Topology of the Na(+)/dicarboxylate cotransporter: the N-terminus and hydrophilic loop 4 are located intracellularly.
Topology of the Na(+)/dicarboxylate cotransporter: the N-terminus and hydrophilic loop 4 are located intracellularly.
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Na(+)/二羧酸协同转运蛋白的拓扑结构：N 末端和亲水环 4 位于细胞内。
DOI：
10.1016/s0005-2736(00)00385-0
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Pajor,AM
中科院分区：
文献类型：
--
作者：
Zhang,FF;Pajor,AM
Na+/二羧酸盐共转运体NaDC-1目前的二级结构模型包含11个跨膜结构域。该模型基于亲水分析和含有n -糖基化位点的羧基端胞外位置。本研究进一步采用COS-7细胞间接免疫荧光法检测模型。将Flag表位标签（DYKDDDDK）融合到NaDC-1的氨基末端（Flag-NaDC-1）上，利用针对Flag表位的单克隆抗体确定其n端位置。利用针对含有NaDC-1的164-233个氨基酸的融合蛋白的多克隆抗体鉴定了NaDC-1的亲水性环4。NaDC-1和Flag-NaDC-1在COS-7细胞中的表达通过琥珀酸转运功能测定和细胞表面生物素化蛋白的Western blot证实。对同时表达NaDC-1和Flag-NaDC-1的细胞进行免疫荧光标记需要洋地黄苷渗透质膜，而在完整细胞中没有可见的免疫荧光。本研究结果表明，NaDC-1的n端和亲水环4都位于细胞内，这支持了目前NaDC-1的结构模型。
The current secondary structure model of the Na+/dicarboxylate cotransporter, NaDC-1, contains 11 transmembrane domains. The model is based on hydropathy analysis and the extracellular location of the carboxy terminus, which contains an N-glycosylation site. In this study, the model was further tested using indirect immunofluorescence of COS-7 cells. The Flag epitope tag (DYKDDDDK) was fused to the amino terminus of NaDC-1 (Flag-NaDC-1), and a monoclonal antibody against the Flag epitope was used to determine the location of the N-terminus. Hydrophilic loop 4 of NaDC-1 was identified using polyclonal antibodies raised against a fusion protein containing amino acids 164–233 of NaDC-1. The expression of NaDC-1 and Flag-NaDC-1 in COS-7 cells was confirmed by functional assays of succinate transport and by Western blots of cell surface biotinylated proteins. Immunofluorescent labeling of cells expressing both NaDC-1 and Flag-NaDC-1 required permeabilization of the plasma membranes with digitonin whereas no immunofluorescence was visible in intact cells. The results of this study show that both the N-terminus and hydrophilic loop 4 of NaDC-1 are located intracellularly, which supports the current model of NaDC-1 structure.