1Yale University School of Medicine, New Haven, Connecticut 06511; and 2Brooklyn College, Brooklyn, New York 11210
Submitted 9 July 2003 ; accepted in final form 10 December 2003
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ABSTRACT |
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GABA transporter; traffic; sorting signal; targeting signal
Whereas glycosyl phosphatidylinositol (GPI) and carbohydrate modification have been shown to serve as signals encoding the apical distribution of several proteins (4, 6, 12, 37), the information responsible for distributing proteins to the basolateral membrane appears to be embedded exclusively within the amino acid sequences of these proteins. Tyrosine- and dileucine-based motifs present in the cytoplasmic tails of a number of proteins specify the basolateral distributions of these proteins (21, 22, 24, 29). In the case of the Caenorhabditis elegans LET-23 receptor (5, 15), a PDZ-binding motif present at the extreme COOH-terminus of the protein mediates stable basolateral localization.
Previous experiments from our laboratory have examined the distribution information in the GAT-2 isoform of the -amino butyric acid (GABA) transporter, a polytopic membrane protein. Initial studies of the basolateral sorting signals of GAT-2 were conducted by deleting and exchanging portions of the basolateral GAT-2 and apical GAT-3 GABA transporters. These two transport proteins are 65% identical, and yet they exhibit dramatically different subcellular localizations. The COOH-terminal 32 amino acids of the GAT-3 transporter are necessary for its apical localization; without them, the transporter is present equally at the apical and basolateral membranes of Madin-Darby canine kidney (MDCK) cells. The COOH-terminal 22 amino acids of GAT-2 are necessary for its basolateral distribution, and their removal results in its random cell surface distribution. When the COOH-terminal 32 amino acids of the GAT-3 transporter are replaced with the COOH-terminal 22 amino acids of GAT-2, the transporter accumulates at the basolateral surface. This observation demonstrates that these 22 amino acids are necessary and sufficient to ensure the basolateral localization of the GAT-2 transporter at steady state in MDCK cells (26). In the present study, we endeavored to determine whether the basolateral signal in the COOH-terminal tail of GAT-2 can function autonomously in the absence of other GABA transporter sequences. We also determined which portions of the tail sequence comprise the basolateral distribution signal and asked whether this information accounts for the localization of the protein by specifying its initial biosynthetic targeting or by stabilizing its basolateral steady-state distribution.
To investigate these questions, we appended the entire COOH-terminal tail of the GAT-2 GABA transporter to PLAP-TMR, a membrane protein construct composed of the transmembrane domain (TMR) of the vesicular stomatitis virus G (VSVG) protein and the human placental alkaline phosphatase (PLAP) extracellular domain. Previous studies have established that the GPI anchor normally attached to PLAP is required for its apical membrane distribution in polarized epithelial cells (6). Without this anchor, the protein is secreted without polarity, implying that the extracellular domain of PLAP has no intrinsic sorting information (6). The transmembrane domain of the chimera, derived from the membrane-spanning domain of the VSVG protein, also has been shown to be devoid of intrinsic sorting information in polarized epithelia (17, 35). The construct combining these two domains (PLAP-TMR) should, therefore, contain no intrinsic sorting information. The COOH-terminal tail of GAT-2 was appended to PLAP-TMR to generate PLAP-GAT-2. All of these constructs can be detected at the surfaces of intact cells with anti-PLAP antibodies. Immunofluorescence confocal microscopy and domain-specific cell surface immunoprecipitation of radioactively labeled PLAP-GAT-2 allowed us to evaluate the polar localization of the tail and tail deletion mutant constructs expressed by transfection in polarized MDCK cells.
We found that the tail has a motif that can function independently, in the absence of other transporter sequences, to mediate both basolateral targeting and distribution. This basolateral distribution motif is encoded in the seven amino acids proximal to the COOH-terminal PDZ domain-interacting motif. Thus a PDZ interaction does not play a significant role in mediating the sorting of GAT-2 or in stabilizing its basolateral distribution.
