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Am J Physiol Cell Physiol 291: C678-C686, 2006. First published April 26, 2006; doi:10.1152/ajpcell.00090.2006
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Wide variety of locations for rodent MATE1, a transporter protein that mediates the final excretion step for toxic organic cations

Miki Hiasa, Takuya Matsumoto, Toshinori Komatsu, and Yoshinori Moriyama

Laboratory of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

Submitted 24 February 2006 ; accepted in final form 14 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
MATE1 was the first mammalian example of the multidrug and toxin extrusion (MATE) protein family to be identified. Human MATE1 (hMATE1) is predominantly expressed and localized to the luminal membranes of the urinary tubules and bile canaliculi and mediates H+-coupled electroneutral excretion of toxic organic cations (OCs) into urine and bile (Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, and Moriyama Y. Proc Natl Acad Sci USA 102: 17923–17928, 2005). mMATE1, a mouse MATE ortholog, is also predominantly expressed in kidney and liver, although its transport properties are not yet characterized. In the present study, we investigated the transport properties and localization of mMATE1. Upon expression of this protein in HEK-293 cells, mMATE1 mediated electroneutral H+/tetraethylammonium exchange and showed a substrate specificity similar to that of hMATE1. Immunological techniques with specific antibodies against mMATE1 combined with RT-PCR revealed that mMATE1 is also expressed in various cells, including brain glia-like cells and capillaries, pancreatic duct cells, urinary bladder epithelium, adrenal gland cortex, {alpha} cells of the islets of Langerhans, Leydig cells, and vitamin A-storing Ito cells. These results indicate that mMATE1 is a polyspecific H+/OC exchanger. The unexpectedly wide distribution of mMATE1 suggests involvement of this transporter protein in diverse biological functions other than excretion of OCs from the body.

multidrug and toxin extrusion; multidrug transport; hydrophobic cation

THE EXPORT OF ORGANIC ELECTROLYTES with extremely diverse chemical structures into the urine and bile plays an essential role in elimination of toxic compounds of exogenous and endogenous origin from the body. The export occurs through transepithelial transport across the basolateral and luminal membranes of renal tubular cells and across the sinusoidal membranes and bile canaliculi of hepatocytes (12, 15, 16, 21, 22, 30, 32). Although expression cloning studies have identified various transporters involved in the transport processes (1, 6, 7, 9, 13, 14, 25, 27, 28), the transporter(s) primarily responsible for the elimination of polyspecific organic cations (OCs) remained unknown. On the basis of a large number of biochemical and physiological studies, it has been concluded that an electroneutral H+/OC exchanger(s) is principally responsible for the final step of OC excretion (12, 15, 16, 21, 22, 30, 32).

The multidrug and toxin extrusion (MATE) family is the most recently identified multidrug resistance-conferring protein family in bacteria (3, 11, 23). Although the overall properties of the MATE family are not yet elucidated, some MATE-type proteins mediate H+- or Na+/cationic drugs exchange (3, 11, 23). Very recently, our laboratory identified the human and mouse orthologs of MATE1 (20). Human MATE1 (hMATE1) is predominantly expressed in kidney and liver. When expressed in HEK-293 cells, hMATE1 is localized in the plasma membrane and mediates electroneutral H+/tetraethylammonium (TEA) and H+/1,4-methylphenylpyridinium (MPP) exchange. Furthermore, in cis-inhibition studies, hMATE1 was shown to exhibit a substrate specificity similar to that of renal OC export, and thus it was concluded that hMATE1 is responsible for the final step of excretion of OCs through kidney and liver.

To establish the concept that MATE1 is generally responsible for the final step of excretion of OCs in mammals, we decided to study the expression, localization, and function of MATE1 counterpart in mice (mMATE1). Such studies are also important from a comparative aspect, because many previous studies on renal and hepatic OC excretion have been carried out in mice or mouse specimens.

In the present study, we have shown that mMATE1 mediates electroneutral H+/TEA exchange and that the substrate specificity is similar to that of hMATE1. Furthermore, we have found that mMATE1 is widely distributed throughout body, especially in epithelial cells and secretory cells.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
cDNA. cDNA of mMATE1 (accession no. AAH31436) was cloned by RT-PCR from mouse kidney RNA (20). The sequence of the mMATE1 clone was confirmed by comparison with the mouse genome sequence.

