Impaired trafficking of distal renal tubular acidosis mutants of the human kidney anion exchanger kAE1

Janne A. Quilty, Jing Li, and Reinhart A. Reithmeier

Canadian Institutes of Health Research Group in Membrane Biology, Departments of Biochemistry and Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8


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ABSTRACT
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Distal renal tubular acidosis (dRTA) is an inherited disease characterized by the failure of the kidneys to appropriately acidify urine and is associated with mutations in the anion exchanger (AE)1 gene. The effect of the R589H dRTA mutation on the expression of the human erythroid AE1 and the truncated kidney form (kAE1) was examined in transfected human embryonic kidney 293 cells. AE1, AE1 R589H, and kAE1 were present at the cell surface, whereas kAE1 R589H was located primarily intracellularly as shown by immunofluorescence, cell surface biotinylation, N-glycosylation, and anion transport assays. Coexpression of kAE1 R589H reduced the cell surface expression of kAE1 and AE1 by a dominant-negative effect, due to heterodimer formation. The mutant AE1 and kAE1 bound to an inhibitor affinity resin, suggesting that they were not grossly misfolded. Other mutations at R589 also prevented the formation of the cell surface form of kAE1, indicating that this conserved arginine residue is important for proper trafficking. The R589H dRTA mutation creates a severe trafficking defect in kAE1 but not in erythroid AE1.

band 3; membrane protein; biosynthesis; human kidney anion exchanger 1


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INTRODUCTION
MATERIALS AND METHODS
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DISTAL RENAL TUBULAR ACIDOSIS (dRTA) is a disease characterized by an impaired proton secretion into the urine, leading to metabolic acidosis and often to hypokalemia, bone disease, and nephrocalcinosis (2, 32). Several mutations in the anion exchanger (AE)1 gene were found to be associated with autosomal dominant dRTA (4, 15, 18, 33, 44), and two mutations were associated with autosomal recessive dRTA (39, 41).

AE1 is a polytopic membrane protein localized in the plasma membrane of red blood cells (16, 31, 38). The 52-kDa COOH-terminal membrane domain of AE1 has been proposed to span the membrane 12 times (28) and catalyzes the electroneutral exchange of chloride for bicarbonate (14). Anion transport is inhibited by stilbene disulfonates, which bind to an external site in the membrane domain of AE1 (34). The 43-kDa NH2-terminal cytoplasmic domain anchors the membrane to the underlying cytoskeleton through interactions with ankyrin, band 4.1, and band 4.2 (24, 46).

A kidney form of AE1 (kAE1) is located in the basolateral membrane of acid-secreting (alpha ) intercalated cells of the collecting duct of the kidney (1, 11, 12, 19, 42). kAE1 mRNA lacks the sequence encoded by the first three exons of the AE1 gene, leading to a protein with a truncated NH2 terminus (3, 19). Human kAE1 lacks amino acid residues 1-65, beginning at Met66 (19). kAE1 plays an important role in acid secretion into the urine by providing a path for bicarbonate across the basolateral membrane, working together with apical proton pumps. AE1 mutants associated with autosomal dominant dRTA were functional when expressed in Xenopus laevis oocytes (5, 15). A current hypothesis is that the mutations cause mistargeting of kAE1 in epithelial cells (5). The AE1 G701D mutant, linked to autosomal recessive dRTA, had impaired trafficking to the plasma membrane in oocytes unless the red cell protein glycophorin A was coexpressed (39). This suggested that kAE1 G701D has impaired trafficking to the basolateral membrane in kidney cells, whereas erythroid AE1 G701D can traffic to the plasma membrane because of the presence of glycophorin A in the red cell precursors.

To examine the effect of dRTA mutations on the trafficking of AE1, we transiently transfected erythroid and kidney forms of wild-type and mutant AE1 into a human kidney cell line, human embryonic kidney (HEK)-293. Our results show that the R589H mutation caused impaired kAE1 trafficking to the plasma membrane, whereas erythroid AE1 trafficking was only slightly affected. Coexpression of mutant kAE1 with wild-type kAE1 impaired trafficking of the wild-type protein, which is consistent with a dominant phenotype. The defect in kAE1 trafficking explains why this mutation causes kidney dysfunction but no serious hematological deficiency.


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Construction of plasmid and mutations. Human AE1 cDNA (a generous gift from Drs. A. M. Garcia and H. Lodish, Whitehead Institute) was inserted into the HindIII and BamHI sites of pcDNA3 (Invitrogen, Carlsbad, CA). Mutagenesis was performed with Stratagene's (La Jolla, CA) QuickChange Site-directed mutagenesis kit and oligonucleotide primers from ACGT (Toronto, ON). The kidney form (kAE1) was constructed by creating a XhoI site at the HindIII site. Another XhoI site was created 20 bases upstream of Met66. AE1 pcDNA3 was cut with XhoI and religated so that Met66 became the initiator methionine. To construct the AE1 N555 and kAE1 N555 mutants, the endogenous N-glycosylation acceptor site was removed by an N642D mutation and a novel N-glycosylation acceptor site was created at Asn555 by Y555N and V557T mutations (22). The Southeast Asian ovalocytosis (SAO) mutation was constructed as previously described (28). A COOH-terminal His6-tagged version of AE1 was constructed by PCR. First, a new XbaI was created in the Ser781 codon of AE1 pcDNA3. The forward primer consisted of a 32-oligonucleotide sequence starting in the Met776 codon and contained the new XbaI site. The reverse primer consisted of a 50-nucleotide sequence encoding the 6 COOH-terminal amino acids of AE1 followed by 6 codons for His, a stop codon, and a HindIII site. The PCR product and the AE1 pcDNA3 were digested with the appropriate restriction enzymes, and the digested PCR product was ligated into the open pcDNA3 vector. The mutations were confirmed by using the T7 Sequencing Kit (Amersham Pharmacia Biotech) and [35S]dATP (NEN Life Sciences Products, Boston, MA). The plasmid DNA for transfections was purified with Qiagen (Valencia, CA) Plasmid Midi columns.

