摘要
目的:鉴定解脂耶氏酵母中Anillin家族蛋白,探究其对Septin细胞骨架组织的调控功能.方法:将模式生物酿酒酵母Anillin家族蛋白Bud4序列在解脂耶氏酵母数据库中比对,找到潜在的Anillin家族蛋白;从解脂耶氏酵母基因组中扩增其序列,将其与绿色荧光蛋白(green fluorescent protein,GFP)融合表达,通过荧光显微镜观察其胞内定位;对酿酒酵母细胞中潜在的Anillin家族蛋白进行过量表达,观察其对细胞形态和Septin组织的影响,从而确定其调控Septin组织的功能.结果:通过解脂耶氏酵母数据库中序列比对,找到两个潜在的Anillin家族蛋白,分别将其命名为YlBud4A(数据库中蛋白编号YALI0D11880)和YlBud4B(数据库中蛋白编号YALI0B07623),其C端与该家族蛋白成员同源性高,含有保守的DUF1709和PH结构域;与酿酒酵母Bud4类似,YlBud4B可以定位在芽颈,在芽体较大细胞中呈明显的双环结构,而YlBud4A没有芽颈定位;在酿酒酵母细胞中过量表达YlBud4B导致细胞产生显著的芽体变长细胞形态,Septin的正常组织被破坏,在变长的芽体中呈圆形结构.结论:解脂耶氏酵母YlBud4B与Anillin家族蛋白在序列和功能上都具有同源性,其可以与Septin细胞骨架相互作用,在调控Septin组织过程中发挥功能.
Abstract
Objective:To identify the anillin-related protein in Yarrowia lipolytica and to study its function in septin organization.Methods:We identified the anillin-related protein in Y.lipolytica by aligning the protein sequence of Saccharomyces cerevisiae Bud4 in the Y.lipolytica genome database.The sequences of these anillin-related proteins were amplified and fused with green fluorescent protein GFP to observe their intracellular localization.In the model organism budding yeast,these anillin-related proteins of Y.lipolytica were overexpressed and their effects on cell morphology and septin organization were determined to investigate their function in this process.Results:Two potential anillin-related proteins,named YlBud4A(protein number YALIOD11880 in the database)and YlBud4B(protein number YALIOB07623),were identified by sequence comparison in the Y.lipolytica genome database,both of which were conserved in their C-terminal containing DUF1709 and PH domain.YlBud4A had no bud neck localization,whereas YlBud4B localized to the bud neck as a double ring in medium and large bud cells,a localization pattern identical to Bud4.Overexpression of YlBud4B in budding yeast caused elongated buds and septin disruption,which clustered into dots.Conclusion:YlBud4B may interact with the septins and it is likely to be the functional homolog of anillin-related protein whereas YlBud4A is not.
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万方数据