Membrane topology of the human dipeptide transporter, hPEPT1, determined by epitope insertions
Membrane topology of the human dipeptide transporter, hPEPT1, determined by epitope insertions
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DOI：
10.1021/bi981128k
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发表时间：
1998-10-27
期刊：
影响因子：
2.9
通讯作者：
Sadée, W
中科院分区：
文献类型：
--
作者：
Covitz, KMY;Amidon, GL;Sadée, W
我们已经使用表位插入来分析人类H+二肽分类器HPEPT1的跨膜拓扑。将表位滞后eympme（EE）插入氨基酸39、78、106、412和708的不同位置，该位置是通过位置定向的诱变的HPEPT1的。通过测量转染的COS7细胞中的二肽转运活性来测试标记蛋白的功能完整性。此外，将表达HPEPT1或EE标记的HPEPT1衍生物的细胞用抗EE-EE-EE-ONOCOLONAL抗体（抗EE-MAB）或针对HPEPT1（抗HPEPT1）的羧基末端升高的抗血清标记，并通过免疫共荧光共聚焦显微镜检查。 EE106-，412-和708-HPEPT1运输二肽示踪剂以及野生型HPEPT1。显示106和412位的标签被证明是细胞外的，因为它们可用于非渗透性细胞中的抗质抗体。相比之下，羧基末端结构域和EE708被证明是细胞内的，因为它们仅在透化细胞中的抗体才能访问。这些结果与PEPT1的12跨膜结构域（TMD）拓扑模型一致。在连接推定的TMD1和TMD2以及TMD2和TMD3（EE39-和EE78-HPEPT1）的区域的表位插入，废除了二肽转运到细胞中。在转染的COS7细胞中，这些标记的蛋白在很大程度上保持细胞内而不是质膜。这些结果表明，这些区域的完整性对于运输者的运输和/或功能至关重要。因此，包括推定的TMD1和-2在内的氨基末端部分的拓扑仍然有待澄清。
We have used epitope insertion to analyze the transmembrane topology of the human H+-dipeptide symporter hPEPT1. An epitope lag, EYMPME (EE), was inserted into different locations at amino acids 39, 78, 106, 412, and 708 of hPEPT1 by site-directed mutagenesis. The functional integrity of the tagged protein was tested by measuring its dipeptide transport activity in transfected Cos7 cells. Further, cells expressing hPEPT1 or EE-tagged hPEPT1 derivatives were labeled with an anti-EE-monoclonal antibody (anti-EE-mAb) or an antiserum raised against the carboxyl terminus of hPEPT1 (anti-hPEPT1) and examined by immunofluorescence confocal microscopy. EE106-, 412-, and 708-hPEPT1 transported the dipeptide tracer as well as wild-type hPEPT1. Tags at position 106 and 412 were shown to be extracellular because they were accessible to anti-epitope antibody in nonpermeabilized cells. In contrast, the carboxyl-terminal domain and EE708 were shown to be intracellular since they were only accessible to the antibodies in permeabilized cells. These results are consistent with a 12-transmembrane domain (TMD) topological model of PEPT1. Epitope insertions at regions linking the putative TMD1 and TMD2, and TMD2 and TMD3 (EE39- and EE78-hPEPT1), abolished the dipeptide transport into the cells. In transfected Cos7 cells, these tagged proteins remained largely intracellular rather than at the plasma membrane. These results suggest that the integrity of these regions is essential for transporter trafficking and/or function. Thus, the topology of the amino-terminal portion, including putative TMD1 and -2, remains to be clarified.