Cell culture. HEK-293 cells were grown in DMEM supplemented with 10% calf serum and 0.5% penicillin and streptomycin (GIBCO Life Technologies, Gaithersburg, MD) in 5% CO2 at 37°C. The day before transfection, the cells were collected by trypsin digest and replated in six-well plates. Either DEAE-dextran (23) or Promega (Madison, WI) Transfast was used to transiently transfect the cells (0.4 µg of DNA/well of 6-well dish). As described below, the cell extracts were prepared by detergent solubilization 24-48 h after transfection.

Immunofluorescence. HEK cells were grown for 1 day on glass coverslips covering the bottom of a 6-cm plate. The cells were transfected by using Promega Transfast. Two days after transfection, the cells were fixed with 3.7% formaldehyde for 10 min. The cells were then rinsed twice in PBS [(in mM) 140 NaCl, 2.7 KCl, 10 Na2HPO4, and 1.8 KH2PO2, pH 7.4] and permeabilized in 0.1% Triton X-100 (Boehringer Mannheim, Indianapolis, IN), 100 mM PIPES, 1 mM EGTA, and 4% polyethylene glycol 8000, pH 6.9. For detection of AE1, the coverslips were incubated with 1:500 dilution of a mouse monoclonal anti-AE1 (IV F12) antibody (17) in PBS containing 3% BSA for 30 min at room temperature, followed by washing with PBS and incubation in 1:50 dilution of rhodamine-labeled anti-mouse IgG (Molecular Probes, Eugene, OR) in PBS containing 3% BSA for 30 min at room temperature. A rabbit anti-calnexin antibody (29) at a 1:500 dilution and a 1:50 dilution of fluorescein isothiocyanate-labeled anti-rabbit IgG (Molecular Probes) were used to localize the endoplasmic reticulum (ER) in cells. The coverslips were washed with PBS and mounted with 90% glycerol, 1 mg/ml p-phenylenediamine, and 0.1× PBS (pH 9.0) onto glass slides. Immunofluoresent staining was carried out at room temperature. Samples were viewed through a LSM410 invert laser-scan microscope (Carl Zeiss).

Cell surface biotinylation. Biotinylation conditions had been previously optimized (30). Forty-eight hours after transfection, HEK cells that had been grown in six-well culture dishes were washed with borate buffer [(in mM) 10 boric acid, 154 NaCl, 7.2 KCl, 1.8 CaCl2, pH 9.0]. Cells were treated twice with 1 ml of freshly prepared 0.8 mM EZ-Link NHS-SS-Biotin (Pierce, Rockford, IL) in borate buffer for 30 min at room temperature to biotinylate the cell surface. The cells were then rinsed in 1 ml of 0.192 M glycine and 25 mM Tris (pH 8.3) solution to quench any unreacted reagent. The cells were lysed with 1 ml radioimmunoprecipitation assay (RIPA) buffer (1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, and 10 mM Tris · HCl, pH 7.5) containing the protease inhibitors (in µM) 200 phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO), 2.8 E64 (Sigma-Aldrich), 1 leupeptin (Boehringer Mannheim), and 1 pepstatin (Boehringer Mannheim). An aliquot of the lysate (total) was saved for Western blotting. ImmunoPure (Pierce) immobilized streptavidin (100 µl) was added to the lysate for 1 h at 0°C to bind the biotinylated proteins. The supernatant was removed, and an aliquot was saved for Western blotting. The streptavidin beads were washed three times with RIPA buffer. SDS sample buffer (containing 5% beta -mercaptoethanol) was added to the beads, and the samples (bound) were incubated for 1 h to cleave the disulfide bond in the biotinylating reagent and release the captured proteins. Samples were analyzed for AE1 content by Western blotting.

Western blotting. SDS-PAGE was used to analyze protein samples (21). The protein was electrophoretically transferred to nitrocellulose (40) that was stained with Ponceau S (Sigma-Aldrich) for protein content. An antibody serum raised in rabbits against a peptide of the last 16 amino acids of human band 3 COOH terminus was used to probe the nitrocellulose at a 1:5,000 dilution (28). A second polyclonal antibody was raised in rabbits against a peptide corresponding to the first 14 amino acids of human erythroid AE1, including the NH2-terminal acetyl group. A horseradish peroxidase-conjugated goat anti-rabbit antibody (New England Biolabs, Beverly, MA) was then used to probe for the primary antibody at a 1:5,000 dilution. A chemiluminescence substrate (Boehringer Mannheim) and exposure to film were used to detect AE1 protein bands.

Enzymatic deglycosylation. Cell extracts (40 µl), prepared in PBS containing 1% C12E8 (Nikko, Tokyo, Japan) and protease inhibitors, were treated with 1,000 units of endoglycosidase H (endo H; New England Biolabs) or 500 units of peptide N-glycosidase F (PNGase F; New England Biolabs) at room temperature for 1 h, followed by the addition of 1 volume of 2× Laemmli sample buffer containing 4% (wt/vol) SDS. AE1 was detected by Western blotting.

Pulse-chase assay. One day after transfection, the cells were pulsed with 200 µCi/ml of L-[35S]methionine (NEN Life Science Products) in methionine-free DMEM media (GIBCO Life Technologies) for 20 min (30). After labeling, the medium was removed and replaced with DMEM. Each chase time point was a single well of a six-well plate. At various time points, the cells were lysed in RIPA buffer containing protease inhibitors (PMSF, leupeptin, pepstatin A, and E64). AE1 was immunoprecipitated with an anti-COOH terminal antibody followed by protein G-Sepharose (Amersham Pharmacia Biotech). The immunoprecipitates were analyzed by electrophoresis using 8% SDS-PAGE. The gels were dried and exposed to film and a PhosphorImager screen to visualize the radiolabeled AE1.

