Apical heterodimeric cystine and cationic amino acid transporter expressed in MDCK cells

Christian Bauch and François Verrey

Institute of Physiology, University of Zürich, CH-8057 Zürich, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The luminal uptake of L-cystine and cationic amino acids by (re)absorptive epithelia, as found in the small intestine and the proximal kidney tubule, is mediated by the transport system b0,+, which is defective in cystinuria. Expression studies in Xenopus laevis oocytes and other nonepithelial cells as well as genetic studies on cystinuria patients have demonstrated that two gene products, the glycoprotein rBAT and the multitransmembrane-domain protein b0,+AT, are required for system b0,+ function. To study the biosynthesis, surface expression, polarity, and function of this heterodimer in an epithelial context, we established stable Madin-Darby canine kidney (MDCK) cell lines expressing rBAT and/or b0,+AT. Confocal immunofluorescence microscopy shows that both subunits depend on each other for apical surface expression. Immunoprecipitation of biosynthetically labeled proteins indicates that b0,+AT is stable in the absence of rBAT, whereas rBAT is rapidly degraded in the absence of b0,+AT. When both are coexpressed, they associate covalently and rBAT becomes fully glycosylated and more stable. Functional experiments show that the expressed transport is of the high-affinity b0,+-type and is restricted to the apical side of the epithelia. In conclusion, coexpression experiments in MDCK cell epithelia strongly suggest that the intracellular association of rBAT and b0,+AT is required for the surface expression of either subunit, which together form a functional heterocomplex at the apical cell membrane.

glycoprotein-associated amino acid transporter; cystinuria; exchanger; epithelial cell polarity; kidney transport; intestinal absorption; Madin-Darby canine kidney cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

STUDIES OF CYSTINURIA PATIENTS have revealed that the underlying defect is a lack of cystine and cationic amino acid (re)absorption across small intestine and proximal kidney tubule epithelia due to missing transport across their luminal membranes (see Ref. 21 for a review). The molecular identification of the defective transporter has been initiated by the cloning of the glycoprotein rBAT (D2) that induces a b0,+-type transport (Na+-independent uptake of dicationic and large neutral amino acids) when expressed in Xenopus laevis oocytes (3, 28, 32). Genetic studies have shown that defects in the gene encoding this glycoprotein (SLC3A1) were indeed responsible for type I cystinuria (6). However, it became clear that rBAT itself was not the entire transporter and that it had to associate with an endogenous oocyte protein to transport amino acids. This conclusion was based on the fact that rBAT had the predicted structure of a type II single transmembrane-domain glycoprotein that was homologous to the heavy chain of 4F2 (CD98), known to form a heterodimer with an unknown hydrophobic protein called "light chain." Furthermore, immunoprecipitation experiments had shown that rBAT similarly formed a covalently bound heterodimer with an unknown protein (see Ref. 21 for a review).

Many functional amino acid uptake and two-electrode voltage-clamp studies were performed in oocytes expressing rBAT, presumably associated with an endogenous X. laevis light chain, that defined the substrate uptake specificity of the expressed amphibian b0,+-type transport and revealed that many of the rBAT mutations found in cystinuria lead to a lack of transporter surface expression (9, 20). In view of the physiological role played by the epithelial b0,+ transport system for vectorial amino acid (re)absorption, it was particularly interesting to learn that it functions as an obligatory exchanger that preferentially exchanges extracellular dicationic amino acids for intracellular neutral amino acids (5, 10, 22). This exchange mode indicates that, for net vectorial amino acid transport, the b0,+ system has to function in parallel with another transporter that unidirectionally recycles the exchanged neutral amino acids back into the cell, such as the B0 system, which has not been clearly identified as yet at a molecular level (see Refs. 30 and 31 for a discussion).

After the identification of light chains of 4F2hc, which define the new family of glycoprotein-associated amino acid transporters (gpaATs) (30), another mammalian member of that family, b0,+AT (BAT1), was shown to associate specifically with rBAT in transient expression systems. The b0,+-type uptake property of the resulting heterodimer was characterized in COS cells (that do not express endogenous b0,+AT) and in X. laevis oocytes (7, 19, 22). In this latter expression system, the problem of the endogenous light chains was circumvented by expressing a functional fusion protein [human rBAT (hrBAT)-mouse b0,+AT (mb0,+AT)] (22). Genetic studies have demonstrated that most cases of non-type I cystinuria could be explained by a mutation at the level of the b0,+AT gene SLC7A9 (11, 12).

