Chararcterisation of fungal protein kinases involved in the regulation of the cell cycle of Saccharomyces cerevisiae and of sexual development in Aspergillus nidulans

Abstract

In this work, fungal model organisms, the single cell budding yeast Saccharomyces cerevisiae and the filamentous fungus Aspergillus nidulans were used to characterize different factors involved cell cycle control and in the regulation of development. In yeast, the cyclin Clb5, a regulatory subunit of cyclin-dependent kinases, and Ime2, a protein kinase with an essential function in the meiotic cell cycle, were studied. In filamentous fungi, the Ime2 homolog protein kinase ImeB, which plays an important role in regulation of asexual and sexual fruiting bodies formation, was characterised. In the first part of the project, the degradation pathways of the yeast S-phase cyclin Clb5 were analyzed. Clb5 is known to be a substrate of the anaphase promoting complex (APC/C), a multi-subunit ubiquitin ligase essential for the cell cycle. Pulse labelling experiments were applied to determine the half-life of Clb5. In contrast to other cyclins, Clb5 was found to be unstable throughout the cell cycle and also in cells lacking APC/C activity. However, in the presence of an active APC/C, the half-life of Clb5 was further decreased. These data suggest that two pathways, an APC/C-dependant and an APC/C -independent mechanism, have overlapping roles in triggering Clb5 proteolysis during the cell cycle. Degradation is mediated by the 26S proteasome. The aim of the second project was a better understanding of the regulation of the yeast protein kinase Ime2. This kinase and the cyclin-dependent kinase Cdk1 have many common functions in the regulation of meiosis. Ime2 is not regulated by cyclins, but is itself a highly unstable protein. By constructing a set of deletions, it was shown that the C-terminal region is essential for Ime2 instability and probably contains multiple overlapping degradation signals. A truncated Ime2 lacking the C-terminal 242 amino acids was stable and active. Expression of this version of Ime2 during meiosis did not interfere with meiotic cell divisions, but resulted in abnormalities in spore production. Frequently, asci had a reduced spore number and were mostly dyads. Thus, Ime2 instability mediated by the C-terminal region is important for the efficient enclosure of nuclei into spore walls and for the formation of normal 4-spore asci. In the final part of this project, the homolog of Ime2 in A. nidulans, ImeB, was characterised. For this purpose an imeB deletion strain was constructed. On agar plates, the imeB mutants displayed a slow growth phenotype, but an increased formation of sexual fruiting bodies, the cleistothecia. In contrast to wild-type strains, imeB delta strains also formed typical sexual structures, Hülle cells, in liquid media. Overexpression of imeB blocks conidiophore formation when grown in the dark. imeB transcript levels increase during both the asexual and the sexual life cycle. These data indicate that ImeB acts a regulator of development in A. nidulans. In contrast to Ime2 protein in yeast, ImeB appears to be an inhibitor of the sexual life cycle. These findings imply that in evolution, these proteins have acquired opposite functions as regulators of sexual development. Ime2 and ImeB belong to the family of mitogen activated protein (MAP) kinases characterised by a TXY motif in their activation loop. A site-directed mutagenesis showed that every single amino acid this of motif is needed for accurate ImeB function.