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MATERIALS AND METHODS |
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To delete the COOH-terminal three amino acids, we modified an oligonucleotide pair encoding the extreme COOH terminus through the deletion of the bases coding for the final three amino acids. The PLAP-GAT-2 full-length (FL) construct was digested to release the nonmutant sequence. Altered oligonucleotides were annealed and then ligated into the cut DNA. Constructs were sequenced to confirm the deletion.
To prepare the PLAP-GAT-2 583592 and PLAP-GAT-2
592602 chimeras, we again employed oligonucleotides encoding an alternate COOH-terminal sequence. Oligonucleotides designed for the PLAP-GAT-2
583592 construct were the same as above but had base pairs encoding amino acids 583592 of the tail deleted. Oligonucleotides generated for the PLAP-GAT-2
592602 chimera had a sequence encoding amino acids 593602 of the tail deleted and replaced with a stop codon. Constructs were sequenced to confirm the mutations. Oligonucleotide sequences are available upon request.
Cell culture. Wild-type MDCK cells were maintained in -MEM (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FBS (Sigma, St. Louis, MO), 50 U/ml penicillin and streptomycin (GIBCO BRL), and 2 mM glutamine (GIBCO BRL). The incubator was kept at 5% CO2, 37°C, and 100% humidity. Stable cell lines were selected and maintained under the same conditions with the addition of 0.9 g/l geneticin (GIBCO BRL) to the medium.
Preparation of stable cell lines. Transfection of MDCK cells for the establishment of stable cell lines was performed with PerFect lipid number 2 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After transfection, cells were split 1:10 into 100-mm dishes containing medium supplemented with 0.9 g/l geneticin. Cells were incubated for 810 days in this selection medium, and clones were isolated by cloning rings (Belco Glass, Vineland, NJ). Cells harvested from the same individual colonies were seeded into both 24- and 96-well plates to facilitate the first round of screening.
This first round of screening was performed by using a colorimetric PLAP enzymatic assay (Kirkegaard and Perry Laboratories, Gaithersburg, MD) in the 96-well plates when the clones reached confluence. Positive colonies were then evaluated by immunofluorescence microscopy after plating on eight-chamber glass slides (Becton Dickinson Labware, Franklin Lakes, NJ). Immunofluorescent detection of the PLAP chimeras was carried out as described previously (1). Cells were incubated with a 1:100 dilution of Fitzgerald rabbit anti-PLAP (Fitzgerald Industries, Concord, MA) followed by a 1:100 dilution of rhodamine-conjugated anti-rabbit IgG (Sigma). Slides were mounted with Aquamount (Lerner Laboratories, Pittsburgh, PA) and examined with a Zeiss Axiophot upright fluorescence microscope (Carl Zeiss, Thornwood, NY).
Immunofluorescence confocal microscopy. Approximately 5 x 105 cells were plated on 2.4-cm polycarbonate Transwell filter 0.45-µm inserts (Corning Costar, Cambridge, MA). These cells were allowed to grow for 510 days under standard conditions. Cells were washed three times in PBS++, and monolayers were then incubated for 1 h on ice in a 1:100 dilution of Fitzgerald rabbit anti-PLAP antibody in PBS++/1% BSA present in both the apical and basolateral media compartments. Cells were then washed three times for 5 min in ice-cold PBS++/1% BSA and fixed for 30 min in 4% paraformaldehyde in PBS++. After fixation, cells were prepared for secondary antibody incubation and stained with a 1:100 dilution of fluorescein- or rhodamine-conjugated goat anti-rabbit secondary antibody (Sigma) as described previously (1). Filters were mounted with Aquamount. Immunofluorescence images were obtained with a Zeiss LSM 410 scanning confocal microscope (Carl Zeiss). Brightness and contrast were set so that all the pixels were within the linear range and all images were the product of eightfold line averaging. We then produced x-z sections with the use of a 0.2-µm motor step.
