This work focused on the further characterisation of Idhp and of the Krebs cycle enzymes citrate synthase 1 (Cit1p) and malate dehydrogenase 1 (Mdh1p) both of which have been identified as RNA-binding proteins without known RNA recognition motifs. Besides analysing their effects on mitochondrial translation and their organisation in protein complexes the work focused on the characterisation of the RNA-binding properties of recombinant Cit1p and Mdh1p: · Cit1p and Mdh1p play no essential role in mitochondrial protein synthesis. · Idhp is in a complex of molecular weight larger than the cytochrome c oxidase (250 kDa). · Cit1p and Mdh1p are in mitochondrial complexes smaller than 250 kDa. · 1000-fold molar excess of tRNA referring to COX2 leader RNA did not inhibit the RNA-binding of Cit1p and Mdh1p. · Cit1p and Mdh1p bind mitochondrial mRNAs (sense and antisense). The influence of cofactors and substrates on RNA-binding was analysed in order to reveal a possible link between the enzymatic function and the property of RNA-binding: · Acetyl-CoA and ATP inhibited the RNA-binding of Cit1p and Mdh1p at a concentration of 5 mM.

In the presented study, dimorphic yeasts were applied as model organisms to study the growth of fungal mycelia. When environmental conditions are chosen appropriately, yeast colonies are built up of well separated individual cells. Thus, in contrast to fungal mycelia the translocation of nutrients and information within the colony can be neglected. The study focuses on the question of how the growth behaviour of a population of single cells is regulated, and which differences can be expected when nutrient translocation actually occurs. To answer this question, at first, an effective method for the highly resolved estimation of biomass distributions inside the colonies was developed. This method facilitates a dynamic non-invasive monitoring of colony development. Furthermore, mathematical models were established which describe the development of the colonies based on the behaviour of discrete individual cells. Growth simulations allow a quantitative prediction, and, thereby, an in silico testing of hypothetic regulatory mechanisms. The growth behaviour of yeast colonies was investigated applying the model organisms Candida boidinii and Yarrowia lipolytica. The yeasts were cultivated on solid agar substrates at various degrees of carbon and nitrogen limitation, respectively. The highest gain of understanding was achieved for the growth of both yeasts on glucose as the limiting carbon source: Investigations showed that mycelial yeast colonies adapt to declining nutrient concentrations by decreasing the cell density in their mycelium while the growth rate of the colony diameter remains constant. Under glucose limitation, the yeast C. boidinii grows diffusion-limited, i.e., the growth of the population is controlled by the amount of nutrient that diffuses towards the colony. The cessation of growth coincides with the depletion of the primary nutrient source glucose from the growth substrate. In contrast to these findings, it was shown that Y. lipolytica colonies continue to extend even after the complete consumption of glucose. In the absence of the primary nutrient source, the yeast assimilates biomass from the inner colony regions to facilitate the growth of the population. The suggested mechanism of coupled extension and decay processes was verified by a number of experiments. However, the mechanism which facilitates the transport of decay products to the growing colony boundary, i.e., the actual nature of the decay process, remains unclear. Mathematical simulations show that a continuous colony extension on the decay products of dying cells cannot be explained by the assumption that colonies are built up of uncoordinatedly growing single cells. Therefore, a hypothesis for the growth of Y. lipolytica colonies was derived which suggests that these