Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence
Transmembrane organization of mouse P-glycoprotein determined by epitope insertion and immunofluorescence
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DOI：
10.1074/jbc.271.16.9240
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发表时间：
1996-04-19
影响因子：
4.8
通讯作者：
Gros, P
中科院分区：
文献类型：
--
作者：
Kast, C;Canfield, V;Gros, P
P-糖蛋白（P-gp）是一种膜蛋白，当在肿瘤细胞中过度表达时会导致多药耐药。迄今为止，确定其12个假定跨膜（TM）结构域的位置和极性的努力未能产生一致的拓扑模型。最近，我们已经描述了一种基于在预测的蛋白质的细胞内或细胞外环中插入小抗原肽表位（YPYDVPDYA）的拓扑映射方法。标记的蛋白质，然后在中国仓鼠卵巢细胞中的功能性表达，和插入的标签相对于质膜的极性推断在完整的或透化的细胞中的免疫荧光。我们先前将TM 1和TM 2之间以及TM 5和TM 6之间的片段定位为细胞外片段，将TM 2和TM 3之间以及TM 6下游的片段定位为细胞内片段（Kast，C.，坎菲尔德，利文森河，和Gros，P.（1995）Biochemistry 34，4402-4411）。我们现在已经在位置207、235、276、741、782、797、815、849、887、961和1024处插入了单表位标签;在位置736、849和961处插入了双表位标签;并且在位置849处插入了三表位标签。在位置235、736、741、849、887、961和1024处插入表位产生功能性蛋白，而在位置207、276、782、797和815处插入则消除了P-gp赋予多药耐药性的能力。在位置736、849和961处插入的表位标签定位于细胞外，而在位置235、887和1024处的标签定位于细胞内。这些结果表明，由TM 4-TM 5、TM 10-TM 11和TM 12下游分隔的中间片段是细胞质的;由TM 7-TM 8、TM 9-TM 10和TM 11-TM 12描绘的片段是细胞外的。我们对P-gp的氨基和羧基末端的半部分的综合分析支持具有细胞内氨基和羧基末端以及ATP结合位点和细胞外糖基化环（TM 1-TM 2）的12-TM结构域拓扑结构与亲水性预测一致。这些结果明显不同于通过在体外和异源表达系统中分析截短的P-GPS所获得的结果。
P-glycoprotein (P-gp) is an integral membrane protein that causes multidrug resistance when overexpressed in tumor cells. Efforts to identify the position and polarity of its 12 putative transmembrane (TM) domains have so far failed to yield a consistent topological model. Recently, we have described a method for topology mapping based on the insertion of a small antigenic peptide epitope (YPYDVPDYA) in predicted intra- or extracellular loops of the protein. The tagged proteins are then functionally expressed in Chinese hamster ovary cells, and the polarity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously localized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellular (Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemistry 34, 4402-4411). We have now inserted single epitope tags at positions 207, 235, 276, 741, 782, 797, 815, 849, 887, 961, and 1024; double epitope tags at positions 736, 849, and 961; and a triple epitope tag at position 849. Insertions of epitopes at positions 235, 736, 741, 849, 887, 961, and 1024 resulted in functional proteins, whereas insertions at positions 207, 276, 782, 797, and 815 abrogated the capacity of P-gp to confer multidrug resistance. The epitope tags inserted at positions 736, 849, and 961 were localized extracellularly, whereas tags at positions 235, 887, and 1024 mapped intracellularly. These results indicate that the intervening segments separated by TM4-TM5, TM10-TM11, and downstream of TM12 are cytoplasmic; segments delineated by TM7-TM8, TM9-TM10, and TM11-TM12 are extracellular. Our combined analysis of the amino- and carboxyl-terminal halves of P-gp supports a 12-TM domain topology with intracellular amino and carboxyl termini and ATP binding sites and an extracellular glycosylated loop (TM1-TM2) in agreement with hydropathy prediction. These results are clearly distinct from those obtained by the analysis of truncated P-gps in vitro and in heterologous expression systems.