CELL SURFACE RESCUE OF KIDNEY ANION EXCHANGER 1 MUTANTS BY DISRUPTION OF CHAPERONE INTERACTIONS

Sian T. Patterson1 and Reinhart A.F. Reithmeier1 From the 1Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8

Running title: Rescuing kAE1 dRTA mutants Address correspondence to: Reinhart A.F. Reithmeier, Department of Biochemistry, MSB5216, 1 King’s College Circle, University of Toronto, Toronto, Ont. Canada, M5S 1A8. Tel: 416-978-7739; Fax: 416-978-8458; email: r.reithmeier@utoronto.ca

Mutations in the human kidney anion exchanger 1 (kAE1) membrane glycoprotein cause impaired urine acidification resulting in distal renal tubular acidosis (dRTA). Dominant and recessive dRTA kAE1 mutants exhibit distinct trafficking defects with retention in the endoplasmic reticulum (ER), Golgi, or mislocalization to the apical membrane in polarized epithelial cells. We examined the interaction of kAE1 with the quality control system responsible for the folding of membrane glycoproteins and the retention and degradation of misfolded mutants. Using small molecule inhibitors to disrupt chaperone interactions, two functional, dominant kAE1 mutants (R589H and R901stop), retained in the ER and targeted to the proteasome for degradation by ubiquitination, were rescued to the basolateral membrane of Madin-Darby canine kidney (MDCK) cells. In contrast, the Golgi-localized, recessive G701D and the severely misfolded, ER-retained dominant Southeast Asian Ovalocytosis (SAO) mutants were not rescued. These results show that functional dRTA mutants are retained in the ER due to their interaction with molecular chaperones, particularly calnexin, and that disruption of these interactions can promote their escape from the ER and cell surface rescue.

Distal renal tubular acidosis (dRTA) is a kidney disease characterized by a defect in urine acidification and metabolic acidosis. Symptoms of dRTA include nephrocalcinosis, hypokalemia, metabolic bone disease (rickets or osteomalacia), deafness, muscle weakness, mental retardation and progressive renal failure (1-3). dRTA arises due to mutations in the genes encoding carbonic anhydrase II, the apical H+-ATPase, or the basolateral kidney anion exchanger 1 (kAE1). Human anion exchanger 1 (AE1, Band 3) is a 911 amino acid glycoprotein that exists as a dimer in

the plasma membrane of red blood cells where it mediates the electro-neutral exchange of chloride for bicarbonate (4). The human kidney isoform, kAE1, lacks the N-terminal 65 amino acids of the erythroid version, is produced from a tissue specific alternative promoter in the third intron of the AE1 (SLC4A1) gene, and also undergoes alternative splicing of the mRNA (5,6). kAE1 is expressed in the basolateral membrane of the acid-secreting (α) intercalated cells of the collecting duct of the kidney where it mediates the exchange of chloride and bicarbonate. The primary cause of dRTA is the inability of kAE1 mutants to traffic efficiently to the cell surface resulting in impaired kAE1 mediated anion transport.

Various autosomal dominant and recessive dRTA mutants of kAE1 have been identified that exhibit a wide variety of clinical severity in the heterozygous, homozygous or compound heterozygous states (7). Dominant dRTA mutants induce either complete or incomplete dRTA and are usually asymptomatic until adulthood. When co-expressed with the WT kAE1 (kWT), the complete dominant mutations (R589H, R901stop) are able to retain the wild type protein intracellularly in the ER by a ‘dominant-negative effect’ (8). Interestingly, these dominant dRTA mutants retain normal transport activity when expressed in Xenopus oocytes (9,10), and the ability to bind the stilbene disulfonate inhibitor (SITS), which binds specifically to properly folded AE1 (11-13), suggesting that they are functional and not grossly misfolded.

Recessive dRTA mutants are also impaired in their trafficking to the cell surface, however they can be rescued to the cell surface in a ‘dominant-positive’ effect by wild type kAE1 (8,14-16). The homozygous state, however, is lethal as the recessive mutants are impaired in their ability to traffic to the cell surface, and in their transport activity (14,17). The presence of recessive dRTA mutations in compound

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heterozygotes with other recessive dRTA mutants also results in severe dRTA due to trafficking defects and lack of function.

Southeast Asian Ovalocytosis (SAO) is a dominantly inherited hematological condition arising from a nine amino acid deletion, Δ400-408 in AE1, resulting in a misfolded and transport-inactive protein (18). The presence of this misfolded protein at the cell surface of red blood cells result in an ovalocytic and rigid shape. However, the presence of sufficient functional wild type AE1 in erythrocytes, or kWT in kidney cells in the heterozygous state, compensates for the lack of kSAO transport activity resulting in asymptomatic anemia or dRTA (15,19). Co-expression of kSAO and dRTA mutants in the compound heterozygote state however, results in severe dRTA due to the enhanced intracellular retention of the dRTA mutant by heterodimer formation with kSAO and the complete lack of transport activity of kSAO (16,19).

The mechanism of intracellular retention of kAE1 mutants has yet to be examined, but may involve interactions with molecular chaperones. Newly-synthesized glycoproteins undergo rounds of binding and release with molecular chaperones, proteins that facilitate folding, suppress aggregation, and mediate the retention and subsequent degradation of misfolded proteins (20,21). Disruption of chaperone interactions may allow for the release of ER–retained membrane glycoproteins and permit their trafficking to the cell surface. We have previously demonstrated that dominant dRTA kAE1 mutants are retained in the ER when expressed in HEK-293 and MDCK cells (14,15). We have also shown that erythroid AE1 is able to interact with calnexin in a glycosylation dependent manner both in vitro, and in HEK and K562 cells (22,23). Chaperones may therefore play a role in the intracellular retention of dRTA mutants and may be therapeutic targets to promote ER exit and trafficking to the cell surface.

