Human Molecular Genetics, 2000, Vol. 9, No. 3 431-438
© 2000 Oxford University Press
Functional analysis of novel mutations in y+LAT-1 amino acid transporter gene causing lysinuric protein intolerance (LPI)
1Department of Medical Genetics, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland, 2Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain, 3Department of Neuropediatrics, Hospital Virgen del camino, Pamplona, Spain, 4Department of Biology, University of Turku, FIN-20500 Turku, Finland, 5Department of Pediatrics, University of Turku, FIN-20520 Turku, Finland, 6Laboratory Department of Helsinki University Hospital and Department of Clinical Chemistry, University of Helsinki, FIN-00014 Helsinki, Finland and 7Department of Medical Genetics, Haartman Institute, University of Helsinki, FIN 00014 Helsinki, Finland
Received 18 October 1999; Revised and Accepted 6 December 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Lysinuric protein intolerance (LPI; MIM 222700) is an autosomal recessive disorder characterized by defective transport of the cationic amino acids lysine, arginine and ornithine at the basolateral membrane of the polar epithelial cells in the intestine and renal tubules, and by hyperammonemia after high-protein meals. LPI is caused by mutations in the SLC7A7 (solute carrier family 7, member 7) gene encoding y+LAT-1 (y+L amino acid transporter-1), which co-induces together with 4F2 heavy chain (4F2hc) system y+L in Xenopus oocytes. All Finnish LPI patients share the same founder mutation 1181-2A
T (LPIFin) not found in LPI patients elsewhere. Mutation screening of 20 non-Finnish LPI patients revealed 10 novel mutations: four deletions, two missense mutations, two nonsense mutations, a splice site mutation and a tandem duplication. Five LPI mutations (L334R, G54V, 1291delCTTT, 1548delC and LPIFin) were studied functionally. All mutant proteins failed to co-induce amino acid transport activity when expressed with 4F2hc in Xenopus oocytes. Immunostaining experiments revealed that frameshift mutants 1291delCTTT, 1548delC and LPIFin remained intracellular on expression with 4F2hc. In contrast, the missense mutants L334R and G54V reached the oocyte plasma membrane when co-expressed with 4F2hc, demonstrating that they are transport-inactivating mutations. This finding, together with the strong degree of conservation among all members of this family of amino acid transporters, indicates that residues L334 and G54 play a crucial role in the function of the y+LAT-1 transporter. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Lysinuric protein intolerance (LPI; MIM 222700) is an autosomal recessive disorder presenting growth failure, hepatosplenomegaly, osteoporosis, postprandial hyperammonemia and aversion to dietary protein. Transport of cationic amino acids at the basolateral membrane of the polar epithelial cells in the intestine and kidney tubules is decreased, and non-polar cells may also express the defect at the plasma membrane (1). LPI is more prevalent in Finland (1 in 60 000) than in other countries of the world. We have previously shown that mutations in the SLC7A7 (solute carrier family 7, member 7) gene encoding the y+L amino acid transporter-1 (y+LAT-1) cause LPI (2,3). All the Finnish LPI patients studied share the same founder mutation: a splice site mutation 1181-2A
T (LPIFin) leading to a 10 bp frameshift deletion in the cDNA. The non-Finnish LPI patients examined so far have had other mutations in the same gene (2,3). y+LAT-1 is a member of the growing family of light subunits of heterodimeric amino acid transporters (LSHAT). This family includes several human light subunits of the heavy chain of the surface antigen 4F2 (4F2hc; CD98) (4–9) and a subunit of rBAT (10). Mutations in the rBAT subunit were recently described in another aminoaciduria, non-type I cystinuria (11), which, in contrast to LPI, is exclusively expressed in the apical membrane of intestinal and tubular epithelial cells, and presents an impaired transport of cystine together with basic and neutral amino acids. We previously showed that y+LAT-1 induces y+L amino acid transport activity in Xenopus oocytes only when it is co-expressed with 4F2hc, but not when it is expressed alone (4,9). We also reported that an LPI-specific y+LAT-1 mutant (L334R) induced no y+L activity when co-expressed with 4F2hc in Xenopus oocytes (2). The fact that mutations in y+LAT-1 cause LPI suggests that this transporter is directly responsible for the efflux of basic amino acids at the basolateral membrane of epithelial cells, and hence plays a crucial role in the intestinal absorption and renal reabsortion of lysine, arginine and ornithine. The efflux of basic amino acids against membrane potential is probably achieved by exchange with neutral amino acids and simultaneous movement of sodium inside the cell (reviewed in refs 12,13).