In the present study, we have addressed in an epithelial expression system the questions of the requirement of rBAT and b0,+AT interaction for their biosynthesis, surface expression, apical polarity, and function.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell culture. MDCK cells (strain II) were cultured at 37°C and 5% CO2 in DMEM (52100-013, Life Technologies, Basel, Switzerland) with 22 mM NaHCO3, 5 × 104 U/l penicillin, 50 mg/l streptomycin, 2 mM L-glutamine, 1% nonessential amino acids (11140-035, Life Technologies), and 10% FCS.

cDNA constructs and transfection. The mb0,+AT cDNA (22) coding sequence was subcloned in the vector pcDNA3.1(-)Hygro (Invitrogen, Carlsbad, CA). The hrBAT cDNA (2) coding sequence was subcloned in the vector pLKneo, in which its transcription is controlled by a glucocorticoid-inducible promoter (16). MDCK cells were transfected with these constructs using the Lipofectamine Plus reagent (Life Technologies), according to the manufacturer's protocol. Selection of transfected cells was carried out with 200 µg/ml of G418 and/or 150 µg/ml of Hygromycine, beginning 48 h after transfection. Resistant clones were isolated after ~3 wk of selection using cloning rings.

Northern blot and RT-PCR analyses. Total cellular RNA was isolated according to the manufacturer's protocol using TRIzol Reagent (Life Technologies). Transcription of hrBAT was tested by Northern blot analysis, as previously described (27). Alternatively, transcription of mb0,+AT was tested by RT-PCR as follows: first-strand cDNA was synthesized from 100 ng of total RNA with Moloney murine leukemia virus RT (Promega, Madison, WI) and 50 pmol of random hexamer primers (Life Technologies). One-tenth of the first-strand cDNA was used as a template for PCR amplification using 50 pmol of mb0,+AT primers [forward primer 5'-TTCACAGTGATGACCCCAACGGAGCT-3', reverse primer 5'-TTTGTGACCGGCCTGGAGATTCTCTG-3' (Microsynth, Balgach, Switzerland)] and 2 U of recombinant Taq polymerase (Biofinix, Praroman, Switzerland) in a total volume of 30 µl of reaction buffer. Transcription of hrBAT was confirmed by performing PCR on the same first-strand cDNA using hrBAT primers [forward primer 5'-GGCACTTTGACGAAGTGCGAAACCA-3', reverse primer 5'-AACGCGAAGTCAGCCGTGAACTGTCT-3' (Microsynth)].

Pulse-labeling of MDCK cells. MDCK cells cultivated to confluency on plastic dishes (Corning-Costar, Acton, MA) were washed three times with PBS containing 2 mM EDTA. Cells were incubated for 30 min in methionine-free DMEM (D3916, Sigma, St. Louis, MO) supplemented with 22 mM NaHCO3, 0.5 × 106 U/l penicillin, 0.5 g/l streptomycin, 62.6 mg/l L-cystine · 2HCl, and 10% FCS. The medium was then exchanged for the above-mentioned medium containing 1.0-2.5 mCi/ml of 35S-methionine (NEN, Boston, MA) for 30 min or 2 h at 37°C, 5% CO2. For chase experiments, cells were subsequently incubated in completed culture medium for the times indicated. The fact that the precipitated bands are stronger after a 2-h chase period (see Fig. 3, A and B, lanes 5 and 8) than just after the 2-h pulse-labeling (see Fig. 3, A and B, lanes 4 and 7) is due to the continuing incorporation, during the chase period, of labeled L-methionine that accumulated within the cell during the pulse period. Cells were then washed three times with PBS containing 0.1 CaCl2 and 1 mM MgCl2 and lysed on ice with (in mM) 50 Tris · HCl, pH 8.0, 120 NaCl, 0.2 polymethylsulfonyl fluoride, and 10 diamide, as well as 0.5% Nonidet P-40 (NP-40) and a 1% protease inhibitor cocktail (Sigma). Incorporated radioactivity was determined by TCA precipitation and liquid scintillation.

Antisera. Polyclonal rabbit antibodies were raised against synthetic peptides corresponding to the NH2 terminus of hrBAT, MAEDKSKRDSIEMSMKGC, and the COOH terminus of mb0,+AT, CHLQMLEVVPEKDPE, coupled to keyhole limpet hemocyanin (Eurogentech, Seraing, Belgium).

Immunoprecipitation of pulse-labeled MDCK cells with polyclonal antibodies (serum). Lysates (each sample containing the same amount of incorporated methionine) were precleared by two incubations with uncoated beads. Polyclonal antibodies were added to the lysates and incubated overnight at 4°C. Beads (protein G plus protein A-agarose, Calbiochem, La Jolla, CA) were added and incubated for 4 h at 4°C. The beads were washed three times each in 20 mM Tris · HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40 with and without 500 mM LiCl. SDS-PAGE sample buffer was added, and samples were heated to 65°C for 15 min, with beta -mercaptoethanol added where indicated, and SDS-PAGE was performed on 10% gels. Gels were stained in Coomassie blue, fixed, incubated in Amplify (Amersham, Arlington Heights, IL), and exposed to film.

