1 Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona 08028, 2 Departament de Bioanàlisi Mèdica, Institut d'Investigaciones Biomédiques Pi y Sunyer, Insituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Científicas, Barcelona 08036; and 3 Servei de Nefrologia Institut d'Urologia, Nefrologia i Andrologia, Fundació Puigvert, Barcelona 08025, Spain
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ABSTRACT |
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Mutations in the rBAT and b0,+AT genes cause type I and non-type I cystinuria, respectively. The disulfide-linked rBAT-b0,+AT heterodimer mediates high-affinity transport of cystine and dibasic amino acids (b0,+-like activity) in heterologous cell systems. However, the significance of this heterodimer for cystine reabsorption is unknown, as direct evidence for such a complex in vivo is lacking and the expression patterns of rBAT and b0,+AT along the proximal tubule are opposite. We addressed this issue by biochemical means. Western blot analysis of mouse and human kidney brush-border membranes showed that rBAT and b0,+AT were solely expressed as heterodimers of identical size and that both proteins coprecipitated. Moreover, quantitative immunopurification of b0,+AT followed by SDS-PAGE and mass spectrometry analysis established that b0,+AT heterodimerizes exclusively with rBAT. Together with cystine reabsorption data, our results demonstrate that a decreasing expression gradient of heterodimeric rBAT-b0,+AT along the proximal tubule is responsible for virtually all apical cystine reabsorption. As a corollary of the above, there should be an excess of rBAT expression over that of b0,+AT protein in the kidney. Indeed, complete immunodepletion of b0,+AT did not coprecipitate >20-30% of rBAT. Therefore, another rBAT-associated subunit may be present in latter parts of the proximal tubule.
proximal tubule; heterodimeric amino acid transporter; expression gradient
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INTRODUCTION |
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CLASSIC CYSTINURIA IS DUE to the impaired renal reabsorption of cystine and dibasic amino acids. Low cystine solubility causes the deposition of cystine calculi, which eventually leads to kidney failure (34). Reabsorption of cystine has been the object of a great research effort (32, 39). Dent and Rose (12) proposed a defective apical cystine transporter shared with dibasic amino acids as the main cause for cystinuria. Evidence for this hypothesis came recently with the identification of the two cystinuria genes: SLC3A1, which codes for the rBAT protein and is responsible for type I cystinuria, and SLC7A9, which codes for b0,+AT and causes non-type I cystinuria (5, 14, 40). Together, mutations in these two genes account for ~80% of cystinuria patients (16, 33).
b0,+AT and rBAT belong to the family of heteromeric amino acid transporters (HAT), which are made up of two structurally different subunits (10, 51, 54). The heavy subunit is a type II membrane glycoprotein, whereas the light subunit is an unglycosylated membrane protein bearing 12 putative transmembrane domains. Two conserved cysteines form a disulfide-linked heterodimer between the two subunits (37). A trafficking role has been proposed for the heavy subunit (30), the light subunit being the carrier itself. 4F2hc (CD98hc) and rBAT are the only known heavy subunits, while at least seven different light subunits have been cloned, each one specifying a distinct amino acid transport activity (7, 27, 38, 44, 45, 50).
The only known rBAT-associated light chain is b0,+AT. The heterodimer travels to the plasma membrane, where it mediates high-affinity transport of cystine and dibasic amino acids in a tightly coupled equimolar exchange with neutral amino acids (b0,+-like system). This activity allows accumulation of cystine and dibasic amino acids (8). A defective b0,+-like system, localized at the apical border of proximal tubule cells, explains the cystinuria phenotype (32). However, several lines of research questioned the role of the rBAT-b0,+AT heterodimer in vivo. First, the phenotypes of type I and non-type I cystinuria are not identical; i.e., type I cystinuria is a completely recessive disease whereas non-type I cystinuria is incompletely recessive (34). Second, both rBAT and b0,+AT are expressed at the apical border of the proximal tubule cells, but they display opposite expression gradients rBAT expression increases from the S1 to the S3 segment, and the reverse holds for b0,+AT (7, 18, 29, 35). Third, Ganapathy and colleagues (42, 43) found b0,+-like amino acid transport induction in cells transiently expressing both b0,+AT and 4F2hc, although other teams did not find this interaction (7, 14, 35). Moreover, rBAT-b0,+AT heterodimers have only been reported on coexpression in cell culture lines and oocytes (Ref. 35; Fernández E, Palacín M, and Chillarón J, unpublished observations). Thus direct in vivo evidence of this heterodimer is still lacking. Because of the above concerns, several authors have speculated that other heavy or light subunits may interact with b0,+AT or rBAT, respectively, in different segments of the proximal tubule (7, 10, 52).
