Charakterisierung kleiner G-Proteine der Ras- und Rho/Rac-Familie in Ustilago maydis: Zentrale Schalter in komplexen Netzwerken und ihr Einfluss auf die Organisation der Zellmorphologie

Small G-proteins have manifold regulatory functions in eukaryotic cells. They serve as molecular switches, which are active in the GTP-bound state, but inactive in the GDP-bound state. In the active state they are able to interact with downstream effectors and thereby to activate them. Small G-proteins are involved in many different processes such as the regulation of gene expression and cell proliferation, the organization of the actin cytoskeleton and of the vesicular traffic. This work investigates thefunctions of small G-proteins of the Rho/Rac- and Ras-family in the phytopathogenic basidiomycete Ustilago maydis. Because of its dimorphic growth pattern, this fungus is a suitable model organism for the investigation of the regulation of cellular morphology. Small G-proteins of the Rho/Rac-family are involved in the organization of the actin cytoskeleton and therefore their influence on cellular morphology is wide. In Ustilago maydis is the absence of the Rho/Rac-protein Cdc42 is not lethal as it is in Saccharomyces cerevisiae. Here, only a defect in cell separation and in the mating reaction is observable. In this work it is demonstrated that the absence of the highly related Rho/Rac-protein Rac1 in U. maydis leads to defects in bud formation and filamentous growth. Despite the defects in bud formation, rac1 deletion mutants are viable and separate by fission at a central septum. Overexpression of rac1 leads to filamentous growth, whereas rac1 deletion mutants are not able to grow as filament. Therefore, Rac1 is essential and sufficient for the morphogenetic switch in U. maydis. Expression of a constitutive active version of Rac1 results in a strong vacuolization and a swelling of the cells and also to a cell cycle arrest. The analysis of a cdc42 rac1 double mutant strain uncovered redundant functions of the proteins in the regulation of vesicle fusion. The investigation of Ras1 in U. maydis demonstrated that this protein is essential. Absence of this Protein results in clusters of large round cells. Additionally these cells show defects in the nuclear distribution. The cell morphology of these mutants is similar to the morphology of cdc42 rac1 double mutant strains. Also the defects in vacuolar fusion are similar here. An epistatic analysis shows that Ras1 regulates the vacuolar fusion by activating Rac1. Overexpression of wildtype ras1 results in filamentous growth. The responsible signal is transmitted independently of Rac1 but the filaments are much thicker in the absence of this protein. The expression of constitutive active ras1 does not result in filamentous growth. This demonstrates that for this process an intact GTPase cycle is crucial. Strong expression of the constitutive active version results in a hyperseptation of the cells. In a weaker form, this morphological phenotype is also observable upon strong overexpression of the wildtype version of ras1. This work also demonstrates that Ras1 is able to activate the MAPK-pathway which was not assumed so far. Strong overexpression of constitutive active ras1 results in activation of this signal cascade. This observation leads to a model in which the MAPK-pathway is activated by both Ras-proteins, Ras1 and Ras2. On the other hand the cAMP-pathway can only be activated by Ras1 but not by Ras2. The results of an analysis of the expression of ras1 and ras2 upon constitutive activation or inactivation of the cAMP-pathway and the MAPK-pathway lead to a model in which the cAMP-pathway influences the activity of the MAPK-pathway by regulating of the expression of ras2.