Double immunofluorescence staining of cotransfected MDCK cells. Cells were seeded on filters (35 mm, Corning Costar Transwell filters) at 100% confluency and cultivated for 7 days preceding the experiments. rBAT expression was induced 3 days before the experiments with 1 µM dexamethasone. Filters were washed three times using PBS containing 2 mM EDTA. Cells were fixed using 3% paraformaldehyde and 0.2% Triton X-100 for 15 min at room temperature. Filters were washed three times and cut into squares. The cells were first incubated with rabbit anti-b0,+AT serum at 1:500 dilution in PBS containing 0.5% BSA overnight at 4°C and, after being washed, were then incubated for 6 h at room temperature with 1 µg/ml of Texas red-labeled anti-rabbit-IgG Fab fragments (Rockland, Gilbertsville, PA). After being washed, free binding sites on anti-b0,+AT antibodies were blocked by incubating the filter pieces overnight at 4°C with 150 µg/ml of unlabeled anti-rabbit-IgG Fab fragments (Rockland). After another round of washes, filter pieces were incubated for 6 h with anti-rBAT serum diluted 1:50 in PBS containing 0.5% BSA and washed again. Filter pieces were subsequently incubated overnight at 4°C with FITC-labeled anti-rabbit-IgG antibody (Sigma), washed, and mounted in DAKO-glycergel (DAKO, Glostrup, Denmark) containing 2.5% 1,4-diazabicyclo[2,2,2]octane (DABCO) as a fading retardant. Confocal images were taken using a Leica laser scanning microscope (TCSSP, Wetzlar, Germany) equipped with a ×63 oil-immersion objective. The appropriate controls were performed without the first and/or second primary (serum) antibodies: omission of the rBAT antibody did not yield any detectable FITC signal, demonstrating the absence of cross-reaction with the b0,+AT antibody.

Filter uptake experiments. MDCK cells were passaged to 35-mm Corning Costar Transwell filters at 100% confluency and cultivated for 7 days. rBAT expression was induced 24 h before the experiments with 1 µM dexamethasone. The integrity of the monolayer was checked by resistance measurement using the Millicell device (Millipore, Bedford, MA). Filters were washed three times with uptake buffer [in mM: 150 NaCl (+Na condition) or cholin-Cl (-Na condition), 10 HEPES, pH 7.4, 1 CaCl2, 5 KCl, 1 MgCl2, and 10 glucose] at 37°C and incubated in uptake buffer for 10 min. The buffer was replaced unilaterally with buffer supplemented with amino acid at the indicated concentrations and the corresponding 3H-labeled L-amino acid as the tracer (except for L-[14C]cystine and L-[14C]isoleucine); the contralateral compartment received the same solution without the labeled L-amino acid tracer. Uptake experiments were performed for 1 min (the linearity of uptake was verified in preliminary time course experiments). Diamide (10 mM) was added to the solution for experiments with L-cystine. The uptake was stopped by replacing the amino acid uptake solution with ice-cold uptake buffer and washed four times. The filters were excised and placed into scintillation vials containing scintillation fluid (Packard, Meriden, CT). After vials were shaked overnight at room temperature, radioactivity was determined by scintillation counting.

Statistics. Data are expressed as means ± SE. The difference between control and test values was evaluated using Student's t-test (2 tailed, unpaired, or 1 sample).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stable cell lines expressing rBAT and/or b0,+AT. MDCK cell lines stably expressing human rBAT under the control of a glucocorticoid-inducible promoter were generated and tested for dexamethasone-inducible rBAT mRNA expression by Northern blotting. In a second round of transfections, mouse b0,+AT cDNA, placed under the control of a constitutively active promoter, was introduced into rBAT-expressing cell lines and wild-type MDCK cells, together with a second selection marker. Double-transfected cell lines with the highest b0,+AT mRNA expression levels were identified by RT-PCR. Another series of MDCK cell lines was generated that constitutively express an rBAT-b0,+AT fusion protein. The function of this fused heterodimer has been characterized previously in X. laevis oocytes (22).

