1 Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029; and 2 Department of Pediatrics and 3 Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ~75% of each protein was delivered to the basolateral surface compared with ~90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.
protein sorting signal; bile acid transport
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INTRODUCTION |
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BILE ACIDS ARE ACIDIC sterols synthesized from cholesterol in the liver. After synthesis, bile acids are the major solutes secreted into bile and then enter the lumen of the small intestine, where they facilitate absorption of fats and fat-soluble vitamins. Bile acids play a critical role in the generation of canalicular bile flow (22, 24, 34). Less than 5% of the bile acid pool is fecally excreted each day, indicating the high efficiency for intestinal reclamation of these compounds (22, 24, 34). The process of vectorial transport of bile acids and other ions across the hepatocyte and ileal enterocytes is highly dependent on the polarized distribution of specific transport mechanisms localized to plasma membranes of these cells (22, 24, 34). Similar to other epithelial cells, the membrane domains of hepatocytes and enterocytes are structurally, biochemically, and physiologically distinct. To ensure rapid and unidirectional transport, the different domains of the epithelial plasma membranes must possess distinct classes of transport proteins to mediate fluxes against concentration gradients. For polarized cells, the maintenance of normal physiological function requires that newly synthesized membrane proteins be differentially sorted to their appropriate domain of the plasma membrane.
Na+-dependent bile acid uptake, critical to the enterohepatic circulation of the bile acid pool, has been demonstrated in the brush-border membrane of the ileum (21) and the sinusoidal membrane of the hepatocyte (5). The cDNAs encoding rat ileal apical Na+-dependent bile acid transporter (ASBT) and liver Na+-taurocholate cotransporting polypeptide (Ntcp) have been cloned (14, 30). These transporters transport conjugated bile acids in an Na+-dependent fashion but are products of two different genes. ASBT and Ntcp are members of a new family of anion transporters. Analysis of the open reading frames of the cloned cDNAs for these two proteins reveals ~35% identity and 62% similarity at the amino acid level. Both transporters exhibit a developmentally regulated pattern of expression (15, 30, 35). There are two potential N-linked glycosylation sites in the NH2 terminus of both proteins. In addition, the two proteins share almost identical hydropathy profiles with seven potential transmembrane domains, suggesting that both proteins assume the same topology in the plasma membrane (17, 40). This seven-transmembrane organization is similar to that of G protein-related seven-transmembrane receptors (40). In spite of these similarities at the protein level, transport and immunochemical localization studies have confirmed that Ntcp and ASBT are sorted exclusively to the basolateral surface of hepatocytes and the apical surface of ileal enterocytes, respectively (2, 30, 33). The mechanisms underlying the differential sorting of these bile acid transporters are unknown. In the current study, potential sorting signals in the COOH-terminal domains of the ileal and hepatic bile acid transporters were sought by expression of mutated and chimeric transport proteins in Madin-Darby canine kidney (MDCK) cells. Our results indicate that the cytoplasmic tail of Ntcp is only of minor importance for the sorting of this protein to the basolateral domain. In contrast, there is a strong apical sorting determinant located in the cytoplasmic tail of ASBT.
