Progressive C-terminal Deletions of the Renal Cystine Transporter, NBAT, Reveal a Novel Bimodal Pattern of Functional Expression
1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês
10.1074/jbc.273.49.32980
ISSN1083-351X
AutoresArun B. Deora, Richik N. Ghosh, Suresh S. Tate,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoNearly 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 Xenopusoocytes (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, Δ588–683, 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, Δ588–683, 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 Δ588–683 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. 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 Xenopusoocytes (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, Δ588–683, 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, Δ588–683, 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 Δ588–683 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. membrane-spanning domain neutral and basic amino acid transporter (NAA-Tr BBM, brush border membrane 2-mercaptoethanol wild-type etc., denote the truncated forms of NBAT polyacrylamide gel electrophoresis polymerase chain reaction phosphate-buffered saline. Plasma membrane transport of amino acids is mediated by several transport systems, Na+-dependent and Na+-independent, with overlapping substrate specificities (1McGivan J.D. Pastor-Anglada M. Biochem. J. 1994; 299: 321-334Crossref PubMed Scopus (340) Google Scholar). Mammalian proteins that catalyze, or are related to, many of these transport systems have now been cloned and characterized (1McGivan J.D. Pastor-Anglada M. Biochem. J. 1994; 299: 321-334Crossref PubMed Scopus (340) Google Scholar, 2Palacin M. J. Exp. Biol. 1994; 196: 123-137Crossref PubMed Google Scholar, 3Malandro M.S. Kilberg M.S. Annu. Rev. Biochem. 1996; 65: 305-336Crossref PubMed Scopus (181) Google Scholar). A majority of these are complex membrane proteins containing from 8 to 12 membrane-spanning domains (MSDs).1 An exception to this general rule is a family of proteins exemplified by NBAT (also denoted NAA-Tr, rBAT, and D2) (4Tate S.S. Yan N. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1-5Crossref PubMed Scopus (151) Google Scholar, 5Bertran J. Werner A. Stange G. Markovich D. Biber J. Testar X. Zorzano A. Palacin M. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5601-5605Crossref PubMed Scopus (197) Google Scholar, 6Bertran J. Werner A. Chillarón J. Nunes A. Biber J. Testar X. Zorzano A. Estivill X. Murer H. Palacin M. J. Biol. Chem. 1993; 268: 14842-14849Abstract Full Text PDF PubMed Google Scholar, 7Lee W.-S. Wells R.G. Sabbag R.V. Mohandas T.K. Hediger M.A. J. Clin. Invest. 1993; 91: 1959-1968Crossref PubMed Scopus (117) Google Scholar, 8Segawa H. Miyamoto K. Ogura Y. Haga H. Morita K. Katai K. Tatsumi S. Nii T. Taketani Y. Takeda E. Biochem. J. 1997; 328: 657-664Crossref PubMed Scopus (17) Google Scholar) and 4F2hc (the heavy chain of a mammalian cell surface antigen, 4F2) (9Wells R.G. Lee W.-S. Kanai Y. Leiden J.M. Hediger M.A. J. Biol. Chem. 1992; 267: 15285-15288Abstract Full Text PDF PubMed Google Scholar, 10Bertran J. Maganin S. Werner A. Markovich D. Biber J. Testar X. Zorzano A. Kuhn L.C. Palacin M. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5606-5610Crossref PubMed Scopus (143) Google Scholar, 11Broër S. Broër A. Hamprecht B. Biochem. J. 1995; 312: 863-870Crossref PubMed Scopus (63) Google Scholar, 12Yao S.Y.M. Muzyka W.R. Elliott J.F. Cheeseman C.I. Young J.D. Biochem. J. 1998; 330: 745-752Crossref PubMed Scopus (18) Google Scholar). These proteins induce transport of basic and certain neutral amino acids when their cRNAs are injected intoXenopus oocytes and are predicted to contain only 1–4 MSDs. For this reason, it has been suggested that NBAT and 4F2hc may not function as transporters themselves but may, in fact, be regulatory/modulatory subunits of larger transporter complexes (2Palacin M. J. Exp. Biol. 1994; 196: 123-137Crossref PubMed Google Scholar, 13Wells R.G. Hediger M.