Coexpression is required for the apical surface expression of both subunits. The subcellular localization of rBAT and b0,+AT was analyzed by confocal immunofluorescence microscopy in selected cell lines cultivated on filters (Fig. 1). In cell lines expressing only b0,+AT, a diffuse cytosolic signal was observed, the intensity of which was strong on average but highly variable among cells (Fig. 1A). Cell lines expressing rBAT alone also showed, after induction with dexamethasone, cytosolic staining that was variable but, on average, of a much lower intensity (Fig. 1B).


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Fig. 1.   Apical colocalization of b0,+AT and rBAT in transfected Madin-Darby canine kidney (MDCK) cells shown by double immunofluorescence. rBAT is shown in green, b0,+AT in red, and colocalization in yellow in the confocal microscopic images. Square panels, images taken parallel to the filter (x-y plane) at the level of the nucleus (A-C) or the apical membrane (D-F); rectangular panels, corresponding z-x and z-y reconstitutions. A and B: single-transfected cells expressing b0,+AT (A) or rBAT (B) show a weak intracellular staining of either protein. C: in coexpressing cells, both proteins colocalize to the apical membrane, as seen by the yellow staining (z-x reconstitution). There is also a large intracellular excess of b0,+AT. D: when cotransfected cells are viewed at a lower magnification, a clear intracellular excess of b0,+AT can be seen in most cells. rBAT is visible essentially colocalized with b0,+AT (yellow) at the apical membrane. E: when rBAT induction by dexamethasone was omitted in the same cell line, b0,+AT staining was no longer visible at the apical membrane but remained intracellularly. F: cells expressing a fusion protein construct of rBAT-b0,+AT showed an exclusively apical localization of the heterodimer. Bars: A-C and E-F = 5 µm; D = 12 µm.

In contrast to these intracellular stainings, cells coexpressing rBAT and b0,+AT display a clear apical labeling for both subunits, which appears as yellow apical staining in the double-labeled cells shown in Fig. 1, C and D (see Z-Y reconstitutions, in particular). Figure 1C shows a cross section through the middle of the cell, and Fig. 1D shows a lower magnification view of a cross section at the level of the apical cell membrane. It is clearly visible that, despite a 3-day induction of rBAT expression with dexamethasone, b0,+AT is present in excess over rBAT in most doubly transfected cells, as indicated by a strong residual cytosolic b0,+AT signal. This suggests the possibility that b0,+AT expressed in the absence of rBAT, or in excess over it, is stable and accumulates in an intracellular compartment, possibly the endoplasmatic reticulum.

Interestingly, on expression of b0,+AT in rBAT-expressing cell lines (Fig. 1, C and D), rBAT staining was not only shifted to the apical membrane but clearly also became stronger. This suggests the possibility that rBAT is unstable in the absence of b0,+AT and that the subunit association does not only promote its translocation to the surface but thereby also increases its half-life.

The fact that the apical expression of b0,+AT depends on rBAT coexpression is confirmed by the observation that in the same cell line in which apical coexpression of both subunits could be observed, b0,+AT was localized exclusively intracellularly when rBAT expression was not induced with dexamethasone (Fig. 1E). Figure 1F shows a cell line expressing the rBAT-b0,+AT fusion protein double-stained for rBAT and b0,+AT. The exclusive apical colocalization of both signals confirms that the heterodimer localizes to the apical plasma membrane.

Stabilization and maturation of rBAT depends on association with b0,+AT. To analyze the interaction between the subunits, rBAT and b0,+AT were immunoprecipitated separately from biosynthetically pulse-labeled cells expressing both proteins and analyzed by SDS-PAGE fluorography (Fig. 2). b0,+AT appears in lanes 2 (sample reduced) and 6 (nonreduced) as a band with a relative mass (Mr) of 40. This value is lower than expected from its calculated molecular weight (53.7 kDa), similarly to what has been observed previously for other gpaATs (for glycoprotein-associated amino acid transporters). The bands observed at ~80 and 90 kDa in lane 2 correspond to the coprecipitated core-glycosylated and terminally glycosylated forms of rBAT, respectively (9, 22). Disulfide linkage of rBAT with b0,+AT can be inferred from the fact that in nonreducing conditions (lane 6), the rBAT bands are replaced by a band at ~135 kDa, which corresponds to the heterodimer (doublet due to differential rBAT glycosylation not resolved because of compression by immunoglobulins). The labeled material close to the top of the separating gel likely represents higher order aggregates. The fact that the b0,+AT band (Mr 40) precipitated by the anti-b0,+AT antibody is nearly as strong in lane 6 (sample not reduced) as in lane 2 (reduced conditions) indicates that much of the precipitated b0,+AT is not associated with rBAT, thus suggesting that b0,+AT is present in great excess over rBAT. On the other hand, when the precipitation is made with an anti-rBAT antibody, all terminally glycosylated and nearly all core-glycosylated rBAT migrate, in nonreducing conditions (lane 8), at a higher Mr corresponding to the heterodimer or higher order aggregates. Thus nearly all of the expressed rBAT is associated with b0,+AT.