SITS-Affi-Gel binding assay. Transfected cells were lysed in PBS containing 1% C12E8 and protease inhibitors (PMSF, leupeptin, pepstatin A, and E64) at 4°C. The lysates were centrifuged (16,000 g) to remove insoluble material. Immunoblotting showed that all of the AE1 was solubilized and that the pellet was devoid of AE1. SITS was conjugated to Affi-Gel 102 (Bio-Rad, Hercules, CA) as described (27). SITS-Affi-Gel (25 µl) and lysate (100 µl) were added to binding buffer (1% C12E8 and 228 mM sodium citrate buffer, pH 7.1, final concentration) with and without 1 mM free anion transport inhibitor H2DIDS (Sigma-Aldrich). The mixture was incubated at 4°C for 15 min. Resin was collected by centrifugation (8,000 g) for 5 s and washed three times with 0.1% C12E8 and 228 mM sodium citrate buffer, pH 7.1. SDS gel electrophoresis sample buffer (4% SDS) was added to the SITS-Affi-Gel to elute bound proteins, and the total and bound fractions were analyzed for AE1 content by Western blotting.

Transport assays. The chloride/bicarbonate exchange activities of AE1, kAE1, AE1 R589H, and kAE1 R589H were determined in transfected HEK cells by monitoring change in intracellular pH as described previously (37).

Purification of His-tagged AE1 and heterodimer formation. To determine whether wild-type AE1 can form heterodimers with mutant AE1 proteins, a His-tagged version of AE1 was constructed. This protein and any associated proteins can be purified from transfected cells by Ni affinity chromatography. Cells were either transfected with AE1 His6 alone, AE1 His6 with AE1, AE1 R589H, kAE1, kAE1 R589H, or kAE1 alone. Transfected cells were lysed in PBS (pH 7.4) containing 1% C12E8, 5 mM imidazole, and protease inhibitors at 0°C. A portion (300 µl) of lysate was incubated with 40 µl of ProBond Ni beads (Invitrogen) for 1 h at 4°C. Beads were washed 3 times with 0.5 ml of PBS, 7.4, 0.1% C12E8, and 40 mM imidazole. Bound proteins were eluted with the same buffer containing 500 mM imidazole. AE1 in the elutant was detected by Western blotting (see Western blotting).


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Immunolocalization of AE1 mutants in transfected HEK-293 cells. Immunofluorescence was used to examine the subcellular distribution of AE1 in transfected HEK cells. Cells transfected with AE1, AE1 R589H, and kAE1 showed bright AE1 staining at the periphery of the cells that did not colocalize with the ER marker calnexin (Fig. 1). This staining represents cell surface expression of AE1, AE1 R589H, and kAE1. There was some colocalization of AE1, AE1 R589H, and kAE1 with calnexin, representing an ER pool of the proteins. In contrast, cells transfected with kAE1 R589H did not show peripheral AE1 staining; rather, they showed predominantly internal staining that partially colocalized with calnexin. These data suggest that kAE1 R589H did not have a high level of cell surface expression and that some of the protein was localized to the ER. Untransfected cells or cells incubated without the primary antibody did not show AE1 staining (data not shown).


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Fig. 1.   Immunolocalization of wild-type and R589H mutant anion exchanger (AE)1 and kAE1 in transfected human embryonic kidney (HEK) cells. HEK cells transfected with wild-type AE1, AE1 R589H, kAE1, or kAE1 R589H were fixed, permeabilized, and incubated with anti-AE1 antibody and anti-calnexin antibody. Rhodamine-conjugated secondary antibody was used against the anti-AE1 antibody, and fluorescein isothiocyante (FITC)-conjugated secondary antibody was used against the anti-calnexin antibody. Left: confocal images visualizing rhodamine from anti-AE1. Middle: confocal images visualizing FITC from the anti-calnexin antibody. Right: merged images.

Cell surface biotinyation of AE1 mutants. To confirm the immunolocalization results and quantitate the level of cell surface expression of AE1, cell surface biotinylation of transfected cells was performed by using a membrane-impermeant biotinylation reagent. Biotinylated (cell surface) proteins in the total cell extract were separated from nonbiotinylated (intracellular) proteins with streptavidin-conjugated beads. The biotinylated proteins were released from the beads with sample buffer containing beta -mercaptoethanol to cleave the disulfide within the biotinylating reagent. Samples of the total cell lysate (Fig. 2, lane T), supernatant from the beads (Fig. 2, lane S), and the beads' eluant (Fig. 2, lane B) were analyzed for AE1 content by Western blotting. AE1 expressed in transfected cells appears as a single 100-kDa band containing a high-mannose oligosaccharide (28), whereas kAE1 appeared as a single 95-kDa band (Fig. 2). To calculate the percentage of cell surface expression, the difference in pixel density between the AE1 bands in the supernatant (internal proteins) and the total (total protein) fractions was determined. The amount of AE1 in the beads' eluant fraction was not used to calculate the cell surface expression because biotinylated AE1 is poorly eluted from the streptavidin beads (22, 30, 37).


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Fig. 2.   Cell surface biotinylation of erythroid and kidney wild-type and R589H mutant AE1 in transfected HEK cells. Immunoblots show the total amount of AE1 in the cell lysate (lane T), the relative amounts of unbiotinylated AE1 not bound to streptavidin beads (lane S), and biotinylated AE1 eluted from the streptavidin beads (lane B). The fraction bound to the beads was overloaded 5 times with respect to the total and supernatant to get comparable band densities within the linear range of chemiluminescence exposure. Location of the 98-kDa molecular mass marker (phosphorylase b) is indicated.

