The outermost layer of the yeast Saccharomyces cerevisiae spore, termed the dityrosine layer, is primarily composed of bisformyl dityrosine. Bisformyl dityrosine is produced in the spore cytosol by crosslinking of two formyl tyrosine molecules, after which it is transported to the nascent spore wall and assembled into the dityrosine layer by an unknown mechanism. A P450 family protein, Dit2, is believed to mediate the crosslinking of bisformyl dityrosine molecules. To characterize Dit2 and gain insight into the biological process of dityrosine layer formation, we performed an in vitro assay to crosslink formyl tyrosine with using permeabilized cells. For an unknown reason, the production of bisformyl dityrosine could not be confirmed under our experimental conditions, but dityrosine was detected in acid hydrolysates of the reaction mixtures in a Dit2 dependent manner. Thus, Dit2 mediated the crosslinking of formyl tyrosine in vitro. Dityrosine was detected when formyl tyrosine, but not tyrosine, was used as a substrate and the reaction required NADPH as a cofactor. Intriguingly, apart from Dit2, we found that the spore wall, but not the vegetative cell wall, contains bisformyl dityrosine crosslinking activity. This activity may be involved in the assembly of the dityrosine layer.

In response to nutrient starvation, diploid cells of the budding yeast differentiate into a dormant form of haploid cell termed a spore. The dityrosine layer forms the outermost layer of the wall of spores and endows them with resistance to environmental stresses. ll-Bisformyl dityrosine is the main constituent of the dityrosine layer, but the mechanism of its assembly remains elusive. Here, we found that ll-bisformyl dityrosine, but not ll-dityrosine, stably associated withΔ spores, which lack the dityrosine layer. No other soluble cytosolic materials were required for this incorporation. In several aspects, the dityrosine incorporated in resembled the dityrosine layer. For example, dityrosine incorporation obscured access of the dye calcofluor white to the underlying chitosan layer, and ll-bisformyl dityrosine molecules bound toΔ spores were partly isomerized to the dl-form. Mutational analyses revealed several spore wall components required for this binding. One was the chitosan layer located immediately below the dityrosine layer in the spore wall. However, ll-bisformyl dityrosine did not stably bind to chitosan particles, indicating that chitosan is not sufficient for this association. Several lines of evidence demonstrated that spore-resident proteins are involved in the incorporation, including the Lds proteins, which are localized to lipid droplets attached to the developing spore wall. In conclusion, our results provide insight into the mechanism of dityrosine layer formation, and the assay described here may be used to investigate additional mechanisms in spore wall assembly.

The cell wall is the interface between the fungal cell and its environment and disruption of cell wall assembly is an effective strategy for antifungal therapies. Therefore, a detailed understanding of how cell walls form is critical to identify potential drug targets and develop therapeutic strategies.

The polysaccharide chitosan is found in the cell wall of specific cell types in a variety of fungal species where it contributes to stress resistance, or in pathogenic fungi, virulence. Under certain growth conditions, the pathogenic yeast Candida dubliniensis forms a cell type termed a chlamydosospore, which has an additional internal layer in its cell wall as compared to hyphal or yeast cell types. We report that this internal layer of the chlamydospore wall is rich in chitosan. The ascospore wall of Saccharomyces cerevisiae also has a distinct chitosan layer. As in S. cerevisiae, formation of the chitosan layer in the C. dubliniensis wall requires the chitin synthase CHS3 and the chitin deacetylase CDA2. In addition, three lipid droplet-localized proteins Rrt8, Srt1, and Mum3, identified in S. cerevisiae as important for chitosan layer assembly in the ascospore wall, are required for the formation of the chitosan layer of the chlamydospore wall in C. dubliniensis. These results reveal that a conserved machinery is required for the synthesis of a distinct chitosan layer in the walls of these two yeasts and may be generally important for incorporation of chitosan into fungal walls.ImportanceThe cell wall is the interface between the fungal cell and its environment and disruption of cell wall assembly is an effective strategy for antifungal therapies. Therefore, a detailed understanding of how cell walls form is critical to identify potential drug targets and develop therapeutic strategies. This work shows that a set of genes required for assembly of a chitosan layer in the cell wall of S. cerevisiae is also necessary for chitosan formation in a different cell type in a different yeast, C. dubliniensis. Because chitosan incorporation into the cell wall can be important for virulence, the conservation of this pathway suggests possible new targets for antifungals aimed at disrupting cell wall function.