In the present paper we describe a spectrum of novel mutations in the SLC7A7 gene, all associated with LPI. Analysis of amino acid transport activity and the cellular distribution of normal and mutant y+LAT-1 in Xenopus oocytes revealed that the frameshift mutants do not reach the plasma membrane, whereas the missense mutations lead to inactivation of amino acid transport. We also refined the physical localization and clarified the genomic structure of the SLC7A7 gene on chromosome 14q.
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Fine mapping and genomic structure of SLC7A7
Fine mapping of the SLC7A7 gene was accomplished by hybridizing fluorescently labeled DNA probes on extended DNA fibers (fiber-FISH), which allows visual high resolution physical characterization of the SLC7A7 locus (14). Fiber-FISH experiments verified that PAC 94G1 and PAC 109J14 clone insert sequences harbor the full genomic sequence of the SLC7A7. Twenty DNA fibers studied showed that the SLC7A7 gene is localized at the 3' end of PAC 94G1 and 109J14, ~240 kb downstream from TCRA, an intragenic microsatellite marker of the T cell receptor
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gene on chromosome 14q11.2 (Fig. 1). The fiber-FISH results suggested a genomic length of ~20 kb for the SLC7A7 gene (Fig. 1), well in line with the polymerase chain reaction (PCR) data (below).
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The genomic structure of SLC7A7 was obtained by using the exon–exon PCR method. The gene was found to comprise 11 exons and 10 introns, harboring ~18 kb of genomic DNA (Fig. 2; Table 1). The sizes of all introns were determined by sequencing both strands of DNA, except for introns 1, 2 and 5, which were analyzed by agarose gel electrophoresis. All exon–intron junctions obeyed the universal GT-AG rule. The information obtained from the genomic structure was utilized to design primers for PCR amplification of each coding exon from genomic DNA to screen LPI patients for mutations.
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Novel mutations in SLC7A7
Twenty non-Finnish LPI patients from 17 families were studied for mutations in SLC7A7 by reverse transcription (RT)–PCR analysis of y+LAT-1 mRNA or PCR amplification of SLC7A7 exons and the corresponding splice sites. SLC7A7 mutations were found in 19 patients in 16 families. In only one case, an 8-year-old Turkish patient with typical clinical findings, no mutations were detected (excluded from Table 2). In a Canadian patient (no. 18), SLC7A7 mutation of one allele only was detected (Table 2). Thus, this study revealed a total of 11 SLC7A7 mutations, 10 of which were novel mutations. The new mutations comprised two nonsense point mutations, two missense mutations, a splice donor mutation, four small frameshift deletions and a tandem duplication (Table 2; Figs 2 and 3). The 1291delCTTT mutation of a Spanish patient (no. 1) had been found earlier in another Spanish patient but in one allele only (2). This study increases the total number of LPI-specific SLC7A7 mutations to 15 (2,3; present study).
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The missense mutation G54V found in a Latvian and in an Estonian patient causes a Gly
Val change. Gly54, which is located at the first transmembrane domain, is conserved in all members of the LSHAT family (Fig. 3). The nonsense mutation 1012GA (W242X) in the third intracellular loop of the protein was found in four unrelated patients from four families with a Moroccan or North African ethnic background. The homozygous splice site mutation 911+1GA in a Turkish patient is interesting because it led to aberrant splicing and skipping of exon 4 in cDNA. All the deletions detected in genomic DNA or cDNA changed the reading frame of the gene. The mutations 539delTT, 1291delCTTT and 1438duplAACTA produce truncated proteins with new stop codons at amino acid positions 97, 351 and 384, respectively. On the contrary, mutant proteins of 1471delTTCT, 1548delC and 1746delG are extended proteins with amino acid residue lengths of 517, 518 and 518, respectively. The normal gene product has a length of 511 amino acids. Interestingly, the 1746delG was found in two unrelated patients, one of Norwegian and the other of Dutch origin. The Swedish missense mutation G338D affects a non-conserved residue in the LSHAT family (Fig. 3). This change was not seen in control chromosomes, and it was present in one allele of both parents (data not shown), and therefore a polymorphism is a very unlikely possibility. During mutation screening, five silent polymorphisms were found in Finnish LPI patients and controls, and two of these were also present in several non-Finnish LPI patients (Table 3).
