Abstract
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.
Highlights
There is, no direct evidence that the transport mediated by these proteins involves such complexes; but it is noteworthy that NBAT in the renal and intestinal brush border membranes (BBMs), its primary sites of localization (14 –15), is associated with a smaller protein, the association involving one or more interprotein disulfide bonds (16 –17)
Several mutations in the human NBAT gene have been found in a substantial number of patients with cystinuria [21,22,23,24,25], an autosomal recessive disease in which excessive amounts of cystine and basic amino acids are excreted in urine [26, 27]
The most striking result of our studies is that progressive C-terminal truncations of rat NBAT are associated with a bimodal pattern for the expression of amino acid transport in Xenopus oocytes
Summary
3H-labeled amino acids were from Amersham Pharmacia Biotech. Oocyte-positive Xenopus laevis females were from Nasco (Fort Atkinson, WI). The fragment to be overlapped was amplified in separate reactions in which the sense mutated primer was paired with a downstream unmutated antisense primer (5Ј-GTCCAGCTTCTCTTGGATACC-3Ј, complementary to bases 459 – 479 in NBAT cDNA), and the antisense mutated primer was paired with an upstream vector sequence (5Ј-TAATACGACTCACTATAGGG-3Ј, T7 promoter primer) In both reactions, 660 ng of pSPORT/ NBAT served as a template. NBAT and its mutants, blotted onto the nitrocellulose membranes, were detected using anti-NBAT antibodies, Ab357 and Ab527, either used individually or in combination, and horseradish peroxidase-conjugated donkey anti-rabbit IgG, from Amersham Pharmacia Biotech, followed by the ECL Western blot detection procedure as described in the protocol supplied by them. The oocytes were incubated in 2 ml of PBS containing rhodamine-conjugated goat anti-rabbit IgG (from Pierce; 1:400, final dilution) to label the surface-bound anti-NBAT antibodies. A ϫ10 objective with a 0.25 numerical aperture was used to obtain an optical section every 20 m. (See Ref. 38 for details regarding confocal microscopy.)
Sign-in/Register to view highlights & summary 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 callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
