Transmembrane Topology and Oligomeric Structure of the High-affinity Choline Transporter
Transmembrane Topology and Oligomeric Structure of the High-affinity Choline Transporter
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DOI：
10.1074/jbc.m112.405027
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发表时间：
2012-12-14
影响因子：
4.8
通讯作者：
Haga, Tatsuya
中科院分区：
文献类型：
--
作者：
Okuda, Takashi;Osawa, Chieko;Haga, Tatsuya
高亲和力胆碱转运蛋白CHT 1介导胆碱能神经末梢中乙酰胆碱合成所必需的胆碱摄取。CHT 1属于Na+/葡萄糖协同转运蛋白家族（SLC 5），该家族被假定具有共同的13跨膜结构域核心;然而，尚未报道CHT 1跨膜拓扑结构的直接实验证据。我们研究了跨膜拓扑结构的人CHT 1使用半胱氨酸扫描分析。单一的半胱氨酸残基被引入到推定的细胞外和细胞内的循环和探测的外部访问标记与膜不可渗透的，巯基特异性的生物素化试剂在完整的细胞表达这些突变体。实验结果为一个13跨膜结构域蛋白质的拓扑模型提供了实验依据。我们还构建了一个三维同源模型的基础上的细菌Na+/半乳糖共转运蛋白的晶体结构，这支持了我们的结论，CHT 1跨膜拓扑结构。此外，我们研究了CHT 1是否作为单体或低聚物存在。化学交联诱导在HEK 293细胞中的细胞表面上形成更高分子量形式的CHT 1。在相同细胞中表达的两种不同表位标记的CHT 1蛋白可以被免疫共沉淀。此外，与野生型的失活突变体I89 A的共表达诱导对总体胆碱摄取活性的显性负效应。这些结果表明，在培养的细胞中，CHT 1在细胞表面上形成同源寡聚体。
The high-affinity choline transporter CHT1 mediates choline uptake essential for acetylcholine synthesis in cholinergic nerve terminals. CHT1 belongs to the Na+/glucose cotransporter family (SLC5), which is postulated to have a common 13-transmembrane domain core; however, no direct experimental evidence for CHT1 transmembrane topology has yet been reported. We examined the transmembrane topology of human CHT1 using cysteine-scanning analysis. Single cysteine residues were introduced into the putative extra- and intracellular loops and probed for external accessibility for labeling with a membrane-impermeable, sulfhydryl-specific biotinylating reagent in intact cells expressing these mutants. The results provide experimental evidence for a topological model of a 13-transmembrane domain protein with an extracellular amino terminus and an intracellular carboxyl terminus. We also constructed a three-dimensional homology model of CHT1 based on the crystal structure of the bacterial Na+/galactose cotransporter, which supports our conclusion of CHT1 transmembrane topology. Furthermore, we examined whether CHT1 exists as a monomer or oligomer. Chemical cross-linking induces the formation of a higher molecular weight form of CHT1 on the cell surface in HEK293 cells. Two different epitope-tagged CHT1 proteins expressed in the same cells can be co-immunoprecipitated. Moreover, co-expression of an inactive mutant I89A with the wild type induces a dominant-negative effect on the overall choline uptake activity. These results indicate that CHT1 forms a homo-oligomer on the cell surface in cultured cells.