In this study, we examined the role of chaperones in the trafficking and retention of kAE1 mutants in Madin-Darby canine kidney (MDCK) cells. Using specific small molecule inhibitors that affect chaperone binding, we have been able to rescue the plasma membrane expression of two dominant ER-retained kAE1 mutants, R589H and R901stop, but not the non-

functional kSAO mutant or the Golgi-retained recessive G701D mutant. The mode of ER retention was glycosylation-dependent, as removal of the single N-glycan found in kAE1 allowed these two mutants to traffic to the cell surface. Quality control in MDCK cells also involves the proteasome-mediated degradation of kAE1 mutants as the proteasomal inhibitor lactacystin slowed their rate of degradation in MDCK cells. Our results demonstrate that functional, dominant dRTA mutants (R598H and R901stop) are retained in the ER due to their interaction with chaperones, notably calnexin, and disruption of this interaction allows the mutants to escape the quality control system and traffic to the cell surface.

EXPERIMENTAL PROCEDURES Antibodies and Reagents-The primary

antibodies used in this study include a polyclonal rabbit anti-CNX directed against the luminal domain (24), a polyclonal rabbit anti-kAE1 directed against the first 15 amino acids located in the N terminus of kAE1, and the commercially available antibodies including anti-hemagglutinin (HA) (MMS-101R), and anti-ubiquitin (MMS-258R) from Covance, Inc., and rat anti-HA (1867423) from Roche. Secondary antibodies include goat anti-mouse Alexa 488 (A-11001) from Molecular Probes, donkey anti-rabbit Cy3 (code711-165-152) and mouse anti-rat Cy5 (code212-176-168) from Jackson ImmunoResearch Laboratories, and horseradish peroxidase-linked anti-mouse IgG antibody (7076) from Cell Signaling. Other reagents include MAL3-101 (MAL3), a kind gift from Dr. Jeff Brodsky, C3 (4-cyclohexyloxy-2-{1-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-yl]-ethyl}-quinazoline, VRT-325) and C4 (N-[2-(5-Chloro-2-methoxy-phenylamino)-4'-methyl-[4,5']bithiazolyl-2'-yl]-benzamide, Verkman Corrector 4a) generously provided by the Cystic Fibrosis Foundation for Therapeutics (CFFT) courtesy of Dr. Robert Bridges, castanospermine (CST, #218775) from Calbiochem, cycloheximide (C-7698) from Sigma, and lactacystin (PI-104) from Enzo Life Sciences.

kAE1 Expression in MDCK cells-MDCK cells expressing HA-tagged kAE1 were obtained using a retroviral system as previously described (14). The external HA epitope is found after Val-557 in the 3rd extracellular loop of kAE1 (12).

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Immunofluorescence and confocal microscopy confirmed the cell surface expression and intracellular localization of the wild type kAE1 and dRTA mutants. These cells continually lose expression of kAE1 and are therefore freshly infected for the experiments described in this paper. The endogenous N-glycosylation side at Asn-642 was mutated to Asp (N642D) in the pFBNeo constructs of kAE1 as previously described (25). Polarized MDCK cells expressing the various kAE1 constructs were grown on Transwell polycarbonate filters (Corning) for 6 days.

Drug treatment-MDCK cells were seeded in 6-well dishes for co-immunoprecipitations, on glass coverslips for non-polarized cells, or on Transwell polycarbonate filters for polarized cells, and treated with 1mM CST, 30µM MAL3, 10µM C3 or C4 overnight (~18h) at 37°C.

Co-immunoprecipitation and immunoblotting-Infected MDCK cells were lysed in PBS containing 1% digitonin detergent and proteasome inhibitors (1 µM leupeptin, aprotinin, pepstatin A and 200 µM PMSF) for 30 min. on ice. Cell lysates were centrifuged (16,000g, 10min) and the supernatant was collected. Co-immunoprecipitation was then performed using either anti-kAE1 Nt or anti-CNX antibodies, and immunoblotting using anti-HA antibodies identified co-immunoprecipitated AE1. For immunoblotting of whole cell lysate, cells were lysed directly in 2 X Sample Buffer, and loaded directly onto 7.5% SDS-PAGE gels, followed by immunoblotting using an anti-HA antibody for protein expression.

Immunofluorescence and Microscopy- Immunofluorescence staining and confocal microscopy of MDCK cells stably expressing kAE1 was performed as previously described (15). Imaging equipment includes a laser scanning confocal Zeiss LSM 510 microscope, Zeiss AxioCam, AxioVision, and LSM image browser. Flow cytometry analysis-MDCK cells stably expressing kAE1 were trypsinized and incubated with mouse anti-HA antibodies for flow cytometry analysis as previously described (14). A secondary anti-mouse Alexa 488 was used to detect cell surface kAE1 by flow cytometry using a FACSCalibur.

Cell surface biotinylation-Cell surface biotinylation of MDCK cells stably expressing kAE1 was performed as previously described (26). The presence of kAE1 in the total, unbound and bound fractions was detected by immunoblotting with anti-HA antibody.

Proteasomal degradation assays-Infected MDCK cells were grown in 6-well culture dishes and pre-treated overnight with 10 µM lactacystin. For the ubiquitination of kAE1, kAE1 expressing MDCK cells were lysed in 1 X RIPA buffer (1% deoxycholate, 1% Triton X-100, 0.1% SDS, 0.15 M NaCl, 10 mM Tris/HCl, pH 7.5, and 1 mM EDTA). Cell lysates were centrifuged (16,000g, 10min) and the supernatant was collected. Co-immunoprecipitation was performed as above and immunoblotting confirmed immunoprecipitated kAE1 or ubiquitination using an anti-ubiquitin (Ub) antibody. For cycloheximide chase experiments, cells were first pre-treated with lactacystin for 1 hour, treated with cycloheximide and then lysed at various times as indicated in 2 X SDS sample buffer, resolved on 7.5% SDS-PAGE gels, and immunoblotted using an anti-HA antibody.