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Fig. 2.   rBAT-b0,+AT association and b0,+AT excess shown by immunoprecipitation from pulse-labeled epithelia. rBAT (rB) and b0,+AT (b) immunoprecipitation was performed on lysate of wild-type (wt) or rBAT-b0,+AT-transfected (rB-b) MDCK cells that were pulse-labeled for 2 h. Precipitations with anti-b0,+AT or anti-rBAT antibody (Ab) were carried out in nonreducing conditions, and SDS-PAGE was then performed with (+) or without (-) prior reduction of the samples with beta -mercaptoethanol (beta ME). Left: molecular mass of marker proteins (in kDa). Right: expected levels of precipitated bands. HD, heterodimer of rBAT-b0,+AT; TrB, terminally glycosylated rBAT; CrB, core-glycosylated rBAT. b0,+AT appears in lanes 2 (reduced) and 6 (nonreduced) as a band with an apparent molecular mass of ~40 kDa. rBAT is coprecipitated by the anti-b0,+AT antibody, as seen by the bands at ~80 and 90 kDa in lane 2. In the absence of the reducing agent, the intact rBAT-b0,+AT heterodimer migrates as an ~135-kDa band (lane 6). The fact that the b0,+AT band is of similar intensity in lane 6 as in lane 2 (reduced sample) indicates that b0,+AT is present in large excess over rBAT, the majority being not associated. The rBAT-doublet precipitated with anti-rBAT antibody (lane 4) is of comparable intensity to that coprecipitated with b0,+AT, suggesting that most or all rBAT is associated with b0,+AT (lane 2). The fact that coprecipitated b0,+AT is not visible is due to the large excess of stable unlabeled b0,+AT that preexisted to the pulse labeling. That b0,+AT is indeed coprecipitated with rBAT is confirmed by the fact that in lane 8, the rBAT antibody precipitates only a band corresponding to the heterodimer.

The fact that the b0,+AT band is much less intense than that of rBAT (a protein that contains a similar number of methionines) is probably due to a proportionally lower biosynthetic rate of b0,+AT. However, because b0,+AT is quite stable in the absence of rBAT (see below), the cells contain a large pool of unlabeled b0,+AT, which can associate with newly synthesized rBAT. Thus because of its low degree of specific labeling, b0,+AT coprecipitated by the rBAT antibody is nearly not visible as a distinct band in reducing conditions (lane 4). However, the fact that b0,+AT is coprecipitated by the anti-rBAT antibody is demonstrated, in the SDS-PAGE analysis made in nonreducing conditions, by the fact that the precipitated material migrates at a level corresponding to the heterodimer (lane 8).

To investigate the effect of b0,+AT association on the stability of rBAT, a pulse-chase experiment was performed (Fig. 3A). When immunoprecipitated from single-transfected cells (lanes 4-6), rBAT appeared exclusively as a ~80-kDa band, corresponding to its core-glycosylated form, and almost completely disappeared within an 8-h chase period. Immunoprecipitated from cells cotransfected with rBAT and b0,+AT, rBAT similarly appeared after the pulse-labeling period as a ~80-kDa band at first. During the chase period, however, it shifted to a ~90-kDa band, the intensity of which remained nearly unchanged over the 8-h chase period. This shift to ~90 kDa most probably corresponds to the terminal glycosylation acquired en route to the cell surface during the passage through the Golgi complex (9, 22). The fact that rBAT requires association with b0,+AT for its maturation and stabilization explains the low level and intracellular localization of rBAT staining found by immunofluorescence in single-transfected cells (Fig. 1).


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Fig. 3.   Pulse-chase labeling followed by immunoprecipitation shows that b0,+AT association leads to rBAT maturation and stabilization. rBAT and b0,+AT immunoprecipitation was performed on lysates of wild-type (wt), rBAT (rB)-, or rBAT-b0,+AT (rB-b)-transfected MDCK cells that were pulse-labeled for 2 h and chased for the number of hours (h) indicated. All samples were treated with the reducing agent beta -mercaptoethanol before SDS-PAGE. A: precipitations with anti-rBAT antibody on cells transfected with rBAT only yielded an ~80-kDa band, corresponding to the core-glycosylated form of rBAT (lanes 4 and 5), which disappeared after an 8-h chase period (lane 6). In cells additionally transfected with b0,+AT, the same band was seen after the 2-h pulse period (0-h chase, lane 7), gradually shifting to an ~90-kDa band in the course of the chase (lane 8), which remained visible after the 8-h chase period (lane 9). This band most probably corresponds to the terminally glycosylated, and thus mature and stabilized, form of rBAT. B: precipitations with anti-b0,+AT antibody on rBAT-b0,+AT-transfected cells without prior induction of rBAT (rB-induction) only yielded a b0,+AT band (lane 4), which remained stable throughout the subsequent chase period (lanes 5 and 6), indicating that b0,+AT stability is not affected by the absence of rBAT. When rBAT expression was induced, rBAT was coprecipitated in its core-glycosylated form at first (lane 7), gradually being terminally glycosylated in the course of the chase (lanes 8 and 9).