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MATERIALS AND METHODS |
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Materials
Cell culture media were from GIBCO BRL (Gaithersburg, MD). [3H]taurocholic acid (2.1-3.47 Ci/mmol) was purchased from DuPont NEN (Boston, MA). Unlabeled taurocholate was purchased from Sigma Chemical (St. Louis, MO). PCR reagents and enzymes were obtained from Perkin Elmer Cetus (Foster City, CA). Subcloning reagents, enzymes, and competent cells were obtained from Stratagene (La Jolla, CA), GIBCO BRL, New England BioLabs (Beverly, MA), and Invitrogen (Carlsbad, CA).Plasmid Construction
Bile acid transporter cDNAs were subcloned into the mammalian expression vector pCMV2, using standard techniques as previously reported (3). The cDNAs encoding the rat ileal ASBT or rat liver Ntcp were digested from pBluescript vectors with EcoR I/Kpn I (ASBT) or BamH I/Hind III (Ntcp) and directly subcloned into the same or compatible (Bgl II/BamH I) sites of pCMV-2, respectively. The positive clones containing the ASBT cDNA insert were identified by restriction enzyme mapping and sequenced on both strands using the ABI automated DNA sequencer (model 373A) at Keck Biotechnology Center, Yale University School of Medicine. Sequence alignment and searches were performed using the Wisconsin Genetics Computer Group sequence analysis package (version 7.1) (10). The ASBT insert was 1,255 bp in length and encoded a 114-nt 5'-untranslated region, a 1,044-nt coding sequence, and a 97-nt 3'-untranslated region. The Ntcp clone was a kind gift of Dr. Paul Dawson (Bowman Gray School of Medicine, Winston-Salem, NC). These verified ASBT and Ntcp expression clones were used for further study.Cell Culture and Transfection
MDCK cells were maintained in a humidified incubator at 37°C under 5% CO2 atmosphere in complete MEM that was supplemented with 10% (vol/vol) fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. Cells were stably transfected by the calcium phosphate method as previously described (1, 6). Briefly, on the day before transfection cells were plated at ~5-10 × 105 cells/100-mm dish. On day 1, cells were ~50% confluent and were transfected by precipitating 10 µg of pCMV2-ASBT or pCMV2-Ntcp plasmid DNAs with calcium phosphate and cotransfected with 1 µg pSV2-neo cDNA plasmid DNA per 100-mm tissue culture dish (6). On day 2, transfected MDCK cells were split at a ratio of 1:3 in complete MEM. Transfected cell lines were selected by growth in the antibiotic G418 (900 µg/ml) (Life Technologies). After ~10 days, large colonies were selected using cloning cylinders and transferred to 12-well plates. In some cases, monoclonal populations of transformants were obtained by limiting the dilution cloning method. For polarity studies, transfected and untransfected MDCK cells were grown to confluence for >5 days on Transwell filter inserts (Costar, Cambridge, MA, and FALCON, Franklin Lakes, NJ) (7, 13). Formation of a tight seal between the upper and lower chambers was measured by transepithelial transport of [14C]mannitol, which was <10%. The expression of the recombinant proteins was assayed by taurocholate uptake and confocal immunofluorescence microscopy. To enhance gene expression, we preincubated the stably transfected MDCK cells in 10 mM sodium butyrate for 15 h at 37°C.COS-7 (SV40 transformed monkey kidney fibroblast) cells were maintained in complete DMEM [DMEM containing 10% (vol/vol) fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine]. Transient DNA transfection was carried out by lipofectin-mediated transfection (GIBCO BRL) according to the manufacturer's suggested protocol. Cells were harvested 24-48 h later for transport and protein assays.
Transport Studies
Na+-dependent taurocholate uptake assays were performed as described by Liang et al. (19). The transfected COS-7 or MDCK cells were incubated in 10 µM taurocholate (containing 1 µM [3H]taurocholate) in the presence of a buffer containing 116 mM NaCl or choline. After 10 or 30 min incubation at 37°C, the medium was removed, and each cell monolayer was washed and processed to determine cell-associated protein and radioactivity for taurocholate uptake. The polarity of taurocholate uptake was performed by using Transwell filter inserts. Transfected and untransfected MDCK cells were grown to confluence for >5 days on Transwell filter inserts. The filters were washed twice with warm uptake buffer [116 mM NaCl (or choline), 5.3 mM KCl, 1.1 mM KH2PO4, 0.8 mM MgSO4, 1.8 mM CaCl2, 11 mM D-glucose, and 10 mM HEPES, pH 7.4], and each well was incubated from the apical (0.2 ml) or basolateral (0.6 ml) side with uptake buffer containing 10 µM taurocholate (containing 1 µM [3H]taurocholate). After incubation, the uptake assay was terminated by aspirating the medium, and the filters were successively dipped into three beakers, each of which contained 100 ml of ice-cold uptake buffer. The filters were excised from the cups and the attached cells were solubilized in 0.2 ml of 1% SDS, and transferred into scintillation vials with 4 ml Optifluor (DuPont NEN). Protein estimations were carried out with the Bio-Rad protein assay kit (Hercules, CA).Antibodies
Rabbit polyclonal antibodies (a gift from Dr. Paul Dawson) were raised to the COOH-terminal 14 amino acids of the hamster ASBT, as previously described (41). Ntcp antibody was generated by immunization of rabbits with a fusion protein where the entire COOH terminus of Ntcp was fused to glutathione S-transferase (2). ASBT and Ntcp antibodies were used at 0.2 and 1 µg/ml of purified IgG in the immunoblotting studies, respectively. An antibody was generated against a synthetic peptide whose sequence corresponds to the NH2-terminal 24 amino acids of rat liver Ntcp (Zymed Laboratories, South San Francisco, CA). This antibody was used to characterize COOH-terminal truncated Ntcp. An Anti-FLAG monoclonal antibody M2 (Eastman Kodak Company, New Haven, CT) was used for the FLAG-fusioned mutant transporters.Blot Hybridization
All blot hybridizations were done according to standard techniques (3).Southern blots.