RESULTS Expression of kAE1 in MDCK cells-In

order to examine the role chaperones play in the intracellular retention of kAE1 mutants, WT kAE1, kSAO and three different kAE1 dRTA mutants (R589H, G701D, R901stop) were expressed in MDCK cells. The wild-type kAE1 traffics efficiently to the cell surface of MDCK cells (10). Three dominant mutations in kAE1 (kSAO, R589H and R901stop) resulted in retention in the ER in non-polarized MDCK cells, while the recessive G701D is localized to the Golgi (10,14,15,27). These mutants are also retained intracellularly in polarized MDCK cells, while the R901stop mutant can also be seen missorted to the apical membrane (27,28). Previous immunofluorescence studies of the kAE1 mutants, kSAO, R589H, and R901stop, colocalize these mutants to the ER, suggesting that the ER chaperone, calnexin, may play a role in folding of kAE1, a membrane glycoprotein (14,15). Indeed, we have shown that erythroid AE1 is able to interact with calnexin in a glycosylation dependent manner both in vitro and in HEK-293 cells (22,23).

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HA-tagged wild type and kAE1 mutants were expressed in MDCK cells using a MMLV viral infection system, and their intracellular localization was confirmed by immunofluorescence (Fig. 1A). We have previously shown that the HA tag does not effect the trafficking or folding of kAE1 (12,14), while others have shown that it has no affect on transport function (29,30). Wild-type kAE1 was localized to the plasma membrane, while the four mutants all exhibited an intracellular localization as expected. Wild type kAE1 expressed in MDCK cells migrates as multiple bands on SDS gels. The major upper bands (>80%) contains complex oligosaccharide and the minor lower band corresponds to kAE1 with high mannose oligosaccharide (Fig. 1B, total and αAE1 immunoprecipitate), as characterized previously (15). During kAE1 biosynthesis, the core Glc3Man9GlcNAc2 oligosaccharide is transferred en bloc to Asn642 in the lumen of the ER and is then trimmed and processed in the ER and as kAE1 traffics through the Golgi adopting a complex oligosaccharide (14,27,31). The upper bands (closed circle) represents kAE1 that has exited the ER, undergone oligosaccharide processing in the Golgi, and trafficked to the cell surface, while the lower band (open circle) represents kAE1 that is still in the ER. In contrast to the wild type kAE1, the immunoblots of the kSAO, R589H and R901stop mutants expressed in MDCK cells show very little upper complex form of kAE1, with the lower high mannose form dominating. This is consistent with their predominant ER localization. The G701D mutant exhibits an intermediate pattern, with a reduced amount of upper complex bands (50%) relative to wild type kAE1, consistent with its impaired ability to exit the ER and predominant Golgi localization.

kAE1 interacts with calnexin in vitro-Co-immunoprecipitation studies using an anti-calnexin antibody (αCNX) demonstrates that calnexin interacts preferentially with the high mannose version of wild type kAE1 and the high mannose versions of kAE1 mutants (Fig. 1B, open circle), consistent with calnexin role as an ER chaperone. Indeed, a heightened interaction of the high mannose-containing mutant glycoproteins, kSAO, R589H, G701D, and R901stop, with calnexin was seen by co-immunoprecipitation

indicative of their ER retention (Fig. 1C, percent kAE1 immunoprecipitated by the anti-calnexin antibody relative to the amount immunoprecipitated by the anti-kAE1 antibody). Calnexin may therefore play a role in the folding of kAE1 and in the retention and degradation of kAE1 mutants.

Castanospermine blocks calnexin recognition of kAE1-To test whether calnexin is responsible for the retention of kAE1 mutants in the ER of MDCK cells, we used castanospermine (CST), an inhibitor of glucosidase I, to block glucose trimming and the subsequent lectin interaction of substrates with calnexin (32). Calnexin recognizes the transient Glc1Man9GlcNAc2 intermediate present on membrane glycoproteins during biosynthesis (32). Calnexin has been shown to bind to monoglucosylated N-linked oligosaccharides present on many glycoproteins in the ER until the mature protein conformation is achieved (33-35). Immunoblotting of whole cell lysate biochemically confirmed the effect of CST as processing of the oligosaccharide is inhibited retaining the initial Glc3Man9GlcNAc2 oligosaccharide (Fig. 2A). In the presence of CST, wild-type kAE1 is mainly in the high-mannose form, compared to wild-type kAE1 in the absence of CST, which is mainly in the complex form (Fig. 1B). A shift in oligosaccharide from high mannose to complex form in the G701D mutant is also effectively inhibited by CST with no detectable effect on the migration of the SAO, R589H or R901stop mutants, which are already predominantly in the high mannose form (Fig. 2A). The presence of some complex oligosaccharide on wild type kAE1 even after overnight CST treatment may represent kAE1 at the cell surface. Previous pulse chase studies done in HEK-293 cells have shown that wild type kAE1 has a half life of 15 hours, indicating that it is quite stable at the cell surface and not targeted for rapid degradation (11).

Co-immunoprecipitation studies of cells treated with CST to inhibit glucosidase I showed impaired calnexin interactions with the wild type and mutant forms of kAE1 as expected (Fig. 2B). Even though overnight treatment of MDCK cells with 1mM CST results in an increase in the high mannose form of wild type and mutant kAE1 (Fig. 2B, total and αAE1), minimal amounts of wild

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type and the various mutants of kAE1 were seen in the co-immunoprecipitation fractions with calnexin (Fig. 2B, αCNX) compared with the amount readily detected in the absence of drug (Fig. 1B). This indicates that blocking oligosaccharide processing of kAE1 effectively inhibits the interaction with the chaperone calnexin.

Castanospermine rescues kAE1 dRTA mutants to the cell surface of MDCK cells-Confocal microscopy was used to examine the effect of calnexin binding inhibition by CST on the localization of kAE1 and dRTA mutants (Fig. 3). Preventing the interaction of kAE1 with calnexin may allow ER-localized mutants to escape this quality control step and traffic to the cell surface. In the first column, the cell surface expression of wild-type kAE1 and the mutants expressed in MDCK cells after overnight treatment with CST as detected using the extracellular HA epitope is shown. Wild-type kAE1 was readily detected at the cell surface of non-polarized MDCK cells indicating that blocking its interaction with calnexin does not prevent its trafficking to the cell surface. Thus, an interaction of kAE1 with calnexin is not required for its trafficking to the plasma membrane.