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5596-5600Crossref PubMed Scopus (202) Google Scholar). There is, however, 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 (14Pickel V.M. Nirenberg M.J. Chan J. Mosckovitz R. Udenfriend S. Tate S.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7779-7783Crossref PubMed Scopus (61) Google Scholar, 15Furriols M. Chillarón J. Mora C. Castello A. Bertran J. Camps M. Testar X. Vilaro S. Zorzano A. Palacin M. J. Biol. Chem. 1993; 268: 27060-27068Abstract Full Text PDF PubMed Google Scholar), is associated with a smaller protein, the association involving one or more interprotein disulfide bonds (16Wang Y. Tate S.S. FEBS Lett. 1995; 368: 389-392Crossref PubMed Scopus (62) Google Scholar, 17Palacin M. Chillarón J. Mora C. Biochem. Soc. Trans. 1996; 24: 856-863Crossref PubMed Scopus (28) Google Scholar). An equivalent complex is detected in oocytes injected with NBAT cRNA, and such a heterodimer has been proposed to constitute the minimal functional unit of NBAT-mediated transport (16Wang Y. Tate S.S. FEBS Lett. 1995; 368: 389-392Crossref PubMed Scopus (62) Google Scholar). In mammalian cells, 4F2hc is also found as part of a heterodimeric complex, 4F2, in which it is disulfide-linked to a smaller subunit (18Hemler M.E. Strominger J.L. J. Immunol. 1982; 129: 623-628PubMed Google Scholar, 19Parmacek M.S. Karpinski B.A. Gottesdiener K.M. Thompson C.B. Leiden J.M. Nucleic Acids Res. 1989; 17: 1915-1931Crossref PubMed Scopus (86) Google Scholar, 20Quackenbush E. Clabby M. Gottesdiener K.M. Barbosa J. Jones N.H. Strominger J.L. Speck S. Leiden J.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6526-6530Crossref PubMed Scopus (100) Google Scholar). However, equivalent heterodimeric complexes have not yet been detected in oocytes injected with 4F2hc cRNA. Although both NBAT and 4F2hc elicit amino acid transport activity in Xenopus oocytes, their precise functions in mammalian cells have still not been defined, although accumulating data indicate that NBAT is involved in renal and intestinal cystine transport. Thus, several mutations in the human NBAT gene have been found in a substantial number of patients with cystinuria (21Calonge M.J. Gasparini P. Chillarón J. Chillon M. Gallucci M. Rousaud F. Zelante L. Testar X. Dallapiccola B. Di Silverio F. et al.Nat. Genet. 1994; 6: 420-425Crossref PubMed Scopus (345) Google Scholar, 22Gasparini P. Calonge M.J. Bisceglia L. Purroy J. Dianzani I. Notarangelo A. Rousaud F. Gallucci M. Testar X. Ponzone A. et al.Am. J. Hum. Genet. 1995; 57: 781-788PubMed Google Scholar, 23Miyamoto K. Katai K. Tatsumi S. Sone K. Segawa H. Yamamoto H. Taketani Y. Takada K. Morita K. Kanayama H. et al.Biochem. J. 1995; 310: 951-955Crossref PubMed Scopus (54) Google Scholar, 24Pras E. Raben N. Golomb E. Arber N. Aksentijevich I. Schapiro J.M. Harel D. Katz G. Liberman U. Pras M. et al.Am. J. Hum. Genet. 1995; 56: 1297-1303PubMed Google Scholar, 25Horsford J. Saadi I. Raelson J. Goodyear P.R. Rosen R. Kidney Int. 1996; 49: 1401-1406Abstract Full Text PDF PubMed Scopus (41) Google Scholar), an autosomal recessive disease in which excessive amounts of cystine and basic amino acids are excreted in urine (26Segal S. Thier S.O. Scriver C.H. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. 7th Ed. III. McGraw-Hill, New York1995: 3581-3601Google Scholar, 27McKusick V.A. Mendelian Inheritance in Man. 11th Ed. The Johns Hopkins University Press, Baltimore1994: 1751-1752Google Scholar). Recent studies indicate that mutations in NBAT occur only in type I cystinurics but not in Types II and III (28Calonge M.J. Volpini V. Bisceglia L. Rousaud F. de Sanctis L. Beccia E. Zelante L. Testar X. Zorzano A. Estivill X. et al.