MEMBRANE TOPOLOGY OF THE RAT-KIDNEY NEUTRAL AND BASIC-AMINO-ACID TRANSPORTER

A recently cloned rat kidney protein (NBAT) mediates the sodium-indepe ndent transport of neutral as well as basic amino acids and cystine wh en expressed in Xenopus laevis oocytes. The human equivalent of this t ransporter may be the one that is defective in cystinuria. Immunocytoc hemical studies have indicated that NBAT is primarily localized in the brush border membranes of rat kidney and intestinal epithelial cells, a localization consistent with its proposed role in amino acid transp ort. Two contrasting topological models have been proposed for NBAT: a four membrane-spanning domain (MSD) Nin-Cin model and a single MSD Ni n-Cout model. We have investigated the topology of this membrane prote in using two different approaches. One method was an immunofluorescent labeling technique in which intact or membrane-permeabilized cells ex pressing NBAT were probed with antibodies directed against putative ex tracellular and intracellular domains of the protein. In the second me thod, fragments generated by limited surface proteolysis of intact bru sh border membrane vesicles were subjected to immunoblot analysis usin g several site-specific antibodies. Both approaches yielded results co nsistent with a four MSD Nin-Cin topological model for NBAT.