Remarkably, the two dominant dRTA mutants, R589H and R901stop were detected at the cell surface after CST treatment of cells (Fig. 3, green staining, first column). kSAO and G701D, however, were not detected at the cell surface following CST treatment. Following fixation and permeabilization, the total intracellular levels of kAE1 were seen, indicating that CST had no effect on the intracellular retention of the kSAO and G701D mutants (Fig. 3, second column). Immunofluorescence of calnexin (Fig. 3, blue) was also used to stain for the ER. These results are not unique to MDCK cells, as similar results were seen when HEK293 cells expressing the various dRTA mutants were treated with CST (data not shown), where the R589H and R901stop mutants were rescued to the cell surface, while the kSAO and G701D mutants were not.

CST was also able to rescue both the R589H and R901stop mutants to the basolateral membrane in polarized MDCK cells (Fig. 4). Note the diffuse intracellular staining of these two mutants in the absence of CST, but the strong basolateral staining after treatment of the MDCK

cells with CST. Again, CST was unable to rescue the trafficking of the SAO and G701D mutants to the basolateral membrane in polarized MDCK cells. Therefore, not only does CST allow the mutants to exit the ER, it also allows them to traffic to their proper site in polarized cells. These results indicate that calnexin plays an essential role in their intracellular retention of these dominant dRTA mutants, resulting in a lack of functional protein at the cell surface.

To confirm that the high-mannose form of kAE1 is able to traffic to the cell surface in MDCK cells treated with CST, cell-surface biotinylation experiments were performed using a membrane-impermeant biotinylation reagent, NHS-SS-biotin. Biotinylated proteins were isolated using streptavidin beads and the unbound (U) and bound (B) fractions were analyzed by immunoblotting for kAE1 using an anti-HA antibody (Fig. 5A&B). In the absence of CST, the wild type kAE1 adopts a complex oligosaccharide (Fig. 5A –CST, closed circle) and this form can be biotinylated, indicative of cell surface expression. Higher molecular weight species migrating near the top of the gel were also found in the biotinylated fraction, likely corresponding to SDS-resistant kAE1 dimers. No biotinylated high mannose form of wild type kAE1 was detected, confirming the intracellular localization of this form of kAE1. In the presence of CST, biotinylation confirmed that the high mannose form of kAE1 (Fig. 4A, +CST, open circle in bound (B) fraction) is able to traffic to the cell surface, showing that cell surface expression is not dependant on oligosaccharide processing. Biotinylation experiments were also performed on the dominant dRTA mutants expressed in MDCK cells, in the presence of CST, however no R589H or R901stop kAE1 was detectable in the bound biotinylated fraction (Fig. 5B, R901stop not shown). The inability to detect rescued dRTA mutants by this method is likely due to the low level of rescue or the inefficiency of the biotinylation reaction.

Flow cytometry was used to determine the changes in cell surface expression of kAE1 mutants following CST treatment. Using the external HA epitope on the 3rd extracellular loop, cell surface expression of kAE1 was monitored on intact transfected cells. In the presence of CST, an increase in cell surface expression corresponding

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to an upward shift in fluorescence was seen for the R589H and R901stop mutants (Fig. 5C, blue histogram). No such shift was observed for the wild-type protein or the SAO mutant. A slight upward shift for the G701D mutant in cells treated with CST was observed in this experiment, however, this effect was not reproducible. The effect of CST on the amount of cell surface expression of kAE1 dRTA mutants is however quite subtle, as the level of cell surface protein does not achieve the same fluorescence intensity as seen in the kWT histograms (Fig. 5C). Thus, the amount of mutant protein rescued to the cell surface was much lower than the level of wild-type protein, as was also indicated by the biotinylation experiments.

The changes in cell surface expression following CST treatment in five separate experiments were quantified by two separate methods. Overton subtraction (36) comparing histograms in the absence and presence of CST shows a 21 ± 8% increase in R589H cell surface expression as well as a 19 ± 4% increase in R901stop cell surface expression. There was also a slight increase in kWT cell surface expression 8 ± 2%, but a negligible change in kSAO and G701D cell surface expression. The percent of cells above background levels of MFI (Fig. 5C, mutants versus MDCK cells alone), were also examine with a 3% and 5% increase in cells positive for R589H and R901stop at the cell surface in the presence of CST. This modest increase in cell surface expression of dRTA mutants in the presence of CST explains the inability to detect rescued dRTA mutants in the cell surface biotinylation experiments.

Removal of the N-linked glycan prevents calnexin interactions and leads to cell surface rescue of kAE1-The effect of CST indicates the involvement of calnexin in the ER retention of these dRTA mutants. The interaction of calnexin with AE1 is N-glycosylation-dependent (23) suggesting that the oligosaccharide found in the fourth extracellular loop of AE1 plays a role in the ER retention of the dominant R589H and R901stop dRTA mutants. In order to confirm the lectin role of calnexin in the ER-mediated retention of the mutants, the asparagine at position 642 was mutated to an aspartate residue to prevent oligosaccharide attachment. The absence of oligosaccharide has been shown to have no effect

on the trafficking of AE1 to the cell surface in transfected HEK-293 cells (37) or when expressed in Xenopus oocytes (38). Mutation of the native N-linked glycosylation site, Asn-642, resulted in expression of unglycosylated kAE1 (N642D) as confirmed by immunoblotting of whole cell lysate from MDCK cells expressing the N642D mutants (Fig. 6A). No complex oligosaccharide could be detected on the N642D mutants, and bands with a slightly faster mobility relative to the high mannose form were seen (Fig. 6A, compare kSAO and kSAO N642D lanes, open circle bands and X). These bands were insensitive to treatment with endoglycosidase H (data not shown), indicating that the N642D kAE1 mutants were indeed non-glycosylated.

