Biologische Hochschulschriften (Goethe-Universität)
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Calcium-activated potassium channels are fundamental regulators of neuron excitability. SK channels are activated by an intracellular increase of Ca++ (such as occurs during an action potential). They have a small single channel conductance (less than 20pS) and show no voltage dependence of activation. To date, there are only a few examples of high-resolution structures of eukaryotic membrane proteins. All of them were purified from natural sources. Since no abundant natural sources of eukaryotic K+ channels are available we overexpressed rSK2 in order to produce the quantities necessary for structural analysis. Unfortunately the Pichia pastoris expression system did not yield sufficient amount of pure protein, mainly because most of the protein was retained by in the ER and was only partially soluble. Subsequently, two constructs were expressed: SK2-FCYENE (containing a specific sequence that promotes surface expression), and SK2-q-CaM a concatamer of SK2 and calmodulin. Although these proved an improvement in terms of solubilisation, little improvement was found in terms of amounts of purified material obtained. For this reason we tested the Semliki Forest virus expression system, since the protein is expressed in a mammalian system where we hoped that it would be trafficked in the same way as in vivo. Using this system it was possible to express rSK2 and solubilise it with several detergents and to achieve much better purification. However, the levels were still not sufficient for high-resolution structural studies, although sufficient for single particle electron microscopy analysis.
Life of Varroa destructor, Anderson and Trueman, an ectoparasitic mite of honeybees, is divided into a reproductive phase in the bee brood and a phoretic phase during which the mite is attached to the adult bee. Phoretic mites leave the colony with workers involved in foraging tasks. Little information is available on the mortality of mites outside the colony. Mites may or not return to the colony as a result of death of the infested foragers, host change by drifting of foragers, or removal of mites outside the colony. That mites do not return to the colony was indicated by substantially higher infestation of outflying workers compared to the infestation of returning workers (Kutschker, 1999). The main objective of the study was to provide information whether V. destructor influences flight behaviour of foragers and consequently returning frequency of foragers to the colony. I first repeated the experiment of Kutschker (1999) examining the infestation of outflying and returning workers. Further, I registered flight duration of foragers using a video method. In this experiment I compared also the infestation and flight duration of bees of different genetic origin, Carnica from Oberursel and bees from Primorsky region. I investigated returning time of workers, returning frequency until evening, drifting to other colonies and orientation toward the nest entrance in the experiments in which workers were released in close vicinity of the colony. At last, I measured the loss of foragers in relation to colony infestation using a Bee Scan. Results from this study, listed below, showed considerable influence of V. destructor on flight behavior of foragers translating into loss of mites. Loss of mites with foragers add substantial component to mite mortality and was underestimated in previous studies. Such loss might be viewed as a mechanism of resistance against V. destructor. a) The mean infestation of outflying workers (0.019±0.018) was twice as the mean infestation of returning workers (0.009±0.018). The difference in the infestation between outflying and returning workers was more marked in highly infested colonies. b) Investigation of individually tagged workers by use of a two camera video recording device showed significantly higher infestation of outflying workers compared to returning workers. Mites were lost by the non returning of infested foragers (22%) and by loss of mites from foragers that returned to the colony without the mite (20%). A small portion of mites (1.8%) was gained. Loss of mites significantly exceeded mite gain. c) The flight duration of infested workers determined by using the same two camera video system was significantly higher in infested compared to uninfested workers of the same age that flew closest at time. The median flight duration of infested workers was 1.7 higher (214s) than the median duration of unifested workers (128s). d) Infested workers took 2.3 times longer to return to the colony than uninfested workers of the same age when released from the same locations, closest at time. The returning time increased with the distance of release. In a group of bees released simultaneously the infestation was higher in bees returning later and in those that did not return in the observation period of 15 min. e) Released workers did not return to the colony 1.5 more frequently than uninfested workers in evening. The difference in returning was significant for locations of 20 and 50m from the colony. No difference in returning between infested and uninfested workers were observed for the most distant location of 400m. f) No significant difference was found in returning time and/or in the returning frequency until evening between workers artificially infested overnight and naturally infested workers. Artificially infested workers returned later and less frequently than a control group indicating rapid influence of V. destructor on flight behavior of foragers. g) The orientation ability of infested workers toward the nest entrance was impaired. Infested workers compared to uninfested workers twice as often approached a dummy entrance before finding the nest entrance. h) No significant differences were found in drifting between infested and uninfested workers. Drifting in the neighboring nucleus colony occurred in about 1% occasions after release of marked workers. Similarly, more infested, but not significantly more infested workers (2.6%) entered a different colored hive than the same colored hive (1.9%). However, the number of drifting bees were to low to make results conclusive. i) The comparison between Carnica and Primorsky workers revealed higher infestation in Carnica compared to Primorsky. Further, Primorsky workers lost more mites during foraging due to mite loss from foragers and non returning of infested workers. No significant differences in flight duration were observed between the two bee stocks. j) Loss of foragers, as determined by the Bee Scan counts of outflying and returning foragers, and the infestation of outflying bees increased significantly over a period of 70 days. A colony with 7.7. higher infestation of outflying foragers lost 2.2. time more bees per flight per day compared to a low infested colony. k) The estimates of mite loss with foragers from mite population per day up to 3.1% exceeds approximately mite mortality of 1% within the colony as represented by counting dead mites on bottom board inserts.