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9667-9671Crossref PubMed Scopus (98) Google Scholar, 29Bisceglia L. Calonge M.J. Totaro A. Feliubadaló L. Melchionda S. Garcia J. Testar X. Gallucci M. Ponzone A. Zelante L. et al.Am. J. Hum. Genet. 1997; 60: 611-616PubMed Google Scholar, 30Wartenfeld R. Golomb E. Katz G. Bale S.J. Goldman B. Pras M. Kastner D.L. Pras E. Am. J. Hum. Genet. 1997; 60: 617-624PubMed Google Scholar). It, therefore, appears that other gene products in addition to NBAT are also involved in renal cystine transport. We have speculated that one of these might be the protein complexed to NBAT in renal and intestinal membranes (16Wang Y. Tate S.S. FEBS Lett. 1995; 368: 389-392Crossref PubMed Scopus (62) Google Scholar). The transport systems induced in oocytes by NBAT and 4F2hc exhibit some similarities and certain significant differences. For example, NBAT induces a high-affinity, largely Na+-independent uptake system for basic amino acids, cystine, and certain neutral amino acids, a system similar to b0,+, a Na+-independent system first detected in mouse blastocysts (31Van Winkle L.J. Campione A.L. Gorman J.M. J. Biol. Chem. 1988; 263: 3150-3163Abstract Full Text PDF PubMed Google Scholar). 4F2hc, in contrast, induces a transport system resembling system y+L, i.e., Na+-independent uptake of basic amino acids and largely Na+-dependent uptake of neutral amino acids (9Wells R.G. Lee W.-S. Kanai Y. Leiden J.M. Hediger M.A. J. Biol. Chem. 1992; 267: 15285-15288Abstract Full Text PDF PubMed Google Scholar, 10Bertran J. Maganin S. Werner A. Markovich D. Biber J. Testar X. Zorzano A. Kuhn L.C. Palacin M. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5606-5610Crossref PubMed Scopus (143) Google Scholar, 11Broër S. Broër A. Hamprecht B. Biochem. J. 1995; 312: 863-870Crossref PubMed Scopus (63) Google Scholar, 12Yao S.Y.M. Muzyka W.R. Elliott J.F. Cheeseman C.I. Young J.D. Biochem. J. 1998; 330: 745-752Crossref PubMed Scopus (18) Google Scholar). The similarities between the transport induced by the two proteins could be a reflection of the fact that alignment of their sequences reveals a significant degree of identity and similarity (12Yao S.Y.M. Muzyka W.R. Elliott J.F. Cheeseman C.I. Young J.D. Biochem. J. 1998; 330: 745-752Crossref PubMed Scopus (18) Google Scholar). Interestingly, a report recently appeared claiming that a truncated form of human NBAT, in which 175 residues from the C-terminal tail were deleted, induced amino acid transport in oocytes that was qualitatively similar to that elicited by 4F2hc, i.e., system y+l-like (32Miyamoto K. Segawa H. Tatsumi S. Katai K. Yamamoto H. Taketani Y. Haga H. Morita K. Takeda E. J. Biol. Chem. 1996; 271: 16758-16763Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). To define more closely the structural boundaries at which this switch in specificity occurs and also to elucidate the domains essential for its functional expression, we generated mutants of rat kidney NBAT containing progressive C-terminal deletions. A striking outcome of our studies is that the expression of the truncated mutants in oocytes shows a bimodal pattern for the induction of amino acid transport activity. A preliminary account of this work has appeared as an abstract (48Deora A.B. Tate S.S. FASEB J. 1998; 12 (abstr.): 1394Google Scholar). 3H-labeled amino acids were from Amersham Pharmacia Biotech. Oocyte-positive Xenopus laevis females were from Nasco (Fort Atkinson, WI). The Riboprobe RNA transcription kit and T7 RNA polymerase were from Promega, and the RNaid kit for the purification of cRNA was from Bio 101, Inc. Restriction enzymes used in this study were purchased from New England Biolabs Inc. and Promega. Cloning of rat kidney NBAT/cDNA has been described previously (4Tate S.S. Yan N. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1-5Crossref PubMed Scopus (151) Google Scholar). The cDNA (cloned into the SalI and NotI sites of pSPORT1 (pSPORT/NBAT)) contains a 2049-base pair open reading frame (683 amino acids) within a 2259-base pair sequence. For cRNA synthesis, the plasmid was linearized with NotI and transcribedin vitro with T7 RNA polymerase in the presence of the GpppG cap, using the protocol supplied with the Riboprobe transcription kit. The RNA transcripts were purified using the RNaid kit. Production and properties of the anti-peptide antibodies directed against NBAT have been described previously (33Mosckovitz R. Udenfriend S. Felix A. Heimer E. Tate S.S. FASEB J. 1994; 8: 1069-1074Crossref PubMed Scopus (46) Google Scholar). In this study, we used the immunoglobulin fractions from antisera directed against NBAT sequences 357–375 (Ab357) and 527–539 (Ab527) (see Fig. 1). Details of the SDS-PAGE and the ECL Western blotting (Amersham Pharmacia Biotech) procedures have been described (16Wang Y. Tate S.S. FEBS Lett. 1995; 368: 389-392Crossref PubMed Scopus (62) Google Scholar). Fig. 1 schematically depicts the proposed 4 MSD topological model for NBAT and the sites of C-terminal truncations (labeled T1 through T5). The truncations were effected by single base changes in pSPORT/NBAT which converted codons for selected amino acid residues to stop codons. The mutations were introduced using reagents and protocol supplied with the QuikChange Site-Directed Mutagenesis Kit from Stratagene and pairs of custom-synthesized complementary mutagenic oligonucleotides (from GeneLink, Thornwood, NY). The deletions (Δ) effected, along with the amino acid residues, codons for which were mutated to stop codons, were as follows: Δ658–683, K658X; Δ615–683, K615X; Δ588–683, K588X; Δ566–683, W566X; and Δ508–683, W508X. Fragments from the mutant cDNAs between the restriction sites for XhoI (base 1393 of the cDNA) and NotI (downstream of cDNA) were purified by agar-gel electrophoresis and ligated into the wild-type (WT) pSPORT/NBAT from which the XhoI/NotI fragment had been deleted. Mutations were confirmed by complete sequencing of the XhoI/NotI cassettes. The C111S (Cys-111 to Ser-111) mutant of the WT/NBAT (NBAT/C111S) was constructed by site-directed mutagenesis using the PCR overlapping extension procedure with primers containing the desired mutation (34Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6849) Google Scholar,35Horton R.M. Cai Z. Ho S.N. Pease L.R. BioTechniques. 1990; 8: 528-535PubMed Google Scholar). The Cys to Ser substitution was used because this substitution is conservative and not likely in and of itself to distort the conformational structure of the protein (36Falke J.J. Dernburg A.F. Sternberg D.A. Zalkin N. Milligan D.L. Koshland Jr., D.E. J. Biol. Chem. 1988; 263: 14850-14858Abstract Full Text PDF PubMed Google Scholar, 37Flitsch S.L. Khorana H.G. Biochemistry. 1989; 28: 7800-7805Crossref PubMed Scopus (40) Google Scholar). The mutagenic primers were 5′-CCAAAAT[C]CCTTGACTGGTGG-3′ and its reverse complement (the mutated nucleotide is shown within square brackets). The mutation was carried out in two steps. In the first step, the fragment to be overlapped was amplified in separate reactions in which the sense mutated primer was paired with a downstream unmutated antisense primer (5′-GTCCAGCTTCTCTTGGATACC-3′, complementary to bases 459–479 in NBAT cDNA), and the antisense mutated primer was paired with an upstream vector sequence (5′-TAATACGACTCACTATAGGG-3′, T7 promoter primer). In both reactions, 660 ng of pSPORT/NBAT served as a template. In the second step, a small amount of each product from the