The role of YBR180w and YDR371 in yeast sporulation

Research project P 14735 The role of YBR180w and YDR371w in yeast sporulation Peter BRIZA 27.11.2000 Sporulation of the lower eukaryote Saccharomyces cerevisiae is a relatively simple model system for cell differentiation. Under appropriate nutritional conditions (presence of a non-fermentable carbon source and lack of nitrogen) diploid cells undergo meiosis and form four haploid spores surrounded by the ascus wall. The surface layers of the spore wall are chemically distinct from the vegetative cell wall and contain large amounts of the rare amino acid, dityrosine. Dityrosine in the spore surface is present in the LL-, DD- and DL configuration. We performed a screen of EUROFAN deletion strains, looking for mutants with deviations of the LL- to DL-dityrosine ratio. In this project we propose to characterize two genes, YBRI 80w and YDR371 w, that were identified in this screen. The phenotype of both mutations was similar. Morphology of asci and spores appears to be normal. The over-all dityrosine content Of the spores is comparable to wild type, as well, but the content of DL-dityrosine in spore walls is drastically reduced. This phenotype indicates that both mutants are impaired in spore wan maturation. Ybr180p belongs to Cluster I of MFS-MDR multidrug resistance proteins. We speculate that Ybr180p acts as dityrosine permease, transporting bisformyl dityrosine from the prospore`s cytoplasm to the maturing prospore wall. Ydr371p has homologies to chitinases. We intend to elucidate the role of both genes in yeast sporulation with the following experiments. 1. Detailed phenotypic analysis (including chemical analysis of the mutant spore was and electron microcopy). 2. Heterologous expression in vegetative cells and characteTization of enzymatic functions in non-sporulation environment. 3. Subcellular localization of the gene products in sporulating cells using GFP fusions. 4. Expression studies and characterization of regulatory regions. 5. Functional domain analysis of Ybr180p and functional comparison of Ybr180p with other members of the NTSMDR family. This work will provide further insights into the yet poorly understood mechanism of spore formation in S.cerevisiae and its regulation. In addition, we expect that this work will contribute to the understanding of multidrug resistance mediated by WS-MDR proteins.

Sporulation is the biological process that leads from growing to dormant cells - the spores. Spores are capable of withstanding environmental stress situations like elevated temperatures or dessication. When conditions turn favorable, spores germinate and the proliferating life cycle starts again. Sporulation of baker`s yeast (Saccharomyces cerevisiae) is a simple model system for cell differentiation. Therefore it is of interest to understand the biochemistry and biology of this process, as it might provide clues otherwise not obtainable in more complex organisms. In this project we focussed on the biosynthesis of the spore surface layer, a chemically unusual structure consisting of polysaccharides and a protein-like macromolecule. We identified and characterized two proteins and their corresponding genes involved in the synthesis of the spore wall surface layers. We showed that Dtr1 was an integral part of the prospore membrane and that its function was to transport dityrosine, the building block of the spore surface. With this finding it is now possible to understand the spatial coordination of spore surface biosynthesis. The prospore membrane separates the immature spores from the surrounding mother cell and prevents the exchange of molecules between them. At the same time it serves as scaffold for the deposition of spore wall building blocks. Dityrosine, a derivative of the amino acid tyrosine, is the major (if not only) building block of the protein-like macromolecule of the spore surface. Synthesis of dityrosine takes place in the cytoplasm of the maturing spore, i.e. within the space enclosed by the prospore membrane. Polymerization of dityrosine to form the surface macromolecule, on the other hand, takes place on the outside of this membrane. Dtr1 bridges the prospore membrane and enables the translocation of dityrosine from the inside of the membrane to the outside. Interestingly, Dtr1 is a member of the family of multidrug resistance (MDR) transporters. Many MDR proteins are of general interest, since they play a role in cell detoxification by removing specific toxins from cells. Although Dtr1 is specialized in dityrosine transport, it is also capable of removing several unrelated toxic compounds from cells in vitro, thereby rendering these cells resistant to the toxins. Cts2, the other protein under investigation, is a chitinase involved in the synthesis of a specific carbohydrate moiety of the spore wall. At first glance it may appear surprising that a degradation process is involved in the formation of a biological structure, but our results show that without Cts2 the assembly of spore walls is severely impared. Obviously this enzyme is necessary to re-shape sugar chains by partial degradation, so that the assembly process can continue.