RT-PCR analysis. Total RNA (1 µg) extracted from isolated organs from wild-type ddY and C57BL/6 mice was transcribed into cDNA in 20 µl of reaction buffer containing 0.2 mM each dNTP, 10 mM dithiothreitol, 100 pmol of random octamers, and 200 units of Moloney murine leukemia virus reverse transcriptase (Amersham). After 1 h of incubation at 42°C, the reaction was terminated by heating at 90°C for 5 min. For PCR amplification, the cDNA solution was added to a PCR buffer, which contained 0.6 mM total dNTP (150 µM each dNTP), 25 pmol of primers, and 1.5 units of AmpliTaq Gold DNA polymerase (PerkinElmer). Thirty-five temperature cycles were conducted. Each cycle comprised denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min. The amplification products were analyzed with polyacrylamide gel electrophoresis. The primers used were based on the database sequence (GenBank accession no. BC031436) 5'-CCTTCAGGCTTCAGTGTGGCT-3' (nucleotides 960–980) and antisense primer 5'-ATGCCTCGAGTTATTGCTGTCCTTTGGACGG-3' (nucleotides 1614–1644). No amplified products were obtained without the RT reaction products. DNA sequencing was performed using the chain termination method (24).

mMATE1-expressing cells. cDNA encoding mMATE1 was subcloned into the expression vector pcDNA3.1(+) (Invitrogen). This plasmid, pcDNA/mMATE1, was used to transfect HEK-293 cells by lipofection using TransIT reagent (Mirus). HEK-293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, penicillin, and streptomycin at 37°C under 5% CO2 as described previously (20). Twenty-four hours later, 1.5 x 106 cells per 10-cm dish were transfected with 10 µg of pcDNA3.1/mMATE1. For selection of cells that stably express mMATE1, the cells were grown for 2 days in the presence of 400 µg/ml geneticin. Colonies expressing mMATE1 were selected by means of immunohistochemistry and the transport assay described below.

Transport assay. After selection with geneticin, mMATE1-expressing cells were harvested and suspended in transport assay medium (125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM Tricine, pH 8.0). Cells were incubated at 37°C for 5 min; the transport assay was initiated by adding 50 µM radiolabeled TEA (5 kBq/assay; PerkinElmer Life Science) as described previously (20). At appropriate times, aliquots of the mixture (200 µl) were filtered through 0.45-µm type HA membrane filters (Millipore). Each filter was washed with 5 ml of ice-cold medium, and the radioactivity remaining on the filter was counted. Amounts of TEA taken up by the cells were expressed as nanomoles per milligram of total cell protein.

Antibodies. Site-specific rabbit polyclonal antibodies against mMATE1 were prepared by repeated injections of glutathione S-transferase fusion polypeptides encoding amino acid residues P495–Q532 of mMATE1 (PESHGEIMMTDLEKKRRDSVGPADEPATSFAYPSKGQQ). Immunological specificity was investigated and described previously (20). The following antibodies were used as cell markers. Mouse monoclonal antibodies against glucagon, insulin, or serotonin were obtained from Sigma, Progen, or NeoMarkers, respectively. Rabbit polyclonal antibodies against gastrin and rat monoclonal antibodies against somatostatin were obtained from Chemicon. Guinea pig polyclonal antibodies against rat pancreatic polypeptide and PYY were from Linco Research. Alexa Fluor 488-labeled anti-rabbit IgG and Alexa Fluor 568-labeled anti-mouse IgG were purchased from Molecular Probes.

Western blot analysis. Total membrane fractions of mouse ddY or C57BL/6 tissues (~0.1–1 g wet weight depending on the organ) were isolated, suspended in ice-cold 20 mM MOPS-Tris, pH. 7.0, containing 0.3 M sucrose, 5 mM EDTA, and protease inhibitors (pepstatin A, leupeptin, antipain, and chymostatin at 10 µg/ml each), homogenized, and centrifuged at 800 g for 8 min at 4°C. The postnuclear supernatant was then centrifuged at 100,000 g for 1 h at 4°C. The pellet was suspended in the same buffer and denatured at room temperature for 30 min in the presence of 1% SDS and 10% beta-mercaptoethanol. Samples (40–300 µg of protein) were subjected to electrophoresis and Western blot analysis as described previously (20). As a positive control, mMATE1 was expressed in sf9 cells transfected with recombinant baculovirus containing cloned mMATE1 (20).