first reactions (purified by agarose-gel electrophoresis), together with the T7 promoter primer and the downstream wild-type antisense primer, was used for the PCR reaction (using a GeneAmp PCR kit from Perkin Elmer). The PCR product was purified by agar-gel electrophoresis, digested withSalI/BglII, and ligated by T4 DNA ligase into the pSPORT/NBAT from which the SalI/BglII fragment had been deleted (BglII restricts NBAT cDNA at nucleotide 402,i.e. downstream of Cys-111). The mutation was confirmed by double-stranded DNA sequencing. The C111S mutant of the Δ588–683 truncated form of NBAT (Δ588–683/C111S) was made as follows: the fragment obtained by treatment of the Δ588–683 mutant with BglII and NotI was purified and ligated into NBAT/C111S plasmid from which the BglII/NotI cassette had been deleted. DNA sequencing was performed either by the Protein/DNA Technology Center of the Rockefeller University, New York, NY or by the DNA Sequencing Facility at the BioResearch Center of Cornell University, Ithaca, NY. Isolation and maintenance of oocytes and amino acid uptake measurements have been described previously (4Tate S.S. Yan N. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1-5Crossref PubMed Scopus (151) Google Scholar). The oocytes were injected with 45 nl of the desired cRNA solution. The rate of uptake of amino acids was measured at 20 °C in a medium containing 0.015 m Tris-HCl, pH 7.5, and either 0.1 m NaCl (for transport in presence of Na+) or 0.2 msucrose (for transport in absence of Na+), and 50 μm (unless otherwise stated) of the3H-labeled amino acid (l-arginine, as a representative of the basic amino acid substrates, and eitherl-leucine or l-phenylalanine as a neutral amino acid substrate). Note that previous studies have shown that transport of l-cystine into NBAT cRNA-injected oocytes displays characteristics similar to those seen for either Arg, Leu, or Phe (6Bertran J. Werner A. Chillarón J. Nunes A. Biber J. Testar X. Zorzano A. Estivill X. Murer H. Palacin M. J. Biol. Chem. 1993; 268: 14842-14849Abstract Full Text PDF PubMed Google Scholar,7Lee W.-S. Wells R.G. Sabbag R.V. Mohandas T.K. Hediger M.A. J. Clin. Invest. 1993; 91: 1959-1968Crossref PubMed Scopus (117) Google Scholar). Generally, six oocytes were incubated in 1 ml of the transport medium. The uptake is expressed as picomoles of amino acid transported into each oocyte (after subtraction of uptake into uninjected oocytes as controls; we have found that amino acid uptake rates with uninjected oocytes were similar to those obtained with water-injected oocytes) and is presented as the average of individual measurements on five to six oocytes ± S.E. Oocyte membranes were isolated as described previously (16Wang Y. Tate S.S. FEBS Lett. 1995; 368: 389-392Crossref PubMed Scopus (62) Google Scholar). The membrane pellet (from two to four oocytes) was suspended either in 25 μl of 0.125 m Tris-HCl buffer (pH 6.8) containing 4% SDS (Tris-SDS) (nonreducing conditions) or in 25 μl of the Tris-SDS buffer containing 5% 2-mercaptoethanol (MSH) (reducing conditions) and heated at 100 °C for 5 min. The SDS extracts were then subjected to SDS-PAGE (10 or 12% gels) followed by Western analyses. 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. Three days after the injection of cRNA, 4–6 oocytes were suspended in 1 ml of PBS containing 3.3% paraformaldehyde and incubated at 20 °C for 15 min. Uninjected oocytes were similarly treated and served as controls. Oocytes were extensively rinsed with PBS and kept overnight at 4 °C in 1 ml of PBS containing 2% non-fat dry milk. The oocytes were then washed and incubated at 20 °C for 1 h in 2 ml of PBS containing 0.5% dry milk and Ab357 (1:1,000, final dilution). After thorough washings, 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. The labeled oocyte