Immunofluorescence staining and confocal microscopy confirmed that the absence of oligosaccharide on the wild type protein did not affect the localization of kAE1 to the cell surface in non-polarized MDCK cells (Fig. 6B, first panel). Thus, N-linked oligosaccharide is not required for the trafficking of kAE1 from the ER to the plasma membrane in MDCK cells. Interestingly, both the R589H/N642D and R901stop/N642D mutants were seen at the cell surface, while the non-functional kSAO/N642D and G701D/N642D also were retained intracellularly (Fig. 6B), as was seen with the CST rescue experiments. This demonstrates that the mode of ER retention of the two dominant mutants is glycosylation-dependent, as removal of the single N-glycan allows trafficking of these two mutants to the cell surface.

To confirm that kAE1 is able to traffic to the cell surface in MDCK cells in the absence of the single N-glycan, cell-surface biotinylation experiments were performed. When the oligosaccharide attachment is prevented, the wild type kAE1 protein is present as a single band and can be biotinylated, indicative of cell surface expression (Fig. 6C, kWT N642D bound fraction). In this experiment, the R589H/N642D and R901stop/N642D mutants were biotinylated and now detectable in the bound fraction, indicative of cell surface expression. The kSAO/N642D and G701D/N642D were not, however, found in the bound fraction, confirming the confocal microscopy data that the lack of oligosaccharide has no effect on the intracellular retention of these two mutants. The ability to detect biotinylation of

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the dominant mutants lacking their N-glycan chain suggests that the level of these mutants at the cell surface is higher than in the CST rescue experiments (Fig. 5).

Co-immunoprecipitation studies of the N642D mutants was also performed to determine whether or not calnexin is able to interact with these unglycosylated kAE1 membrane proteins via calnexin’s polypeptide binding site. It has been proposed that calnexin has two mechanisms of action, a lectin-only and a dual-binding, lectin- and protein-mediated chaperone function (32). We found that a small amount of kWT N642D can be co-immunoprecipitated with calnexin (Fig. 6D, kWT N642D αCNX), confirming calnexin’s ability to mediate protein-protein interactions. We have previously shown that the amount of AE1 N642D that was co-immunoprecipitated with the anti-calnexin antibody was however, lower than what was found for wild-type AE1 (23). Similarly, little or no N642D forms of the kAE1 mutants were detectable in the anti-calnexin immunoprecipitates relative to the anti-kAE1 immunoprecipitates. We conclude that the interaction of the polytopic kAE1 membrane glycoprotein with calnexin in MDCK cells is highly N-glycosylation dependent.

kAE1 can be rescued with other small molecule inhibitors-A variety of small molecules and pharmacological drugs have been used to rescue the trafficking of intracellularly retained mutant proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) (39). Confocal microscopy was used to examine the effect of small molecules on the cell surface expression of wild type and kAE1 dRTA mutants in MDCK cells. MAL3, a small molecule modulator of Hsc70 binding, and two small molecule correctors of CFTR, C3 and C4, were also used to examine their effect on the trafficking of dRTA mutants. MAL3 is a structural analog of NSC 630668-R/1 and 15-deoxyspergualine that inhibits the Hsp40 stimulating ATPase activity of Hsc70, resulting in decreased peptide affinity (40). Although the mechanism of action of the small molecule correctors, C3 and C4, is unknown, they have also been shown to promote trafficking of CFTR to the cell surface (41,42). By confocal microscopy, all three compounds were able to rescue the R589H and R901stop mutants to the cell surface (Fig. 7, columns 2-4, green staining),

similar to the effect observed by disruption of the calnexin interaction. The severely misfolded, non-functional kSAO and the Golgi localized G701D mutants however, were not rescued to the cell surface following treatment with the various drugs, as can be seen by the complete absence of these mutants at the cell surface (Fig. 7, green staining). To confirm that these MDCK cells are indeed expressing the mutant dRTA kAE1 proteins despite their lack of cell surface expression, these cells were permeabilized and stained for intracellular kAE1 (Fig. 7, red staining). Calnexin was also visualized for co-localization of intracellular kAE1 in the ER, but was omitted for clarity. The effect of these drugs on the trafficking of these dRTA mutants was also examined in polarized MDCK cells by confocal microscopy. The cell surface rescue of R589H and R901stop by MAL3, C3 and C4 to the basolateral membrane in polarized MDCK cells was confirmed (data not shown).

kAE1 is ubiquitinated and degraded by the proteasome-To determine the fate of the kAE1 mutants in MDCK cells, we used the highly specific and irreversible proteasome inhibitor, lactacystin (43), to examine whether or not the proteasome plays a role in the degradation of wild-type or mutant forms of kAE1. Proteins that are targeted for degradation by the proteasome are typically covalently modified by the attachment of one or more ubiquitin molecules. We set out to see if kAE1 is ubiquitinated by immunoprecipitating kAE1 from MDCK cells treated with lactacystin to inhibit the degradation of ubiquitinated proteins, and then blotting the immunoprecipitated kAE1 for covalently-attached ubiquitin. Wild type and dRTA mutants were immunoprecipitated from MDCK cells and then resolved on SDS-PAGE gels. Western blotting was then used to identify either kAE1 (Fig. 8A, left panel), or ubiquitinated proteins (Fig. 8A, right panel) in the immunoprecipitated fractions. Both the wild type and mutant kAE1 proteins were found to be ubiquitinated in cells treated with lactacystin, which would indicate that they could be targeted for degradation. Deglycosylation of ubiquitinated kAE1 using Endoglycosidase Hf (data not shown), showed that the form of kAE1 that was ubiquitinated does not contain high mannose oligosaccharide, suggesting that the oligosaccharide had been removed from kAE1. A

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cytosolic N-glycanase may be responsible for the removal of kAE1 oligosaccharides as ER associated degradation (ERAD) substrate intermediates have been found to be deglycosylated when they reach the cytosol prior to their degradation by the proteasome (44).