To investigate the effect of rBAT coexpression on b0,+AT stability, pulse-chase experiments were performed on a double-transfected cell line (Fig. 3B). The lack of rBAT expression without its prior induction (lanes 4-6) was demonstrated by the fact that no coprecipitated band was seen when b0,+AT was precipitated. However, in contrast to rBAT in the converse experiment (Fig. 3A), b0,+AT remained stable throughout the course of the chase period, even in the absence of its heterodimerization partner. This long half-life of b0,+AT in the absence of rBAT explains the large intracellular b0,+AT pool observed by immunofluorescence in single- and double-transfected cell lines. When rBAT expression was induced (lanes 7-9), rBAT protein was coprecipitated with b0,+AT, yielding the same pattern of core- and terminally glycosylated forms as seen in Fig. 2.

Apically restricted amino acid transport by the b0,+AT-rBAT heterodimer. To demonstrate that the apically localized rBAT-b0,+AT heterodimer is functional, we performed amino acid uptake experiments on epithelia cultivated on permeable supports (Fig. 4). All assays were carried out for 1 min, a time period that is within the linear phase of uptake, as determined in preliminary time course experiments (data not shown). The polarity of the expressed transport was tested by measuring the L-arginine-inhibitable L-cystine uptake from both sides of the epithelia in the absence of Na+. This typical b0,+ transport was observed only at the apical side of epithelia expressing both rBAT and b0,+AT. As expected, cells transfected with either rBAT or b0,+AT alone did not exhibit any notable b0,+-type transport. Expression of the fusion protein rBAT-b0,+AT induced apical L-cystine transport similarly to the coexpression of both subunits. However, this construct also induced some transport at the basolateral side, indicating that the link constructed between b0,+AT and rBAT somehow interferes with mechanisms leading to polar distribution.


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Fig. 4.   Apically restricted amino acid uptake by the rBAT-b0,+AT heterodimer. Wild-type MDCK cells or cells transfected with either b0,+AT (b), rBAT (rB), both (rB-b), or an rBAT-b0,+AT fusion protein construct (FP) were cultivated on permeable supports. The apical or basolateral uptake of 10 µM L-cystine (solid bars) or 10 µM L-cystine with 1 mM of unlabeled L-arginine (open bars) was measured for 1 min in the absence of Na+. Significant uptake could only be measured in cells in which both rBAT and b0,+AT were present, either separately transfected (rBAT-b0,+AT) or as an FP. This transport was inhibitable by an excess of another, more soluble b0,+ substrate, L-arginine. Error bars, SE (n = 4).

To test the substrate specificity and Na+ independence of the apically expressed heterodimeric transporter, cotransfected cells cultivated on permeable supports were incubated with various amino acids given apically at a concentration of 100 µM, in either the presence or absence of Na+ (Fig. 5). Uptake by endogenous MDCK cell transporters was measured in parallel on untransfected cells (open bars). As expected for b0,+-type transport, high uptake rates (1.5-3.5 nmol · h-1 · cm-2) were induced by the expression of rBAT and b0,+AT for the diamino acids L-arginine, L-lysine, and L-cystine as well as for the large neutral amino acids L-tyrosine and L-leucine in both the presence and absence of Na+. The induced transport of L-isoleucine, L-valine, and L-alanine were much slower but nonetheless significant (paired t-test on pooled results from Fig. 5, A and B: P = 0.005, 0.001, and 0.029, respectively). No significant induced glycine transport was observed. The meaning of the differences in transport rates observed among the various efficiently transported substrates (for instance, larger L-arginine than L-lysine uptake in the presence of Na+ only) is not clear, as there were relatively large experiment-to-experiment variations.


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Fig. 5.   Substrate specificity and Na+ dependence of the apical amino acid uptake by rBAT-b0,+AT heterodimer. Apical uptake of 100 µM of L-amino acid was measured for 1 min in the presence (A) or the absence (B) of Na+. Solid bars, rBAT-b0,+AT-transfected cells; open bars, wild-type MDCK cells; CssC, L-cystine. Error bars, SE (n = 7 from 4 experiments).