Genomic DNAs from transfected MDCK cells were purified by TRIzol
reagent (GIBCO BRL) and digested with
EcoR I and
Kpn I. Fragments (10 µg/lane) were
separated on 1% agarose gel, blotted onto a GeneScreen membrane
(DuPont NEN), and hybridized to a
32P-labeled full-length cDNA probe
encoding ASBT or Ntcp. The blots were washed twice and exposed to X-ray
films for 4 h or overnight at 70°C. For quantitation of the
signals, autoradiographs were scanned on a PhosphorImager using
ImageQuant software (Molecular Dynamics).
Northern blots. Total RNA was extracted from stably transfected MDCK cells by TRIzol reagent (GIBCO BRL). RNAs (10 µg/lane) were subjected to electrophoresis on a 1% agarose gel and blotted onto a GeneScreen membrane (DuPont NEN). Hybridization was carried out in 50% formamide buffer at 42°C overnight with a 32P-labeled full-length cDNA probe (1 × 107 counts/min) encoding ASBT or Ntcp protein and detected by autoradiography. Quantitation of autoradiographic signals was done as described above. Total RNAs harvested from rat adult ileum or liver were used as positive control.
Western blots. Total protein homogenates (10-20 µg/lane) from stably transfected MDCK cells were separated by 10% SDS-PAGE and transferred to nitrocellulose. ASBT and Ntcp proteins were detected by incubation with a specific COOH-terminal antipeptide antibody and COOH-terminal fusion protein antibody, respectively, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma Chemical). HRP activity was visualized using enhanced chemiluminescence assay (Amersham, Arlington Heights, IL). Rat ileal brush-border membrane protein and rat liver basolateral plasma membrane proteins were used as positive controls. Quantitation of the protein bands was performed using a laser densitometer (Molecular Dynamics).
Confocal Immunomicroscopy
Indirect immunofluorescence microscopy was performed on a confluent monolayer of transfected cells. Transfected cells were cultured on either glass coverslips or Transwell filters. The cells were fixed and permeabilized for 10 min in methanol atConstruction of Truncated and Chimeric Transporter cDNAs
To generate truncated and chimeric transporters, PCR amplifications were performed with rat ASBT and Ntcp cDNAs as templates (4, 26). All PCR amplified products were purified by QIAquick column (QIAGEN, Valencia, CA; according to manufacturer's suggested protocol), digested with Mlu I and Hind III, and gel purified. Fragments were subcloned into the Mlu I and Hind III sites of pCMV 2 vectors. Subcloning and restriction enzyme analysis were carried out according to previously established methods (3). Proper orientation of the inserts was further verified by DNA sequencing (ABI automated DNA sequencer model 373A) (3, 16).NL (truncated Ntcp). The COOH-terminal 56-amino-acid coding sequence of Ntcp was removed by PCR, using Ntcp cDNA as template. A TAG stop codon was inserted to the position just after the 306Cys of Ntcp. The PCR product was digested with Mlu I and Hind III and ligated into similarly digested pCMV2.