populations are built up of tube-like hyphal cells. Accordingly, the measured drop of biomass density in the inner colony areas is the consequence of a cytoplasm transport towards the growing edge of the mycelium where it is assimilated as a secondary nutrient resource in the absence of glucose. It has to be emphasized that this hypothesis also provides a mechanistic explanation for the vacuolisation of hyphae in mycelia of higher fungi. / In der vorgestellten Arbeit wurden dimorphe Hefen als Modellorganismen für die Untersuchung des Wachstums von Pilzmyzelien eingesetzt. Bei geeigneter Wahl der Umgebungsbedingungen sind Hefekolonien aus Einzelzellen aufgebaut, wodurch im Gegensatz zu Myzelien höherer Pilze der Transport von Nährstoffen und Informationen innerhalb der Kolonie vernachlässigt werden kann. Im Mittelpunkt der Untersuchungen stand die Frage, wie das Wachstumsverhalten einer Population individueller Zellen reguliert ist, bzw. welche Unterschiede sich ergeben, wenn ein Nährstofftransport tatsächlich stattfindet. Um diese Fragestellungen bearbeiten zu können, wurde zunächst eine effektive Methode zur hoch ortsaufgelösten Bestimmung der Biomasseverteilung innerhalb der Kolonien entwickelt. Diese Methode ermöglicht ein dynamisches nichtinvasives Monitoring der Entwicklung einer Kolonie. Weiterhin wurden mathematische Modelle entwickelt, die das Wachstumsverhalteeiner Population auf der Grundlage des Verhaltens von diskreten Einzelzellen beschreibt. Die Wachstumssimulationen erlauben quantitative Vorhersagen und damit ein in silico Testen der Auswirkungen von hypothetischen Regulationsmechanismen. Das Wachstumsverhalten von Hefekolonien wurde anhand der Modellorganismen Candida boidinii und Yarrowia lipolytica untersucht. Die Hefen wurden auf festen Agar-Nährböden bei verschieden starker Kohlenstoff- und Stickstofflimitation kultviert. Der größte Erkenntnisgewinn wurde dabei für das Wachstum beider Hefen auf Glukose als limitierender Kohlenstoffquelle erzielt: Die Untersuchungen ergaben, dass myzelartig wachsende Hefekolonien bei sinkenden Nährstoffkonzentrationen eine geringere Zelldichte aber einen konstante Wachstumsgeschwindigkeit des Koloniedurchmessers aufweisen. Die Hefe C. boidinii wächst unter Glukoselimitation diffusionslimitiert, d.h. das Wachstum der Population wird durch die Menge der zur Kolonie diffundierenden Nährstoffe bestimmt. Der Abbruch des Koloniewachstums fällt mit dem Verbrauch der primären Nähstoffquelle Glukose zusammen. Im Gegensatz dazu konnte für das Wachstum von Y. lipolytica gezeigt werden, dass sich die Kolonien auch nach dem vollständigen Verbrauch von Glukose weiter ausdehnen. Im Abwesenheit der primären Nährstoffquelle nutzt die Hefe Zerfallsprodukte eigener Zellmasse aus dem Inneren der Kolonie als Nährstoff, um das weitere Wachstum der Population zu gewährleisten. Während der vorgeschlagene gekoppelte Ausdehnungs- und Zerfallprozess durch eine Reihe von Versuchen experimentell abgesichert wurde, bleibt der Mechanismus des Transports der Zerfallsprodukte zum Kolonierand, bzw. die eigentliche Natur des Zerfallsprozesses unklar. Simulationsrechnungen ergaben, dass eine kontinuierliche Ausdehnung der Kolonie auf Zellzerfallsprodukten sterbender Zellen nicht durch die Annahme erklärt werden kann, dass die Kolonien aus unkoordiniert wachsenden Einzelzellen aufgebaut sind. Aus diesem Grunde wurde für das Wachstum von Y. lipolytica die Hypothese abgeleitet, dass das Myzelium dieser Hefe aus schlauchartigen Hyphenzellen aufgebaut ist. Der gemessene Abfall der Biomassekonzentration im Kolonieinneren ist demnach die Konsequenz des Transports von Zytoplasma hin zum wachsenden Kolonierand, wo es in Abwesenheit von Glukose als sekundäre interne Nährstoffquelle assimiliert wird. Es ist zu beachten, dass diese Hypothese auch eine mechanistische Erklärung für die Ursachen der Vakuolisierung in Myzelien höherer filamentöser Pilze gibt.