If the mutations cause kAE1 to misfold, the mutants may be more sensitive to degradation and turnover. To compare the rates of degradation of the dRTA mutants relative to the wild type protein, cycloheximide was used to block protein expression, and then immunoblotting was used to assess the levels of protein remaining over time. The wild type protein was found to be quite stable, with protein being detected even 17 hours later (Fig. 8B, kWT - lactacystin). This is in agreement to our previously published results indicating that kAE1 has a half life of 15 hours as determined by pulse-chase studies in HEK-293 cells (11). The levels of the mutants, kSAO and R589H, were also assessed following 17 hours of cycloheximide treatment and were found to be completely absent at that time indicating their more rapid degradation (Fig. 8, kSAO and R589H –lactacystin). Lactacystin was also used to reduce the proteasome-mediated degradation of kAE1. It was found to have a protective effect on the levels of wild type and mutant kAE1 indicating that the proteasome is responsible for the degradation of kAE1 and the ER-localized mutants (SAO, R589H, R901stop), but not the Golgi-localized mutant (G701D) in MDCK cells.

DISCUSSION We have previously shown (8,11,12,14-

16) that in polarized and non-polarized MDCK cells, kAE1 mutants (SAO and dRTA) are retained intracellularly and are unable to traffic efficiently to the plasma membrane to carry out their transport function. In order to assess the role molecular chaperones play in the intracellular retention of dRTA mutants, we used a variety of drugs and non-glycosylated mutants that inhibit chaperone binding to substrate glycoprotein intermediates. Of the mutants studied, the dominant R589H and R901stop showed plasma membrane expression upon treatment with various drugs whereas kSAO and G701D did not (Fig. 3, 4 & 7). This indicates that ER retention is chaperone-mediated, as disruption of chaperone binding allowed these two dominant mutants to

escape to the plasma membrane. Disruption of the interaction of the dominant kAE1 mutants with calnexin, either by blocking N-glycan processing with CST or by removal of the N-glycosylation site, N642D, promoted rescue of the trafficking of these mutants to the cell surface. By preventing the interactions of the mutant protein with the quality control chaperones, the newly synthesized, slightly misfolded, but functional kAE1 protein can escape recognition by the mechanisms responsible for its retention and its ultimate degradation.

The use of small molecules as correctors of ER retention of dominant dRTA mutants is potentially feasible, as the R589H and R901stop mutants retain anion-transport activity in patients’ red blood cells and when expressed in Xenopus oocytes (9,10). These dRTA mutants can be classified as class 2 mutants, mutations that arise due to defective protein processing yet are still functionally active (45). dRTA caused by these mutations arise due to the retention of functional kAE1 protein in the ER. The Hsc70 inhibitor and small molecule correctors (MAL3, C3 and C4) were also able to rescue cell surface expression of the two dominant mutants confirming the role of the quality control system of chaperones in the retention of these dRTA mutants. MAL3, a disruptor of Hsc70 action, suggests that this cytosolic chaperone may play a role in the folding of kAE1, likely by interaction with its large N-terminal cytosolic domain or cytosolic loops located in the membrane domain. We have also been able to demonstrate an interaction between Hsc70 and kAE1 by co-immunoprecipitation (data not shown). The Vertex compounds, C3 and C4, were also shown to rescue the cell surface expression of R589H and R901stop, and although their exact mechanism of action is unknown, these compounds have been shown to interact directly with CFTR in the ER increasing its folding efficiency (41,46). Our results indicate that these compounds can also rescue dRTA mutants of kAE1 and are therefore not specific for CFTR indicating a more general rescue mechanism. Immunoblots of MDCK cells expressing dRTA mutants showed no change in glycosylation pattern in the presence of MAL3 or the small molecule correctors (data not shown). As in previous studies with erythroid AE1 (47), and as we have now shown with the CST results,

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formation of complex oligosaccharide is not required for the trafficking of kAE1 to the cell surface. It is therefore the interaction with the quality control mechanisms in the cell that determine the intracellular localization of this membrane glycoprotein. It is not surprising that disruption of either calnexin or Hsc70 interaction with kAE1 can rescue it to the cell surface, as kAE1 is a polytopic membrane protein with regions exposed on both the ER lumen and cytosol. As previously suggested (48), protein folding pathways are both intertwined and redundant, and these results suggest that multiple chaperones are involved in the folding and intracellular retention of kAE1.

We have shown by flow cytometry that only a small fraction of dominant dRTA mutants can be rescued to the cell surface. In the case of CFTR, the minimal requirements for gene therapy have demonstrated that as few as 6-10% of corrected cells can generated sufficient chloride transport in vivo, suggesting that low levels of CFTR rescue are sufficient to correct the CF epithelial chloride transport defect (49,50). It remains controversial however, that disruption of the calnexin interaction with ER retained forms of mutant CFTR leads to the maturation of CFTR (51,52). Nevertheless, small molecule drugs may be used to therapeutically target chaperone interactions in diseases associated with defective protein folding and trafficking. In the case of dRTA, symptoms arise due to a reduced amount of functional kAE1 at the basolateral membrane of the acid-secreting (α) intercalated cells of the collecting duct of the kidney, not only due to the lack of mutant kAE1 at the cell surface, but also due to the dominant R589H protein ability to retain the wild type protein in the ER (11). The use of these drugs would therefore be able to promote the cell surface expression of these dRTA mutants and the retained wild-type protein that arise to due simple trafficking defects, increasing the overall level of functional protein at the cell surface.

The severely misfolded kSAO is however retained in the ER and is not rescued by CST or the other compounds. Thus, disruption of calnexin interactions is not sufficient to rescue this severely misfolded mutant in MDCK cells. We have now demonstrated that kAE1 is targeted for degradation by the proteasome via ubiquitination, consistent with the theory that excessive

interactions of unfolded proteins with molecular chaperones target them for degradation when the native conformation is not achieved. Misfolded kAE1 may therefore be a substrate for ER-association protein degradation (ERAD). This is not surprising, since the ER chaperone calnexin, and the cytosolic chaperone, Hsc70, which we have shown to play a role in the retention of dRTA mutants, have been implicated in the recognition of misfolded proteins for the ERAD pathway (53). We have now confirmed the protective effect of the proteasome inhibitor lactacystin on the degradation of kAE1 in MDCK cells; lactacystin has previously been shown to protect the erythroid SAO protein against degradation in HEK-293 cells (15). The lack of cell surface rescue of kSAO through disruption of chaperone interactions and the fact that lactacystin provides protection against proteasomal degradation agrees with previous studies (15,18,38) that have shown that kSAO is severely misfolded, non-functional, and reinforces the idea that there are multiple mechanisms of quality control active within the cell to retain misfolded proteins. Quality control in MDCK cells is therefore highly effective in recognizing severely misfolded membrane proteins and targeting them for retention.