NLF (truncated Ntcp with FLAG tail). The COOH-terminal 56-amino-acid coding sequence of Ntcp was removed. A sequence encoding a FLAG tag (DYKDDDDY) was inserted between the 306Cys and the TAG stop codon by PCR amplification. The PCR product was digested with Mlu I and Hind III and ligated into similarly digested pCMV2.
NLCI (Ntcp/ASBT chimera). The COOH-terminal 56-amino-acid coding sequence of Ntcp was replaced with the 40-amino-acid COOH-terminal cytoplasmic tail coding sequence of ASBT by two rounds of sequential PCR amplification. In the first round (reaction 1), Ntcp cDNA was used as a template with primer A corresponding to nt 122-146 of the Ntcp sequence, with an Mlu I restriction site and primer B containing 19 nt complementary to nt 1042-1024 of Ntcp, followed by 24 nt complementary to nt 1062-1039 of ASBT. In reaction 2, ASBT was used as a template with primer C containing 19 nt corresponding to nt 1024-1042 of Ntcp, followed by 24 nt corresponding to nt 1039-1062 of ASBT and primer D complementary to bases 1190-1160 of the ASBT, containing a Hind III restriction site. In the second round of amplification, the products of the first round of PCR were used as a template and oligonucleotides A and D as primers. The fusion product was generated by virtue of the 43-nt overlap between the fragments generated in the first round. The PCR product was digested with Mlu I and Hind III and ligated into similarly digested pCMV2.
NI (truncated ASBT). The COOH-terminal 40-amino-acid coding sequence of ASBT was removed by PCR with a primer containing stop codon (TAG) inserted just after 307Met of ASBT. This mutant was generated by PCR using ASBT cDNA as template. The PCR product was digested with Mlu I and Hind III and ligated into similarly digested pCMV2.
NIF (truncated ASBT with FLAG tail). The DNA sequence encoding the last COOH-terminal 40 amino acids of Ntcp was removed. The sequence encoding a FLAG tag (DYKDDDDY) was inserted between 307Met and the TAG stop codon by PCR amplification. The PCR product was digested with Mlu I and Hind III and ligated into similarly digested pCMV2.
NICL (ASBT/Ntcp chimera). The DNA sequence encoding the last COOH-terminal 40 amino acids of ASBT was replaced with the sequence encoding the 56-amino-acid COOH-terminal cytoplasmic tail of Ntcp by two rounds of sequential PCR amplification. In the first round, reaction 1, ASBT cDNA was used as a template with primers E (corresponding to nt 116-139 of the ASBT sequence, containing the Mlu I restriction site) and F (containing 21 nt complementary to nt 1019-1039 of ASBT, followed by 20 nt complementary to nt 1059-1040 of Ntcp). In reaction 2, Ntcp was used as a template with primers G (containing 21 nt corresponding to nt 1019-1039 of ASBT, followed by 20 nt corresponding to nt 1040-1059 of Ntcp) and H (complementary to bases 1210-1187 of Ntcp, containing the Hind III restriction site). In the second round of amplification, the products of the first round of PCR were used as a template and oligonucleotides E and H as primers. The fusion product was generated by virtue of the 21-nt overlap between the fragments generated in the first round. The PCR product was digested with Mlu I and Hind III and ligated into similarly digested pCMV2.
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RESULTS |
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Expression of Liver and Ileal Na+-Dependent Bile Acid Transporters in MDCK Cells
Ntcp and ASBT, despite their high structural and functional similarity, are sorted to opposite membrane domains in native cells expressing these proteins. As a prerequisite for establishing a model system to identify signals determining apical and basolateral sorting, MDCK cells were first stably transfected with cDNAs encoding Ntcp and ASBT. To verify stable transfection and functional expression of transport proteins on the appropriate membrane domain, cells grown to confluence on Transwell filter inserts were tested by 1) Northern and Western blotting to confirm stable transfection and gene expression and by 2) confocal immunofluorescence microscopy and Na+-dependent taurocholate transport across the apical and basolateral membrane to establish the polarity of protein expression.Figure 1A shows that on Northern analysis the Ntcp mRNA transcript in stably transfected MDCK cells is similar in size to that present in rat liver (14). The ASBT mRNA transcript in transfected MDCK cells was ~2 kb in length, which is smaller than that described in adult rat ileum (~5 kb) (30). The smaller size of the mRNA is related to the fact that only the coding region of the cDNA was employed for these studies.
