Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae
Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae
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使用酿酒酵母对人类芳香氨基酸转运蛋白 MCT10/TAT1 进行功能分析
DOI：
10.1016/j.bbamem.2017.07.013
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Abe Fumiyoshi
中科院分区：
文献类型：
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作者：
Uemura Satoshi;Mochizuki Takahiro;Kurosaka Goyu;Hashimoto Takanori;Masukawa Yuki;Abe Fumiyoshi
色氨酸是人体必需氨基酸，也是重要的血清素和褪黑激素前体。单羧酸转运蛋白MCT10是SLC16A家族蛋白质的一员，介导低亲和力色氨酸转运穿过肾脏、小肠和肝脏上皮细胞的基底外侧膜，尽管精确的转运机制仍不清楚。我们开发了一种简单的功能分析方法，利用酵母中高亲和力色氨酸通透酶Tat 2的缺失突变体分析人MCT 10对色氨酸的转运。由于培养基中存在的酪氨酸与色氨酸竞争低亲和力色氨酸通透酶Tat 1，因此Tat 2 Δ trp 1细胞在YPD培养基中生长缺陷。MCT 10似乎允许tat 2 Δ trp 1细胞在YPD培养基中生长，并在Rsp 5泛素连接酶缺陷的细胞中积累。这些结果表明，MCT 10在酵母中是功能性的，并且受到泛素依赖性质量控制。而Tat2表达细胞的生长显着受损的中性pH值，MCT 10表达细胞几乎不受影响。该性质与MCT 10通过易化扩散的转运机制一致，无需跨质膜的pH梯度。已知单核苷酸多态性（SNP）发生在人MCT 10编码区。在MCT10中的8个SNP氨基酸变化中，N81K突变完全废除色氨酸输入，而在表达或定位中没有任何异常。在MCT 10模型结构中，N81似乎突出到色氨酸的推定轨迹中。还在人胚肾293 T细胞中验证了MCT 10和变体蛋白的质膜定位。
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 inSaccharomyces cerevisiae.tat2Δtrp1cells 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 oftat2Δtrp1cells 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.