Immunohistochemistry. Immunohistochemical analysis was performed using indirect immunofluorescence microscopy as described previously (10). In brief, male ddY mice were anesthetized with ether and then perfused intracardially with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The organs were isolated, and frozen sections were prepared. In the case of cultured cells, cells on poly-L-lysine-coated coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. After being washed with PBS, the specimens were incubated for either 20 min (cells) or 30 min (organs) in the same buffer containing 0.1% Triton X-100, followed by PBS containing 2% goat serum and 0.5% bovine serum albumin. The specimens were incubated with antibodies diluted to 1 µg/ml or 1,000-fold (anti-mMATE1 or other antibody) with PBS containing 0.5% bovine serum albumin for 1 h at room temperature. Samples were washed four times with PBS and then reacted with the secondary antibody or Alexa Fluor 568-labeled anti-mouse IgG (1 µg/ml) or Alexa Fluor 488-labeled anti-rabbit IgG (2 µg/ml) for 1 h at room temperature. Finally, the immunoreactivity was examined under either an Olympus BX60 microscope or an Olympus FV300 confocal laser microscope.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
mMATE1 as a polyspecific OC transporter. The cDNA for mMATE1 encodes a protein of 532 amino acids with 78.1 and 24.1% sequence identity to that of human MATE1 and Vibrio parahaemolyticus NorM Na+/multidrug antiporter, a prototype of the MATE family (17), respectively (Fig. 1A). A hydropathy plot of mMATE1 predicts 12 transmembrane domains (Fig. 1B).


Figure 1
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  		Fig. 1. Amino acid sequence of mouse multidrug and toxin extrusion 1 (mMATE1). A: amino acid sequences of the proteins are aligned with that of NorM (17). Identical amino acid residues are indicated by asterisks. Predicted transmembrane regions are boxed. hMATE1, human MATE1. B: putative secondary structure of mMATE1. The membrane topology of mMATE1 was predicted by the combined procedure of Kyte and Doolittle and TMPred. A glutamate residue (E273) that is conserved in the MATE transporter family and that is essential for activity is circled (19). N, NH2 terminus; C, COOH terminus.

 
To characterize the transport properties of mMATE1, we measured the pH-dependent translocation of OCs across the plasma membranes of mMATE1-expressing HEK-293 cells. This approach allowed us to study the luminal efflux of OCs as classic cellular uptake (20, 28). Upon expression of mMATE1, the transporter proteins are predominantly localized in the plasma membrane region (Fig, 2A). The mMATE1-expressing cells exhibited time-dependent transport activity toward TEA, a typical substrate for the H+-coupled OC exporter (Fig. 2B) (3, 25). The transport activity of mMATE1 was saturable with respect to substrate concentration with Km and Vmax values for TEA of 410 µM and 600 pmol·min–1·mg protein–1, respectively (Fig. 2C). The transport also showed pH dependence. The transport activity was lower at pH 6.0 and increased at higher extracellular pH values; it was maximal at around pH 8.0–8.5 (Fig. 2D). Na+ was not required for transport activity (Fig. 2E). The addition of 1 µM 3,5-di-tert-butyl-4-hydroxybenzylidene malononitrile (SF6847), a proton conductor, and 5 µM nigericin in the presence of KCl, which dissipates the pH gradient, both strongly inhibited the uptake, whereas 1 µM valinomycin in the presence of 65 mM KCl, which causes membrane depolarization, did not have much effect (Fig. 2E). Furthermore, TEA taken up by the cells was released after being transferred to pH 6.0 (Fig. 2F). As a whole, these results are essentially the same as those of hMATE1 (20) and suggest that mMATE1 mediates electroneutral H+/TEA exchange.