The studies presented here were focused on the understanding of the principles for protein sorting from the Golgi to the cell surface. As a marker protein we used Fus1p, a type I plasma membrane protein that is O-glycosylated on the extracellular domain and plays a role in cell fusion during yeast mating. Additionally, we analyzed mechanisms responsible for asymmetric distribution of Fus1p in mating cells. We demonstrated that the glycans attached to the protein act as a sorting determinant for protein transport to the cell surface. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A similar defect in exocytosis was observed when a Fus1p mutant lacking the O-glycosylated domain was expressed in wild-type cells, however, the cell surface delivery could be rescued if the 33 amino acid portion of the Fus1p ectodomain, containing 15 potentially glycosylated sites was added to the protein. It was previously well documented in epithelial cells that different types of protein glycosylation and association with lipid rafts play a role of determinants for protein delivery to the apical plasma membrane. However, otherwise the machinery responsible for cargo sorting to the apical membrane is poorly understood. Our finding that also in yeast, protein glycosylation can function as a sorting determinant provides a new possibility to investigate underlying mechanisms...

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.

The studies presented here were focused on the understanding of the principles for protein sorting from the Golgi to the cell surface. As a marker protein we used Fus1p, a type I plasma membrane protein that is O-glycosylated on the extracellular domain and plays a role in cell fusion during yeast mating. Additionally, we analyzed mechanisms responsible for asymmetric distribution of Fus1p in mating cells. We demonstrated that the glycans attached to the protein act as a sorting determinant for protein transport to the cell surface. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A similar defect in exocytosis was observed when a Fus1p mutant lacking the O-glycosylated domain was expressed in wild-type cells, however, the cell surface delivery could be rescued if the 33 amino acid portion of the Fus1p ectodomain, containing 15 potentially glycosylated sites was added to the protein. It was previously well documented in epithelial cells that different types of protein glycosylation and association with lipid rafts play a role of determinants for protein delivery to the apical plasma membrane. However, otherwise the machinery responsible for cargo sorting to the apical membrane is poorly understood. Our finding that also in yeast, protein glycosylation can function as a sorting determinant provides a new possibility to investigate underlying mechanisms...

