Hyperactive mutation occurs adjacent to the essential glutamate 286 for transport in the yeast tryptophan permease Tat2
Hyperactive mutation occurs adjacent to the essential glutamate 286 for transport in the yeast tryptophan permease Tat2
复制标题
过度活跃突变发生在酵母色氨酸通透酶 Tat2 中转运所必需的谷氨酸 286 附近
DOI：
10.1016/j.bbrc.2019.01.038
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发表时间：
2019
影响因子：
3.1
通讯作者：
Abe Fumiyoshi
中科院分区：
文献类型：
--
作者：
Amano Kaori;Ishii Ryoga;Mochizuki Takahiro;Takatsu Shiori;Abe Fumiyoshi
在酿酒酵母中，高亲和力色氨酸的输入是由质膜通透酶Tat2介导的。在这里，我们发现了高度活跃的TAT2突变，I285V和I285T，它们允许细胞在非常低的色氨酸浓度(<；4 μg/mL)下生长。野生型Tat2对色氨酸的Km值为24 μg/mL，Tat2I285V和Tat2I285T的Km值分别为17和11 μg/mL。Tat2I285V和Tat2I285T介导的色氨酸输入的归一化值Vmax/Km是Tat2的2倍，这表明这些突变增加了对色氨酸的亲和力，并在很低的色氨酸浓度下介导了色氨酸的转运。I285位于E286附近，E286是氨基酸酶中的一个完全保守的残基。根据对Tat2的结构同源物E208(pKA∼8.3-11.7)的预测，Tat2的E286羧链在色氨酸/H+转运体过程中可能被加载一个质子。因此，I285V和I285T突变可能会影响Tat2的埋藏残基环境，从而促进色氨酸的输入。此外，Tat2I285V和Tat2I285T的表达水平迅速升高，在低色氨酸(0.5 μg/mL)条件下，Tat2I285V和Tat2I285T有效地定位于细胞表面。我们的发现为深入了解高亲和力转运机制的性质提供了线索，并提供了一种独特的方法来改善广泛类型的氨基酸渗透的功能。
InSaccharomyces cerevisiae, high-affinity tryptophan import is mediated by the plasma membrane permease Tat2. Herein, we identified hyperactive Tat2 mutations, I285V and I285T, which allowed the cells to grow at very low tryptophan concentrations (<4 μg/mL). TheKmvalue of wild-type Tat2 for tryptophan appeared to be 24 μg/mL, whereas that of Tat2I285Vand Tat2I285Twas 17 and 11 μg/mL, respectively. Normalized values ofVmax/Kmfor Tat2I285V- and Tat2I285T-mediated tryptophan import were 2-fold higher than that for Tat2, suggesting that these mutations increase the affinity for tryptophan, and mediate transport at very low tryptophan concentrations. I285 resides adjacent to E286, a fully conserved residue among amino acid pemreases. According to a pKa prediction for E208 (pKa ∼8.3–11.7) ofEscherichia coliAdiC antiporter, a structural homologue of Tat2, the E286 carboxyl chain of Tat2 could get loaded with a proton during tryptophan/H+symport. Hence, I285V and I285T mutations might affect the buried residue environment of Tat2, thereby facilitating tryptophan import. Additionally, Tat2I285Vand Tat2I285Tlevels increased rapidly, and were efficiently localized to the cell surface after transferring the cells to low tryptophan medium (0.5 μg/mL). Our findings provide a clue to gain insights into the property of high-affinity transport mechanisms, and offer a unique approach to improve the functionality of broad types of amino acid permeases.