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The Ntcp protein expressed in MDCK cells has the same molecular mass (~50 kDa) as that found in rat liver (Fig. 1B). However, ASBT protein was expressed with a molecular mass of ~45 kDa, slightly less than that of the rat ileum (~50 kDa). Additional bands of higher molecular mass were also detected and represent apparent aggregation products described previously for the rat ASBT protein (30). After removal of the N-linked carbohydrates, ASBT expressed in transfected MDCK cells comigrated with an apparent molecular mass of 38 kDa similar to that of the deglycosylated ASBT bands from ileal brush-border membranes (data not shown).
The polarized distribution of these transporters in stably transfected MDCK cells was verified by confocal indirect immunofluorescence microscopy. First, the identity of the basolateral domain was established by double labeling with an antibody directed against the basolateral marker, Na+-K+-ATPase, in the stably transfected MDCK cells (8, 13, 27). Labeled cells were examined by en face and X-Z cross sections generated by confocal microscopy. Na+-K+-ATPase retained its normal basolateral localization, demonstrating that the transfected cells remain appropriately polarized (data not shown). In Fig. 2F, when viewed en face, ASBT staining is distributed in a punctate pattern, characteristic of apical microvillar labeling. X-Z cross-sectional images confirm that ASBT is distributed to the apical surface (Fig. 2I). In contrast to ASBT, Ntcp was clearly limited to the basolateral membrane in a pattern identical to that observed with Na+-K+-ATPase (Fig. 2, B and D).
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To quantitate the polarized distribution of bile acid transporters, stably transfected and untransfected MDCK cells were grown on permeable Transwell filter inserts until confluence. Uptake of 10 µM [3H]taurocholate was studied by incubation in apical or basolateral Transwell compartments. As shown in Fig. 3, Na+-dependent taurocholate uptake was detected at the basolateral surface of Ntcp-transfected MDCK at a rate that was 6- to 10-fold higher than that at the apical surface. In contrast, ASBT-transfected cells mediated a rapid taurocholate influx, which was more than ninefold greater at the apical vs. the basolateral surface. These studies confirmed that MDCK cells are an appropriate model system to study the plasma membrane targeting of these bile acid transporters.
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The expression of certain genes has been reported to respond positively to induction by sodium butyrate. In transfected cell lines, the transfected gene is expressed more efficiently after the addition of butyrate to the medium and the butyrate-induced cells maintained a normal polarized pattern (18, 28). Our immunofluorescence studies demonstrated that only <5% of ASBT-transfected MDCK cells expressed the bile acid transporter (Fig. 2, F and I). To enhance the gene expression, we preincubated stably transfected MDCK cells in 10 mM sodium butyrate for 15 h at 37°C. Immunofluorescence microscopy demonstrated that after sodium butyrate treatment, 50-80% of transfected MDCK cells showed expression of ASBT, and this expression was restricted to the apical surface of transfected cells (Fig. 2, G and J). This was further confirmed by transport studies that showed that the apical Na+-dependent taurocholate uptake of ASBT-transfected MDCK cells was increased ~10-fold compared with noninduced cells (data not shown). To determine the differences between noninduced and induced cells, DNA, mRNA, and protein levels were quantitated using Southern, Northern, and Western blotting studies. After sodium butyrate induction, ASBT mRNA and protein levels increased ~10- and ~15-fold, respectively. In contrast, similar amounts of DNA were detected in the sodium butyrate-induced and noninduced MDCK cells (Fig. 4). Similar results were found in the Ntcp-transfected MDCK cells after incubation with sodium butyrate (data not shown). These data suggest that sodium butyrate stimulated the transporter gene expression presumably by enhanced transcription, reconfirming earlier observations (18, 28). Subsequent studies were done in transfected cells incubated with 10 mM sodium butyrate.
