酿酒酵母组氨酸转运蛋白Hip1p的泛素化水平对组氨酸利用的影响

Ubiquitination regulation of histidine transporter Hip1p on histidine utilization in Saccharomyces cerevisiae

摘要:

[目的]为了研究泛素化对组氨酸转运及调控的影响。[方法]应用泛素化位点预测及定点突变等技术手段，Hip1p的3个潜在的泛素化位点K30、K42和K52被突变。这些突变的Hip1p被克隆到泛素化检测质粒中，检测泛素化位点突变对Hip1p的泛素化水平的影响。同时这些突变对细胞生长及组氨酸利用的影响也进一步做了检测。[结果]Hip1p的3个赖氨酸位点K30、K42和K52突变能有效降低其泛素化水平。同时，双重突变对其泛素化水平有明显的协同作用，并进一步影响了细胞生长和组氨酸利用。[结论]泛素化水平调控能有效调节组氨酸代谢，引起细胞对组氨酸利用的改变，为进一步研究氨基酸转运蛋白的调控机制提供了重要的依据。

Abstract:

[Objective] In order to demonstrate the ubiquitination regulation mechanism of histidine transporters. [Methods] By both ubiquitination sites prediction and site-directed mutagenesis, 3 potential ubiquitination sites, K30, K42 and K42 of Hip1p were mutated. These Hip1p mutants were cloned into the ubiquitination detection plasmid for measuring its ubiquitination level change. Effects of these mutants on both cell growth and histidine utilization were measured. [Results] By comparing the relative fluorescence of Hip1p and its mutants, ubiquitination sites mutation reduced the ubiquitination levels of Hip1p. Furthermore, double mutation of ubiquitination sites showed a synergy effect on reducing ubiquitination level. The ubiquitination sites mutants also influenced cell growth and enhance histidine utilization when histidine was used as the sole nitrogen source. [Conclusion] The ubiquitination levels could regulate the histidine metabolism and change the pattern for histidine metabolism, and provide clues for further investigation of regulation mechanisms involved in the amino acids transporter proteins.