was then placed on a 1-cm2glass slide. Sequential focal planes of the fluorescence-labeled oocyte were imaged with an MRC600 laser scanning confocal unit (Bio-Rad, Hercules, CA) attached to a Zeiss Axiovert microscope. The oocyte was oriented such that its vegetal (white) hemisphere faced the light source. The oocyte was excited with the 514-nm line from a 25-mW Argon ion laser, and standard rhodamine optics were employed. A ×10 objective with a 0.25 numerical aperture was used to obtain an optical section every 20 μm. (See Ref. 38Ghosh R.N. Gelman D.L. Maxfield F.R. J. Cell Sci. 1994; 107: 2177-2189Crossref PubMed Google Scholar for details regarding confocal microscopy.) The proposed four MSD topological model for NBAT is schematically depicted in Fig. 1 (adapted from Ref. 33Mosckovitz R. Udenfriend S. Felix A. Heimer E. Tate S.S. FASEB J. 1994; 8: 1069-1074Crossref PubMed Scopus (46) Google Scholar). The sites at which the truncations were effected are labeled T1 through T5. Fig. 2 is a representative Western blot of an SDS gel, run under reducing conditions, showing the monomers of the WT/NBAT and its truncated forms in membranes of oocytes injected with the appropriate cRNA (10 ng/oocyte). Blot A was probed with Ab357 directed against an epitope preserved in all of the truncated forms of NBAT. Blot B was probed with Ab527 which is directed against an epitope deleted from the mutant Δ508–683. Therefore, the band corresponding to this mutant is not seen (lane 2), whereas the other mutants are readily observed. Ab527 also reacts with an approximately 62-kDa protein in uninjected as well as the injected oocytes. This protein, thus, appears to be an endogenous oocyte membrane protein whose identity is as yet unknown. As noted previously (39Mosckovitz R. Yan N. Heimer E. Felix A. Tate S.S. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4022-4026Crossref PubMed Scopus (39) Google Scholar), the WT/NBAT monomers migrate as two closely spaced bands (approximately 80 and 83 kDa, respectively) (lane 1 in each of the two blots), shown to be because of differential glycosylation. The monomeric species of the truncated forms exhibit the approximate expected mass, Δ508–683, the smallest of the mutants being approximately 58 kDa (this form lacks two of the five potential N-glycosylation sites of WT/NBAT). Noticeable also is a progressive decrease in the amount of expressed protein with longer truncations. Fig. 3 shows the induced Arg uptake activity in oocytes at 3 and 5 days after injection of the cRNAs. A surprising bimodal pattern for the induction of transport activity is revealed. Deletion of as few as 25 C-terminal amino acids virtually abolished the ability of the resultant truncated form (Δ658–683 mutant) to induce amino acid transport. The next in the series of truncated mutants (Δ615–683) was also devoid of transport activity. However, further deletion of 27 amino acids (Δ588–683) restored the ability of the mutant to induce transport (to about 75% of that elicited by the WT/NBAT). Note that in this truncated form, the fourth putative MSD has been deleted. Further truncations resulting in the Δ566–683 and Δ508–683 forms again abrogate the ability to induce transport. It is noteworthy that the mutation to a stop codon that generates the Δ566–683 form also disrupts the third heptad repeat in the leucine zipper motif (residues 545 to 572 in the WT/NBAT), an α-helical coiled-coil structure shown to be involved in oligomerization of a wide variety of proteins (see Ref. 40Hu J.C. Newell N.E. Tidor B. Sauer R.T. Protein Sci. 1993; 2: 1072-1084Crossref PubMed Scopus (84) Google Scholar and references contained therein). A parallel bimodal pattern of expression of transport activity was also seen when either Phe or Leu was used as a transport substrate instead of Arg (data not shown). A more detailed investigation of the transport induced in oocytes by NBAT has shown that this transport is in fact an exchange transport in which uptake of a cationic amino acid is enhanced by the outward flux of neutral amino acids and uptake of a neutral amino acid is stimulated by counter-transport of a cationic substrate (41Coady M.J. Jalal F. Chen X. Lemay G. Berteloot A. Lapointe J.