Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan; Division of Medical Biochemistry, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai, Japan.
Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan.
Biochim Biophys Acta Biomembr. 2017 Oct;1859(10):2076-2085. doi: 10.1016/j.bbamem.2017.07.013. Epub 2017 Jul 25.
Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells.
色氨酸是人体必需的氨基酸，也是 5-羟色胺和褪黑素的重要前体。单羧酸转运蛋白 MCT10 是 SLC16A 家族蛋白的成员，可介导肾脏、小肠和肝上皮细胞基底外侧膜对低亲和力色氨酸的转运，尽管确切的转运机制尚不清楚。在这里，我们开发了一种简单的功能测定法，使用酿酒酵母中高亲和力色氨酸转运蛋白 Tat2 的缺失突变体来分析人 MCT10 的色氨酸转运。tat2Δtrp1 细胞在 YPD 培养基中生长不良，因为培养基中的酪氨酸与色氨酸竞争低亲和力色氨酸转运蛋白 Tat1。MCT10 似乎允许 tat2Δtrp1 细胞在 YPD 培养基中生长，并在缺乏 Rsp5 泛素连接酶的细胞中积累。这些结果表明 MCT10 在酵母中具有功能，并受泛素依赖性质量控制。虽然 Tat2 表达细胞的生长在中性 pH 值下受到显著抑制，但 MCT10 表达细胞的生长几乎不受影响。这一特性与 MCT10 通过易化扩散而无需跨质膜形成 pH 梯度的转运机制一致。人 MCT10 编码区已知存在单核苷酸多态性 (SNP)。在 MCT10 的 8 个 SNP 氨基酸变化中，N81K 突变完全消除了色氨酸的摄取，而表达或定位没有任何异常。在 MCT10 建模结构中，N81 似乎突出到色氨酸的可能轨迹中。在人胚肾 293T 细胞中还验证了 MCT10 和变体蛋白的质膜定位。