Dose-response experiments were performed with the cotransfected cells cultivated on permeable supports (Fig. 6). The apical uptakes were made with three representative amino acids for system b0,+, i.e., L-cystine, L-arginine, and L-leucine. Due to the insolubility of L-cystine, maximal velocity rates could not be directly measured and were extrapolated by curve fitting (see below). L-Leucine uptake rates could also not be accurately measured at high concentrations, due to a high-capacity, low-affinity endogenous transporter of MDCK cells. Curves corresponding to Michaelis-Menten kinetics were fitted to the experimental points, and apparent affinity values (Km) were derived. The Km values for L-cystine, L-arginine, and L-leucine were 94, 179, and 258 µM, respectively. The order of magnitude of these values is similar, and their rank order is identical to those obtained previously for the rBAT-b0,+AT fusion protein-mediated transport measured in X. laevis oocytes (22).


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Fig. 6.   Concentration dependence of apical uptake of rBAT-b0,+AT substrates. Apical uptakes were measured for 1 min in the absence of Na+. Sigmoidal curves corresponding to Michaelis-Menten kinetics (Km) were fitted to the experimental data using a nonlinear regression routine, and apparent Km values were derived. , L-cystine [Km = 94 µM (95% confidence interval: 85-103 µM)]; open circle , L-arginine [Km = 179 µM (95% confidence interval: 121-265 µM)]; black-down-triangle , L-leucine [Km = 258 µM (95% confidence interval: 203-328 µM)]. L-arginine and L-cystine, n = 6 from 2 experiments. L-leucine, n = 9 from 3 experiments. High L-cystine concentrations could not be tested due to lack of cystine solubility, and L-leucine uptake via rBAT-b0,+AT could not be measured precisely at high concentrations due to the presence of a low-affinity, high-capacity endogenous transporter of MDCK cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To address questions of rBAT and b0,+AT heterooligomerization, maturation, surface expression, polarity, and transport properties in the context of an epithelium, we expressed these proteins in MDCK cells. We chose this cell line of distal nephron origin as the recipient because it has no endogenous proximal tubule-like amino acid transport properties and represents a well-established model for epithelial polarity studies. In terms of amino acid transport, untransfected MDCK cell epithelia cultured on filters display only very low apical Na+-independent uptake rates of the dicationic amino acids L-cystine, L-arginine, and L-lysine, when tested at the concentration of 100 µM (Fig. 5). In addition, they express some partially Na+-dependent apical transport of neutral amino acids (Fig. 5) that probably corresponds to that attributed earlier to system ASC plus an operationally defined "general transport system G" (4). On the basolateral side, wild-type MDCK cells are known to express systems L and ASC, as well as the inducible transporters A, TAUT (taurine), and BGT (betain and GABA) which do not interfere with short apical uptake experiments (4, 29, 33).

In contrast to the present study with transfected MDCK epithelia, previous studies of the heterogeneously expressed rBAT-b0,+AT complex were made in nonepithelial systems, i.e., X. laevis oocytes (22) (with an rBAT-b0,+AT fusion protein to prevent heterodimerization with an endogenous b0,+AT), COS cells (7, 11, 19), and HELA cells (12), such that it was not clear whether heterodimerization of rBAT and b0,+AT in epithelial cells would also be a condition for surface expression and the resulting transport properties would be identical. We have to mention, however, that one other group has described transport that surprisingly depends on coexpression of b0,+AT with 4F2hc (23, 24). The physiological relevance of this observation is questionable in view of the strict basolateral localization of 4F2hc and the clear apical restriction of b0,+AT in the same cells (proximal tubule, small intestine) (13, 22, 25) as well as the lack of biochemical interaction of the two transporter chains (22).

In the present study, we show by immunofluorescence that both rBAT and b0,+AT remain intracellular unless they are coexpressed, at which point they reach the apical cell surface (Fig. 1). From the immunoprecipitation experiments, we can infer that it is the heterooligomerization that permits exit from the endoplasmatic reticulum (ER) and surface expression. Indeed, in the absence of b0,+AT, rBAT is rapidly degraded, presumably in the ER, as none of it is terminally glycosylated (Fig. 2). In contrast, in the presence of b0,+AT, it is rapidly shifted to a terminally glycosylated form that remains stable over an 8-h chase period (Fig. 3). This mature rBAT form is shown to be entirely heterooligomerized on nonreducing gels (Fig. 2).

