epiglucan and dityrosine

The ascospores of Saccharomyces cerevisiae are surrounded by a complex wall that protects the spores from environmental stresses. The outermost layer of the spore wall is composed of a polymer that contains the cross-linked amino acid dityrosine. This dityrosine layer is important for stress resistance of the spore. This work reports that the dityrosine layer acts as a barrier blocking the diffusion of soluble proteins out of the spore wall into the cytoplasm of the ascus. Diffusion of a fluorescent protein out of the spore wall was used as an assay to screen for mutants affecting spore wall permeability. One of the genes identified in this screen, OSW3 (RRT12/YCR045c), encodes a subtilisin-family protease localized to the spore wall. Mutation of the active site serine of Osw3 results in spores with permeable walls, indicating that the catalytic activity of Osw3 is necessary for proper construction of the dityrosine layer. These results indicate that dityrosine promotes stress resistance by acting as a protective shell around the spore. OSW3 and other OSW genes identified in this screen are strong candidates to encode enzymes involved in assembly of this protective dityrosine coat.

Topics: beta-Glucans; Catalytic Domain; Cell Wall; Cytoplasm; Diffusion; Gene Expression Regulation, Fungal; Luminescent Proteins; Mutation; Peptide Hydrolases; Permeability; Phenotype; Plasmids; Saccharomyces cerevisiae; Spores, Fungal; Tyrosine

The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.

Topics: beta-Galactosidase; beta-Glucans; Cell Membrane; Chitosan; DNA Mutational Analysis; Fungal Proteins; Gene Expression Regulation, Fungal; Genotype; Glucosamine; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Models, Biological; Mutation; Plasmids; Saccharomyces cerevisiae; Sensitivity and Specificity; Spores, Fungal; Time Factors; Tyrosine