Cell surface immunoprecipitation. Approximately 5 x 105 cells were plated per 2.4-cm polycarbonate Transwell filter 0.45-µm insert (Corning Costar). These cells were allowed to grow for 510 days under standard conditions. For targeting experiments, cells were labeled for 1 h at 37°C with 500700 µl of cysteine/methionine-free medium supplemented to 0.2 mCi/ml with [S35]cysteine/methionine (Amersham Biosciences, Piscataway, NJ) at the basolateral side of the filter and 500700 µl of cysteine/methionine-free medium at the apical side. For steady-state labeling experiments, the labeling medium contained cold cysteine and methionine at 0.01 mM (10% of the level in growth medium). This medium was supplemented to 0.1 mCi/ml with [S35]cysteine/methionine, and the cells were labeled overnight. After labeling, the plates holding the filter inserts were placed on wet ice and washed twice with ice-cold PBS++. Filters were incubated at either the apical or basolateral side with 500700 µl of ice-cold PBS++ supplemented with 1% BSA to block nonspecific antibody binding. A 1:200 dilution of Fitzgerald rabbit anti-PLAP antibody was added to the either the apical or basolateral medium compartment. Filters were incubated on ice with antibody for 2 h, washed three times with ice-cold PBS++, cut out of the holder, and lysed in ice-cold TBS (150 mM NaCl, 5.0 mM EDTA, 50 mM Tris, pH 7.4) with 1% Triton X-100 for 30 min. Cells were scraped off the filters and transferred into microcentrifuge tubes. Lysates were spun at maximum speed for 10 min to pellet cell debris, and the cleared supernatant was transferred to another microfuge tube. Forty microliters of a 50:50 (vol/vol) slurry of protein A-Sepharose beads (Pierce Biotechnology, Rockford, IL) were added to each supernatant, and the samples were rotated overnight at 4°C. Samples were washed three times in 1 ml of TBS plus 1% Triton X-100, and proteins were eluted from the beads with SDS-PAGE sample buffer before being run on SDS-PAGE gels. Gels were fixed for 1 h in 10% acetic acid and 20% methanol and then washed for at least 2 h in several changes of distilled water before being dried under a vacuum. Radioactivity was detected by phosphorimaging (Amersham) and quantitated using ImageQuant software (Amersham). Each cell line was assayed in triplicate.
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RESULTS |
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To obtain a quantitative biochemical measurement of the distribution of PLAP-TMR, we performed cell surface immunoprecipitation. Cells were metabolically labeled to steady state with [S35]methionine for 16 h at 37°C. After the temperature was reduced to 4°C to prevent the redistribution of plasmalemmal proteins, cells were incubated with anti-PLAP antibody added to either the apical or basolateral medium compartment. Unbound antibody was removed by washing, after which cells were solubilized in 1% Triton X-100. Antibody-bound PLAP-TMR was then precipitated through the addition of protein A-Sepharose beads to the detergent lysate. Analysis of the immunoprecipitated, radiolabeled PLAP-TMR protein revealed an apical-to-basolateral ratio of 0.99 ± 0.21 (Fig. 3). These experiments demonstrate that the populations of PLAP-TMR at the apical and basolateral membranes are approximately equal and, thus, that the chimera has no intrinsic localization information. This property makes this construct a useful tool for studying sorting and targeting information in sequences appended to its cytoplasmic COOH terminus.
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The sequence of the entire GAT-2 tail was appended to PLAP-TMR to make PLAP-GAT-2 FL (Fig. 1). Stable MDCK cell lines expressing this construct were generated, and the distribution of PLAP-GAT-2 FL was evaluated initially by immunofluorescence microscopy. Cells were grown to confluence on 0.4-µm polycarbonate filter supports, and anti-PLAP antibody staining and confocal microscopy were carried out as described above. The en face view (Fig. 2B) indicated that the distribution of this construct was predominantly basolateral. This finding was confirmed through analysis of the x-z sections, which show almost exclusively lateral staining. Further confirmation was provided by domain-specific steady-state immunoprecipitation experiments. These experiments yielded an apical-to-basolateral ratio of 0.21 ± 0.03 (Fig. 3). These results indicate that the COOH-terminal tail of GAT-2 contains a basolateral localization motif that can function independently and without contributions from the rest of the transporter.