Figure 2
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  		Fig. 2. mMATE1 mediates electroneutral H+/tetraethylammonium (TEA) exchange. A: presence of mMATE1 in HEK-293 cells, as revealed by indirect immunofluorescence microscopy (left). No immunoreactivity was observed in a mock control (HEK-293 cells transfected with the pcDNA3.1 vector, right). B: time course of TEA (50 µM) uptake at pH 8.0 by HEK-293 cells expressing mMATE1. C: dose dependence of TEA uptake at pH 8.0. Values were obtained at the indicated concentrations at 5 min after the corresponding mock control cell values were subtracted from mMATE1-expressing cell values. D: pH dependence of TEA uptake. TEA uptake at 20 min was measured in HEK-293 cells expressing mMATE1 or control cells incubated at the indicated pH. E: 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 for a membrane potential or pH gradient for TEA uptake was also examined at pH 8.0 in the absence or presence of 1 µM nigericin, 1 µM SF6847, or 0.5 µM valinomycin in buffer containing 65 mM KCl and 65 mM NaCl (control). Assays were terminated after 20 min of incubation. F: pH-dependent extrusion of TEA from mMATE1-expressing HEK-293 cells. mMATE1-expressing HEK293 cells were incubated with 50 µM radiolabeled TEA as in B for 10 min. The cells were then transferred to fresh buffer with the indicated pH (time 0) and incubated for a further 10 min, and the remaining radioactivity was assayed. Error bars indicate SD of 3 samples.

 
The pharmacology of the cis-inhibition of TEA transport was similar to that of renal H+/OC exchange (12, 15, 16, 21, 22, 30–32) and human MATE1 (20). It is strongly inhibited by cimetidine, MPP, or rhodamine-123, less so by nicotine, choline, or quercetin, and not at all by organic anions such as p-aminohippurate and uric acid (Table 1). Steroid hormones (corticosterone, testosterone, progesterone, and androstenedione) also inhibited the TEA transport. We concluded that mMATE1 is an electroneutral polyspecific H+/OC exchanger. A significant difference between mMATE1 and hMATE1 is the lower sensitivity of mMATE1 toward quinidine, nicotine, and corticosterone.


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  		Table 1. Cis inhibition of TEA transport by mMATE1

 
mMATE1 is expressed in a wide variety of locations. In a previously published Northern blot (20), we showed that the gene encoding mMATE1 is expressed in kidney, liver, and, at lower levels, in skeletal muscle as a 3.8-kilobase (kb) transcript, whereas the expression in other organs such as brain and testes was below the detection limit (20). Immunohistochemical staining showed that mMATE1 is predominantly present in the luminal membranes of renal urinary tubules and bile canaliculi (20). However, this does not mean that mMATE1 is not present in organs other than kidney and liver. In RT-PCR analyses, a 695-bp MATE1-specific transcript was amplified in tissues from brain, heart, stomach, small intestine, urinary bladder, thyroid gland, adrenal gland, and testes as well as kidney and liver (Fig. 3A). Furthermore, immunoblotting using specific polyclonal antibodies against mMATE1 (20) indicated the presence of an ~50-kDa immunological counterpart in crude membrane fractions of the heart, stomach, small intestine, bladder, thyroid gland, adrenal gland, and testes, indicating the presence of mMATE1 at the protein level in these organs (Fig. 3B). In contrast, an immunoreactive polypeptide was not detected in the membrane fraction of brain, indicating that content of mMATE1 in this organ is either below the detection limit of our system or is degraded by proteases (Fig. 3B).


Figure 3
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  		Fig. 3. Expression of mMATE1 gene and protein in various organs. A: results of RT-PCR analysis of total cellular RNA prepared from various organs (top) after RT reaction (+) and without RT reaction (–). Expression of glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) gene is also shown as a RNA quality control (bottom). B: Western blot analysis. Crude membrane fractions from various organs were examined: 5 µg of mMATE1 protein in sf9 cells, 40 µg of kidney protein; 40 µg of liver protein; 300 µg of brain cortex; 200 µg of heart and stomach; 300 µg of small intestine, bladder, thyroid gland, and adrenal gland; 200 µg of testis protein. Immunodecoration was performed in the presence of 2 mg of antigenic polypeptide (bottom).

