Progressive C-terminal deletions of the renal cystine transporter, NBAT, reveal a novel bimodal pattern of functional expression.
Nearly identical proteins (denoted NAA-Tr, rBAT, D2, NBAT), cloned from mammalian kidneys, induce a largely sodium-independent high-affinity transport system for cystine, basic amino acids, and some neutral amino acids in Xenopus oocytes (system b0,+-like). Mutations in the human NBAT gene have been found in several type I cystinurics. In kidney, NBAT is associated with a second, smaller protein (approximately 45 kDa), and this heterodimer has been proposed to be the minimal functional unit of the renal cystine transporter (Wang, Y., and Tate, S. S. (1995) FEBS Lett. 368, 389-392). To delineate regions minimally required for functional expression in oocytes, we constructed a series of C-terminal truncated mutants of rat kidney NBAT (wild-type (WT), 683 amino acids). Expression of these mutants in oocytes yielded an unusual bimodal pattern for the induction of amino acid transport activity. Thus, initial C-terminal truncations aborted elicitation of transport activity. The next mutant in the series, Delta588-683, exhibited most of the transport-inducing potential inherent in the WT/NBAT. Further deletions again attenuated transport activity. Although both the WT/NBAT and the truncated mutant, Delta588-683, induce qualitatively similar transport systems, the two forms of the protein exhibit contrasting sensitivities toward a point mutation in which the cysteine residue at position 111 was mutated to serine. This mutation did not greatly affect induction of transport by the WT/NBAT; however, the Delta588-683 mutant was inactivated by this mutation. Our data further suggest that cysteine 111 is probably the site of disulfide linkage with an approximately 45-kDa oocyte protein producing a complex equivalent to that seen in kidney membranes.
Deora
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Department of Biochemistry, Cornell University Medical College, New York, New York 10021, USA.
Ghosh
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D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003553:Cystine / Q000378:metabolism*; D017931:DNA Primers; D005260:Female; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017384:Sequence Deletion; D014982:Xenopus laevis
D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D017931:DNA Primers; C489857:SLC3A1 protein, human; C081009:Slc3a1 protein, rat; D003553:Cystine