Chromosomal localization and gene organization of hMATE1 and hMATE2.

Amino acid sequences of hMATE1 and hMATE2. (A) The amino acid sequences of the proteins are aligned with that of NorM (14). Identical sequences are indicated by asterisks. Predicted transmembrane regions are shaded. (B) Putative secondary structure of hMATE1. A glutamate residue (E273) that is essential for the transport activity is shown in red (16).

Expression of hMATE1 and hMATE2 in humans. Northern blot analysis revealed that the expression of hMATE1 (Top) was predominantly in the kidney, liver, and skeletal muscle, and that of hMATE2 (Middle) was primarily in kidney. Expression of G3PDH was also shown as a loading control (Bottom).

MATE1 is a membrane protein localized to the apical membrane of renal tubule cells and bile canaliculi. (A) Western blot analysis of hMATE1. The antibodies recognized hMATE1 expressed in transfected HEK293 cells and identified an immunological counterpart in human kidney tissue. The preabsorption test was performed by incubating the antibody with hMATE1 polypeptides (N461–R546) (20 μg/ml). Proteins from HEK293 cells transfected with pcDNA3.1 vector alone were used as the mock control. (B and C) Immunohistochemical detection of hMATE1 in kidney (B) and liver (C). Sections of human samples were immunostained by the HRP method and counterstained with hematoxylin. (Insets) Background staining with preimmune serum. PCT, proximal convoluted tubule; DCT, distal convoluted tubule. (Scale bars, 100 μm.)

Expression and localization of mMATE1 and mMATE2. (A) Northern blotting revealed that the expression of mMATE1 was predominantly in the kidney and liver (Left) and that of mMATE2 in testis (Right). kb, kilobases. (B) Western blot analysis indicated that the site-specific antibody recognized the mMATE1 polypeptide in kidney and liver of mice (arrow). Little immunoreactivity was observed in testis, and no mMATE1 was observed in membrane fractions prepared from other organs. (C) HRP-DAB immunostaining revealed that mMATE1 was localized to the renal cortex (a) and medulla (b). GL, glomerulus; PCT, proximal convoluted tubule; DCT, distal convoluted tubule; CCD, cortical collecting duct. (Scale bars, 100 μm.) (D) In immunoelectron micrographs, mMATE1 was observed in the apical membrane of renal proximal tubules. BBM, brush border membrane; BM, basal membrane; M, mitochondrion; L, lumen. (Scale bar, 1 μm.) (E and F) In liver, mMATE1 (green) was localized to the canalicular membrane (E) and the apical membrane of the bile duct (F), as revealed by indirect immunofluorescent microscopy. BD, interlobular bile duct; HPV, hepatic portal vein. Pictures merged with Nomarski images are shown. (Scale bars, 10 μm.)

MATE1 mediates electroneutral H⁺/TEA exchange. (A) Presence of wild-type and E273Q mutated hMATE1 in HEK293 cells, as revealed by indirect immunofluorescence microscopy. No immunoreactivity was observed in a mock control, which was from HEK293 cells transfected with pcDNA3.1 vector alone. (B) Time course of TEA (50 μM) uptake at pH 8.0 by HEK293 cells expressing hMATE1, the E273Q mutant, or a mock control. (C) Dose dependence of TEA uptake at pH 8.0. The values obtained at the indicated concentrations from the mock control cells were subtracted from the corresponding values obtained from cells expressing wild-type hMATE1. (D) pH dependence of TEA uptake. HEK293 cells expressing wild-type hMATE or a mock control were incubated at the indicated pH, and TEA uptake was then measured. (E) The effect of Na⁺ on TEA uptake was examined in buffer containing 65 mM KCl and 65 mM NaCl (control) or in buffer containing 130 mM KCl (Na⁺ free). The requirement of a membrane potential or pH gradient for TEA uptake was also examined at pH 8.0 in the absence or presence of 10 mM ammonium chloride, 5 μM nigericin, 10 μM SF6847, or 5 μM valinomycin in buffer containing 65 mM KCl and 65 mM NaCl (control). (F) pH-dependent extrusion of TEA. hMATE1-expressing cells were incubated with 50 μM radiolabeled TEA as in Fig. 2B for 10 min. Then, the cells were transferred to buffer with the indicated pH (time 0) and incubated for a further 10 min and the remaining radioactivity then assayed. Error bars are the standard deviation of three samples.

MATE1 is a polyspecific OC exporter at the final step of OC excretion. In liver, hepatocytes take up OCs at the sinusoidal membrane through an organic cation transporter 1 (OCT1), and then extrude them across the bile canaliculi by a combination of MATE1 and a multidrug resistance protein 1 (MDR1). In kidney, OCs are taken up by the renal tubular cells mainly through an organic cation transporter 2 (OCT2) located in the basolateral membrane and secreted by different transporters including MATE1, MDR1, and OCTN2 (OCT isoform).