Here, we use antibodies directed against b0,+AT and rBAT to demonstrate for the first time that they indeed form a rBAT-b0,+AT heterodimer in kidney brush-border membranes. Quantification of the interaction shows that rBAT is the only b0,+AT-associated heavy subunit and suggests that there is another rBAT-associated light chain. On the basis of these results and the genetic and early physiological data from cystinuria patients, we conclude that the rBAT-b0,+AT heterodimer is responsible for most, if not all, renal cystine reabsorption.
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MATERIALS AND METHODS |
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Antibodies and reagents. The monoclonal antibody 6-1-13 against human 4F2hc was a kind gift from Drs. Y. Ito and M. Tsurudome (Dept. of Microbiology, Mie Univ. School of Medicine, Mie-Ken, Japan). The polyclonal antibodies against mouse rBAT and human b0,+AT are described elsewhere (16, 18). The polyclonal antibodies against human rBAT and mouse b0,+AT were produced in rabbits. The peptides used as antigens were 1-MAEDKSKRDSIEMSMKG-17 and 1-MEETSLRRRREDEKSTHS-18, respectively. Cysteines were introduced to facilitate keyhole limpet hemocyanin conjugation. The preimmune antisera used in the study were obtained from the same rabbits. The P4N1 polyclonal antibody against the peptide 13-PEVETSPLGDGASPGPEQVK-32 from human y+LAT1 was produced at Research Genetics and used as a nonspecific control in the immunopurification experiments.
CnBr-activated Sepharose CL-4B was purchased from Amersham Pharmacia Biotech. Other general laboratory products were purchased from Sigma.Brush-border membrane preparations.
Preliminary evidence showed that rBAT and b0,+AT were
unequivocally detected only after enrichment in brush-border membranes. These were prepared by the Ca2+ precipitation method
(25). N-ethylmaleimide at 5 mM was present in
all buffers used (except in the resuspension buffer) following Tate and
colleagues (55), to prevent artifactual
reduction/shuffling of disulfides. The membranes were kept at 80°C
until use.
Western blot analysis and immunoprecipitation.
The protein content of the membrane preparation was measured by the
method of Bradford (4) using -globulin as a standard. SDS-PAGE was performed according to Laemmli (23) in the
absence or presence of 100 mM dithiothreitol (DTT). Proteins were
transferred to an Immobilon membrane (Millipore), which was blocked
with 5% nonfat dry milk and incubated with the various antibodies. The immune complexes were detected with a horseradish peroxidase-conjugated secondary antibody (Jackson Immunochemicals) followed by enhanced chemiluminescence (Amersham Pharmacia Biotech). Measurements were performed with Phoretix software under conditions in which detection was in the linear response range.
Immunopurification of b0,+AT from mouse brush-border membranes. Mouse brush-border membranes (50 mg) were solubilized at 1 mg/ml in RIPA as above. The solubilized membranes were incubated overnight at 4°C with 7.5 mg of P4N1 antibody IgGs covalently coupled to Sepharose CL-4B at 5 mg/ml according to the manufacturer's instructions. The cleared material was then incubated overnight at 4°C with 7.5 mg of anti-mouse b0,+AT IgGs coupled as before. The P4N1 and the anti-b0,+AT beads were applied to columns and washed in 40 vol of RIPA buffer, 20 vol of RIPA containing 0.3 M NaCl, and 20 vol of preelution buffer (10 mM Tris, pH 8, 0.5% sodium deoxycholate, 0.1% SDS). Elution was performed at room temperature with 2 vol of 50 mM diethylamine, pH 11.5, and 0.1% sodium deoxycholate and collected in 200-µl fractions. Purification was verified by Western blot analysis of 2% of each fraction with the anti-mouse b0,+AT antibody. The appropriate fractions were pooled, concentrated to 40 µl using ultrafiltration with a Centricon with a 10-kDa cutoff, subjected to reducing SDS-PAGE, and silver stained (21).
