Abstract
A renal cDNA clone (rBAT) that induces system bo,+-like amino acid transport activity in Xenopus oocytes has recently been isolated (Bertran, J., Werner, A., Moore, M. L., Strange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacín, and Murer, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5601-5605). Here we show the isolation of a cDNA clone by screening a human kidney cortex cDNA library for expression of sodium-independent transport of L-[3H]arginine in Xenopus oocytes. The cRNA of this clone induces in oocytes, in addition to the uptake of L-arginine, that of L-[35S]cystine and L-[3H]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 Km in the micromolar range. All these characteristics resemble induction of system bo,+ related to rBAT in the oocytes. Human rBAT mRNA (approximately 2.5 kilobases) is found in kidney, small intestine (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 oligonucleotide of the isolated clone greatly prevents (80-97%) human kidney mRNA-dependent induction of the uptake of these amino acids (i.e., L-cystine, L-arginine, and L-leucine). The isolated clone (2304 base pairs 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, respectively. This predicted protein corresponds to a membrane glycoprotein, and contains six potential N-glycosylation sites which might be functional in the oocyte: [35S] methionine labeling of oocytes shows a specific band of 94 kDa in crude membranes of these human cRNA-injected oocytes; treatment of these oocytes with tunicamycin shifts the cRNA-specific translation product to approximately 72 kDa. We conclude that we have isolated a functional cDNA corresponding to human rBAT. The isolation of this human cDNA would lead to the study of the possible involvement of rBAT in human hyperaminoacidurias.
Highlights
AND DISCUSSIONWater-injected oocytes and the rBAT-induced transport activities legend to Fig. 2). Dataaremean 2 S.E.from 6-8 oocytes in a (see A single clone (human rBAT) was isolated by screening a representative experiment
Expression Cloning ofa Human Renal cDNA That Induces High Affinity Transport of L-CystineShared with Dibasic Amino Acidsin Xenopus Oocytes*
To the study of the possible involvement of rBAT in we show the isolation of a cDNA clone by screening a human hyperaminoacidurias
Summary
Water-injected oocytes and the rBAT-induced transport activities legend to Fig. 2). Dataaremean 2 S.E.from 6-8 oocytes in a (see A single clone (human rBAT) was isolated by screening a representative experiment. This long transcript was not detected when total RNA was used ( i e .in jejunum and pancreas; Fig. 4, or kidney and liver; data not shown).rabbit and ratbrain and rat heart show transcripts that hybridize with rBAT (or rat NAA-Tr) of-5 kb in length (1,5) It has been shown in rat tissues, by RNaseprotection assay, that these transcripts represent homologous NAA-Tr cross-reacting mRNA species (5). To test furthfeorr a relationship of these induced uptake activities to rBAT-system W+-like transport, we performed hybrid-depletion experiments of human kidney poly(A)+ RNA (25 ng/oocyte) with 40 PM of an antisense oligonucleotide (to a coding region of human clone rBAT; see legend to Fig. 5 and “Experimental Procedures” fordetails). As shown in Fig.[5], rBAT hybrid depletion of human kidney mRNA (prior to oocyte injection) resulted in an almost complete block in mRNA-induced L-cystine, Larginine, and L-leucine uptake.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