Western blots showed that AE1, AE1 R589H, and kAE1 could be detected in the biotinylated fraction (Fig. 2). Only a very faint band of kAE1 R589H could be detected in the biotinylated fraction. By using the difference in the amount of AE1 in the supernatant (lane S) and total (lane T) fractions, the percentage of cell surface expression was calculated to be 34 ± 6% AE1, 32 ± 7% AE1 R589H, 41 ± 7% kAE1, and 12 ± 5% kAE1 R589H (n = 3 ± SD). Controls with transfected cells that were not biotinylated showed no AE1 in the biotinylated fraction and only a minor decrease (2 ± 5%) in the supernatant fraction compared with the total, indicating that there was only a small amount of nonspecific binding to the streptavidin beads (data not shown). As an internal control, Western blotting with an anti-actin antibody showed that no actin was biotinylated (data not shown). The biotinylation data show that kAE1 R589H had approximately threefold lower cell surface expression compared with AE1, AE1 R589H, and kAE1.

Oligosaccharide processing of AE1 mutants. AE1 in transfected HEK-293 or COS-7 cells contains only a high-mannose oligosaccharide at the endogenous glycosylation acceptor site N642, even though AE1 trafficks to the plasma membrane (22, 28). Moving the acceptor site to position 555 (AE1 N555) on the preceding extracellular loop enables the oligosaccharide to be processed from a high-mannose to a complex structure (22). Trafficking of AE1 N555 from its site of synthesis in the ER to the plasma membrane can be monitored by following the conversion of the N-linked oligosaccharide from a high-mannose to a complex structure.

Figure 3 is a Western blot of lysates from transfected cells. The lysates were treated with no enzyme (lane C), endo H (lane H), or PNGase F (lane F). Endo H can remove a high-mannose oligosaccharide, whereas PNGase F can remove all N-linked oligosaccharides. Removal of the oligosaccharide will cause a shift in the protein to a lower molecular weight on SDS gels. AE1 N555-transfected cells had two immunoreactive bands, as previously described (22). The bottom band was endo H sensitive, indicating it contained a high-mannose oligosaccharide, whereas the top band could only be shifted by PNGase F, indicating it contained a complex oligosaccharide (Fig. 3). The top complex band has been previously shown to be at the cell surface (22). The percentage of complex (cell surface) AE1 N555 was 36 ± 1%, which is similar to the value obtained above (see Cell surface biotinylation) by cell surface biotinylation of AE1 with the endogenous glycosylation site and previous measurements of cell surface expression of AE1 (30, 37).


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Fig. 3.   Enzymatic deglycosylation of erythroid and kidney wild-type and R589H mutant AE1 N555. Transfected cells were lysed in 1% C12E8 and incubated with no enzyme (lane C), endoglycosidase H (endo H; lane H), or peptide N-glycosidase F (lane F). Western blotting was used to detect AE1. , Position of the complex form; open circle , position of the high-mannose form.

kAE1 N555 could also be resolved into two bands (Fig. 3). The percentage of complex kAE1 N555 was 31 ± 8%, indicating that the oligosaccharide can be processed and the protein was present at the plasma membrane at a level similar to AE1 N555. Again, the percentage of complex agrees with the results in kAE1 with the endogenous glycosylation site (see Cell surface biotinylation). Thus the amount of kAE1 at the plasma membrane in transfected HEK cells is similar to AE1.

The AE1 N555/R589H mutant did not have a prominent top complex band that could be readily resolved from the bottom high-mannose band. After endo H digestion, however, it was seen that a small portion was resistant to digestion, indicating the presence of some complex oligosaccharide. In some Western blots, this blurry top band was visible without endo H digestion (see Fig. 5). Although the mobility of the high-mannose form of AE1 N555/R589H was similar to the high-mannose form of AE1 N555, the top band had a different mobility. This indicates that oligosaccharide processing may differ in this mutant. This top band was sensitive to PNGase F and accounted for 8 ± 2% of total AE1 N555/R589H. This level of the complex form is less than expected compared with the cell surface biotinylation results that indicated 32% of AE1 R589H at the plasma membrane. Because the shift on the gel is smaller than that for AE1 N555, the high-mannose and complex bands overlapped. The poor resolution of the two bands caused difficulties in accurately calculating the percentage of the complex form. This may account for the difference between the oligosaccharide processing and biotinylation results.

The kAE1 N555/R589H showed a single band on Western blots (Fig. 3) that comigrated with the high-mannose form of kAE1 N555. This band could be shifted with endo H and PNGase F, indicating it contained a high-mannose oligosaccharide. After endo H digestion, there was no detectable complex form, which suggests a lack of cell surface expression. The biotinylation results indicated a low level (12%) of kAE1 R589H at the cell surface, which may be too low to be detected by glycosylation processing. It is also possible that if a small amount of kAE1 N555/R589H is converted to the complex form, the extent of processing may be decreased, as seen with AE1 N555/R589H. Again, the reduced processing could give an underestimation of the amount of the complex form.

Pulse-chase analysis of AE1 mutant trafficking. The rate of trafficking of AE1 from the ER to the plasma membrane was monitored by observing the conversion of the N555 constructs from a high-mannose to a complex form in a pulse-chase experiment (22). At the initial time point of the chase (t = 0 h), AE1 N555 is seen as a single endo H-sensitive band, representing newly synthesized protein (Fig. 4A). As the chase progressed, an upper endo H-insensitive band appears while there was a corresponding decrease in the endo H-sensitive band. This represents the conversion of the AE1 N555 high-mannose form to the complex form as it trafficked through the Golgi apparatus and was modified by mannosidase II and subsequent oligosaccharide-processing enzymes. Densitometric analysis was used to quantitate the amount of complex AE1 at each time point. Figure 4B shows a plot of the percentage of complex AE1 as a function of time. At the 10-h chase point, ~50% of the remaining labeled AE1 N555 was in the complex form. Very similar results were seen with kAE1 N555, indicating a similar trafficking pattern.