 
To examine the exact expression and localization of mMATE1 in the tissues other than kidney and liver, we conducted immunohistochemical studies. In brain, mMATE1 immunoreactivity was observed in glial-like cells (Fig. 4A) and capillaries (Fig. 4B). In glandula submandibularis, mMATE1 was localized to the striated duct in the mixed gland (Fig. 4C). In heart, mMATE1 immunoreactivity was observed in the endothelial cells of blood vessels (Fig. 4D). In stomach, mMATE1-positive cells were identified in the mucosa, especially in the gastric antrum and pylorus (Fig. 4, E and F). In the antrum, gastric glands contained several mMATE1-positive small cells (Fig. 4E). The cells were not parietal cells or chief cells, because the cells did not contain pepsinogen and H+/K+-ATPase (not shown) and were morphologically similar to auxiliary cells that secrete mucin. In the pylorus, mMATE1 was expressed in endocrine-like cells (Fig. 4F). The cells do not correspond to G cells, because they did not contain gastrin (data not shown). In pancreas, mMATE1 immunoreactivity is localized to the centroacinar cells and intercalated duct, from which bicarbonate and mucin are secreted (Fig. 4G). The islets of Langerhans were also mMATE1 positive; its precise localization is described below. In the small intestine, mMATE1-positive cells were also frequently observed in the ileum (Fig. 4H). Double immunohistochemical analysis indicated that a subpopulation of mMATE1-positive cells contain serotonin, glucagon-like peptide 1, or somatostatin (data not shown). This indicates that a subpopulation of mMATE1-positive cells corresponds to enterochromaffine cells and neuroendocrine cells, including L cells. In urinary bladder, strong mMATE1 immunoreactivity was observed in the intermediate cells of the epithelium (Fig. 4I). Essentially all intermediate cells were mMATE1 immunoreactive, suggesting that electroneutral H+/OC exchange occurs not only in luminal membranes of renal urinary tubules and bile canaliculi but also in the bladder and that this transport activity plays an important role in bladder function.


Figure 4
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  		Fig. 4. Localization of mMATE1 in various tissues. Indirect immunofluorescence microscopy revealed that mMATE1 was localized in various tissues. Pictures merged with Nomarski images are shown. A: brain glial-like cells. B: brain capillaries. BV, blood vessel. C: glandula submandibularis. D: heart. E: gastric antrum. F: pylorus. G: exocrine part of pancreas. H: small intestine. I: urinary bladder epithelium. Su, superficial cell; In, intermediate cell; Ba, basal cell. Bars, 10 µm.

 
mMATE1 immunoreactivity was also observed in various endocrine cells. In thyroid follicular cells, strong mMATE1 immunoreactivity was observed in the plasma membrane facing both the follicle lumen (Fig. 5A, arrows) and blood vessels (Fig. 5A, arrowhead). The parathyroid gland also possessed mMATE1-positive cells (Fig. 5B). Furthermore, strong mMATE1 immunoreactivity was observed throughout the cortex of adrenal gland (Fig. 5C). In the islets of Langerhans, mMATE1-positive cells coincided with the location of glucagons (Fig. 5, G–I) but not with that of insulin or somatostatin (data not shown), indicating that mMATE1-positive cells were {alpha} cells. The pancreatic polypeptide also partly colocalized with mMATE1, indicating that a population of pancreatic F cells expressed mMATE1 (Fig. 5, G–I). In testes, mMATE1 is specifically present in Leydig cells (Fig. 5J). Finally, we found that Ito cells, liver-specific pericytes that store vitamin A (8), also contained strong mMATE1 immunoreactivity (Fig. 6).


Figure 5
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  		Fig. 5. Various endocrine cells express mMATE1. A: in thyroid gland, mMATE1 is present in follicle cells. F, follicle. B: parathyroid gland. C: cortex of the adrenal gland expresses mMATE1. D–F: an {alpha} cell of islets of Langerhans contains mMATE1. Specimens were doubly immunostained for mMATE1 (D) and glucagon (E). A merged image of glucagon and mMATE1 immunoreactivity is shown in F. G–I: a subpopulation of F cells contains mMATE1. Specimens were doubly immunostained for mMATE1 (G) and pancreatic polypeptide (H). A merged image of pancreatic polypeptide and mMATE1 is shown in I. J: in testes, mMATE1 is expressed in Leydig cells. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. Ito cells show mMATE1 immunoreactivity. In liver, mMATE1 is predominantly localized with Ito cells (arrows) as well as in the canalicular membrane (arrowheads) as revealed on indirect immunofluorescence microscopy (20). A picture merged with Nomarski images is shown. Bar, 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Role of H+/OC exchange in physiological function. In this study, we have shown that mMATE1, the second member of mammalian MATE family to be identified, may act as an electroneutral polyspecific H+/OC antiporter. Because mMATE1 possessed a substrate specificity similar to that of hMATE1 (Table 1) and is predominantly localized to the luminal membrane of the renal urinary duct and bile canaliculi (20), mMATE1 is proposed to mediate the final excretion step for toxic OCs into urine and bile as in the case for hMATE1. After OCs are taken up by the organic cation transporter 1 (OCT1) or OCT2 in the renal tubule cells and hepatocytes (12, 15, 16, 21, 22, 30, 32), they are excreted out of the cells through a cooperation between MATE1 and P-glycoprotein.

