A Novel Assay Reveals a Maturation Process during Ascospore Wall Formation

The ascospore wall of the budding yeast Saccharomyces cerevisiae consists of inner layers of similar composition to the vegetative cell wall and outer layers made of spore-specific components that confer increased stress resistance on the spore. The primary constituents of the outer spore wall are chitosan, dityrosine, and a third component termed Chi that has been identified by spectrometry but whose chemical structure is not known. The lipophilic dye monodansylpentane readily stains lipid droplets inside of newly formed ascospores but, over the course of several days, the spores become impermeable to the dye. The generation of this permeability barrier requires the chitosan layer, but not dityrosine layer, of the spore wall. Screening of a set of mutants with different outer spore wall defects reveals that impermeability to the dye requires not just the presence of chitosan, but another factor as well, possibly Chi, and suggests that the OSW2 gene product is required for synthesis of this factor. Testing of mutants that block synthesis of specific aromatic amino acids indicates that de novo synthesis of tyrosine contributes not only to formation of the dityrosine layer but to impermeability of the wall as well, suggesting a second role for aromatic amino acids in spore wall synthesis.