While disruption of chaperone interactions may allow rescue of dominant dRTA mutants to the cell surface but not the severely misfolded kSAO, chaperones likely do not play a major role in the Golgi retention of recessive mutants like G701D. Even though the G701D dRTA mutant was shown to have a heightened interaction with calnexin, castanospermine treatment did not rescue its cell surface expression. This is not surprising, since G701D can exit the ER and traffic to the Golgi, yet is severely impaired in moving from the Golgi to the cell surface. When co-expressed with glycophorin A (GPA) in Xenopus oocytes or in erythrocyte precursors that endogenously express GPA, the G701D mutant is able to traffic to the cell surface showing that GPA facilitates the cell surface expression of G701D kAE1 (17). Kidney cells, however, do not contain GPA, which explains the lack of cell surface expression of this mutant and the development of dRTA. Thus, the cellular context and nature of interacting proteins is very important in producing a phenotype of mutations with the same genotype. Indeed, human red cell precursors lose specific chaperones like

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calnexin during differentiation (22). This loss of protein quality control may allow mis-folded forms of AE1 like the SAO mutant to traffic to the cell surface during erythropoiesis and be expressed in the mature red cell. Plasma membrane rescue is therefore not a general phenomenon for a protein but is specific for a particular set of mutants in a particular cellular context. This study adds significant new insight into the role chaperones

play in the molecular basis and manifestation of certain forms of dRTA. Small molecules that inhibit chaperone-mediated retention and promote cell surface expression of certain dominant dRTA mutants (R5899H, R901stop) in kidney cells are therefore potential therapeutic agents which may be used to treat this group of dRTA patients.

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FOOTNOTES

The authors would like to thank their colleagues David Williams (University of Toronto), Jeff Brodsky (University of Pittsburgh), Dr. Robert Bridges (Cystic Fibrosis Foundation for Therapeutics) and Christine Bear (SickKids, Toronto) for their generous gifts of antibodies and small molecule correctors. Jing Li is thanked for her help in the construction of the N642D kAE1 mutants. This work was supported by a grant (MOP102493) from the Canadian Institutes of Health Research and funding from the Faculty of Medicine, University of Toronto. S.T.P. was the recipient of a studentship award from the CIHR Strategic Training Program in Membrane Proteins in Health and Disease. The abbreviations used are: AE1/Band 3, Anion Exchanger 1; BSA, bovine serum albumin; CFTR, Cystic Fibrosis Transmembrane Regulator; CHX, cycloheximide; CNX, calnexin; CST, castanospermine; dRTA, distal Renal Tubular Acidosis; ER, endoplasmic reticulum; ERAD, ER associated degradation; FACS, fluorescence-activated cell sorting; GPA, Glycophorin A; HA, hemagglutinin; HEK-293, human embryonic kidney 293 cells; HS, Hereditary Spherocytosis; kAE1, kidney Anion Exchanger 1; kSAO, kidney Anion Exchanger 1; MDCK, Madin Darby Canine Kidney cells; MMLV, Moloney Murine Leukemia Viral; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SAO, Southeast Asian Ovalocytosis; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SITS, stilbene disulfonate inhibitor; Ub, ubiquitin.

FIGURE LEGENDS

Fig.1. Expression of HA-tagged kAE1 mutants in MDCK cells. (A) Localization of wild type and kAE1 mutants in permeabilized non-polarized MDCK cells by confocal microscopy using anti-HA antibodies. Scale bar 10µm. (B) Co-immunoprecipitation of wild type and kAE1 mutants with calnexin. Confluent MDCK cells were lysed in 1% digitonin for co-immunoprecipitation with rabbit antibodies against the N-terminus of human kAE1 (αkAE1) or the luminal domain of human calnexin (αCNX). Immunoprecipitated proteins were run on 7.5% SDS-polyacrylamide gels and immunoblotted using a mouse monoclonal anti-HA antibody to detect HA-tagged AE1. Closed circles correspond to kAE1 carrying complex oligosaccharide while open circles correspond to high mannose oligosaccharide. (C) Comparison on the percent of high mannose kAE1 interacting with calnexin by co-immunoprecipitation relative to the amount of high mannose kAE1 present in kAE1 immunoprecipitates. Fig.2. Castanospermine (CST) treatment of MDCK cells expressing wild type and kAE1. (A) Immunoblot of total cell lysate of CST treated MDCK cells using an anti-HA antibody. CST blocks oligosaccharide processing resulting in the presence of the high mannose oligosaccharide on kAE1. (B) Co-immunoprecipitation of CST treated MDCK cells. Detergent extracts were immunoprecipitated using either rabbit anti-kAE1 or anti-CNX antibodies. Immunoprecipitated kAE1 was detected by

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immunoblotting using a mouse anti-HA antibody. CST treatment blocks the interaction of kAE1 with calnexin resulting in a decrease in kAE1 co-immunoprecipitated with anti-calnexin antibodies. Closed circles correspond to kAE1 carrying complex oligosaccharide while open circles correspond to high mannose oligosaccharide.