In the presented study, dimorphic yeasts were applied as model organisms to study the growth of fungal mycelia. When environmental conditions are chosen appropriately, yeast colonies are built up of well separated individual cells. Thus, in contrast to fungal mycelia the translocation of nutrients and information within the colony can be neglected. The study focuses on the question of how the growth behaviour of a population of single cells is regulated, and which differences can be expected when nutrient translocation actually occurs. To answer this question, at first, an effective method for the highly resolved estimation of biomass distributions inside the colonies was developed. This method facilitates a dynamic non-invasive monitoring of colony development. Furthermore, mathematical models were established which describe the development of the colonies based on the behaviour of discrete individual cells. Growth simulations allow a quantitative prediction, and, thereby, an in silico testing of hypothetic regulatory mechanisms. The growth behaviour of yeast colonies was investigated applying the model organisms Candida boidinii and Yarrowia lipolytica. The yeasts were cultivated on solid agar substrates at various degrees of carbon and nitrogen limitation, respectively. The highest gain of understanding was achieved for the growth of both yeasts on glucose as the limiting carbon source: Investigations showed that mycelial yeast colonies adapt to declining nutrient concentrations by decreasing the cell density in their mycelium while the growth rate of the colony diameter remains constant. Under glucose limitation, the yeast C. boidinii grows diffusion-limited, i.e., the growth of the population is controlled by the amount of nutrient that diffuses towards the colony. The cessation of growth coincides with the depletion of the primary nutrient source glucose from the growth substrate. In contrast to these findings, it was shown that Y. lipolytica colonies continue to extend even after the complete consumption of glucose. In the absence of the primary nutrient source, the yeast assimilates biomass from the inner colony regions to facilitate the growth of the population. The suggested mechanism of coupled extension and decay processes was verified by a number of experiments. However, the mechanism which facilitates the transport of decay products to the growing colony boundary, i.e., the actual nature of the decay process, remains unclear. Mathematical simulations show that a continuous colony extension on the decay products of dying cells cannot be explained by the assumption that colonies are built up of uncoordinatedly growing single cells. Therefore, a hypothesis for the growth of Y. lipolytica colonies was derived which suggests that these populations are built up of tube-like hyphal cells. Accordingly, the measured drop of biomass density in the inner colony areas is the consequence of a cytoplasm transport towards the growing edge of the mycelium where it is assimilated as a secondary nutrient resource in the absence of glucose. It has to be emphasized that this hypothesis also provides a mechanistic explanation for the vacuolisation of hyphae in mycelia of higher fungi. / In der vorgestellten Arbeit wurden dimorphe Hefen als Modellorganismen für die Untersuchung des Wachstums von Pilzmyzelien eingesetzt. Bei geeigneter Wahl der Umgebungsbedingungen sind Hefekolonien aus Einzelzellen aufgebaut, wodurch im Gegensatz zu Myzelien höherer Pilze der Transport von Nährstoffen und Informationen innerhalb der Kolonie vernachlässigt werden kann. Im Mittelpunkt der Untersuchungen stand die Frage, wie das Wachstumsverhalten einer Population individueller Zellen reguliert ist, bzw. welche Unterschiede sich ergeben, wenn ein Nährstofftransport tatsächlich stattfindet. Um diese Fragestellungen bearbeiten zu können, wurde zunächst eine effektive Methode zur hoch ortsaufgelösten Bestimmung der Biomasseverteilung innerhalb der Kolonien entwickelt. Diese Methode ermöglicht ein dynamisches nichtinvasives Monitoring der Entwicklung einer Kolonie. Weiterhin wurden mathematische Modelle entwickelt, die das Wachstumsverhalteeiner Population auf der Grundlage des Verhaltens von diskreten Einzelzellen beschreibt. Die Wachstumssimulationen erlauben quantitative Vorhersagen und damit ein in silico Testen der Auswirkungen von hypothetischen Regulationsmechanismen. Das Wachstumsverhalten von Hefekolonien wurde anhand der Modellorganismen Candida boidinii und Yarrowia lipolytica untersucht. Die Hefen wurden auf festen Agar-Nährböden bei verschieden starker Kohlenstoff- und Stickstofflimitation kultviert. Der größte Erkenntnisgewinn wurde dabei für das Wachstum beider Hefen auf Glukose als limitierender Kohlenstoffquelle erzielt: Die Untersuchungen ergaben, dass myzelartig wachsende Hefekolonien bei sinkenden Nährstoffkonzentrationen eine geringere Zelldichte aber einen konstante Wachstumsgeschwindigkeit des Koloniedurchmessers aufweisen. Die Hefe C. boidinii wächst unter Glukoselimitation diffusionslimitiert, d.h. das Wachstum der Population wird durch die Menge der zur Kolonie diffundierenden Nährstoffe bestimmt. Der Abbruch des Koloniewachstums fällt mit dem Verbrauch der primären Nähstoffquelle Glukose zusammen. Im Gegensatz dazu konnte für das Wachstum von Y. lipolytica gezeigt werden, dass sich die Kolonien auch nach dem vollständigen Verbrauch von Glukose weiter ausdehnen. Im Abwesenheit der primären Nährstoffquelle nutzt die Hefe Zerfallsprodukte eigener Zellmasse aus dem Inneren der Kolonie als Nährstoff, um das weitere Wachstum der Population zu gewährleisten. Während der vorgeschlagene gekoppelte Ausdehnungs- und Zerfallprozess durch eine Reihe von Versuchen experimentell abgesichert wurde, bleibt der Mechanismus des Transports der Zerfallsprodukte zum Kolonierand, bzw. die eigentliche Natur des Zerfallsprozesses unklar. Simulationsrechnungen ergaben, dass eine kontinuierliche Ausdehnung der Kolonie auf Zellzerfallsprodukten sterbender Zellen nicht durch die Annahme erklärt werden kann, dass die Kolonien aus unkoordiniert wachsenden Einzelzellen aufgebaut sind. Aus diesem Grunde wurde für das Wachstum von Y. lipolytica die Hypothese abgeleitet, dass das Myzelium dieser Hefe aus schlauchartigen Hyphenzellen aufgebaut ist. Der gemessene Abfall der Biomassekonzentration im Kolonieinneren ist demnach die Konsequenz des Transports von Zytoplasma hin zum wachsenden Kolonierand, wo es in Abwesenheit von Glukose als sekundäre interne Nährstoffquelle assimiliert wird. Es ist zu beachten, dass diese Hypothese auch eine mechanistische Erklärung für die Ursachen der Vakuolisierung in Myzelien höherer filamentöser Pilze gibt.

