Analysis Of Yeast Cell Wall Synthesis-associated Genes In Saccharomyces Cerevisiae

In response to the absence of nitrogen and the presence of a nonfermentable carbon source such as acetate,diploid cells of the budding yeast Saccharomyces cerevisiae undergo meiosis,and sporulate and then finally produce four haploid daughter spores.The spore wall differs from the vegetative cell wall in two important respects: it contains additional components,and it must be assembled de novo.Newly formed vegetative cell walls can be formed by extension of the mother cell wall to cover newly inserted plasma membrane.The vegetative cell wall consists of two major components.The inner layer is mainly beta-glucan layer and outside of these beta-glucans is mannan layer.By contrast,for the spore wall there is no preexisting structure available to act as a template,and it assembly begins within the cytoplasm of the mother cell after completion of the meiosis.The spore wall is composed of four layers;they are,from inside to outside,mannan,glucan,chitosan,and dityrosine layers.The chitosan and dityrosine layers are specifically formed in the spore wall and cause the spores to be resistant to environmental stresses.Despite ?-1,6-glucan plays critical glue roles to maintain the organization of the vegetative cell wall,the process of ?-1,6-glucan synthesis is largely unknown,and the functional characterization of ?-1,6-glucan in the spore wall and its effect on the spore wall has not yet been performed.OSW2 is a sporulation specific expression gene,and perhaps involved in spore wall formation but its function remains elusive.Because the mammalian cells do not have cell wall,fungal cell wall has become the target for screening of antifungal agents.Given that almost all the genes in the spore cells have corresponding conserved homologous genes in vegetative cells.Therefore,to investigate the ?-1,6-glucan and Osw2 in the spore wall can provide broder insight into other fungal cell wall assembly and the theoretical basis for screening antifungal drugs.Since yeast spores exhibit strong resistance to the environmental stresses and the permeability of the spore wall can be improved by using genetic engineering techniques,yeast spores can be applied as an enzyme-immobilized carrier.All strains used in this study are SK-1 strain background which sporulate with a high efficiency.According to the gene homologus recombination,a series of ?-1,6-glucan defective mutants were constructed and OSW2 was disrupted based on the chs3? mutant which lacks the outer two(chitosan and dityrosine)layers to form osw2?chs3? double mutant.Then,the sporulation defect phenotype of these mutants was examined and the mutant spores were used to encapsulate enzyme.The main results of this study are summarized as follows:(1)?-1,6-glucan is required for the integrity of spore wall.The mutants defective in ?-1,6-glucan synthesis,including kre1?,kre6?,kre9?,and big1?,were sporulated to analyze the effect of ?-1,6-glucan defects on the spore wall.Morphologically,the mutant spores were indistinguishable from wild-type spores.Except for kre6?,these mutant spores were sensitive to treatment with ether,suggesting that the mutations perturb the integrity of the spore wall.Eosin Y staining for the chitosan indicated that mutations which cause a deficiency of ?-1,6-glucan result in an increase of chitosan deposition in the spore wall.They lacked significant sporulation defects partly because the chitosan layer,which covers the glucan layer,compensated for the damages.The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer.Among the double mutants,the most severe spore wall deficiency was observed in big1? spores.The majority of the big1?chs3? mutants failed to form visible spores at a higher temperature and the viability of the sporulation cells were decreased.Given that the ?-1,6-glucan mutants caused a failure to attach a GPI-anchored reporter,Cwp2-GFP,to the spore wall,?-1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall.Thus,?-1,6-glucan is required for proper organization of the spore wall.(2)Osw2 is required for proper assembly of glucan and/or mannan layers of the yeast spore wall.The effect of Osw2 on the inner layers of spore wall was examined by making osw2?chs3? diploid double mutant.We found that deletion of both OSW2 and CHS3 results in a synthetic sporulation defect.Localization of GPI-anchored protein indicated that a GPI-anchored protein reporter GFP-Cwp2 mislocalizes in osw2?chs3? spores.The osw2? mutation also exhibited a severe synthetic sporulation defect when combined with the deletion of a ?-1,6-glucan synthesis-related gene,BIG1.Osw2 is localized to the prospore membrane during sporulation.However,it disappears in mature spores,indicating that it is not a structural component of the spore wall.Given that Osw2 contains a probable 2-dehydropantoate 2-reductase domain,it may mediate an enzymatic reaction.Osw2 shows a weak similarity to other 2-dehydropantoate 2-reductase domain-containing proteins,Svl3 and Pam1.A pam1?svl3? mutant exhibits vegetative cell and spore wall defects.Taken together,these results suggested that Osw2 is involved in the assembly of the inner(glucan and mannan)layers,and the 2-dehydropantoate 2-reductase domain-containing proteins may have a similar function in glucan and/or mannan layer assembly.(3)kre1? spores can be used as a superior carrier of enzyme capsules.The creatininase was immobilized by using kre1? spores and its enzymatic properties were investigated.The results suggested the activity of the creatininase encapsulated in kre1? spores was similar to osw2? spores but was significantly higher than that of wild type spores.Compared with free enzymes,like osw2?,encapsulated creatininase in kre1? spores exhibits resistance to environmental stresses and has a good reusability.Moreover,the kre1? spore-encapsulated enzymes are resistant to high temperature and exhibit a wide range of pH tolerance as same as that of osw2? spores.Therefore,as well as osw2? spores,kre1? spores also can be used as a superior carrier of enzyme capsules.