Apical heterodimeric cystine and cationic amino acid transporter expressed in MDCK cells

Abstract

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.