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Construction of Truncated Transporter Chimeras and Their Expression in Transfected MDCK Cells
The mutant proteins and cytoplasmic tail sequences of ASBT and Ntcp proteins are shown schematically in Fig. 5. PCR-generated constructs were subcloned into the pCMV2 vector and stably transfected into MDCK cells. For each of these proteins, several independently derived, cloned, polarized MDCK cell lines were established as described under MATERIALS AND METHODS. Northern blot analysis confirmed that these mutant constructs were successfully transcribed in stably transfected MDCK cells (Fig. 6). In limited studies, the functional expression of mutated and chimeric transporters was initially tested by transient transfection in COS-7 cells.
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Ntcp contains two potential Tyr-based basolateral sorting sequences at its COOH-terminal end (Y307-E-K-I and Y321-K-A-A). In addition, the COOH-terminal portion of the transporter has a low degree of similarity with that of the ASBT protein. In initial experiments, truncated and chimeric transporters were used to determine the importance of the cytoplasmic tail to bile acid transport activity and for sorting to the appropriate domain of the plasma membrane. We first removed the 56-amino-acid COOH-terminal tail from Ntcp to create a truncated mutant, designated NL (Fig. 5). When MDCK cells stably transfected with this construct were assessed for Na+-dependent [3H]taurocholate uptake, 74.6 ± 3.4% of transport activity occurred across the basolateral domain and 25.4 ± 3.4% was detected at the apical surface (Fig. 7). Thus deletion of the cytoplasmic tail from Ntcp protein resulted in partial loss of its fidelity of basolateral polarity in MDCK cells, with ~75% being delivered to the basolateral surface, which was significantly different from the distribution of wild-type Ntcp protein (~90% located in the basolateral surface, P < 0.05). Similar results were achieved when the cytoplasmic tail of Ntcp was replaced by an eight-amino-acid epitope tag (NLF, Fig. 7). The results from confocal microscopy further confirmed that these two mutant proteins were largely detected in the basolateral membrane in stably transfected MDCK cells (Fig. 8). These studies indicate that information contained within the COOH-terminal tail of Ntcp was not predominant for exclusive basolateral targeting and that additional sorting signals may be present.
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Apical Sorting Information is Contained Within Cytosolic COOH-Terminal 40 Amino Acids of ASBT
MDCK cells were stably transfected with a cDNA encoding an ASBT protein lacking its cytoplasmic tail (NI, Fig. 5). In these cells Na+-dependent [3H]taurocholate uptake could not be demonstrated across the apical or basolateral domains. Transport activity was also absent in COS-7 cells transiently transfected with the same construct (data not shown). These results suggested that the truncated protein was not functional or not sorted to the plasma membrane. To allow localization of the mutant protein, a FLAG epitope, which could be detected with a specific antibody, was inserted in place of the 40-amino-acid cytoplasmic tail (NIF, Fig. 5). Confocal immunofluorescence experiments carried out in epitope-tagged ASBT transfected COS-7 and MDCK cells showed an intracellular distribution of the tailless ASBT that was typical of localization in the endoplasmic reticulum (data not shown).To test the possibility that a potential internal endoplasmic reticulum retrieval motif was uncovered by removal of the cytoplasmic tail of ASBT, a chimeric protein was created in which the 40-amino-acid tail of ASBT was replaced by the 56-amino-acid COOH tail of Ntcp (NICL, Fig. 5). Confocal immunofluorescence analysis of transfected MDCK cells showed a predominant intracellular distribution of the chimeric protein with slight staining of the basolateral membrane. Na+-dependent [3H]taurocholate uptake was not detected in transfected COS-7 cells or across the apical or basolateral surfaces of MDCK cells (data not shown).