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Fig. 4.   Pulse-chase analysis of erythroid and kidney wild-type and R589H mutant AE1 N555. A: transfected HEK cells were pulsed with 200 µCi/ml of [35S]methionine for 20 min. Cells were collected at 0, 1, 2, 5, and 10 h of the chase and AE1 was immunoprecipitated. Samples were treated with endo H to improve the separation of complex and high-mannose forms. Radiolabeled AE1 was visualized by SDS-PAGE and autoradiography. , Position of the endo H-insensitive (complex) form; open circle , position of the endo H-sensitive (high-mannose) form. B: percentage of complex form relative to total (complex + high-mannose) was calculated at each time point by densitometric analysis of the autoradiography film. Percentage of complex was expressed as a function of chase time: AE1 N555 (), AE1 N555/R589H (diamond ), kAE1 N555 (open circle ), and kAE1 N555/R589H (triangle ).

The AE1 N555/R589H also began as an endo H-sensitive band and, as the chase continued, was converted to an endo H-insensitive band at a similar rate as AE1 N555 and kAE1 N555. As seen with the Western blots, the extent of glycosylation processing was decreased with AE1 N555/R589H, which is indicated by a smaller shift on the gel. However, the complex form was more easily resolved on the pulse-chase autoradiograph than on Western blots. In contrast, only a small fraction of kAE1 N555/R589H was converted to a complex form. This complex form could be seen as a fuzzy endo H-insensitive band. The pulse-chase results again suggest that the R589H mutation caused a trafficking defect in kAE1. The smaller molecular weight shift seen with both erythroid and kAE1 R589H mutants suggests that the mutation may have caused altered oligosaccharide processing.

Previously, wild-type AE1 was shown to have a half-life of 15 h in transfected HEK cells (30) and COS-7 cells (22). At the 10-h time point, the amount of total (high-mannose+complex) radiolabeled AE1 or kAE1 immunoprecipitated was similar (>50% compared with to the 0-h time point) for wild-type and mutant proteins, indicating similar half-lives. This suggests that the R589H mutation did not cause AE1 or kAE1 to be targeted for rapid degradation.

Anion transport and inhibitor binding of AE1 mutants. Anion transport assays carried out in transfected HEK cells showed that AE1 and kAE1 had similar chloride/bicarbonate transport rates. The AE1 R589H mutant also exhibited a high (75% of wild-type AE1, n = 3) transport rate, which is not statistically different from wild-type. However, no transport activity could be detected for the kAE1 R589H mutant in transfected HEK cells, which is consistent with a lack of cell surface expression. The transport assays carried out in intact cells do not provide information on the functional state of the protein retained within the cell. ER retention is often caused by misfolding or incomplete folding of mutant proteins (20). The ability of the mutant AE1 to bind to an inhibitor affinity resin was used to test this possibility. The SITS-Affi-Gel has been shown to specifically bind to properly folded AE1 (27) and provides an assay for the integrity of the membrane domain of AE1. Cell lysates prepared with a nondenaturing detergent, C12E8, were incubated with SITS-Affi-Gel resin in the absence and presence of free inhibitor (1 mM H2DIDS). The total cell lysate and the portions bound to the SITS-Affi-Gel in the absence or in the presence of free inhibitor were analyzed by Western blotting to detect AE1 (Fig. 5A; total lysate, lane T; fraction bound, lane B; fraction bound in presence of H2DIDS, lane D). The pixel density of the AE1 bands was determined, and the amount bound in the absence and presence of free inhibitor was expressed as a percentage of the total amount of AE1 (Fig. 5B).


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Fig. 5.   SITS-Affi-Gel binding of erythroid and kidney wild-type and R589H mutant AE1 N555 and Southeast Asian ovalocytosis (SAO) AE1. A: immunoblot of the total AE1 N555 produced in transfected cells (lane T), the amount of AE1 N555 bound to the SITS-Affi-Gel resin in the absence (lane B) or in the presence of 1 mM H2DIDS (lane D). B: the relative amounts of AE1 N555 from transfected HEK cells that bound to the SITS-Affi-Gel resin in the absence (dark gray bars) or in the presence (light gray bars) of 1 mM H2DIDS. SAO AE1 is unable to bind inhibitors and is used as a nonbinding negative control. Error bars, SD (n = 3).

Under the conditions used, ~80% of AE1 N555 could bind the affinity resin. Both high-mannose and complex forms of the protein could bind to the resin. This indicates that the ER and plasma membrane forms of AE1 had a native structure. This binding could be completely blocked by the presence of free inhibitor, indicating that the binding was specific. SAO AE1 is an AE1 variant that has a nine-amino acid deletion in the first transmembrane segment and has been previously characterized to be a nonfunctional anion transporter and unable to bind to stilbene disulfonate inhibitors (25, 35, 36). As a negative control, SAO AE1 bound poorly to the inhibitor affinity resin.

AE1 N555/R589H, kAE1 N555, and kAE1 N555/R589H all bound to the SITS-Affi-Gel in a manner similar to AE1 N555. This binding was blockable by a free inhibitor showing specificity. Similar binding was observed for AE1, AE1 R589H, kAE1, and kAE1 R589H containing the endogenous N-glycosylation site (data not shown), indicating that the location of the oligosaccharide did not affect binding to the affinity resin. A small amount of kAE1 N555/R589H bound to the resin in the presence of 1 mM H2DIDS. This residual binding was sometimes observed with wild-type AE1 (30) and likely represents nonspecific binding. The ability of dRTA mutant AE1 to bind to the inhibitor affinity resin suggests that the membrane domain of AE1 is correctly folded and able to bind classic inhibitors of anion exchange.

