Determination of external loop topology in the serotonin transporter by site-directed chemical labeling
Determination of external loop topology in the serotonin transporter by site-directed chemical labeling
复制标题
DOI：
10.1074/jbc.273.20.12675
复制
发表时间：
1998-05-15
影响因子：
4.8
通讯作者：
Rudnick, G
中科院分区：
文献类型：
--
作者：
Chen, JG;Liu-Chen, S;Rudnick, G
通过测量选定的赖氨酸和半胱氨酸残基与细胞外试剂的反应性，检查了5-羟色胺转运蛋白（SERT）的跨膜拓扑结构。一种不渗透的生物素化试剂，磺基琥珀酰亚胺基2-（生物素酰胺基）乙基-1，3-二硫代丙酸酯（NHS-SS-biotin），被证明可以标记在培养细胞中瞬时表达的SERT。预测位于外部亲水环（eK-少）中的四个赖氨酸残基的替换在很大程度上阻止了生物素化反应。同样，半胱氨酸特异性生物素化试剂N-生物素基氨基乙基甲硫基磺酸盐（MTSEA-生物素）标记的野生型SERT，而不是突变体，其中预测位于第一外环的Cys-109被丙氨酸取代。这两种突变体转运蛋白在毛地黄皂苷透化的细胞中与生物素化试剂反应，这证明了预测位于细胞内亲水结构域中的丰富的赖氨酸和半胱氨酸残基是反应性的，但在完整细胞中不可接近。在位置111、194、243、319、399、490和571处含有单个外部赖氨酸的突变体比eK较少的突变体更容易与NHS-SS-生物素反应。类似地，在位置109、310、406、489和564处具有单个半胱氨酸的突变体比C109 A突变体更容易与MTSEA-生物素反应。所有这些突变体都是有活性的，因此可能正确折叠。这些结果支持原来的跨膜拓扑结构，并反对最近提出的替代拓扑结构的相关甘氨酸和γ-氨基丁酸转运蛋白。
The transmembrane topology of the serotonin transporter (SERT) has been examined by measuring the reactivity of selected lysine and cysteine residues with extracellular reagents. An impermeant biotinylating reagent, sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-SS-biotin), was shown to label SERT transiently expressed in cultured cells. Replacement of four lysine residues that were predicted to lie in external hydrophilic loops (eK-less) largely prevented the biotinylation reaction. Likewise, the cysteine-specific biotinylation reagent N-biotinylaminoethylmethanethiosulfonate (MTSEA-biotin) labeled wild type SERT but not a mutant in which Cys-109, predicted to lie in the first external loop, was replaced with alanine, These two mutant transporters reacted with the biotinylating reagents in digitonin-permeabilized cells, demonstrating that the abundant lysine and cysteine residues predicted to lie in intracellular hydrophilic domains were reactive but not accessible in intact cells. Mutants containing a single external lysine at positions 111, 194, 243, 319, 399, 490, and 571 reacted more readily with NHS-SS-biotin than did the eK-less mutant. Similarly, mutants with a single cysteine at positions 109, 310, 406, 489, and 564 reacted more readily with MTSEA-biotin than did the C109A mutant. All of these mutants were active and therefore likely to be folded correctly. These results support the original transmembrane topology and argue against an alternative topology proposed recently for the related glycine and gamma-aminobutyric acid transporters.