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首页> 外文期刊>American Journal of Physiology >Apical heterodimeric cystine and cationic amino acid transporter expressed in MDCK cells.
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Apical heterodimeric cystine and cationic amino acid transporter expressed in MDCK cells.

机译：顶端异源二聚体胱氨酸和阳离子氨基酸转运蛋白在MDCK细胞中表达。

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摘要

The luminal uptake of L-cystine and cationic amino acids by (re)absorptive epithelia, as found in the small intestine and the proximal kidney tubule, is mediated by the transport system b(0,+), which is defective in cystinuria. Expression studies in Xenopus laevis oocytes and other nonepithelial cells as well as genetic studies on cystinuria patients have demonstrated that two gene products, the glycoprotein rBAT and the multitransmembrane-domain protein b(0,+)AT, are required for system b(0,+) function. To study the biosynthesis, surface expression, polarity, and function of this heterodimer in an epithelial context, we established stable Madin-Darby canine kidney (MDCK) cell lines expressing rBAT and/or b(0,+)AT. Confocal immunofluorescence microscopy shows that both subunits depend on each other for apical surface expression. Immunoprecipitation of biosynthetically labeled proteins indicates that b(0,+)AT is stable in the absence of rBAT, whereas rBAT is rapidly degraded in the absence of b(0,+)AT. When both are coexpressed, they associate covalently and rBAT becomes fully glycosylated and more stable. Functional experiments show that the expressed transport is of the high-affinity b(0,+)-type and is restricted to the apical side of the epithelia. In conclusion, coexpression experiments in MDCK cell epithelia strongly suggest that the intracellular association of rBAT and b(0,+)AT is required for the surface expression of either subunit, which together form a functional heterocomplex at the apical cell membrane.
机译：在小肠和近端肾小管中发现的（再）吸收性上皮细胞对L-胱氨酸和阳离子氨基酸的腔内吸收是由运输系统b（0，+）介导的，该系统在胱氨酸尿症中是有缺陷的。在非洲爪蟾卵母细胞和其他非上皮细胞中的表达研究以及对胱氨酸尿症患者的遗传研究表明，系统b（0，需要糖蛋白rBAT和多跨膜结构域蛋白b（0，+）AT这两个基因产物+）功能。为了研究这种异二聚体在上皮中的生物合成，表面表达，极性和功能，我们建立了稳定的表达rBAT和/或b（0，+）AT的Madin-Darby犬肾（MDCK）细胞系。共聚焦免疫荧光显微镜检查显示，两个亚基的顶表面表达相互依赖。生物合成标记蛋白的免疫沉淀表明，在不存在rBAT的情况下b（0，+）AT稳定，而在不存在b（0，+）AT的情况下rBAT迅速降解。当二者共表达时，它们共价结合并且rBAT变得完全糖基化且更稳定。功能实验表明，表达的转运是高亲和力的b（0，+）型，并限于上皮细胞的顶侧。总之，在MDCK细胞上皮细胞中的共表达实验强烈表明，rBAT和b（0，+）AT的细胞内缔合是任一亚基表面表达所必需的，亚基在顶端细胞膜上共同形成功能性杂合物。

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