Coexpression of mutant and wild-type AE1. Because the R589H mutation causes autosomal dominant dRTA, we examined the possibility of a dominant-negative effect of the mutant AE1 on the trafficking of wild-type AE1. This was studied first by coexpressing of kAE1 R589H with AE1 N555 and monitoring the amount of AE1 N555 complex form. Because these two proteins can be resolved on Western blots, we could quantitate the amount of each expressed. Figure 6A shows an immunoblot of HEK cell lysate 2 days after transfection probed with an anti-AE1 COOH-terminal antibody. Figure 6B is a plot of the percentage of AE1 N555 complex form (relative to total AE1 N555 expression) as a function of relative kAE1 wild-type or mutant expression (kAE1/total AE1 N555 expression). Lane 1 contains AE1 wild-type transfected cells to show the position of the AE1 high-mannose form. Lane 2 contains AE1 N555 transfected cells and two bands representing the high-mannose (internal) and complex (cell surface) forms. Lanes 6-8 contain lysates of cells cotransfected with a constant amount of AE1 N555 cDNA and increasing amounts of kAE1 R589H cDNA. kAE1 R589H is seen as the bottom band, AE1 N555 high-mannose form as the middle band, and AE1 N555 complex form as the top band. With increasing amounts of kAE1 R589H cDNA, an increase in kAE1 R589H expression is observed. With the increase in kAE1 R589H, there is a decrease in the amount of AE1 N555 complex form, indicating reduced cell surface expression.


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Fig. 6.   Coexpression of AE1 N555 and kAE1 N555 with kAE1 R589H. A: immunoblot of cells transfected with AE1 alone (lane 1; 0.4 µg cDNA/well); AE1 N555 alone (lane 2, 0.4 µg cDNA/well); AE1 N555 (0.4 µg cDNA/well) with increasing amounts of kAE1 R589H (lanes 3-5; 0.02, 0.08, and 0.2 µg cDNA/well, respectively); or AE1 N555 (0.4 µg cDNA/well) with increasing amounts of kAE1 (lanes 6-8; 0.02, 0.08, and 0.2 µg cDNA/well, respectively). Transfected cells were lysed with 1% C12E8, and AE1 was detected by Western blotting. , Position of the complex form; open circle , position of the high-mannose form; arrow, position of kAE1. B: percentage of complex AE1 N555 as a function of relative kAE1 () or kAE1 R589H (open circle ) expression. C: immunoblot showing the effect of coexpression of kAE1 R589H on cell surface biotinylation of kAE1 N555. Cells were transfected with kAE1 N555 alone (lanes 1 and 4; 0.4 µg cDNA/well), kAE1 N555 and kAE1 (lanes 2 and 5; 0.4 and 0.2 µg cDNA/well, respectively), or kAE1 N555 and kAE1 R589H (lanes 3 and 5; 0.4 and 0.2 µg cDNA/well, respectively). Lanes 1-3, total expression of kAE1; lanes 4-6, kAE1 that eluted from the streptavidin beads.

As a control, coexpression was also performed with wild-type kAE1 to rule out the possibility that overexpression of the two proteins inhibited AE1 N555 cell surface expression by flooding the required cellular translocation and trafficking machinery. Lanes 3-5 (Fig. 6A) contain lysates of cells transfected with a constant amount of AE1 N555 and increasing amounts of kAE1. Again, there is an increase in the amount of kAE1 expression with increased kAE1 cDNA. However, there is only a small decrease in the amount of AE1 N555 complex form (Fig. 6B), indicating that the dominant-negative effect was specific for the kAE1 mutant.

We next examined the effect of coexpression of kAE1 R589H on the trafficking of kAE1 N555 to better mimic the situation in the kidneys of patients with dominant dRTA. In this experiment, the high-mannose forms of kAE1 and kAE1 R589H comigrate. As seen in Fig. 6C (left), expression of kAE1 N555 alone produced two bands, a bottom high-mannose form and a top complex form. Coexpression of kAE1 with kAE1 N555 did not decrease the amount of complex form produced. In contrast, coexpression of kAE1 R589H with kAE1 N555 blocked production of the top complex form of the protein. Thus coexpressing the kAE1 R589H mutant resulted in ER retention of kAE1.

To confirm the loss of cell surface expression of kAE1 N555 by the kAE1 R589H mutant, cell surface biotinylation experiments were carried out. The gel in Fig. 6C (right) shows the biotinylated kAE1 proteins expressed at the cell surface. Biotinylation of cells expressing the kAE1 N555 alone resulted in biotinylation of the top band (Fig. 6C, lane 4). This is consistent with the observation that this protein contains a complex oligosaccharide and has therefore exited the ER by trafficking through the Golgi to the plasma membrane. Coexpression of kAE1 with kAE1 N555 resulted in a similar level of cell surface biotinylation of the top band of kAE1 N555 (Fig. 6C, lane 5). This shows that the presence of kAE1 does not impair the trafficking of kAE1 N555. This sample also shows a bottom biotinylated band, which corresponds to kAE1 that has trafficked to the cell surface without conversion to a complex oligosaccharide. No biotinylated kAE1 could be detected in cells coexpressing kAE1 N555 and kAE1 R589H (Fig. 6C, lane 6). This result confirms the processing results and shows that the kAE1 R589H mutant inhibits the appearance of kAE1 at the plasma membrane.

AE1 exists predominantly as a dimer in erythrocytes (7) and in transfected HEK cells (6, 30). The ability of the kAE1 R589H mutant to retain kAE1 and AE1 within the cell may be due to heterodimer formation. To directly test for heterodimer formation, a copurification experiment using a COOH-terminal His6-tagged version of AE1 was employed. AE1 His6 was expressed alone or in combination with AE1, AE1 R589H, kAE1, or kAE1 R589H. Figure 7 shows duplicate immunoblots probed with an antibody against the COOH terminus of AE1 (A) or an antibody against the NH2 terminus of the erythroid form of AE1 (B). AE1 His6 could be detected with the NH2-terminal antibody (Fig. 7, lane 1) but could not be detected by using the COOH-terminal antibody because of the presence of the His6 tag (Fig. 7, lane 7). kAE1 could be detected by the COOH-terminal antibody (Fig. 7B) but not by the NH2-terminal antibody, because it is missing the NH2-terminal 65 residues found in AE1 (Fig. 7A).