This work focused on the further characterisation of Idhp and of the Krebs cycle enzymes citrate synthase 1 (Cit1p) and malate dehydrogenase 1 (Mdh1p) both of which have been identified as RNA-binding proteins without known RNA recognition motifs. Besides analysing their effects on mitochondrial translation and their organisation in protein complexes the work focused on the characterisation of the RNA-binding properties of recombinant Cit1p and Mdh1p: · Cit1p and Mdh1p play no essential role in mitochondrial protein synthesis. · Idhp is in a complex of molecular weight larger than the cytochrome c oxidase (250 kDa). · Cit1p and Mdh1p are in mitochondrial complexes smaller than 250 kDa. · 1000-fold molar excess of tRNA referring to COX2 leader RNA did not inhibit the RNA-binding of Cit1p and Mdh1p. · Cit1p and Mdh1p bind mitochondrial mRNAs (sense and antisense). The influence of cofactors and substrates on RNA-binding was analysed in order to reveal a possible link between the enzymatic function and the property of RNA-binding: · Acetyl-CoA and ATP inhibited the RNA-binding of Cit1p and Mdh1p at a concentration of 5 mM.

In Saccharomyces cerevisiae, experiments with temperature-sensitive mutants of the PI4-kinase Pik1p revealed that the PI4P pool generated by this enzyme is essential for Golgi morphology and normal secretory function and that the PI4P pool at the Golgi represents a regulatory signal on its own. In order to function as a spatial and temporal regulator of membrane traffic, PI4P synthesis and turnover must be tightly regulated. It remains elusive which factors are involved in the targeting and regulation of Pik1p. Little is also known about PI4P binding proteins mediating the effects of this phosphoinositide on Golgi function. Since it has been shown that multiple pathways leave the Golgi towards the plasma membrane one can ask the question whether Pik1p and its product PI4P specifically control one pathway? Here we demonstrate an interaction of Pik1p with the 14-3-3 proteins Bmh1p and Bmh2p. Interestingly, overexpression of Bmh1p and Bmh2p results in multiple genetic interactions with genes involved in late steps of exocytosis and it affects the forward transport of the general amino acid permease Gap1p. The detected interaction depends on the phosphorylation state of Pik1p and Pik1p phosphorylation accompanies its shuttling out of the nucleus into the cytoplasm where presumably the binding to Bmh1/2p occurs. Therefore, we reason that these interactions might serve the sequestration of Pik1p away from the Golgi. This study reveals that Pik1p shows a strong effect on the delivery of Gap1p to the surface whereas the transport of exocytosis markers implicated in the direct Golgi-to-plasma membrane pathway are not significantly disturbed. Cells carrying a deletion of gga2 also show a strong defect in delivery of Gap1p to the surface. In addition, pik1-101 gga2[delta]double mutants display synthetic genetic and membrane transport phenotypes and recruitment of Gga2 to the TGN partially depends on functional Pik1p. Therefore, our results suggest a role of Pik1p in the TGN to endosome pathway.

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.

Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research.