The last 10 amino acids of the GAT-2 tail are necessary for basolateral steady-state localization. We next wanted to define the portion of the GAT-2 tail necessary for basolateral sorting. Earlier experiments employing the entire transporter demonstrated that critical basolateral distribution information is contained in the final 22 amino acids (26). Alanine mutagenesis experiments indicated that a dileucine motif at amino acids 592 and 593 does not play any detectable role in generating basolateral distribution of the entire GAT-2 transporter protein (26). The polar distributions of several membrane proteins, including CFTR (25), syndecan (9), and the C. elegans LET-23 epidermal growth factor receptor (36), are at least partly dependent on PDZ-binding motifs displayed at their extreme COOH termini. The final three amino acids of the GAT-2 tailserine, asparagine, and cysteine (SNC)resemble a type I PDZ-binding motif. To investigate the potential contribution of this putative PDZ domain-binding motif to the basolateral sorting information in the GAT-2 tail, we made a truncated PLAP-GAT-2 construct in which these COOH-terminal three amino acids were removed: PLAP-GAT-2 SNC (Fig. 1). To assay the distribution of this construct in MDCK cells, we created stable cell lines and performed cell surface immunoprecipitation and immunofluorescence confocal microscopy as described for the PLAP-TMR and PLAP-GAT-2 FL constructs. The en face immunofluorescence image (Fig. 2B) indicates that the construct exhibits a basolateral distribution. This assessment is borne out by the lateral staining pattern revealed on the x-z sections (Fig. 2B). Domain-specific steadystate surface immunoprecipitation experiments also showed that elimination of these three amino acids causes no statistically significant increase in the apical-to-basolateral ratio of the chimera (Fig. 3), indicating that these amino acids play no role in restricting the chimera to the basolateral membrane.
Because the apical-to-basolateral distribution ratio of the PLAP-GAT-2 SNC construct was not significantly different from that of the full-length chimera, we concluded that there must be basolateral sorting information in the tail that is not dependent on the final three amino acids. To define where this information might reside, we made two additional deletion constructs within the context of the PLAP-GAT-2 FL chimera (Fig. 1). These new protein constructs contain complementary halves of the COOH-terminal 22 amino acids of the GAT-2 tail. This 22-residue sequence was shown to be necessary and sufficient to ensure the basolateral localization of the full-length transporter (26). We deleted the most membrane distal 10 amino acids of the tail, including the SNC motif, to create PLAP-GAT-2
592602. We deleted the 10 amino acids just proximal to these distal 10 to create PLAP-GAT-2
583592 (Fig. 1). Stable MDCK cell lines expressing these chimeras were prepared. Confocal immunofluorescence microscopy and domain-specific steady-state cell surface immunoprecipitation were carried out as described previously.
The results of these assays were markedly different for the two constructs. En face confocal micrographs of MDCK cells expressing PLAP-GAT-2 592602 revealed a principally apical distribution of the construct (Fig. 2D). This was confirmed by the predominance of apical staining in the x-z section (Fig. 2D). Domain-specific steady-state immunoprecipitation assays yielded an apical-to-basolateral ratio of 1.8 ± 0.3 (Fig. 3), demonstrating that the final 10 amino acids of the tail are vital for its basolateral sorting activity. Deletion of the more proximal 10 amino acids had little effect on the distribution of the chimera. En face confocal micrographs of the PLAP-GAT-2
583592-expressing cell lines (Fig. 2E) are consistent with a basolateral distribution. Similarly, x-z sections (Fig. 2E) show principally basolateral staining. Domain-specific steady-state immunoprecipitation assays revealed an apical-to-basolateral ratio for this chimera of 0.4 ± 0.02 (Fig. 3), confirming that the chimera is primarily present at the basolateral membrane. In summary, the steady-state analysis of all the PLAP-GAT-2 chimeras argues that the final 10 amino acids of the tail contain necessary basolateral sorting information.
