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 |
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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 () 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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MATERIALS AND METHODS |
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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% -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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RESULTS |
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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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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 -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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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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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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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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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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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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DISCUSSION |
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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 -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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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