We identified an amino acid transporter that is associated with the cystinuria-related type II membrane glycoprotein, rBAT (related to b 0,؉ amino acid transporter). The transporter designated BAT1 (b 0,؉ -type amino acid transporter 1) from rat kidney was found to be structurally related to recently identified amino acid transporters for system L, system y ؉ L, and system x ؊ C, which are linked, via a disulfide bond, to the other type II membrane glycoprotein, 4F2hc (4F2 heavy chain). In the nonreducing condition, a 125-kDa band, which seems to correspond to the heterodimeric complex of BAT1 and rBAT, was detected in rat kidney with anti-BAT1 antibody. The band was shifted to 41 kDa in the reducing condition, confirming that BAT1 and rBAT are linked via a disulfide bond. The BAT1 and rBAT proteins were shown to be colocalized in the apical membrane of the renal proximal tubules where massive cystine transport had been proposed. When expressed in COS-7 cells with rBAT, but not with 4F2hc, BAT1 exhibited a Na ؉ -independent transport of cystine as well as basic and neutral amino acids with the properties of system b 0,؉ . The results from the present investigation were used to establish a family of amino acid transporters associated with type II membrane glycoproteins.

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Alteration of the dietary intake of phosphate (P(i)) leads to rapid changes in renal P(i) transport activity. The present study, examined the underlying cellular mechanisms of the rapid regulation, with special reference to renal P(i) cotransporter. Rats were fed either a low-P(i) (0.02%) diet (CLP rats), the low-P(i) diet followed by a high-P(i) (1.2%) diet (AHP rats), or a normal (0.6%) diet (control rats). Na(+)-dependent P(i) transport activity in the brush border membrane was significantly increased in CLP rats compared with control rats, and this activity decreased rapidly within 2 h after the change of diet in AHP rats. Kinetic analysis of P(i) transport in the AHP rats indicated that the reduction was accompanied by a decrease in the apparent Vmax for Na(+)-dependent P(i) uptake. Northern blot analysis showed no difference in the abundance of NaP(i)-2 mRNA of the kidney between AHP and CLP rats. In contrast, Western blot analysis of renal brush border membrane proteins of AHP rats indicated a significant decrease in the abundance of NaP(i)-2 protein as compared with CLP rats. Immunoreactive signals for NaP(i)-2 were detected in lysosomal fractions of AHP and CLP rats. Immunohistochemical analysis showed that, NaP(i)-2 immunoreactivity in AHP rats was largely reduced in the apical membrane of the proximal tubular epithelial cells. Neither cycloheximide nor actinomycin D affected high-P(i)-induced reduction of NaP(i)-2 protein in the brush border membrane of AHP rats, indicating that de novo protein synthesis of an unidentified regulator protein was not involved in the mechanism of this reduction. In contrast, treatment with colchicine, which disrupts microtubulers, abolished the effect of high-P(i) diet on NaP(i)-2 expression. These results suggested that rapid endocytotic internalization of NaP(i)-2 may occur specifically in the brush border membrane following an acute increase in dietary P(i) intake.

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