酿酒酵母SPS途径调控因子Stp1的泛素化过程

研究酿酒酵母SPS途径关键调控因子Stp1p泛素化调控机制。通过构建基于双分子荧光互补技术的泛素化检测载体、泛素化位点定点突变及突变体氨基酸利用这三个层面来研究Stp1p的泛素化过程。通过对Stp1p的潜在的4个泛素化位点突变结果表明，与Stp1p相比，各突变体的荧光强度均有一定程度的减弱，其中三重突变体Stp1K49，129，113R荧光强度明显弱化。表明潜在的泛素化位点突变后，Stp1p的泛素化过程受到阻遏。进一步的氨基酸利用实验表明，泛素化位点突变对于调控菌体氮源利用具有重要的作用。上述结果说明，泛素化位点突变可以调控Stp1p的泛素化过程，进而影响菌体氮源的利用。
The ubiquitination mechanism of key regulator Stp1p was investigated in Saccharomyces cerevisiae. The ubiquitination detection vector was constructed based on the BiFC（Bimolecular fluorescence complementation） technology to assay the ubiquitination process of Stp1p. The site-directed mutagenesis on the potential ubiquitination sites were performed to verify the effect on its ubiquitination regulation and the nitrogen utilization. The fluorescence levels of mutant strains were down-regulated compared to the wild type strain. Furthermore，the triple mutant Stp1K49，129，113R achieved the lowest level among the all of the strains. The results indicated that the process of ubiquitination was significantly repressed by removing the potential ubiquitination residues. In addition，the amino acid utilization assay implied the ubiquitination sites played a vital role on its ubiquitination process. The results presented here demonstrated that the ubiquitination process was invovled in the regulation of Stp1p by site-directed mutagenesis of potential ubiquitination sites and thus impact the nitrogen utilization process