Effects of transporter Agp1p ubiquitination on nitrogen utilization in Saccharomyces cerevisiae

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．