The intracellular accumulation of the truncated ASBT protein suggested the novel possibility that the cytoplasmic tail of ASBT could be critical for apical sorting of the transporter. Therefore, an Ntcp chimera (NLCI, Fig. 5) was generated by exchanging the 56-amino-acid COOH tail of Ntcp for the 40-amino-acid tail of ASBT. The cellular distribution of NLCI was first defined by the transport studies. The apical-to-basolateral ratio of polarity of the NLCI chimera was determined by measuring Na+-dependent [3H]taurocholate uptake. Bile acid uptake was predominantly across the apical domain of MDCK cells (82.4 ± 3.9%) and similar to the localization of transport activity that we had found in MDCK cells transfected with wild-type ASBT (Fig. 7).
Confocal images obtained at the horizontal plane showed a punctate pattern of apical microvillar staining of the NLCI mutant proteins similar to cells transfected with wild-type ASBT (Fig. 8). X-Z cross-sectional images confirmed the apical distribution of NLCI (Fig. 8). There was no significant staining of the basolateral surface or of intracellular membranes. Thus these data suggest the presence of an apical sorting signal in the cytoplasmic tail of ASBT that is capable of completely reversing the polarity of a protein normally sorted to the basolateral membrane.
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DISCUSSION |
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The polarity of epithelial cells is critically dependent on the selective delivery and insertion of lipids and proteins into the apical and basolateral domains of the plasma membrane. In the current study, the possibility was considered that signals determining sorting to the plasma membrane reside within the amino acid sequences of the COOH-terminal cytoplasmic tails of two Na+-dependent bile acid transporters, Ntcp and ASBT. These transporters display high structural and functional similarity but are sorted to the opposite membrane domains in native cells expressing these proteins. The MDCK epithelial cell line was used in our studies since much of the current knowledge concerning epithelial cell polarity has been gained using these kidney epithelial cells (11, 23, 32). Stably transfected MDCK cells appropriately localize ASBT and Ntcp to apical and basolateral domains, respectively, based on the polarity of taurocholate transport and immunocytochemical localization studies. Thus MDCK cells are an appropriate model system to study the mechanisms underlying the sorting of these bile acid transporters to the plasma membrane.
Mutational analysis, performed using chimeric and truncated transmembrane proteins, has suggested that short amino acid sequences in the cytoplasmic domains of proteins may function as basolateral targeting signals. With the use of systematic, site-directed mutagenesis, one of the basolateral targeting signals in the cytoplasmic domains of several proteins has been identified as a tyrosine-based signal represented by the sequence Y-X-X-aliphatic (9, 36, 37). Thus it was predicted that two potential Tyr-based basolateral sorting sequences in the COOH-terminal cytosolic domain of Ntcp would be important determinants for basolateral targeting. To investigate the importance of this Tyr motif for the localization of Ntcp, a series of stably transfected MDCK cell lines was prepared that expressed either chimeric or truncated Ntcp proteins. We hypothesized that, if the cytoplasmic tail of Ntcp contains critically important signals for basolateral localization as shown for other basolateral membrane proteins, deletion of this portion of the transporter would essentially inactivate the signal and change the surface distribution of the protein. The results of taurocholate uptake studies across the basolateral and apical domains of the MDCK cells grown to confluence and expressing either a truncated (NL) or a chimeric transporter with a nonspecific epitope FLAG as a tail (NLF) indicated some loss of normal cell polarity for Ntcp expression. In these cells ~75% of the transporter was delivered to the basolateral surface compared with 90% or more in cells transfected with wild-type Ntcp. Although the FLAG tail has a tyrosine residue, the sequence of this tail bears no similarity to any proposed targeting motif. These results are surprising because, despite some heterogeneity, most or all of the identified basolateral sorting determinants have been found to be located in the cytoplasmic domains of basolaterally expressed plasma membrane proteins. Thus it is likely that additional and more predominant sorting signals reside elsewhere within this protein, possibly within transmembrane domains.
