In vitro reconstitution of the yeast spore wall dityrosine layer discloses the mechanism of its assembly

In response to nutrient starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a dormant form of haploid cell termed a spore. The dityrosine layer forms the outermost layer of the wall of S. cerevisiae 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 in vitro with dit1 spores, which lack the dityrosine layer. No other soluble cytosolic materials were required for this incorporation. In several aspects, the dityrosine incorporated in trans 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 to dit1 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 in vitro assay described here may be used to investigate additional mechanisms in spore wall assembly.