Mass spectrometry and peptide sequencing. Bands were manually excised, reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide, and digested with trypsin (Promega, Madison, WI) at 37°C. The peptides were extracted with acetonitrile/trifluoroacetic acid (21, 48), and the sample was evaporated to a volume of 20 µl. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) was performed using a Voyager DE-PRO (Applied Biosystems, Barcelona, Spain) in the reflectron mode. Spectra were externally mass calibrated using a standard peptide mixture. For the analysis, 0.5 µl of the peptide extract and 0.5 µl of matrix were loaded in the matrix-assisted laser desorption/ionization plate.
Automated protein sequencing was performed on a Finnigan LCQ ion trap mass spectrometer (ThermoQuest, Barcelona, Spain) equipped with a homemade microspray source. For chromatography, a custom-made fused silica capillary column (Kromasil C8, 5 cm × 150 µm) was used (31). Chromatographic solvents consisted of 1% acetic acid in water (solvent A) and 1% acetic acid in acetonitrile/water, 80:20 (vol/vol), (solvent B). The peptide extract was analyzed using a linear gradient from 0 to 55% of solvent B at a flow rate of 1 µl/min. Tandem MS was performed using an automated data-dependent MS/MS procedure, which consisted of three scan events: a full MS scan, a high-resolution zoom scan on the most abundant ion from the full MS scan, and an MS/MS experiment on this ion (15). Protein Prospector v 3.4.1 software (UCSF Mass Spectrometry Facility, University of California) was used for protein identification. GenBank was used for the search. ![]() |
RESULTS |
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The expression of rBAT and b0,+AT and their possible interaction in the kidney were analyzed by Western blotting and immunoprecipitation of human and mouse kidney brush-border membranes.
After Western blot analysis in reducing conditions, b0,+AT
appeared as two main bands of 38 and 82 kDa (Fig.
1). The highly hydrophobic character of
the protein may account for its faster mobility in SDS gels (its
predicted size is 53.5 kDa), as has been shown for other members of the
same family (27). The 82-kDa band is consistent with an
SDS-resistant dimer. Other light subunits behave similarly (Fernández E and Palacín M, unpublished
observations). The relative amount of the two bands varied
widely depending on the buffer used for resuspension or solubilization
of the membranes before SDS-PAGE. In nonreducing conditions,
b0,+AT ran as two bands of 130 and 250 kDa (Fig. 1). Their
relative amount also varied depending on the resuspension buffer
or the detergents used for solubilization. Both bands were sensitive to
N-glycosidase F (data not shown), suggesting that they were complexes of b0,+AT, which itself is unglycosylated, and a
glycoprotein. In nonreducing conditions, rBAT was also seen as two
bands of 130 and 250 kDa, which shifted to a single 96-kDa band in
reducing conditions (Fig. 1). In the absence of DTT, neither rBAT nor
b0,+AT was found in monomeric form, even with high
exposures. No significant differences were found between the two
species tested (Fig. 1, A and B). These results
confirmed earlier data from rodents (7) and extend it to
humans but did not demonstrate heterodimerization of b0,+AT
and rBAT. It also has to be kept in mind that despite the fact that
there are six different light subunits that heterodimerize with 4F2hc
(10, 52) and at least some of them should be coexpressed in the same cell, SDS-PAGE analysis did not distinguish between different 4F2hc-light chain complexes (19, 26). Direct
coimmunoprecipitation experiments must be performed to demonstrate
heterodimerization between a given pair of light and heavy subunits.
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Solubilized human brush-border membranes were subjected to
immunoprecipitation. Antisera against human b0,+AT, but not
its preimmune antisera, immunoprecipitated b0,+AT protein
(Fig. 2A). Under these
conditions, rBAT was coprecipitated. The reverse experiment was also
performed with an antibody directed against rBAT, which also
coprecipitated the b0,+AT protein (Fig. 2B).
Immunoprecipitation under denaturing conditions gave the same results
(not shown), indicating that the interaction was of a covalent nature.