-Y. FEBS Lett. 1994; 356: 174-178Crossref PubMed Scopus (47) Google Scholar, 42Chillarón J. Estévez R. Mora C. Wagner C.A. Suessbrich H. Lang F. Gelpi J.L. Testar X. Busch A.E. Zorzano A. Palacin M. J. Biol. Chem. 1996; 271: 17761-17770Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). It was, therefore, essential to determine whether the bimodal pattern of expression also applies to the exchange transport activity. cRNA-injected oocytes were pre-loaded with either [3H]Arg or [3H]Leu. Stimulation of the Arg and Leu efflux by addition of unlabeled Leu and Arg, respectively, to the extracellular medium was then determined (Fig. 4). With both substrates, the stimulated efflux from oocytes expressing the truncated mutant, Δ588–683, was about 70% of that seen with the oocytes expressing the WT/NBAT. No difference in the rates of efflux of either Arg or Leu were seen between the uninjected oocytes and the oocytes expressing the inactive truncated mutants; also, no significant stimulation of efflux was observed upon addition of an extracellular amino acid to these oocytes (Fig. 4, solid circles). The efflux rates observed in these oocytes were about 1/7th–1/6th of the stimulated rates seen with either the WT/NBAT or the Δ588–683 mutant expressing oocytes. Thus, the bimodal pattern of functional expression of the C-terminal truncated forms also applies to the exchange transport. Fig. 5 shows that both the WT/NBAT and its Δ588–683 mutant induce the same transport phenotype in oocytes (system b0,+-like). Thus, a large fraction of the induced uptake of both Arg and Phe (as well as of Leu) with both forms of this protein is Na+-independent. As also noted in several previous studies (4Tate S.S. Yan N. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1-5Crossref PubMed Scopus (151) Google Scholar, 8Segawa H. Miyamoto K. Ogura Y. Haga H. Morita K. Katai K. Tatsumi S. Nii T. Taketani Y. Takeda E. Biochem. J. 1997; 328: 657-664Crossref PubMed Scopus (17) Google Scholar, 13Wells R.G. Hediger M.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5596-5600Crossref PubMed Scopus (202) Google Scholar, 23Miyamoto K. Katai K. Tatsumi S. Sone K. Segawa H. Yamamoto H. Taketani Y. Takada K. Morita K. Kanayama H. et al.Biochem. J. 1995; 310: 951-955Crossref PubMed Scopus (54) Google Scholar), the NBAT-induced transport is stimulated about 20–30% by Na+ (thus, the term "system b0,+-like" used to describe the NBAT-induced transport). Whether this is a consequence of induction in oocytes of more than one amino acid transport system by NBAT is not clear at present. It is pertinent, however, that the transport activities induced by the Δ588–683 mutant are also similarly affected by Na+. With both forms of the protein, the uptake of Arg is inhibited by either Phe or Leu, and the uptake of Phe is inhibited by Arg. No significant difference was found in the K m values for Arg (K m values, 20 to 30 μm) and Leu (K m values, 30 to 40 μm) between the WT/NBAT and its Δ588–683 mutant. However, the transportV max for the truncated form with either Arg, Leu, or Phe was consistently about 75% of that observed for the WT/NBAT. In separate experiments (data not shown), we showed that the rate of induced transport in oocytes by the WT/NBAT cRNA reaches a maximum value between 2.5 and 5 ng of cRNA per oocyte although the amount of protein expressed
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