Previous studies in MDCK cells have revealed that glycophosphatidylinositol (GPI)-anchored proteins are predominantly directed to the apical plasma membrane with their GPI anchors functioning as a possible apical sorting signal (20, 29, 31, 38). Changes in the carbohydrate composition of the GPI anchor may significantly alter the sorting of apical proteins (25, 29, 39). Moreover, recent studies (12) also indicated that apically secreted glycoproteins, such as gp80 (clusterin), were not included in detergent-insoluble complexes in MDCK cells and suggested that this secretory glycoprotein and GPI-linked proteins may use different mechanisms to reach the apical membrane. Whether a previously identified mechanism or a novel sorting signal is involved in trafficking of ASBT was examined in our studies.
The least conserved sequences between ASBT and Ntcp reside within the cytoplasmic tail of the proteins. We initially thought that these differences might be critical for the function of the proteins since the affinity for bile salts is somewhat lower for ASBT compared with Ntcp. It did not seem likely that the cytoplasmic tail of ASBT would be of importance for sorting to the apical domain since apical sorting signals located within the cytoplasmic tail are unprecedented to our knowledge. To evaluate the importance of the cytoplasmic tail to functioning and membrane targeting, a truncated ASBT mutant (NI) was created in which the cytoplasmic tail of ASBT was removed. Expression of this mutant transcript could be documented in both transiently transfected COS-7 cells and stably transfected MDCK cells, but taurocholate transport activity could not be detected in either cell line. Because our antibody was directed against the cytoplasmic tail of ASBT, a suitable reagent was not available to detect protein expression or localization in transfected cells. It is possible that the truncated protein did not reach the cell surface or that the deletion so changed protein structure that bile acid transport activity was lost. To test these possibilities further, we created a chimera in which the cytoplasmic tail of ASBT was replaced with a nonspecific epitope FLAG tail (NIF). In addition, to further explore the importance of the cytoplasmic tail of ASBT as a possible apical sorting signal, the 56-amino-acid COOH-terminal tail of Ntcp was replaced by the 40-amino-acid tail of ASBT (NLCI).
Chimeric transporters in stably transfected MDCK cells were analyzed by taurocholate uptake studies at the basolateral and apical surface and by immunofluorescence microscopy. In MDCK cells stably transfected with the ASBT chimera containing a FLAG tail, no taurocholate transport activity could be detected at the basolateral or apical domains. Immunofluorescence microscopy of transiently transfected COS-7 cells and stably transfected MDCK cells indicated that the chimeric ASBT protein was expressed but largely retained within the cell. Little or no protein could be detected on either domain of the plasma membrane of either cell line. There was also predominant intracellular distribution of a chimeric protein in which the 40-amino-acid tail of ASBT was replaced by the 56-amino-acid COOH tail of Ntcp.
In MDCK cells expressing the chimeric Ntcp with the ASBT tail (NLCI), Na+-dependent taurocholate uptake was detected predominantly from the apical domain (82.4 ± 3.9%). Immunofluorescence microscopy confirmed that the Ntcp protein, which normally is directed to the basolateral domain of the hepatocyte and still demonstrates predominantly basolateral targeting after removal of the cytoplasmic tail, could be completely redirected to the apical domain by protein sequences contained within the cytoplasmic tail of ASBT. These data indicate that a novel apical sorting system is present in the cytoplasmic tail of the ASBT protein that is different from previously reported ectodomain and GPI apical sorting signals.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael J. Caplan (Dept. Of Cellular and Molecular Physiology, Yale University) for fruitful discussions and for assistance with confocal microscopy and Dr. Paul A. Dawson (Dept. Of Medicine, The Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC) for providing pCMV2-Ntcp cDNA.
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FOOTNOTES |
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This study was partially supported by National Institute of Child Health and Human Development Grant HD-20632 (to F. J. Suchy) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34989 (to the Liver Center, Yale University School of Medicine).
A portion of this study was presented at the 46th annual meeting of the American Association for the Study of Liver Diseases, in November 1995, in Chicago, IL, and at PAGE-WOOD International Symposium on Membrane Traffic in Health and Disease, in September 1996, in Cleveland, OH.
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. §1734 solely to indicate this fact.
Address for reprint requests: F. J. Suchy, Dept. of Pediatrics, Box 1198, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574.
Received 28 April 1998; accepted in final form 31 July 1998.
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