The most important finding of the study is that MATE1 is expressed and localized in various tissues other than kidney and liver. Among the organs tested, mMATE1 transcript was not detected in the submandibular gland and pancreas (data not shown), even though mMATE1-positive cells are present in these organs (Figs. 4 and 5). This may be due to degradation of RNA during preparation, given that both organs contain high levels of RNases.

The combined immunohistochemical and molecular biological approaches revealed that mMATE1-expressing cells can be classified into three categories. The first is epithelial cells surrounding internal cavities. We have shown that mMATE1 is expressed in brain capillaries and pancreatic duct cells as well as epithelial cells of the urinary bladder. This suggests that MATE1-mediated excretion of OCs occurs in these MATE1-expressing cells in addition to the urinary tract and bile canaliculi. Striated duct in the glandula submandibularis and auxiliary cells in the stomach also belong to this category. In other words, excretion of toxic OCs, that is metabolic wastes, may occur in all organs in which MATE1 is present. Because MATE1 is an H+/OC exchanger, a slightly acidic pH (~6–6.5) extracellular environment is necessary to drive excretion. Thus the extracellular pH near MATE1-expressing cells is somehow made acidic, probably through vacuolar H+-ATPases and/or Na+/H+ antiporters. In rodent kidney and bladder, such an acidic environment for the excretion of OCs can be achieved by vacuolar H+-ATPase at plasma membrane (2, 29).

The second category includes certain type of endocrine cells in the stomach, small intestine, and islets of Langerhans. The pancreas and the gastrointestinal tract contain more than 18 types of endocrine cells (26). We have ascertained that {alpha} cells and F cells contain mMATE1. These cells are known to secrete peptide hormones and/or transmitters through exocytosis. Although the true function of mMATE1 in these cells is not known, it is possible that these endocrine cells extrude physiologically important cationic transmitters through MATE1-mediated transport.

More interestingly, the third category of MATE1-expressing cells store or secrete hydrophobic hormones and vitamins. Ito cells store vitamin A. Leydig cells synthesize and secrete testosterone. The cells of the adrenal cortex synthesize and secrete corticosterones and follicle cells in thyroid gland secrete thyroxine. On the basis of results of cis-inhibition of TEA transport (Table 1), we suggest that MATE1 is responsible for secretion of these steroid hormones through the plasma membrane. It should be stressed that the molecular mechanism of secretion of steroid hormones remains unknown.

Thus, although further studies are necessary, it appears that the function of mammalian MATE-type transporters is not limited to the excretion of OCs but also may have a role in the homeostasis of electrolytes through efficient and regulated transportation/release of physiological metabolites of various sizes, structures, and hydrophobicity. This possibility is now under investigation in our laboratory.

It is quite likely that the resistance to drugs and endogenous toxic metabolites observed in plants can be attributed to their MATE homologs (4, 5, 18, 33). The observation that both mMATE1 and hMATE1 seem to recognize quercetin as a transport substrate (Table 1) is consistent with the idea that quercetin may be transported into plant vacuoles through a MATE-type transporter (4). Our results support the conservative and ubiquitous nature of the MATE superfamily as a polyspecific OC exporter and its wide variety of roles in the excretion or sequestration of OCs and related compounds.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Grant-in-Aid for Research 16017264 from the Japanese Ministry of Education, Science, Sport, and Culture (to Y. Moriyama).


   ACKNOWLEDGMENTS
 
We thank Drs. H. Omote, R. Morimoto, and M. Otsuka for discussion and S. Arioka, S. Ishimura, and K. Shimizu for help in the initial stage of the study. We also thank Prof. A. Yamamoto (Nagahama Institute of Technology) for discussion and critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Moriyama, Laboratory of Membrane Biochemistry, Okayama Univ. Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan (e-mail: moriyama{at}pheasant.pharm.okayama-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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