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Fig. 7.   Copurification of R589H mutant AE1 and kAE1 with His-tagged AE1. Immunoblots showing the AE1 proteins that bound to the Ni-affinity resin. Extracts were prepared from cells transfected (0.4 µg cDNA each/well) with AE1-His (lanes 1 and 7), AE1-His and AE1 (lanes 2 and 8), AE1-His and AE1 R589H (lanes 3 and 9), AE1-His and kAE1 (lanes 4 and 10), AE1-His and kAE1 R589H (lanes 5 and 11), or kAE1 (lanes 6 and 12). A: immunoblot probed with an antiserum against the NH2 terminus of AE1 (anti-AE1 nt), which detects AE1 and AE1 His6 but not kAE1. B: immunoblot probed with an antiserum against the COOH terminus of AE1 (anti-AE1 ct), which detects AE1 and kAE1 but not AE1 His6.

AE1 His6 expressed in transfected HEK cells could be purified by using a Ni-affinity resin and detected by the NH2-terminal antibody (Fig. 7, lane 1). AE1 without the His6 tag did not bind to the Ni resin (data not shown). Coexpression of AE1 with AE1 His6 showed heterodimer formation, because the Ni resin copurified AE1, as revealed by the COOH-terminal antibody (Fig. 7, lane 8). A heterodimer was also observed for AE1 His6 and AE1 R589H (Fig. 7, lanes 3 and 9), showing that the mutant can dimerize with wild-type AE1. AE1 His6 can also form a heterodimer with kAE1 (Fig. 7, lanes and 10) and with kAE1 R589H (Fig. 7, lanes 5 and 11). kAE1 expressed alone did not bind to the Ni-affinity resin (Fig. 7, lane 12). The intracellular retention of the kAE1 R589H mutant and its ability to form heterodimers with wild-type AE1 accounts for the retention of the wild-type protein within the cell.

Expression of other R589 mutants. Two other mutations at Arg589 (R589C and R589S) have also been associated with autosomal dominant dRTA (4, 18). Therefore, we constructed these two mutations in kAE1 N555 to see whether they had a similar effect on oligosaccharide processing of kAE1. Cell lysates were incubated with no enzyme (lane C), endo H (lane H), or PNGase F (lane F), and samples were then analyzed by Western blotting to detect kAE1 (Fig. 7). As seen before, kAE1 N555 cell lysate had two immunoreactive bands. The top band was endo H insensitive and PNGase F sensitive, whereas the bottom band was sensitive to both endo H and PNGase F. Cells transfected with both R589C and R589S mutants show only a single immunoreactive band that was sensitive to both endo H and PNGase F, which is identical to results seen for the R589H mutant (Fig. 8). Lack of a complex oligosaccharide indicates that the other dRTA mutations at R589 also caused impaired trafficking of kAE1 N555.


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Fig. 8.   Oligosaccharide processing of kAE1 N555 mutants at Arg589. Transfected cells were lysed in 1% C12E8 and incubated with no enzyme (lane C), endo H (lane H), and PNGase F (lane F). Western blotting was used to detect AE1. , Position of the complex form; open circle , position of the high-mannose form.

The known dRTA mutations (R589H, R589C, R589S) result in the partial (R589H) or complete loss (R589C and R589S) of a positive charge at this position. Two novel mutations were also made at position 589 (R589K and R589A) to see whether other mutations could be tolerated. The Lys mutant would retain a positive charge at position 589, whereas the Ala mutant would not. Both of these mutants also showed a single endo H-sensitive band on Western blots (Fig. 8). All R589 mutants were able to specifically bind to SITS-Affi-Gel (data not shown), indicating that these mutations did not greatly alter the folding of the protein. All mutations we examined at position R589 decreased the level of complex oligosaccharide in kAE1 N555, showing that R589 is an important residue for proper trafficking of AE1, which suggests other mutations at this position may also cause dRTA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The R589H mutation caused a severely reduced level of cell surface expression of kAE1 at the plasma membrane of transfected HEK-293 cells. This was shown by immunolocalization, cell surface biotinylation, oligosaccharide processing, and transport assays. The immunolocalization and pulse chase data suggest that the reduced cell surface expression was likely due to retention of kAE1 in the ER. A small amount of kAE1 R589H could be labeled at the plasma membrane by the biotinylating reagent, and there was some conversion of the kAE1 N555/R589H to a complex form, indicating that the mutant was not completely retained within the ER. The portion that did reach the cell surface may be unstable, because no complex kAE1 N555/R589H could be detected by Western blotting. However, the pulse-chase experiment showed that the majority of mutant kAE1 was not rapidly degraded (half-life >10 h). Interestingly, the R589H mutation did not cause a profound decrease in cell surface expression of erythroid AE1. Immunolocalization, cell surface biotinylation, and transport assays showed plasma membrane expression levels similar to those for wild-type AE1. The carbohydrate processing suggested that there was some reduction in cell surface expression, although this may reflect a lack of sensitivity of the assay. X. laevis oocytes expressing recombinant kAE1 R589H showed a 20-50% reduction of chloride transport, whereas introduction of the R589H mutation into erythroid AE1 had no effect on transport (15). These results are in agreement with our findings that the R589H mutation introduced into kAE1 has a more profound effect than when introduced into erythroid AE1. However, in an earlier study also using X. laevis oocytes, the erythroid form of the R589H mutant exhibited impaired (40% of wild-type) chloride transport activity, whereas the kidney form did not show any statistical difference from wild-type kAE1 (4).