This demonstrated that rBAT and b0,+AT form a heterodimer
in vivo. We attempted to quantify the rBAT-b0,+AT
heterodimer: two consecutive rounds of immunoprecipitation with the
antibody against rBAT depleted the protein from the membranes. Under
these conditions, no more than 5% of b0,+AT remained in
the supernatant (Fig. 2B). Although the antibody against
b0,+AT also fully depleted the protein from the membranes,
the amount of coprecipitated rBAT widely varied among the three
different kidney biopsies used (from 30% to >80% of the rBAT present
in the membranes). Certainly, interpretation of these data is hampered by the heterogeneity of the biopsies. Nevertheless, the results suggest
that most of the b0,+AT protein was associated with rBAT,
whereas a significant fraction of rBAT may not heterodimerize with
b0,+AT.
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4F2hc protein is localized to the basolateral membrane of tubule cells in the kidney (41, 44). Ganapathy and co-workers (42, 43) reported a functional association of transiently expressed b0,+AT and 4F2hc, although this result awaits confirmation (7, 14, 35). Our brush-border preparations contained 4F2hc, most likely from basolateral membranes, because it was not enriched compared with a preparation of total membranes. Under the conditions in our study, we were unable to coprecipitate any 4F2hc with either b0,+AT or rBAT (Fig. 2).
Because the human kidney samples were not homogeneous, we performed
similar experiments with solubilized mouse brush-border membranes (Fig.
3). It is in this model, and in rats,
that the opposite expression gradients of rBAT and b0,+AT
are well documented (7, 18, 35). In humans
(29), the data for b0,+AT agree with the
rodent model, whereas no information is available on rBAT. As with
human membranes, we obtained similar results when the solubilization of
mouse membranes was done in denaturing conditions. Antisera against
mouse b0,+AT, but not its preimmune antisera,
immunoprecipitated all the protein, as shown by two consecutive
immunoprecipitations and analysis of the supernatant (Fig.
3A). About 70-80% of the total rBAT present in
different brush-border preparations was not coprecipitated (Fig.
3A). This was not due to rBAT dissociation from the
heterodimer in the solubilization conditions used (which are not
expected to reduce disulfides), because the rBAT protein in the
supernatant ran, in nonreducing SDS-PAGE, with an identical size to
solubilized b0,+AT and rBAT before the immunoprecipitation
(Fig. 3B). Therefore, a significant amount of rBAT (~75%)
may heterodimerize with a light chain other than b0,+AT. On
the basis of the localization data (7, 8, 18, 35), we
conclude that the putative rBAT-X heterodimer (see Fig. 5 and DISCUSSION) may be expressed in the S3 segment.
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Because the antisera did not precipitate mouse rBAT, we were unable to
measure the amount of b0,+AT that formed a heterodimer with
rBAT. We thus affinity purified b0,+AT with the antisera
against the mouse protein in sufficient amounts to analyze copurified
proteins by MS and peptide sequencing. The purification was done
essentially by scaling up the immunoprecipitation procedure, under
conditions in which mouse b0,+AT was fully depleted from
the sample. The brush-border preparation was the same as that used in
the experiment shown in Fig. 3. The eluted proteins were subjected to
preparative SDS-PAGE and silver stained (Fig.
4A). Only two specifically
purified bands appeared in the silver-stained gel (Fig. 4A,
arrows). No other protein copurified with b0,+AT under
these stringent conditions (see MATERIALS AND METHODS). The
bottom band corresponded to the immunopurified
b0,+AT. The top band, with a size consistent
with rBAT, was analyzed by MS and peptide sequencing. Forty-one tryptic
peptides were found that completely matched the mouse rBAT sequence
(Fig. 4B). The peptides covered 38% of the sequence and
were distributed all along the protein sequence. No other protein
peptide was found. Therefore, b0,+AT heterodimerizes in the
kidney only with rBAT.
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DISCUSSION |
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The main purpose of this study was to solve the expression paradox of rBAT and b0,+AT in the kidney, which arose in immunolocalization and in situ hybridization experiments (7, 18, 29, 35, 42). The expression of rBAT decreases from the S3 to the S1 segment of the proximal tubule, whereas b0,+AT expression increases. Moreover, both proteins colocalize only in a few tubule sections (7, 35, 42). The rBAT-b0,+AT heterodimer had just been shown after overexpression in cell lines and in oocytes (14, 35), but not in the kidney.