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Functional analysis of LPI mutants
The transport activity of five different y+LAT-1 mutants, including the previously reported L334R and LPIFin, was studied in the Xenopus oocyte expression system. In order to analyze transport activity and the subcellular distribution of the y+LAT-1 mutants simultaneously, oocytes were injected with 4F2hc cRNA, together with the cRNA from N-myc wild-type or N-myc mutated y+LAT-1. The N-myc epitope did not significantly affect amino acid transport activity (80–100% of the y+LAT-1-induced transport in three separate experiments; data not shown). Sodium-independent L-arginine uptake and sodium-dependent L-leucine uptake were measured 3 days after cRNA injection (Fig. 4). Co-expression of human N-myc-y+LAT-1 and 4F2hc cRNAs increased the y+L activity 3- to 4-fold above the activity induced by 4F2hc expressed alone. On the contrary, when 4F2hc was co-injected with N-myc-L334R, N-myc-G54V, N-myc-1291- delCTTT, N-myc-1548delC or N-myc-LPIFin, no increase in transport activity was recognized either for L-arginine or for L-leucine uptake. These results demonstrate complete loss of function of both the missense and frameshift types of LPI mutation analyzed. The lack of any residual activity does not allow further characterization of these mutations.
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In order to study intracellular expression of LPI mutants, we performed immunofluorescence detection of wild-type and mutated N-myc-tagged y+LAT-1 after co-expression with 4F2hc in Xenopus oocytes. Oocytes were treated with anti-myc antibodies 3 days after injection and studied in a confocal microscope. Wild-type N-myc-y+LAT-1 reached the plasma membrane when co-injected with 4F2hc, but remained intracellular when injected alone (Fig. 5). This is in agreement with previous results showing that 4F2hc brings the corresponding LSHAT (e.g. SPRM1 from Schistosoma mansoni and human LAT-2) to the oocyte plasma membrane (5,8). The expression of the studied missense or frameshift LPI-specific SLC7A7 mutations differed strikingly. The missense mutants L334R and G54V, when co-expressed with 4F2hc, reached the plasma membrane identically to the expression of the wild-type construct (Fig. 5) despite the lack of amino acid transport activity, as shown in Figure 4. On the contrary, expression of the frameshift y+LAT-1 mutants 1291delCTTT, 1548delC and LPIFin was seen as dispersed granules in the cytoplasm without any staining at the plasma membrane. Intensity and expression pattern were identical with all three frameshift mutants studied.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present study, we extend our knowledge of the SLC7A7 gene and its mutations responsible for LPI. Fine localization of the gene was determined by fiber-FISH, and the genomic structure was accomplished by PCR. Several novel mutations in LPI patients were detected, and expression studies in Xenopus oocytes revealed, for the first time, the y+L transport-inactivating mutations in the SLC7A7 gene.
Mutation screening of SLC7A7 revealed 10 novel mutations located in the transmembrane and intracellular domains of the y+LAT-1 protein: five frameshift, two nonsense and two missense mutations, and a tandem duplication. In addition, five apparent polymorphisms were found. The nonsense mutation W242X was found in four unrelated families, three with Moroccan and one with a North African ethnic background. This mutation therefore represents another founder mutation of LPI in addition to the LPIFin mutation in the Finnish population (2). The G54V mutation was found in two unrelated families from neighboring Baltic countries (Estonia and Latvia), and the 1291delCTTT mutation was found in two unrelated Spanish patients. None of these families are known to be related, but a common origin of these mutations in the Baltic area and in Spain is still plausible. The frameshift deletion 1746delG was found in two unrelated European families, one of Dutch and the other of Norwegian origin. All other mutations were private pathogenic mutation events in the SLC7A7 gene. The missing mutations in one allele of a compound heterozygote patient 18 and in both alleles of one Turkish patient may lie in a yet uncharacterized promoter region of the SLC7A7 gene.
