酿酒酵母转运蛋白Agp1p泛素化对氮源利用的影响

Effects of transporter Agp1p ubiquitination on nitrogen utilization in Saccharomyces cerevisiae

摘要:

摘要:【目的】研究酿酒酵母细胞膜关键氮源转运蛋白Agp1p的泛素化对其氮源利用的影响。【方法】构建一个基于双分子荧光互补技术的泛素化检测载体，检测Agp1p是否受到泛素化。采取定点突变的方法对Agp1p中可能的泛素化位点进行突变，研究其泛素化的关键作用位点及其对酿酒酵母氮源利用的影响。【结果】在谷氨酰胺、精氨酸、脯氨酸及铵盐为唯一氮源的培养基中，转运蛋白Agp1p均受到泛素化调控。定点突变实验结果显示，与出发菌株相比，潜在泛素化位点突变后，菌体荧光强度均有一定程度减弱。其中四重突变体Agp1pK11，14，98，112R荧光强度最弱，表明泛素化过程被显著抑制。系列突变菌株在9种氨基酸为复合氮源以及尿素为单一氮源的发酵实验表明，四重突变体菌体氮源利用率提高效果显著。【结论】转运蛋白Agp1p的泛素化受到不同氮源的影响，泛素化位点突变可以显著影响其泛素化过程，从而改变细胞对氮源的利用过程。

Abstract:

Abstract:［Objective］The purpose of this work is to studythe effects of ubiquitination of key nitrogen transporter Agp1p on nitrogen utilization in Saccharomyces cerevisiae．［Methods］ The ubiquitination detection vector to examine the ubiquitination process of Agp1p was constructed based on the bimolecular fluorescence complementation technology．The site-directed mutagenesis on the potential ubiquitination sites were performed to verify the effect on its ubiquitination regulation and nitrogen utilization．［Results］ Agp1p can be ubiquitinated on the medium with glutamine，arginine，proline or ammonium． The fluorescence levels of mutant strains were down-regulated compared to the wild type strain．The quadruple mutant Agp1pK11-14-98-112Ｒ achieved the lowest level among all strains．The ubiuitination process could be significantly repressed by removing the potential ubiquitination residues． Furthermore，flask-shaking experiments with nineamino acids or urea as sole nitrogen source showed that the effect of nitrogen utilization efficiencyinthe quadruple mutant was the highest．［Conclusion］Ubiquitination was involved in the regulation of Agp1p．Site-directed mutagenesis of potential ubiquitination sites of the transporter could significantly affect the nitrogen utilization process by altering the ubiquitination process．