Proton-translocating NADH:ubiquinone oxidoreductase (complex I) transports two electrons from NADH to membranal ubiquinone: in this process protons are translocated across the membrane, producing 40% of the total proton gradient between matrix side and intermembrane space. Mitochondrial complex I contains at least 46 subunits in mammals, and has a molecular weight of around 1000 kDa. Electronic microscopy analysis showed that complex I has an L-form, which consists of two domains: a peripheral “arm” (hydrophilic domain) and a membrane “arm” (hydrophobic domain). The peripheral domain, which protrudes into the matrix, contains one non-covalently bound flavin mononucleotide (FMN) and the iron-sulfur clusters N1a, N1b, N2, N3, N4 and N5 as redox active groups. They transport electrons from NADH to ubiquinone. Cluster N2 is supposed to be the immediate electron donor to ubiquinone by virtue of its highest and pH dependent redox midpoint potential (Em,7 –150 mV). The exact location of the tetra-nuclear cluster N2 is still object of discussion. The TYKY and the PSST subunits contain three binding motifs for tetranuclear clusters which are formed by twelve cysteins. In an effort to investigate the “ubiquinone reduction module” of complex I, in the first part of this work site directed mutagenesis of the TYKY and PSST subunits has been carried out. Mutant strains were characterised in terms of complex I content, catalytic activity and EPR signature of cluster N2. The second part of this work was aimed at developing a substrate inducible version of the internal alternative NADH:ubiquinone oxidoreductase (NDH2i). A substrate inducible NDH2i is expected to offer a “switch” between complex I activity dependent (no NDH2i activity) and independent (NDH2i activity) cell growth, by changing between activating and non-activating substrates. This strategy would allow the screening for two types of complex I mutants, which is a prerequisite for realising a random PCR mutagenesis of single subunits of complex I, that allows the production of a high number of point mutations in relatively short time. Y. lipolytica complex I deficiency mutant strains could be easily identified, by virtue of their inability to survive under complex I dependent growth conditions (no NDH2i activity). By this way, amino acids that have an important role for complex I structure or function could be identified by subsequent sequence analysis. Each of the twelve cysteines that form the above mentioned three binding motifs for iron-sulfur cluster have been mutagenised. In mutant mitochondrial membranes, no assembled complex I could be detected. From these data one may conclude that the mutagenised 6 SUMMARY 92 cysteines play an important role for complex I stability, or that are a prerequisite for complex I assembly in Y. lipolytica, but there is not direct evidence indicating that any of the four mutagenised residues acts as a ligand. Two aspartates in the PSST subunit, Asp-99 and Asp-115, were found to be essential for complex I catalytic activity. EPR spectroscopic analysis indicated that the electron transfer to N2 cluster was not blocked and implied that this was not the reason for the loss of catalytic activity. From these data it can be concluded that D99 and D115 play a vital role for complex I NADH:ubiquinone reductase activity, but are not ligands for cluster N2 and that their position is not close enough to the cluster to influence directly its electromagnetic environment. Three mutations, identified in the PSST and TYKY homologous subunits of patients affected with Leigh syndrome (V119M in PSST, P78L and R101H in TYKY) were reconstructed in the obligate aerobic yeast Y. lipolytica. This approach may help to understand the aetiology of the Leigh syndrome, in terms of the ability of complex I to oxidize NADH and to transport electrons. In fact, all three mutations showed effects on electron transport, reducing the VMax by about 50%. Mutant V119M in the PSST subunit, which had a lethal effect in two patients that were homozygous for this mutation, affects a fully conserved residue. Overall, the results from site directed mutagenesis carried out so far support the theory that the “catalytic core ” (N2 cluster and quinone binding site) of complex I has been evolved from the electron transfer module of the [Ni-Fe] hydrogenases. In fact, mutagenesis of residues that are fully conserved between complex I and [Ni-Fe] hydrogenases, showed dramatic effects on complex I in terms of assembly (cysteine mutants) or catalytic activity (D99-D115). Differently, changing aspartate 174 and glutamic acid 185 (not fully conserved, Fig 4.1A) had little or no effect on the Michaelis-Menten parameters and N2 EPR signal. In recent years Y. lipolytica has been developed as a yeast genetic system to study mitochondrial complex I. The present work introduced the promoter for the isocitrate lyase (pICL1) as a useful tool for the substrate selective expression of the internal version of the alternative NADH:ubiquinone oxidoreductase (pICL1-NDH2i). This allows to rescue complex I deficiencies “in vivo” selectively by growth on acetate (or ethanol) medium. The integration of the pICL1-NDH2i construct into the genome of Y. lipolytica and subsequent deletion of nuclear-coded subunits like PSST, TYKY and 49 kDa, would contribute to further develop this organism as a useful genetic model for studying subunits of mitochondrial complex I by site directed mutagenesis.