Of the 15 LPI-specific SLC7A7 mutations described up to now (2,3; present study), most produce dramatic changes in the structure of the y+LAT-1 amino acid transporter (i.e. seven frameshift, three nonsense and one exon-skipping mutations, and one large amino acid deletion). Only three mutations lead to an amino acid change. The missense (L334R and G54V) and the frameshift (1291delCTTT, 1548delC and LPIFin) LPI mutations analyzed here show a complete loss of amino acid transport function with 4F2hc. Mutants 1291delCTTT and LPIFin lack the last four and five transmembrane domains of the transporter, respectively, whereas the 1548delC mutation distorts the amino acid sequence of the last two transmembrane domains. The fact that neither of these frameshift mutants reached the plasma membrane after co-expression with 4F2hc in the oocyte could be explained as follows: (i) the truncated proteins may fold incorrectly and end up in the degradation pathway in the cell; or (ii) these mutated proteins may be stable in reticular membrane but completely fail to interact with 4F2hc.
On the contrary, missense mutations L334R and G54V do not present any amino acid transport activity despite reaching the oocyte plasma membrane properly, and should therefore be considered transport-inactivating mutations. L334R drastically alters a non-polar amino acid to a positively charged amino acid. Leucine in position 334, located in an internal loop between the putative transmembrane domains VIII and IX, is conserved in all mammalian species in which the LSHAT family of transporters has been studied. It is similarly conserved in some related prokaryote transporters. The G54V mutation is located at the first predicted transmembrane domain (residues 39–59) of the transporter. Gly54 and its surrounding residues constitute a motif, GSGIF (position 54 in bold), that is one of the largest conserved motifs among all members of the LSHAT family (with the exception of human xCT transporter that has GAGIF). Extension of this analysis to other sequence homology related GenBank entries shows that Gly54 is conserved in many prokaryote amino acid and amine transporters as well as in the mammalian cationic amino acid transporter (CAT) family. The G54V mutant represents an apparently subtle change from a non-polar to a non-polar amino acid residue, but the hydrophobicity values of these two amino acids differ markedly [values for glycine and valine are –0.4 and 4.2, respectively (17)]. The hydrophobicity analysis of y+LAT-1 (4) implies that the change from glycine to valine in position 54 induces a dramatic increase in the hydrophobicity (from 74–179 to 120–225 in range), which will affect the amino acid sequence from position 49 to 59. The strong degree of conservation among all members of this family of amino acid transporters and the drastic loss of function of L334R and G54V demonstrate that Leu334 and Gly54 are crucial for the function of y+LAT-1. This information also offers important clues for further experiments towards the elucidation of the structure–function relationship of the y+LAT-1 amino acid transporter.
We were unable to evaluate genotype–phenotype correlations in this study. However, taking into account that all Finnish LPI patients share the same founder mutation (LPIFin) and yet show extensive clinical variation (1), the genotype–phenotype correlation is probably rather loose in LPI. The clinical presentation of the Finnish LPI patients ranges from almost normal growth with minimal protein intolerance to severe cases with visceral enlargement, osteoporosis and development of alveolar proteinosis combined with severe protein intolerance. Evidently, other hitherto unknown factors in addition to SLC7A7 mutations exert an effect on the pathogenesis and clinical manifestations of LPI.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Patients
Twenty LPI patients from 17 families with varied ethnic backgrounds were studied (Table 2). According to the referring centers, the clinical findings of the patients were compatible with LPI. All patients had plasma concentrations and urinary excretion patterns of amino acids characteristic for LPI. They had shown hyperammonemia and orotic aciduria after high-protein meals, and they also had elevated serum ferritin and lactate dehydrogenase concentrations. Lymphoblastoid or skin fibroblast cell lines were available from patients 1–5, 7, 10, 11, 16 and 18, and peripheral blood samples from patients 6, 8, 9, 12–15 and 17–19. We also studied DNA samples (peripheral blood) from the parents (obligate carriers) of patients 1–3, 8–10, 13–15, 17 and 18, and from the healthy siblings of patients 2, 8–10, 13–15, 17 and 18. The parents of patients 7–9, 11 and 16 were closely consanguineous. The samples and clinical information were kindly provided as follows. Patient 1: Dr Yoldi, Pamplona, Spain; patient 2: Dr Mikitis, Valmiera, Latvia; patient 3: Dr Õunap, Tartu, Estonia; patient 4: Dr Leonard, London, UK; patients 5, 6 and 11: Dr Mancini, Rotterdam, The Netherlands; patient 7: Dr Rubio-Gozalbo, Nijmegen, The Netherlands; patients 8 and 9: Dr Bakken, Amsterdam, The Netherlands; patients 10 and 16: Dr Poll-The, Utrecht, The Netherlands; patient 12: Dr Mönch, Berlin, Germany; patients 13 and 14: Dr Kågström, Sandviken, Sweden; patient 15: Dr Berget, Stavanger, Norway; patient 17: Dr Filler, Berlin, Germany; patient 18: Dr Rutledge, Birmingham, AL; and patient 19: Dr Peters, Melbourne, Australia.