The last 10 amino acids of the GAT-2 tail are necessary for targeting. For a membrane protein to achieve a polar distribution, it must either be delivered directly to the appropriate plasma membrane domain or be preferentially maintained there after random surface delivery. We wanted to determine whether the localization information present in the GAT-2 COOH terminus is capable of mediating delivery from the biosynthetic pathway directly to the basolateral surface. The polarity of the initial cell surface delivery was assayed after a pulse-labeling period of 1 h (8). The protocol is essentially the same as that described above for domain-specific cell surface immunoprecipitation at steady state, but a shorter labeling period allowed us to examine a cohort of newly synthesized and delivered protein. The distribution of newly delivered PLAP-GAT-2 FL and PLAP-TMR was not significantly different from their distribution at steady state: PLAP-GAT-2 was delivered in an apical-to-basolateral ratio of 0.21 ± 0.05, whereas for PLAP-TMR, this ratio was 0.8 ± 0.36 (Fig. 4). We found that disruption of the final three amino acids had little or no effect on the apical-to-basolateral ratio of newly delivered chimeric protein (Fig. 4), indicating that this sequence does not play an important role in the initial targeting of the protein.
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Deletion of the distal 10 amino acids perturbed the polarity of the cell surface delivery of the construct, just as it disrupted the basolateral distribution of the protein at steady state. This chimera was initially delivered in an apical-to-basolateral ratio of 3.5 ± 0.24 (Fig. 4), demonstrating that these 10 amino acids contain information necessary for the initial polar delivery of the protein as well as for ensuring its steady-state distribution.
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DISCUSSION |
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By making deletion mutants of the GAT-2 tail in the context of the PLAP-TMR chimera (Fig. 1) and assaying their polarity in MDCK cells by immunofluorescence confocal microscopy and domain-specific cell surface immunoprecipitation, we were able to define a sequence domain required for basolateral localization. Two of the deletion constructs, PLAP-GAT-2 583592 and PLAP-GAT-2
592602, each contained approximately half of the 22-amino acid signal that was initially shown to be necessary and sufficient for basolateral localization of the full-length transporter (Fig. 1). A third mutant construct deleted a PDZ-binding motif at the extreme COOH terminus (Fig. 1). For each of these constructs, the distribution at steady state was similar to the distribution upon delivery to the cell surface, indicating that the sorting signal in the tail encodes biosynthetic sorting information (Figs. 2, 3, 4). Deletion of amino acids 583592, the 10 amino acids proximal to the most COOH-terminal 10 amino acids, did not disturb basolateral distribution of the PLAP-GAT-2 chimera (Figs. 2 and 3). Deletion of a putative PDZ-binding motif at the extreme COOH terminus of the tail to create the PLAP-GAT-2
SNC chimera (Fig. 1) also had no effect on the basolateral distribution of the protein, either upon delivery (Fig. 4) or at steady state (Figs. 2 and 3). However, deletion of these three amino acids along with the seven residues NH2-terminal to them had a profound effect on the sorting of the construct, resulting in an apical-to-basolateral distribution of
2.5:1, compared with the 1:4 apical-to-basolateral ratio characteristic of the full-length chimera (Figs. 2, 3, 4). This led us to conclude that amino acids 593599 are required for the basolateral distribution of the GAT-2 protein.
Given the role that PDZ domain-containing proteins have been shown to play in establishing or maintaining the polar distribution of some membrane proteins, including Kir 2.3 (19) and the LET-23 epithelial growth factor receptor (15, 32), it is perhaps surprising that the deletion of the putative PDZ-interacting motif played no role in the polar delivery or steady-state distribution of the PLAP-GAT-2 chimera. In fact, we have identified a PDZ domain-containing protein that is a binding partner for GAT-2. The protein identified as Gi-interacting protein COOH terminus (GIPC) interacts with the GAT-2 COOH terminus as well as with cytoskeletal motor proteins (7), transmembrane proteins including
6A/
6B-integrins (11, 34), and various soluble proteins (10). The role of the GIPC interaction in regulating the function or distribution of GAT-2 remains to be established. It may be that the GAT-2 PDZ-binding motif is important for extending the lifetime of the protein at the plasma membrane. The ability of GIPC to interact with integrins, proteins known to cluster molecules at the plasma membrane (for review, see Ref. 3), is also consistent with this role. Another GABA transporter family member, the basolateral betaine transporter BGT (27), clearly depends on its PDZ domain-interacting motif to ensure its stable residence at the plasma membrane. The PDZ domain-interacting motif, perhaps through its interaction with GIPC and, in turn, with cytoskeletal motor proteins, may also be important for moving GAT-2 through the secretory pathway. The PDZ interaction motif does not, however, constitute a vital part of the anisotropic distribution information embedded in the GAT-2 tail.
