| In response to nutrient starvation,diploid cells of Saccharomyces cerevisiae differentiate into a dormant form of haploid cell termed a spore.The chitosan layer and dityrosine layer are specific layers of the wall of S.cerevisiae spores and endows them with resistance to environmental stresses.Chitin is the precursor of chitosan layer,and N,N’-bisformyl-LL-dityrosine is the main constituent of the dityrosine layer,but the mechanism of their assembly remains elusive.In previos study,adding formyl-tyrosine into spores in vitro,dityrosine still could be formed on the spore wall.The result suggested that the spore wall of Saccharomyces cerevisiae showed obvious oxidation activity during sporulation,which maybe involved in crosslinking of chitosan or dityrosine,but the specific mechanism was not yet clear.In order to clarify the mechanism of spore wall oxidative activity,three polyoxygenases with strong oxidative activity,Gmc1p/Fet5p/Fet3 p,were selected for studying.In Saccharomyces cerevisiae,GMC1/FET5/FET3 are homologous genes,and GMC1/FET5 are sporulating-induced genes.Meanwhile,GMC1 participates in the synthesis of spore wall,but the specific mechanism is still unclear.By detecting the medium color changes of dit1Δ and dit1Δgmc1Δfet5Δfet3Δ expressed human gene HPD and analyzing the dityrosine amount after adding formyl-tyrosine to dit1Δ and dit1Δgmc1Δfet5Δfet3Δ spores in vitro,we determined the spore wall has obvious oxidation in the process of spore wall formation,which was indeed associated with Gmc1p/Fet5p/Fet3 p.The expression level of Gmc1p/Fet5p/Fet3 p in vegetative cells and spores was analyzed by Western Blot,and it was found that although the expression level of Gmc1 p was significantly lower than that of the other two proteins,the expression level of Gmc1p/Fet5p/Fet3 p was significantly increased during sporulation compared with vegetative cells,that means,it could be determined that Gmc1p/Fet5p/Fet3 p were sporulating-induced proteins.In order to further clarify the specific mechanism of the oxidative effect of Gmc1p/Fet5p/Fet3 p in the sporulation process,the localization analysis were carried out.It was found that GMC1/FET5/FET3 were located around the spore wall during sporulation,and could play an oxidative role on the spore wall,thus affecting the formation of spore wall.Meanwhile,HPLC results indicated that after purification and washing,the dityrosine amount of gmc1Δfet5Δfet3Δspores decreased about 50% than wild-type spores,which meant that GMC1/FET5/FET3 could affect the assembly of the outermost layer dityrosine layer of the spore wall.Given that dityrosine was formed on the basis of chitosan layer the synthetic,we detected the amount of chitosan layer,and further analysis found that chitosan content of dit1Δgmc1Δfet5Δfet3Δ spores is reduced over 60% lower than dit Δ,that affect the formation of the chitosan layer that had an impact on the two layers of tyrosine assembly.In addition,since chitin is the precondition of chitosan,this study detected the content of chitin in the vegetative cells of Saccharides cerevisiae.Intriguingly,it was found that the content of chitin in the wildtype background of each mutant strain was significantly lower than that of the wild type.Therefore,the results of this study suggested that GMC1/FET5/FET3 would be in volve in oxidizing chitin cross-linked with each other to form a stable linear macromolecule,and during sporulation,the precursor of chitosan,chitin,could be oxidized by Gmc1p/Fet5p/Fet3 p,leading to chitosan linear polymers and the correct assembly for chitosan layer.Therefore,a tight protection layer structure,dityrosine layer can be formed well,resulting in a strong spore wall which shows unique stress resistence.In conclusion,the results provide insight into the mechanism of chitosan layer and dityrosine layer formation,and the assay described here may be used to investigate additional assembly mechanisms in other organization catalysed by multicopper oxidase. |