Fine mapping of SLC7A7 using fiber-FISH
To characterize further the genomic structure of the SLC7A7 gene, a P1-derived genomic library (PAC) was screened using PCR with primers LPI-1087F and LPI-1268R to amplify a 440 bp fragment containing the critical region of the Finnish LPI mutation (2). End fragment sequencing of the clones showed that the two recognized insert clones, 94G1 and 109J14, were identical. In fiber-FISH experiments, preparation of target slides, probe labeling, hybridization, detection and fluorescence microscopy were performed as described (18) except for minor modifications. Briefly, free DNA target slides were created by applying agarose-embedded human lymphocytes as described (18,19). The PAC [clones 94G1, 109J14 and 85G14 as distance standard (15)] and y+LAT-1 cDNA probes were labeled with biotin or digoxigenin according to a standard nick translation protocol (Gibco BRL, Paisley, UK). The probes (100 ng in 2x SSC with 50% formamide and 10% dextran sulfate) were hybridized on target slides at 37°C for 16–20 h. After hybridization, the slides were washed (three times at 43°C each) at high stringency with 50% formamide in 2x SSC. The probes were detected using tetramethylrhodamine-conjugated avidin D (biotin) and fluorescein isothiocyanate–antidigoxigenin anti- bodies (digoxigenin). A multicolor image analysis was used for acquisition, display and quantification of the hybridization signals of DNA fibers. Twenty-five images were acquired for distance measurements. Photometrics PXL camera (Photometrics, Tucson, AZ) operation, image acquisition, probe size and distance measurements were performed using IPLab software. A PAC clone with known insert size [PAC 85G14, 140 kb (15)] containing microsatellite marker TCRA was used as a calibration standard in the distance measurements.
Genomic structure
The genomic structure of the SLC7A7 gene was studied by using PCR with PAC 94G1, PAC 109J14 or total genomic DNA as template, and by using SLC7A7-specific primers (available on request). The PCR amplification of the SLC7A7 introns was performed either using a standard protocol or Expand Long Template PCR System (Roche Molecular Biochemicals, Mannheim, Germany) for a Perkin Elmer 2400 Thermal Cycler (Perkin Elmer, Palo Alto, CA).
Mutation screening
For mutation analysis, we used RT–PCR of the SLC7A7 cDNA and genomic PCR of all exons and exon–intron boundaries. mRNA extraction and RT–PCR amplification were performed as described (2). All RT–PCR products were run on a 0.8% agarose gel, purified with the Gel Extraction kit (Qiagen, Hilden, Germany), and sequenced using the BigDye terminator sequencing kit and ABI 377 automated DNA sequencer (Perkin Elmer Applied Biosystems, Foster City, CA). cDNA mutations were verified and patients with only genomic DNA available were screened using genomic PCR amplification and sequencing the exon of the mutation by using 5' and 3' intronic primers (Table 1). The PCR reaction mixture contained dNTPs (200 µM of each), forward and reverse primers (20 pmol each), and Taq DNA polymerase (2 U; Promega, Mannheim, Germany) in a final volume of 50 µl. PCR conditions were 95°C for 5 min; 30–35 cycles of 95°C for 45 s, 53–59°C for 45 s and 72°C for 1 min; and 72°C for 7 min (Perkin Elmer 9700 Thermal Cycler). PCR products were either run on a 1.0–1.5% agarose gel and purified, or purified directly using GFX PCR DNA and the Gel Band Purification kit (Amersham Pharmacia Biotech, Piscataway, NJ). Sequencing was performed as described above. Sequence alignment and comparison were performed by using Sequencher 3.11/4.0 software (Gene Codes, Ann Arbor, MI).
