EXPRESSION CLONING OF A HUMAN RENAL CDNA THAT INDUCES HIGH-AFFINITY TRANSPORT OF L-CYSTINE SHARED WITH DIBASIC AMINO-ACIDS IN XENOPUS-OOCYTES

A renal cDNA clone (rBAT) that induces system b(o,+)-like amino acid t ransport activity in Xenopus oocytes has recently been isolated (Bertr an, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J. , Testar, X., Zorzano, A., Palacin, and Murer, H. (1992) Proc. Natl. A cad. Sci. U. S. A. 89, 5601-5605). Here we show the isolation of a cDN A clone by screening a human kidney cortex cDNA library for expression of sodium-independent transport of L-[H-3]arginine in Xenopus oocytes . The cRNA of this clone induces in oocytes, in addition to the uptake Of L-arginine, that of L-[S-35]cystine and L-[H-3]leucine. Expressed uptake of these amino acids is mutually cis-inhibitable by the other 2 amino acids. Expressed uptake of L-cystine is saturable and shows an apparent K(m) in the micromolar range. All these characteristics resem ble induction of system b(o,+) related to rBAT in the oocytes. Human r BAT mRNA (approximately 2.5 kilobases) is found in kidney, small intes tine (i.e., jejunum), pancreas, and liver. Human kidney poly(A)+ RNA ( mRNA) induces sodium-independent uptake Of L-cystine, L-arginine, and L-leucine in Xenopus oocytes. Hybrid depletion with an antisense oligo nucleotide of the isolated clone greatly prevents (80-97%) human kidne y mRNA-dependent induction of the uptake of these amino acids (i.e. L- cystine, L-arginine, and L-leucine). The isolated-clone (2304 base pai rs in length) contains a poly(A) tail and encodes a predicted 78.8-kDa protein which is 85 and 80% identical to the rabbit and rat rBAT, res pectively. This predicted protein corresponds to a membrane glycoprote in, and contains six potential N-glycosylation sites which might be fu nctional in the oocyte: [S-35] methionine labeling of oocytes shows a specific band of 94 kDa in crude membranes of these human cRNA-injecte d oocytes; treatment of these oocytes with tunicamycin shifts the cRNA -specific translation product to approximately 72 kDa. We conclude tha t we have isolated a functional cDNA corresponding to human rBAT. The isolation of this human cDNA would lead to the study of the possible i nvolvement of rBAT in human hyperaminoacidurias.