The need for association of b0,+AT to a glycoprotein for surface expression is analogous to that of other glycoprotein-associated amino acid transporters that require heterooligomerization with 4F2hc to reach the basolateral surface (18). However, unlike rBAT, 4F2hc can reach the cell surface of X. laevis oocytes also without an associated light chain.

The requirement for heterodimeric association for surface expression of both rBAT and b0,+AT subunits is reminiscent of the situation of the Na,K-ATPase that is also composed of a type II glycoprotein (beta -subunit) and a catalytic multimembrane-spanning protein (alpha -subunit) (14). In the case of the Na,K-ATPase, the site of intracellular subunit retention (ER), the involved chaperones and the intersubunit interaction sites have been studied extensively, such that the Na,K-ATPase represents a useful paradigm for heterooligomeric amino acid transporters (1, 15).

Besides the data presented in this study, transport specificity and kinetic data from the cloned rBAT-b0,+AT complex have been previously generated in our laboratory using a fusion protein made of mouse b0,+AT linked to human rBAT. In terms of the amino acid uptake specificity range (measured at fixed amino acid concentrations), there was no difference between the results obtained previously with the fusion protein expressed in oocytes and the same chains coexpressed in MDCK cells. Two other publications (7, 19) show other sets of kinetic values obtained in COS cells expressing rat and human rBAT-b0,+AT, respectively. In terms of amino acid uptake, all four studies agree on the fact that, relative to L-cystine, cationic amino acids (L-lysine, L-arginine) as well as some large neutral amino acids (L-leucine, L-tyrosine) are transported with a similar efficiency (±3-fold difference) and that glycine is (nearly) not transported. There is, however, a clear difference in the transport of the beta -branched amino acids L-isoleucine and L-valine, which are not significantly transported in our two studies (mouse b0,+AT-human rBAT in oocytes and MDCK cells), whereas they are well transported by the rat and human complexes expressed in COS cells. The significance of this difference is not yet clear.

Our results obtained in MDCK epithelia show a requirement of rBAT- b0,+AT association for the surface expression of each subunit. However, the general validity of this result is questioned by the differential pattern of rBAT and b0,+AT localization in the proximal tubule. Indeed, we and others have observed opposed (and not parallel) expression gradients of b0,+AT and rBAT in the brush-border membrane of the proximal tubule, with a maximum of b0,+AT in the first portions (S1, S2) (7, 19, 22) and a maximum of rBAT in the last part (S3) of the proximal tubule (13, 17, 22). This suggests the possibility that yet another protein that allows the surface expression of b0,+AT in the absence of rBAT, or vice versa, is present in the proximal tubule and not in MDCK cells.

Older in vivo and ex vivo studies suggest the possibility that in the early portions of the proximal tubule (S1, S2), L-cystine is transported by a low-affinity transport system and that a higher affinity system is situated in the later portion (S3) (see Ref. 26). In view of the relatively high apparent affinities measured for the L-cystine transport by the rBAT-b0,+AT complex, it has been suggested that this heterooligomer would be the transporter expressed in S3 (see discussion in Ref. 31). This raises the possibility that another b0,+AT-associated protein is expressed in the S1 and S2 segments, also revealing the necessity of reevaluating amino acid transport in in vivo and/or ex vivo systems.

The functional role of rBAT-b0,+AT in vectorial amino acid transport can only be understood when one considers its complementarity with other transporters. The fact that it functions as an exchanger of extracellular cationic against intracellular neutral amino acids (8, 31) already indicates that there must be, in the same membrane, a unidirectional transporter that recycles the neutral amino acids which the exchanger transports out of the cells (for instance, system B0, the molecular identity of which is not yet clear) (31). Furthermore, amino acids brought into the cell via these apical transporters have then to leave across the basolateral membrane into the extracellular space. Two rBAT-b0,+AT-related exchangers, 4F2-y+LAT1 and 4F2-LAT2, have been localized to the basolateral membrane of the proximal tubule (25, 30), and a parallel unidirectional transport system has not yet been described. Expressing these transporters in an epithelium already expressing rBAT-b0,+AT, such as the cell line presented in this study, will allow us to investigate the functional interactions of these transporters.


    ACKNOWLEDGEMENTS

The authors thank Christian Gasser for the artwork.


    FOOTNOTES

This work was supported by Swiss National Science Foundation Grant 31-59141.99 (to F. Verrey).

Address for reprint requests and other correspondence: F. Verrey, Institute of Physiology, Univ. of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland (E-mail: verrey{at}access.unizh.ch).

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.

First published February 12, 2002;10.1152/ajprenal.00212.2001

Received 6 July 2001; accepted in final form 6 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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