Construction of mutant cDNA
The mutant y+LAT-1 cDNA containing G54V, 1548delC or LPIFin mutant 1181-2AT was constructed using the QuickChange site-directed mutagenesis kit (Stratagene, Cambridge, UK) according to the manufacturer’s protocol. The mutagenic oligonucleotides for G54V and 1548delC were 5'-CATGATCGGCTCAG(T)CATCTTTGTTTCCCCCAA-3' and 5'-CTCAGC- GTTTTCTTCC(C)GATTGTCTTCTGCCTC-3' (sense strand; the mutated sites are in parentheses). Because of the limitations of the method, the Finnish 10 bp deletion of y+LAT-1 had to be constructed in three successive mutagenesis reactions using primers 5'-GCCAGTGATGCTGTTGCTGTG(ACT)TTTGC- AGATCAGATATTTGG-3', 5'-CTTGGCCAGTGATGCTGTTGCTGTGTTTG(CAG)ATCAGATATTTGG-3' and 5'-GAGACATCTTGGCCAGTGATGCTGTTGCTGTG(TTTG)ATCAGATATTTGG-3' (nucleotides in parentheses were omitted). The 1291delCTTT mutant was obtained by the RT–PCR specific amplification of the y+LAT-1 from the RNA of the Spanish patient described elsewhere (2). Construction of the L334R mutant was as described elsewhere (2). The sequences of all mutated cDNAs were confirmed by complete sequencing of the products. To create an N-myc-tagged y+LAT-1 cDNA, the pT7T3D-y+LAT-1 (4) was cut with NcoI, filled with Klenow polymerase and cut with NotI. The resulting fragment was subcloned into pNKS2-myc ‘NotI’. The N-myc-tagged y+LAT-1 cDNA was tested by sequencing. The N-myc-y+LAT-1 mutants were constructed by substituting the cDNA fragment containing the mutations in wild-type pNKS2-N-myc-y+LAT-1 cDNA.
Oocytes, injections and uptake measurements
Oocyte origin, management and injections were as described (20,21). Defolliculated stage VI X.laevis oocytes were injected with 10 ng/oocyte of human 4F2hc, human y+LAT-1, N-myc-y+LAT-1 or N-myc-y+LAT-1 mutants. Synthesis of human 4F2hc cRNA was as described (21). N-myc wild-type and mutated y+LAT-1 cRNAs were obtained by cutting the cDNA with NotI and using SP6 polymerase. Influx rates of L-[3H]arginine or L-[3H]leucine (Amersham Pharmacia Biotech) were measured in 100 mM NaCl or in 100 mM choline chloride medium at day 3 after injection as described (21). Amino acid transport rates obtained with oocytes injected with water (50 nl) were similar to those of non-injected oocytes (data not shown).
Localization of expression of the y+LAT-1 mutants by confocal microscopy
Groups of five oocytes were prepared for immunofluorescence 3 days after injection with 10 ng/oocyte of human 4F2hc cRNA alone or in combination with the same amount of wild-type or mutated N-myc-y+LAT-1 cRNA. Immunocytochemistry experi- ments were performed as described (8).
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ACKNOWLEDGEMENTS |
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We thank Arja Reinikainen, Jenni Leppävuori and Judith García for their technical assistance. This study was supported in part by the Foundation for Pediatric Research (Ulla Hjelt Fund) and by Dirección General de Investigación Científica y Técnica Research Grant PM 96/0060 and by Grant 1995 SRG 537 from the Generalitat de Catalunya (Spain). D.T. and M.P. are recipients of fellowships from the Ministerio de Educación y Cultura (Spain) and from the Comissió Interdepartamental de Recerca i Innovació Tecnològica (Catalonia, Spain).
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FOOTNOTES |
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+ These authors contributed equally to this work
§ To whom correspondence should be addressed. Tel: +358 2 333 7235; Fax: +358 2 333 7300; Email: jmykkane@utu.fi
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REFERENCES |
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