The R589H mutation caused an increase in the mobility of the complex form of both AE1 N555 and kAE1 N555 relative to wild-type AE1 and kAE1 as detected by SDS-gel electrophoresis. This suggests that the R589H mutation resulted in a change in oligosaccharide processing. Perhaps the conformation of the third extracellular loop was changed and made less accessible to oligosaccharide-processing enzymes. The possibility that the mutations caused the protein to traffic faster and therefore have less time for modification is unlikely because the rate of processing of AE1 N555/R589H was similar to that for AE1 N555 or kAE1 N555 (Fig. 5). Altered glycosylation has previously been noted on red cell AE1 from dRTA patients with R589H and R589C mutations (4). The carbohydrate on the endogenous glycosylation acceptor site (N642) located on the fourth extracellular loop was longer than normal, suggesting prolonged processing and perhaps a reduced rate of AE1 trafficking. Interestingly, Ghosh et al. (13) have shown that AE1 in chicken erythroblasts initially moves from the ER to the plasma membrane in the high-mannose form. The protein is recycled to the Golgi, where its oligosaccharide is converted to the complex form. Differences in the oligosaccharide in the human AE1 dRTA mutants may be due to changes in this recycling pathway.

The coexpression of kAE1 R589H with AE1 N555 or kAE1 N555 caused a reduction in the level of complex AE1 N555 and kAE1 N555, indicating a dominant-negative trafficking effect. Because AE1 is a dimer, heterodimerization of wild-type and mutant AE1 is a possible cause of the dominant-negative effect. That is, the mutant AE1 may retain its wild-type dimer partner in the ER. Copurification experiments using His6-tagged AE1 showed that the mutant AE1 R589H protein can indeed form a heterodimer with the normal protein. The inhibition of wild-type kAE1 trafficking would account for the autosomal dominant form of inheritance of dRTA associated with this mutation. Previously, coexpression of AE1 and AE1 R589H or kAE1 and kAE1 R589H in X. laevis oocytes did not cause a dominant-negative effect on chloride transport (4, 15). The differences observed between HEK cells and oocytes may be due to the different types of cells used or the different assays (biosynthesis vs. transport) used. HEK cells are a transformed human cell line that provides a homologous cellular expression system as opposed to the heterologous X. laevis oocyte.

The ability of the R589 mutants to bind to the inhibitor affinity resin suggests that the proteins were correctly folded. The erythroid AE1 R589H mutant had near-normal transport activity in transfected cells. Studies in X. laevis oocytes showed that AE1 R589H could transport radiolabeled chloride (4, 15). Also, red blood cells from dRTA patients with the R589H mutation showed only slightly reduced sulfate influx (76-79% of normal) and normal sensitivity to DIDS inhibition.

There are two other mutations at Arg589 (R589C and R589S) that have also been associated with dRTA (4). In transfected HEK cells, the R589C and R589S mutations resulted in the absence of complex oligosaccharide on kAE1 N555, indicating they also cause defective trafficking of the protein. Two novel mutations that were created in kAE1 N555 (R589K and R589A) also resulted in a lack of complex oligosaccharide on kAE1 N555. Arg589 appears to be essential for the proper trafficking of kAE1, because even substitution of another basic residue (Lys) did not permit proper trafficking.

Basic residues have been shown to be important in trafficking of membrane proteins. KDEL (26), KKXX-COOH, and RXR (8, 45) motifs have been shown to cause ER retention of luminal and membrane proteins. The third intracellular loop of AE1 contains six basic residues (R589, K590, K592, K600, R602, and R603) and may create a motif that is required for proper trafficking. In fact, all six basic residues are highly conserved in known AE1, AE2, and AE3 sequences.

Somehow, the lack of the first 65 amino acids prevented efficient cell surface accumulation of the kidney mutant. The crystal structure of the cytoplasmic NH2-terminal domain of erythroid AE1 (46) provides some insight into possible reasons. The highly acidic NH2 terminus (residues 1-54) was not visible in the crystal structure. A central beta -strand (residues 57-66) of the structure of the erythroid cytoplasmic domain would not be present in the truncated kidney form. The absence of this strand would greatly influence the overall structure and function of the cytosolic domain. Unlike the erythroid NH2-terminal domain, the kidney NH2-terminal domain does not bind to ankyrin, band 4.1, or aldolase (10, 43). Different protein-protein interactions may be required for trafficking of the two forms of AE1. For example, kanadaptin binds the NH2-terminal domain of kAE1, but not erythroid AE1, and colocalizes with an intracellular pool of kAE1 in kidney intercalated cells (9). Another possibility is that the acidic NH2-terminal region of AE1 interacts with the basic loop containing R589 and this interaction facilitates the proper folding and trafficking of erythroid AE1.

The trafficking impairment caused by the R589H mutation in kAE1 but not in erythroid AE1 provides an explanation for the kidney-specific dysfunction seen in dRTA. Erythroid dRTA AE1 is likely able to traffic to the plasma membrane in erythrocyte precursors, so there is no AE1 deficiency in red cells. kAE1 is likely retained in the ER in acid-secreting intercalated cells of the kidney. The dominant-negative effect would account for the autosomal dominant inheritance pattern. With little or no kAE1 at the basolateral membrane, bicarbonate could not be effectively removed from the cell, reducing the production of bicarbonate and protons by carbonic anhydrase. The reduced production of protons would result in less acidification of the urine by the action of apical proton pumps.


    ACKNOWLEDGEMENTS

We thank Drs. Rongmin Zhao for assistance with the confocal microscopy, David Williams for generously supplying the calnexin antibody, and Michael Jennings for generously supplying the mouse monoclonal AE1 antibody. The transport assays were performed by Deborah Sterling in the laboratory of Dr. Joseph Casey, University of Alberta.


    FOOTNOTES

This work was supported by grants from the Kidney Foundation of Canada and the Canadian Institutes of Health Research. J. A. Quilty is supported by a University of Toronto Open Scholarship.

Address for reprint requests and other correspondence: R. A. F. Reithmeier, CIHR Group in Membrane Biology, Dept. of Medicine, Rm. 7344, Medical Sciences Bldg., Univ. of Toronto, Toronto, Ontario, Canada M5S 1A8 (E-mail: r.reithmeier{at}utoronto.ca).

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 December 18, 2001;10.1152/ajprenal.00216.2001

Received 9 September 2001; accepted in final form 10 December 2001.


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