Ligands of Iron-Sulphur Cluster N2: In this work the ubiquinone reducing catalytic core of NADH:ubiquinone oxidoreductase (complex I) from Y. lipolytica was studied by a series of point mutations replacing conserved histidines or arginines in the 49-kDa subunit. Although the missing 4th ligand of cluster N2 could not be found in the 49-kDa subunit of complex I, it was clearly demonstrated that iron-sulphur cluster N2 resides directly on the interface between the PSST and 49-kDa subunits. The results presented in this work show that residues in the 49-kDa subunit have strong influence on this redox centre and also on catalytic activity. The strong influence of Arg-141 and His-226 residues in 49-kDa subunit on this cluster can be deducted from complete loss of N2 signals in EPR spectra such as in case of mutants H226A and R141A. In the case of mutant H226M the EPR signal from cluster N2 was shifted and cluster N2 even lost the pH dependence of its redox midpoint potential and became more similar to the other so called 'isopotential' clusters. Specifically in the case of mutants R141M and R141K the characteristic signature of cluster N2 became undetectable in EPR spectra. However, specific dNADH:DBQ oxidoreductase activity that could be inhibited with the specific complex I inhibitors DQA and rotenone was not absolutely abolished but rather reduced. These reductions in complex I activity did not correspond to similar reductions in the specific EPR signal of cluster N2 as it was observed in the His-226 mutant series. No indications could be found that these mutations had modified the magnetic properties of cluster N2, resulting in different EPR spectra. From these observations it could be concluded that both mutants R141K and R141M virtually or entirely lack iron-sulphur cluster N2. The rates in complex I activity could be reconciled with electron transfer theory: After removal of a single redox centre in a chain, electron transfer rates are predicted to be still much faster than steady-state turnover of complex I. These results from mutants R141K, R141M and also the result from mutant H226M that protons are being pumped even if the redox midpoint potential of cluster N2 is not pH dependent questions the prominent role in the catalytic mechanism of complex I that has been ascribed to cluster N2. Histidine 91 and 95 were found to be absolutely essential for activity of complex I since in both mutants complex I was fully assembled and artificial NADH:HAR activity was parental whereas complex I specific dNADH:DBQ activity was abolished. The signal from cluster N2 in EPR spectra was parental for all His-91 and -95 mutants. Mutations at the C-terminal arginine 466 affected ubiquinone affinity and inhibitor sensitivity but also destabilised complex I. All these results provide further support for a high degree of structural conservation between the 49-kDa subunit of complex I and the large subunit of water soluble [NiFe] hydrogenases. Remodelling of Human Pathogenic 49-kDa Mutations in Y. lipolytica: Y. lipolytica has been proven a good system for studying complex I properties and thus also for studying defects that occur in humans. In this work pathogenic mutations in the 49-kDa subunit of complex I were recreated and studied. The P232Q mutant showed non-assembly of complex I and this is probably the cause why this mutation was lethal in patients. The mutants R231Q and S416P were parental for the content, artificial and also specific complex I activity, Km for DBQ and IC50 for DQA. From these results we can conclude that these two residues Arg-228 and Ser-413 in mammalian cells have specific structural importance for the 49-kDa subunit even if they are not directly involved in catalytic process.