Study On Exocytosis Pathway Of Proteinase A In Saccharomyces Cerevisiae Under Stressful Conditions

Proteinase A(PrA),a kind of aspartic proteinase,is located in the vacuole of yeast cells.PrA is involved in the maturation process of many cacuolar proteases.PrA can be secreted outside yeast cells under stressful conditions.Vps21p in Golgi-to-vacuole pathways,Ypt32p,Sec4p and one of the subunits of exocyst Sec5p in the constitutive pathways were studied.These results indicate that proteinase A was transported outside yeast cells by constitutive pathway tentatively.With Saccharomyces cerevisiae W303-1A as the starting strain,the VPS21,SEC5 and YPT32 genes deletion mutant strains WV21,WS5 and WY2 were constructed by the homologous recombination method and seting W303-1A as the starting strain,and SEC4 deletion mutants RS4 was constructed using another S.cerevisiae RY-A as the starting strain.Fermentation with the mutant strains above to study their effects on the secretion of proteinase A.Studies shown that:(1)PrA acitivity of VPS21 gene deletion mutant WV21 showed an increase to 3.04-fold extracellularly and reduced by 15.2%intracellularly,which demonstrated that Vps21p played important roles in the Golgi-to-vacuole pathways of PrA;(2)After deleting YPT32 gene,PrA activity increased by 17.5%and 55.3%,intracellularly and extracellularly,respectively.These results indicate that Ypt31/32 is essential for the exocytosis pathway of PrA under stressful conditions.Depletion of Sec5p,one of the key subunits of exocyst complexes,triggered an increase by 36.7%intracellularly and an decrease by 29.4%extracellularly,which indicate that the exocyst complexes are crutial for the exocytosis of PrA;fermentation with SEC4 deletion mutant RS4 increased PrA activity by 49.7%extracellularly,with no difference intracellularly,indicating Sec4p plays some roles in the exocytosis of PrA under stressful conditions.(3)In transport pathways of vesicles,Ypt31/32 mediates budding event of vesicles from trans-Golgi network,and exocyst complexes play important roles in tethering and fusion process of vesicles to plasma membrane.According to the research for Ypt31/32 and exocyst complexes,preliminary confirmation could be conducted that PrA excret outside yeast cells by constitutive pathways.