[Effects of transporter Agp1p ubiquitination on nitrogen utilization in Saccharomyces cerevisiae].

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.

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