Our results indicate that rBAT and b0,+AT are expressed solely as heterodimers in brush-border membranes (where they are localized). Coprecipitation experiments showed that the rBAT-b0,+AT heterodimer is present in human and mouse brush-border membranes. Most of the human b0,+AT was associated with rBAT. Finally, the immunopurification of mouse b0,+AT and MS of copurified proteins directly showed that it heterodimerizes exclusively with rBAT. In contrast, rBAT may have another light subunit partner. The hypothesis that a different heavy subunit formed a heterodimer with b0,+AT in S1 and S2, proposed by several authors including ourselves (7, 10, 52), is ruled out. Moreover, we were unable to coprecipitate any 4F2hc protein from the brush-border membranes (where it was still present) with either rBAT or b0,+AT, as might be expected given the basolateral localization of 4F2hc in the kidney (41, 44). This argues strongly against any physiological significance for the functional interaction of 4F2hc and b0,+AT in heterologous cell systems (42, 43), which may be interpreted as the result of the overexpression of both proteins. In any case, our data do not exclude the presence of additional heavy subunit homologs that could bind other light subunits, as neither the mouse nor the human genome is complete (20). In this sense, Kanai and colleagues (6) reported the cloning of a new light subunit, not expressed in the proximal tubule, which seemed to need a partner other than 4F2hc or rBAT.
Implications for cystine transport and the cystinuria phenotype.
Earlier studies in brush-border membrane vesicles and isolated tubule
fragments established that the apical transport of cystine in the
mammalian kidney is mediated by two systems: one with a low
Km for cystine shared with dibasic amino acids
and Na+ independent and the other with a high
Km unshared with dibasic amino acids and with a
degree of Na+ dependence (reviewed in Ref.
49). After functional characterization of rBAT (2,
46, 56), the demonstration that it is the type I cystinuria gene
(5, 40), and its main localization to the S3 segment of
the proximal tubule (18) (where the
low-Km system had been functionally localized)
(46), it appeared that rBAT, together with an unidentified
light subunit, constituted this low-Km system
(1, 13) [note that the S3 expression of rBAT did not fit
with cystine reabsorption (see below)]. Although b0,+AT
was later shown to be the non-type I cystinuria gene (14), and it induced the low-Km system on coexpression
with rBAT in cell lines and oocytes (7, 14, 35), the low
expression overlap with rBAT obscured the role of each protein in
cystine transport in vivo. Our results are consistent with the
translation of rBAT and b0,+AT expression along the
proximal tubule into the expression gradients of two different rBAT
heterodimers: rBAT-b0,+AT and rBAT-X (where X is most
likely another light subunit) (Fig. 5).
The rBAT-b0,+AT heterodimer is expressed in decreasing
amounts from the S1 to the S3 segment, like b0,+AT itself.
Because >90% of cystine reabsorption occurs in early parts of the
proximal tubule (49, 53), the rBAT-b0,+AT
heterodimer is the main apical cystine reabsorption system in the
kidney. This conclusion is also consistent with the finding that
cystinuria patients have clearance ratios of cystine/inulin (or
creatinine) of ~1 or even slightly higher, and they reabsorb very
little cystine (only occasionally >25%) (11, 17).
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ACKNOWLEDGEMENTS |
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We are grateful to Antonio Rosales, Fundació Puigvert, for help during the obtaining of the biopsies, and Dr. Luc Martí for help in obtaining mice kidneys. We also thank Drs. Y. Ito and M. Tsurudome for the 6-1-13 monoclonal antibody and Robin Rycroft for editorial help.
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FOOTNOTES |
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This research was supported by the research grant PM99/0172 from the Dirección General de Investigación Científica y Técnica (Spain) and a BIOMED2 CT98-BMH4-3514 European Commission grant (to M. Palacín). J. Chillarón received a reincorporation contract from the Ministry of Science and Education (Spain). E. Fernández received a predoctoral fellowship from the Ministry of Science and Education (Spain).
Address for reprint requests and other correspondence: J. Chillarón, Dept. of Biochemistry and Molecular Biology, Faculty of Biology, Univ. of Barcelona, Avda. Diagonal, 645, Barcelona 08028, Spain (E-mail: chillaro{at}worldonline.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 19, 2002;10.1152/ajprenal.00071.2002
Received 19 February 2002; accepted in final form 11 March 2002.
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