The portion of the GAT-2 tail required to specify basolateral distribution appears to reside within the seven-amino acid sequence immediately NH2-terminal to the PDZ interaction motif: LRLTELE. Two classes of motifs have been characterized as important for basolateral localization. Tyrosine-based motifs, such as those found in the LDL receptor and the -subunit of the H+-K+-ATPase, can mediate basolateral localization in MDCK cells (21, 29). The interpretation of these motifs is thought to depend on interactions with adaptor complexes (for review, see Ref. 13). Dileucine motifs in the cytoplasmic tails of the macrophage Fc receptor (23) and epithelial adhesion molecule E-cadherin (24) direct basolateral sorting. Similarly, other dihydrophobic amino acid motifs in several proteins, including the furin receptor (31) and major histocompatability complex class I chain-related protein MICA (33), are necessary for basolateral localization in various cell systems. Dileucine motifs have also been shown to interact with adaptor complexes (16).
Non-tyrosine-based and nondihydrophobic basolateral sorting information also has been described (14, 18). Residues 749788 of the cytoplasmic tail of a neural cell adhesion molecule, N-CAM-140, contain necessary basolateral sorting information that involves neither tyrosine nor dihydrophobic residues. Similarly, a 23-amino acid juxtamembrane sequence (residues Lys652 to Ala674) in the cytoplasmic portion of human epidermal growth factor receptor is necessary and sufficient to ensure the basolateral distribution of this protein. Neither of the two dileucine motifs of this protein plays a role in establishing its basolateral distribution. The HRXXV motif in the COOH-terminal tail of the polymeric immunoglobulin receptor is required for its initial basolateral delivery from the trans-Golgi network in MDCK cells (2, 28), although its subsequent transcytosis to the apical membrane involves other sequences (20). The basolateral sorting information in the GAT-2 tail also does not involve tyrosine or dileucine motifs and is not similar in any obvious way to the other novel sequences discussed above. Thus it constitutes a new class of basolateral sorting motif and is interpreted by as-yet unidentified mechanisms within the cell.
Deleting the LRLTELE motif results in an apical-to-basolateral ratio that is 1020 times higher than that of the full-length construct, producing an apical distribution ratio of 1.8 ± 0.27 at steady state and 3.5 ± 0.24 upon initial delivery. This not only demonstrates that the COOH-terminal 10 amino acids are vital for the basolateral sorting activity of the GAT-2 tail but also implies that some cryptic apical sorting information may be uncovered by this deletion. Deleting the final 24 amino acids of the full-length GAT-2 transporter also leads to an apically biased distribution, associated with an apical-to-basolateral ratio of 4 (26). It is possible, therefore, that apical localization information is present in the 26 residues of the GAT-2 COOH-terminal tail that resides between the membrane and the extreme COOH-terminal 22 amino acids deleted by Muth et al. (26). The characteristics and physiological role of this putative second silent signal remain to be determined.
Whatever cryptic apical targeting information may be contained in the GAT-2 tail is entirely subordinate to the basolateral message embodied in the LRLTELE sequence under normal circumstances. This basolateral targeting sequence does not correspond to any known basolateral sorting signal, suggesting that it belongs to a novel class of basolateral targeting motif. Because it plays its role in the delivery of the protein to the appropriate plasma membrane domain rather than simply mediating its retention at the basolateral surface of the plasma membrane, this motif might be involved in sorting the protein into appropriately targeted membrane vesicles during its biosynthetic passage through the trans-Golgi network. It also might recruit basolateral targeting machinery to the vesicles into which it is sorted. Future work is required to determine the nature of the cellular components that actually interpret this novel motif and guide the COOH-terminal tail of the GABA transporter to the basolateral domain of the epithelial plasma membrane.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institutes of Health Grants GM-42136 and DK-17433.
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FOOTNOTES |
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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.
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[GenBank]
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