Fig.3. Cell surface rescue of two dRTA mutants in non-polarized MDCK cells. Non-polarized MDCK cells expressing wild type and dRTA mutants were incubated with 1mM CST overnight. A mouse anti-HA antibody was used to detect cell surface kAE1 using an engineered HA tag in the 3rd extracellular loop, seen as green staining in kWT, R589H and R901stop in intact cells. Cells were then fixed and permeabilized and the total kAE1 pool and calnexin were detected by immunofluorescence, seen in red and blue respectively. Fig.4. Cell surface rescue using castanospermine of R589H and R901stop in polarized MDCK cells. MDCK cells expressing wild type and dRTA mutants were grown on polycarbonate filters, fixed and permeabilized and the probed with and anti-HA antibody to detect kAE1. The top panel, X-Z, corresponds to the side view of the cells, while the X-Y panel shows the middle perpendicular sectional view of the cells. Fig.5. Castanospermine (CST) treatment of MDCK cells expressing wild type and kAE1. (A) Cell surface biotinylation of HA-tagged wild type kAE1 and R589H (B) expressed in MDCK cells grown in the absence (-CST) or presence (+CST) of 1mM castanospermine for 16 hours. Double the amount of total protein was loaded for quantification (2T), T-total, U-unbound, B-bound biotinylated kAE1. Biotinylated protein was detected using a mouse monoclonal anti-HA antibody. Closed circles correspond to kAE1 carrying complex oligosaccharide while open circles correspond to high mannose oligosaccharide. (C) Flow cytometry analysis of cell surface rescue of HA-tagged dRTA kAE1 mutants following CST treatment. Shown are histograms of cell surface kAE1 expression in MDCK cells in the absence of (blue) and presence of (red) CST. Intact MDCK cells were incubated with mouse anti-HA antibody followed by Alexa Fluor 488-conjugated goat anti-mouse antibody. Numbers shown are mean fluorescence intensities for cell surface expression of HA-tagged kAE1. Fig.6. Removal of the N-linked glycan at Asn642 promotes the trafficking of R589H and R901stop to the cell surface. (A) Immunoblot of total protein levels from MDCK cells expressing kAE1 with and without (N642D) N-glycan. Closed circles correspond to kAE1 carrying complex oligosaccharide, open circles correspond to high mannose oligosaccharide, and X is non-glycosylated kAE1. (B) Cell surface expression of N642D wild type and kAE1 mutants in non-polarized MDCK cells by confocal microscopy using an anti-HA antibody. Scale bar 10 µm. (C) Cell surface biotinylation of N642D mutants. In the absence of N-glycan, the wild type, R589H and R901stop can be detected at the cell surface by biotinylation. (D) Calnexin interacts weakly with non-glycosylated kAE1. Confluent MDCK cells were lysed in 1% digitonin for co-immunoprecipitation with antibodies against kAE1 (αkAE1) and calnexin (αCNX). Immunoprecipitated proteins were run on 7.5% SDS-polyacrylamide gels and immunoblotted using an anti-HA antibody to detect AE1. Fig.7. Cell surface rescue of dRTA mutants using small molecules in non-polarized MDCK cells. Non-polarized MDCK cells expressing wild type and dRTA mutants were incubated with MAL3, C3 or C4 overnight. Cell surface expression of kAE1 using an external HA epitope was detected in non-permeabilized cells by immunofluorescence (green). Cells were then fixed and permeabilized and stained for total kAE1 (red) and the ER chaperone, calnexin (omitted for clarity). Note the presence of cell surface kAE1 (green) in MDCK cells expressing R589H and R901stop treated with MAL3, C3 and C4. Fig.8. HA-tagged kAE1 expressed in MDCK cells is ubiquitinated and degraded by the proteasome. (A) Wild type kAE1 and kAE1 mutants are ubiquitinated after lactacystin-mediated inhibition of the

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proteasome. MDCK cells expressing wild type and kAE1 mutants were solubilized in RIPA buffer and kAE1 was immunoprecipitated using an antibody against the N terminus of kAE1. Immunoprecipitated proteins were run on a 7.5% SDS-PAGE gel and immunoblotted for kAE1 using an anti-HA antibody (left) or ubiquitin (right). Closed circles correspond to kAE1 carrying complex oligosaccharide, open circles correspond to high mannose oligosaccharide, and X is non-glycosylated kAE1. (B) Cycloheximide chase experiment to monitor protein degradation. Control cells, or cells that were pre-treated for one hour with 10uM lactacystin, were then incubated with 10uM cycloheximide to inhibit protein synthesis. Cells were lysed at various time points in 2X sample buffer and ran on SDS-PAGE gels. Immunoblotting for AE1 was then used to monitor protein degradation over time. (C) Quantification of degradation rates in the absence (red) and presence (blue) of lactacystin (y-axis in logarithmic scale). Following cycloheximide treatment, the only protein seen after 17 hours was the wild type kAE1 protein and this time point has been omitted in the quantification of degradation for the various mutants.

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AkWT kSAO R589H G701D R901stop

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kWT

no drug MAL3 C3 C4

R901stop

G701D

R589H

kSAO

Figure 7 - Patterson & Reithmeier

21

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Blot α AE1

A

B

kWT

kSAO

R589H

G701D

R901stop

0 1 2 5 17 0 1 2 5 17 hours

0 1 2 5 0 1 2 5 hours

- lactacystin + lactacystin

- lactacystin + lactacystin

C

Figure 8 - Patterson & Reithmeier

X

kWT

10

100

0 5 10 15 20

time

% protein remaining

kWT +CHX kWT +CHX/lactacystin

R589H

10

100

0 1 2 3 4 5

time

% protein remaining

R589H CHX R589H +CHX/lactacystin

kSAO

10

100

0 1 2 3 4 5

time

% protein remaining

kSAO +CHX kSAO +CHX/lactacystin

G701D

10

100

0 1 2 3 4 5

time

% protein remaining

G701D +CHX G701D +CHX/lactacystin

R901stop

10

100

0 1 2 3 4 5

time

% protein remaining

R901stop +CHX R901stop +CHX/lactacystin

MDCK kWT kSAO R589H G701D R901stop

MDCK kWT kSAO R589H G701D R901stop

Blot α Ubiquitin

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Sian T. Patterson and Reinhart A. F. Reithmeierinteractions

Cell surface rescue of kidney anion exchanger 1 mutants by disruption of chaperone

published online July 13, 2010J. Biol. Chem. 

  10.1074/jbc.M110.144261Access the most updated version of this article at doi:

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