für das Native Immunoblotting von Blau-Nativen Gelen (NIBN). Die neue Technik sollte die Identifizierung von konformationsspezifischen Antikörpern ermöglichen und die Unterscheidung von Antikörpern erlauben, die lineare Epitope von denaturierten Proteinen erkennen. Dies wird immer wichtiger für die elektronenmikroskopische Identifizierung von nativen Proteinen und generell für Strukturuntersuchungen. Zu diesem Zweck wurde ein allgemein verwendetes Protokoll für den Western Blott von Blau-Nativen Gelen auf solche Art und Weise modifiziert, dass der native Zustand von Proteinen und Protein-Komplexen in jedem Teilschritt des Protokolls bewahrt wurde. Anstelle des in Western Blott üblicherweise verwendeten Methanols, der leicht zur Proteindenaturierung führt, wurden milde Detergenzien wie Tween 20, Digitonin oder Brij 35 zur Entfärbung des Blotts verwendet. Als Modell für die Methodenentwicklung diente der größte und komplizierteste Atmungskettenkomplex aus Y. lipolytica, die NADH:Ubichinon Oxidoreduktase (Komplex I). Der native Zustand des Enzyms auf der Blott-Membran wurde anschließend durch Aktivitätsfärbung überprüft. Aktivitätsfärbungen des Komplex I auf der Membran, die mit milden Detergentien gewaschen wurden zeigten, dass die NADH Oxidationsdomäne des Enzymes nach Entfernung des Coomassie-Farbstoffes immer noch funktionell blieb. Dies wurde auch durch Verwendung monoklonaler Antikörper, die ausschließlich unter nativen Bedingungen an Komplex I binden, bestätigt. NIBN kann als eine einfache alternative Methode an Stelle des technisch anspruchsvollen und aufwändigen nativen ELISA verwendet werden, wenn ein Fundus von Antikörpern nach konformationsspezifischen Antikörpern für strukturelle, meist elektronenmikroskopische Studien durchsucht werden soll. Im Vergleich zur aufwändigen Probenvorbereitung beim nativen ELISA mit Affinitätschromatographie und Proteinaufreinigung, spart NIBN viel Zeit, weil keine “affinity-tags” in Proteine eingebaut werden müssen und keine Affinitätschromatograpie gebraucht wird. Die native Elektrophorese übernimmt selbst die Isolierung des interessierenden Proteins oder Proteinkomplexes. Besonders wertvoll ist die NIBN-Technik für Proben, die nicht leicht einer genetischen Manipulation und der Einführung von “affinity-tags” zugänglich sind. Der dritte Teil der Arbeit hatte zum Ziel, die direkten Protein-Protein Kontakte von Cox26, einem hydrophoben Protein, zu ermitteln, das Schägger und Kollegen in Hefe-Superkomplexen (Respirasome) identifiziert hatten. Die Superkomplexe von S. cerevisiae, die aus den Komplexen III und IV zusammengesetzt sind, enthalten mindestens 21 Untereinheiten. Zehn Untereinheiten davon sind dem Komplex III zuzurechnen und elf Untereinheiten dem Komplex IV. All diese Untereinheiten waren zunächst als potentielle Nachbarn des Cox26 Proteins anzusehen. Um die tatsächlich vorliegenden Topologien zu ermitteln, wurden multi-dimensionale elektrophoretische Techniken angewendet, die geeignet sind kovalente und nicht-kovalente Protein-Protein Wechselwirkungen von Cox26 zu identifizieren. Drei-dimensionale Elektrophorese (BN/BN/SDS-PAGE) zeigte, dass der Hauptanteil aller Cox26 Proteine nicht-kovalent an die Komplex IV-Komponente der Respirasome gebunden war. Vier-dimensionale Elektrophorese (BN/BN/SDS/SDS-PAGE) unter reduzierenden und nicht-reduzierenden Bedingungen zeigte, dass ein geringer Anteil der Cox26 Proteine an einen Cysteinrest der Cox2-Untereinheit des Komplex IV unter Ausbildung einer Disulfid-Brücke gebunden war. Dies legte nahe, dass Cox26 eine spezielle Rolle für den Komplex IV spielt, obwohl bisher keine konkrete Funktion nachgewiesen werden konnte und bei Cox26-Defizienz kein spezieller Phänotyp auftritt. Auf der Grundlage eines Strukturmodells des Hefe-Respirasoms und mit der neuen Erkenntnis, dass eine Disulfid-Brücke zwischen Cox2 und Cox 26 existiert, wurde eine strukturelle Rolle des Cox26 Proteins für die Assemblierung und/oder Stabilität von respiratorischen Ketten oder Netzen vorgeschlagen. Der vierte Abschnitt dieser Arbeit hatte die Isolierung und Charakterisierung von nativen und morphologisch intakten Nucleoiden aus Rinderherzmitochondrien zum Ziel. Mitochondrien besitzen eine eigene mitochondriale DNA (mtDNA), die im Matrixraum lokalisiert ist. ..

Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs