Correspondence to: John V. Cox, Department of Microbiology and Immunology, University of Tennessee, 858 Madison Avenue, Memphis, TN 38163. Tel:(901) 448-7080 Fax:(901) 448-8462 E-mail:jcox{at}utmem1.utmem.edu.
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Abstract |
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The variant chicken kidney AE1 anion exchangers differ only at the NH2 terminus of their cytoplasmic domains. Transfection studies have indicated that the variant chicken AE1-4 anion exchanger accumulates in the basolateral membrane of polarized MDCK kidney epithelial cells, while the AE1-3 variant, which lacks the NH2-terminal 63 amino acids of AE1-4, primarily accumulates in the apical membrane. Mutagenesis studies have shown that the basolateral accumulation of AE1-4 is dependent upon two tyrosine residues at amino acids 44 and 47 of the polypeptide. Interestingly, either of these tyrosines is sufficient to direct efficient basolateral sorting of AE1-4. However, in the absence of both tyrosine residues, AE1-4 accumulates in the apical membrane of MDCK cells. Pulsechase studies have shown that after delivery to the cell surface, newly synthesized AE1-4 is recycled to the Golgi where it acquires additional N-linked sugar modifications. This Golgi recycling activity is dependent upon the same cytoplasmic tyrosine residues that are required for the basolateral sorting of this variant transporter. Furthermore, mutants of AE1-4 that are defective in Golgi recycling are unable to associate with the detergent insoluble actin cytoskeleton and are rapidly turned over. These studies, which represent the first description of tyrosine-dependent cytoplasmic sorting signal for a type III membrane protein, have suggested a critical role for the actin cytoskeleton in regulating AE1 anion exchanger localization and stability in this epithelial cell type.
Key Words: epithelial, band 3 protein, Golgi, polarity, actin
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Introduction |
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THE kidney plays an essential role in the regulation of pH equilibrium within the body. This regulatory function is accomplished by the wide variety of transporters and channels that mediate the vectorial transport of ions within the cells of the kidney. This selective transport of ions requires the presence of different complements of polypeptides in the apical and basolateral membrane domains of the polarized epithelial cells of the kidney. Although cytoplasmic (
A well-characterized example of vectorial transport by the cells of the kidney is the -intercalated cell of the mammalian kidney collecting duct. In this acid-excreting cell type, basolateral chloride-bicarbonate exchangers and apical proton pumps function coordinately to dispose of bicarbonate and protons generated by intracellular carbonic anhydrase (
-intercalated cells is encoded by the AE1 anion exchanger gene. In contrast to the acid-excreting,
-intercalated cell of the collecting duct, the base-excreting, ß-intercalated cell of the collecting duct possesses an apical chloride-bicarbonate exchanger and a basolateral proton pump (
Molecular analyses have shown that mammalian kidney AE1 anion exchangers are identical to AE1 anion exchangers from erythroid cells except at the NH2 terminus of their cytoplasmic domains. In rats (
Additional diversity has been detected among the anion exchangers encoded by the AE1 gene in chicken kidney. Three kidney AE1 transcripts, AE1-3, AE1-4, and AE1-5, are derived from a single promoter by alternative splicing (
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Materials and Methods |
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Generation of Chicken Kidney AE1 Anion Exchanger Antibodies
Polyclonal antibodies have been generated in rabbit against the cytoplasmic domain of the chicken kidney AE1-4 anion exchanger that was expressed as a bacterial fusion protein in E. coli. In brief, the region of AE1-4 encompassing amino acids 1359 was subcloned into the pFLAG-ATS prokaryotic expression vector (IBI) downstream of the eightamino acid FLAG epitope sequence. The expression of this bacterial fusion protein is driven by the inducible tac promoter. After induction with IPTG, the fusion protein was purified from bacterial extracts by immunoaffinity chromatography using a monoclonal antibody to the FLAG epitope. The purified fusion protein was used as a source of antigen to inject rabbits.
Stable and Transient Expression of Variant AE1 Anion Exchangers in MDCK Cells
Madin-Darby canine kidney (MDCK) cells were maintained in DME supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2. Cells were transfected with various AE1 anion exchanger cDNAs subcloned into the pcDNA3 eukaryotic expression vector (Invitrogen). Media was changed 24 h after transfection, and 48 h after transfection cells were analyzed for AE1 expression. Alternatively, at 48 h after transfection cells were split 1:10 in complete media containing 600 µM G418. Cells that could grow in the presence of G418 were selected and used for various assays.
Construction of AE1-4 Anion Exchanger Mutants
Sense primers complementary to nucleotides 89103, 137151, or 185198 of the chicken AE1-4 anion exchanger cDNA (21, AE1-4
37, and AE1-4
53 constructs were generated by using these PCR products to replace the 5' end of the AE1-4 cDNA in pcDNA3 that had been removed by digestion with HindIII and BamHI.
Point mutants were constructed using the Altered Sites II site-directed mutagenesis kit (Promega). In brief, the AE1-4 cDNA was subcloned into the pALTER-1 vector. After alkaline denaturation, a mutagenic oligonucleotide was annealed to the full-length AE1-4 cDNA template. The sequences of the mutagenic oligonucleotides are as follows: Y44A (5'-TCCACGTAGCCCTCAGCGGTGTCCCTGTGGGC-3'), Y47A (5'-TCG-TGCAGCTCCACGGCGCCCTCATAGGTGTC-3'), Y44A,Y47A (5'-TCGTGCAGCTCCACGGCGCCCTCAGCGGTGTCCCTGTGGGC-3'), and N638T (5'-CCGCGGGCGGTGCCGGTGGTCACCTCCAGCCC-3'). The mutant strand was elongated and ligated using T4 DNA polymerase and T4 DNA ligase, and cotransformed into a repair-minus E. coli strain (ES1301 mut S) along with helper phage DNA. All mutants were confirmed by sequencing using the dideoxy method. The mutant cDNAs were subcloned into pcDNA3 for transfection.
Immunoblotting Analysis of AE1 Anion Exchanger Polypeptides
MDCK cells transiently or stably expressing wild-type or mutant AE1 anion exchangers were harvested and detergent lysed by incubation in 150 mM NaCl, 10 mM Tris (pH 7.5), 5 mM MgCl2, 2 mM EGTA, 6 mM ß-mercaptoethanol, and 1% (vol/vol) Triton X-100 for 5 min on ice. In some instances, the cells were treated with 25 µg/ml of the actin depolymerizing drug, latrunculin B, for 1 h before lysis. Immunolocalization analyses have shown that phalloidin-stained microfilaments are absent in MDCK cells treated with this drug (data not shown). The lysate from treated or untreated cells was microcentrifuged for 5 min to separate the detergent soluble and insoluble fractions. The detergent insoluble pellet was resuspended in immunoprecipitation buffer containing 170 mM NaCl, 20 mM Tris (pH 7.5), 5 mM EGTA, 5 mM EDTA, 0.1% (wt/vol) SDS, 1% (vol/vol) Triton X-100, and 1% (wt/vol) sodium deoxycholate and sonicated three times. After sonication, the insoluble fraction was microcentrifuged for 5 min and the pellet discarded. The detergent soluble and insoluble fractions were then electrophoresed on a 6% SDS polyacrylamide gel, and the proteins were electrophoretically transferred to nitrocellulose. The filters were blocked with 5% powdered milk and incubated overnight with a 1:30,000 dilution of a chicken AE1-specific peptide antibody (
Immunoprecipitation of AE1 Anion Exchanger Polypeptides
Cells expressing the various anion exchanger polypeptides were grown in 100-mm culture dishes or on 24-mm polycarbonate filters (Costar). The cells were washed once with methionine-free DME and incubated with this media for 15 min at 37°C. At this time, 65 µCi/ml 35S-TranslabelTM (ICN) was added to the cells and they were incubated an additional 15 min at 37°C. After washing, the cells were incubated in DME containing 5% fetal calf serum for times ranging from 0 to 48 h. At each time point, the cells were detergent extracted as described above and separated into detergent soluble and insoluble fractions. In some instances, total cell lysates were prepared by directly lysing cells in immunoprecipitation buffer. Protein Aagarose beads that had been preloaded with rabbit polyclonal antibodies directed against amino acids 1359 of chicken AE1-4 were added to these fractions and incubated at 4°C overnight. The protein Aagarose beads were then washed three times with immunoprecipitation buffer, and the immune complexes were released by incubation in SDS sample buffer. Immunoprecipitates were resolved on a 6% SDS polyacrylamide gel. The gels were treated with PPO in DMSO, dried, and exposed to Kodak Biomax MR film at -80°C.
Endoglycosidase H and N-Glycosidase F Digestion of AE1 Anion Exchanger Immunoprecipitates
MDCK cells stably expressing the various chicken AE1 anion exchanger polypeptides were pulsed with 35S-Translabel, chased for 0, 1, or 4 h, and AE1 immunoprecipitates were prepared as described above. After washing of the protein Aagarose beads with immunoprecipitation buffer, the beads were washed with 10 mM Tris (pH 7.4), and 150 mM NaCl (TBS). The beads were then resuspended and digested either with 2.5 mU endoglycosidase H (endo H)1, or 0.1 U N-glycosidase F as described previously (
Cell Surface Delivery of the AE1-4 Anion Exchanger
Three different labeling schemes were used to label MDCK cells stably expressing the chicken AE1-4 anion exchanger. (a) The cells were pulsed with 35S-TranslabelTM in methionine-free DME for 15 min, followed by 15 min of chase in methionine-free DME. The cells were then chased an additional 45 min in methionine-free DME in the absence or presence of 1.5 mg/ml chymotrypsin. (b) The cells were incubated continuously with 1.5 mg/ml chymotrypsin during a 15-min preincubation in methionine-free DME, a 15-min pulse with 35S-TranslabelTM, and a 15-min chase (a total of 45 min). (c) The cells were pulsed for 15 min and chased for 1 h at 37°C. The cells were then shifted to 4°C and incubated an additional 45 min in the presence of 1.5 mg/ml chymotrypsin. For each labeling scheme, AE1 immunoprecipitates were prepared from total cell lysates as described above. A fraction of the total cell lysate was analyzed by immunoblotting analysis using a polyclonal antibody raised against chicken erythroid -spectrin (Sigma) that recognizes
-fodrin or a chicken AE1-specific peptide antibody (
Effects of Various Reagents on AE1-4 Anion Exchanger Processing
MDCK cells stably expressing the AE1-4 anion exchanger were pulsed with 35S-TranslabelTM as described above. After a 45-min chase, either 10 µg/ml brefeldin A (BFA), 25 mM ammonium chloride, or 0.4 M sucrose was added to the media, and the cells were incubated until a total chase period of 4 h had elapsed. At each time point, AE1 immunoprecipitates were prepared from total cell lysates as described above. Immunoprecipitates were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.
Quantitative Densitometry
X-ray films from the immunoblotting analyses and the immunoprecipitation experiments were scanned using DeskScan II 2.1 software, and quantitative densitometry was performed using NIH Image. Each value presented in the text represents the average from three independent experiments in which the exposures of individual bands were not yet saturating.
Immunolocalization Analyses
MDCK cells transiently or stably expressing the various chicken AE1 anion exchanger polypeptides were either grown on coverslips or on polycarbonate filters (Costar). The cells were washed in PBS, and fixed by incubating with 1% paraformaldehyde in PBS for 10 min at room temperature. The cells were then permeabilized in PBS containing 0.5% Triton X-100 or with acetone. After permeabilization, cells were incubated with a chicken AE1-specific peptide antibody, followed by incubation with donkey antirabbit (DAR) IgG conjugated to lissamine. After washing, immunoreactive polypeptides were visualized using a Zeiss Axiophot microscope or a BioRad laser scanning confocal microscope. Background levels of staining were observed in control experiments in which the peptide antigen used to generate the AE1-specific peptide antibody were included as a competitor. In some instances, actin microfilaments were stained by incubating cells with 1 µg/ml phalloidin conjugated to fluorescein isothiocyanate (FITC; Sigma). Additional assays also examined whether the localization of AE1 anion exchangers was affected by pre-extracting MDCK cells with PBS containing 0.5% Triton X-100 before fixation in paraformaldehyde.
The kidney from a 17-d-old chicken embryo was isolated and fixed by incubation for 30 min in 2% paraformaldehyde in PBS at 4°C. After fixation, the tissue was rinsed in PBS and incubated overnight at 4°C in 0.5 M sucrose in PBS. The tissue was then frozen in embedding media and 3-µm tissue sections were cut using a cryotome. The tissue sections were postfixed in 1% paraformaldehyde in PBS for 5 min, and extracted in PBS containing 0.5% Triton X-100. The sections were then incubated with a chicken AE1-specific peptide antibody and a monoclonal antibody directed against the H+-ATPase (the kind gift of Dr. S. Gluck). After washing, sections were incubated with donkey antirabbit (DAR) IgG conjugated to lissamine and donkey antimouse (DAM) IgG conjugated to fluorescein. Immunoreactive polypeptides were visualized using a BioRad laser scanning confocal microscope.
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Results |
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Variant Chicken AE1 Anion Exchangers Accumulate in the Basolateral and Apical Membrane Domains of Kidney Epithelial Cells
Previous studies have indicated that three variant AE1 anion exchanger transcripts, AE1-3, AE1-4, and AE1-5, are expressed in chicken kidney (-intercalated cells (
-intercalated cells that have not yet fully differentiated is not known.
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The AE1-specific peptide antibodies used in these immunolocalization studies recognized a sequence present in both AE1-3 and AE1-4. Therefore, we were unable to determine whether the alternative NH2 termini of the chicken kidney AE1 variants affect their localization in the cells of the kidney collecting duct. To investigate whether the variant cytoplasmic domains of these electroneutral transporters may be involved in directing their intracellular localization in kidney epithelial cells, the AE1-3 and AE1-4 anion exchangers were transiently expressed in MDCK cells. After the establishment of a polarized epithelial phenotype, transfected cells were fixed and stained with AE1-specific antibodies, and immunoreactive polypeptides were visualized by confocal microscopy. This analysis revealed that AE1-4 primarily accumulates in the basolateral membrane of transfected MDCK cells (Figure 2B and Figure D). In contrast, AE1-3 accumulates in or near the apical membrane where it exhibits a diffuse pattern of localization (Figure 2A and Figure C). Although AE1-3 is primarily apical in the cells shown in Figure 2 A, this variant transporter also accumulated in an undefined intracellular compartment in some of the transfected cells (data not shown). These data suggest that the alternative NH2-terminal cytoplasmic domains of AE1-3 and AE1-4 serve as signals that direct these variant transporters to opposite membrane domains in this polarized epithelial cell type.
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Alternative NH2 Termini Affect the Posttranslational Modifications and Detergent Solubility Properties of the Kidney AE1 Variants
Fractionation studies have examined whether the differences in intracellular localization of the AE1-3 and AE1-4 anion exchangers correlated with differences in other properties of the variant transporters. Confluent MDCK cells transiently expressing AE1-3, AE1-4, or a point mutant of AE1-4 that eliminates its single N-linked glycosylation site, AE1-4N638T, were lysed with isotonic buffer containing 1% Triton X-100, and separated into soluble and insoluble fractions by centrifugation. Immunoblotting analysis of these fractions with AE1-specific antibodies detected a discrete polypeptide of ~80 kD in AE1-3 transfected cells that was primarily detergent soluble (Figure 3). In contrast to AE1-3 expressing cells, several polypeptides ranging in size from ~97 to ~115 kD were detected in both the detergent soluble and insoluble fractions of cells transfected with AE1-4 (Figure 3). The polypeptides detected in AE1-4 expressing cells are similar in size to the array of AE1 anion exchangers detected in chicken kidney membrane preparations (
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Pulsechase analyses have investigated the mechanisms involved in generating the steady state profile of anion exchangers detected in AE1-3 and AE1-4 transfected cells. MDCK cells stably transfected with AE1-3 or AE1-4 were pulsed with 35S-TranslabelTM for 15 min, and chased for 1 or 4 h. At each time point, the cells were lysed and immunoprecipitates prepared using AE1-specific peptide antibodies were either undigested, digested with endo H, or digested with N-glycosidase. These studies revealed an AE1-3 polypeptide of ~80 kD accumulated in MDCK cells at the end of a 15-min pulse (Figure 4). This species was susceptible to digestion with endo H and N-glycosidase yielding a polypeptide of ~78 kD. The ~80-kD AE1-3 polypeptide underwent no further modification, and was completely turned over by the end of a 4-h chase. Analysis of AE1-4 transfected cells revealed a discrete AE1-4 species of ~97 kD accumulated in MDCK cells after the 15-min pulse (Figure 4). This polypeptide was susceptible to digestion with endo H and N-glycosidase yielding a polypeptide of ~95 kD. Quantitation of three independent experiments has shown that ~50% of the newly synthesized AE1-4 polypeptides are turned over during a 4-h chase. The remaining AE1-4 polypeptides acquired additional modifications that eventually resulted in a diffuse array of polypeptides ranging in size from ~105 to ~112 kD (Figure 4). The ~105- to ~112-kD polypeptides were still sensitive to digestion with N-glycosidase yielding a polypeptide of ~95 kD. However, the N-linked sugar modifications of the ~105- to ~112-kD polypeptides were insensitive to digestion with endo H (Figure 4). This suggests that AE1-4 initially receives high mannose or biantennary hybrid sugars on its single N-linked site. By 1 h after synthesis, ~10% of these modifications are converted to endo Hresistant complex sugars, and by 4 h after synthesis the bulk of the newly synthesized AE1-4 polypeptides have received these more complex endo H-resistant sugar modifications. The addition of these complex sugars to AE1-4 requires the activity of -mannosidase II and N-acetylglucosamine transferase, markers of the medial compartment of the Golgi. This suggests that AE1-4 passes through the medial compartment of the Golgi between 1 and 4 h after synthesis.
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Newly Synthesized AE1-4 Polypeptides Undergo Recycling from the Plasma Membrane to the Golgi
The acquisition of mature N-linked sugars by chicken erythroid AE1 anion exchangers occurs via recycling of newly synthesized polypeptides from the plasma membrane to the Golgi (-fodrin was not susceptible to chymotrypsin digestion at either of the time points (Figure 5 A) indicating the susceptibility of newly synthesized AE1-4 to chymotrypsin was not due to the cells becoming leaky during digestion. These results indicate that essentially all of the newly synthesized polypeptides arrive at the plasma membrane by 1 h after synthesis. Furthermore, these data suggest that the acquisition of endo Hresistant N-linked sugars by AE1-4 between 1 and 4 h of chase primarily occurs via recycling of cell surface polypeptides to the Golgi. Interestingly, when cells were shifted to 4°C after 1 h of chase to block vesicular trafficking and incubated with chymotrypsin for an additional 45 min at 4°C, newly synthesized AE1-4 was resistant to chymotrypsin digestion (Figure 5 A, lane 5). This result together with the fact that newly synthesized polypeptides have undergone cell surface delivery by 1 h of chase suggests that newly synthesized AE1-4 is rapidly internalized after surface delivery.
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Previous studies (
The Variant AE1-4 Anion Exchanger Colocalizes with the Actin Cytoskeleton in MDCK Cells
During the establishment of epithelial polarity, the actin cytoskeleton of MDCK cells undergoes a dramatic reorganization. Cells grown in subconfluent monolayers possess discrete actin stress fibers in the basal membrane. During polarization, these stress fibers become much less prevalent as actin is redistributed for the most part to sites of cellcell contact. To assess the potential role of the actin cytoskeleton in directing the intracellular localization of AE1-3 and AE1-4 in MDCK cells, subconfluent monolayers of cells transfected with these variant transporters were double stained with AE1-specific antibodies and FITC-phalloidin. These studies revealed that the localization of AE1-4 in transfected cells substantially overlapped the distribution of both phalloidin-stained stress fibers in the basal membrane and cortical actin at sites of cellcell contact (Figure 6A, Figure C, and Figure E). In contrast, there was minimal if any overlap of AE1-3 with phalloidin-stained microfilaments in transfected cells (data not shown).
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To determine whether association with filamentous actin could account for the detergent solubility properties of AE1-4 (Figure 3), transfected cells were extracted in situ with 0.5% Triton X-100 before fixation and staining with AE1-specific antibodies and phalloidin. This analysis revealed that AE1-4 still colocalized with phalloidin-stained stress fibers and filamentous actin at cellcell junctions in these preextracted cells (Figure 6B, Figure D, and Figure F) suggesting that AE1-4 is associated with the actin cytoskeleton. In contrast to AE1-4, there was essentially no detectable fluorescence in AE1-3 expressing cells that were detergent extracted before fixation and staining with AE1 antibodies (data not shown).
The Acquisition of Detergent Insolubility by Newly Synthesized AE1-4
Pulsechase studies have investigated the kinetics with which newly synthesized AE1-4 associates with the detergent insoluble fraction of MDCK cells. Subconfluent MDCK cells stably transfected with AE1-4 were pulsed for 15 min and chased for times ranging from 1 to 48 h (Figure 7 A). At each time point, the cells were detergent fractionated and immunoprecipitates were prepared from the detergent soluble and insoluble fractions using AE1-specific antibodies. Quantitation of three independent experiments identical to the one shown in Figure 7 A has revealed that newly synthesized AE1-4 was almost entirely (>95%) detergent soluble after 1 h of chase. By 2 h of chase, ~50% of the AE1-4 polypeptides have acquired complex N-linked sugars, and ~15% of the polypeptides have become detergent insoluble. The pool of polypeptides with complex N-linked sugars does not increase between 2 and 4 h of chase. However, there is a substantial reduction in the amount of the ~97-kD polypeptide during this time period suggesting that most of the polypeptides that have not acquired complex N-linked sugars by 2 h of chase are turned over. In addition, there is a gradual shift of the remaining polypeptides from the detergent soluble to the insoluble pool such that by 8 h of chase ~45% of the polypeptides are detergent insoluble. By 12 h of chase, ~90% of the newly synthesized polypeptides have turned over, and immunoprecipitable AE1-4 was almost undetectable after 48 h of chase. These pulsechase studies suggest that association of newly synthesized AE1-4 with the detergent insoluble fraction does not provide a mechanism for preferentially stabilizing AE1-4 in MDCK cells, since detergent soluble and insoluble AE1-4 turned over at a similar rate at the later time points of the analysis. Similar studies with MDCK cells that had been grown on permeable supports 5 d after cellcell contact yielded identical results (data not shown) indicating the detergent fractionation properties and turnover rate of AE1-4 are not affected by the state of polarity of these epithelial cells.
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The Role of the Actin Cytoskeleton in the Maintenance of AE1-4 Detergent Insolubility Varies as a Function of MDCK Cell Polarity
The in situ extraction studies described above (Figure 6) suggest that the acquisition of detergent insolubility by newly synthesized AE1-4 is at least in part due to its association with the insoluble actin cytoskeleton. To directly assess the role of the actin cytoskeleton in the maintenance of AE1-4 insolubility, transfected cells grown in subconfluent monolayers were treated with the actin-depolymerizing drug, latrunculin B, for 1 h before detergent lysis. Immunoblotting analysis revealed that the detergent insoluble pool of AE1-4 was dramatically reduced in cells treated with this reagent (Figure 7 B). Interestingly, similar studies with cells grown in confluent monolayers revealed that latrunculin B treatment only had a small but reproducible effect on the detergent insolubility of AE1-4 (Figure 7 B). These results indicate that the maintenance of AE1-4 insolubility is almost entirely dependent upon an intact actin cytoskeleton in subconfluent cells. However, as cells develop a more polarized phenotype, alternative mechanisms are primarily responsible for the insolubility of AE1-4.
Identification of Cytoplasmic Tyrosine Residues Required for the Basolateral Accumulation and Cytoskeletal Association of AE1-4
Other investigators have identified dominant cytoplasmic sorting signals that direct the basolateral sorting of integral membrane polypeptides in epithelial cells (21) or 37 (AE1-4
37) amino acids of AE1-4 primarily accumulated in the basolateral membrane of stably transfected MDCK cells (Figure 8B and Figure C). In contrast, AE1-4
53, which lacks the NH2-terminal 53 amino acids of AE1-4, exhibited a diffuse pattern of localization in or near the apical membrane, and accumulated in an undefined intracellular compartment of transfected cells (Figure 8 D). This indicated that sequences between amino acids 37 and 53 of AE1-4 are required for its basolateral accumulation in MDCK cells.
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The region between amino acids 37 and 53 of AE1-4 contains two tyrosine residues, at positions 44 and 47. Other investigators have shown that tyrosine residues are critical for the basolateral sorting of several type I integral membrane proteins in MDCK cells (
To determine whether the basolateral accumulation of the AE1-4 mutants correlated with their ability to colocalize with the actin cytoskeleton, subconfluent monolayers of MDCK cells expressing the mutant transporters were double stained with AE1-specific antibodies and FITC-phalloidin. This analysis revealed that only those mutants that were efficiently targeted to the basolateral membrane in confluent monolayers, including AE1-421, AE1-4
37, AE1-4(Y44A), and AE1-4(Y47A), colocalized both with stress fibers in the basal membrane of subconfluent MDCK cells, and with cortical actin at sites of cell-cell contact (Figure 9). In addition to colocalizing with stress fibers in the basal membrane of cells, AE1-4
37 often accumulated in clusters in the basal membrane that were also stained by FITC-phalloidin (arrows in Figure 9 L). These actin-containing clusters were rarely observed with the other mutant constructs, and they were negative for staining with talin antibodies, a marker for focal adhesions (data not shown). Although AE1-4(Y44A,Y47A) colocalized with cortical actin at sites of cell-cell contact in a small percentage of transfected cells (arrowheads in Figure 9 O), this mutant transporter was never observed to colocalize with stress fibers in the basal membrane of cells. These results suggest that association of AE1-4 with specific elements of the actin cytoskeleton, like efficient basolateral targeting, requires at least one of the tyrosine residues at amino acids 44 and 47 of the polypeptide. At this time, we can not distinguish whether the association of AE1-4 with the detergent insoluble actin cytoskeleton is a prerequisite for or a consequence of the basolateral sorting of this variant transporter in MDCK cells.
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The Acquisition of Detergent Insolubility and the Stability of AE1-4 Is Linked to Efficient Golgi Recycling
To determine whether the NH2-terminal truncation and point mutants of AE1-4 affected the detergent solubility properties and posttranslational modifications of this variant transporter, confluent MDCK cells stably transfected with each construct were detergent lysed, and separated into soluble and insoluble fractions by centrifugation. Immunoblotting analysis of these fractions with AE1-specific antibodies revealed that the AE1-4 mutants could be grouped into three classes. Quantitation of three independent experiments identical to the one shown in Figure 10 has revealed that the first class of mutants, AE1-421 and AE1-4(Y44A), was similar to wild-type AE1-4 in terms of the percent of the steady state population that was detergent insoluble (~40%), and the percent of the molecules that acquired complex N-linked sugars (~95%). The second class of mutants, AE1-4
37 and AE1-4(Y47A), exhibited partial defects in their posttranslational processing (Figure 10). The percentage of AE1-4(Y47A) polypeptides that acquired complex N-linked sugars (~45%) was reduced relative to that observed with AE1-4, and both AE1-4
37 and AE1-4(Y47A) were substantially reduced in their ability to associate with the detergent insoluble fraction of MDCK cells (~5% of total protein). Interestingly, unlike AE1-4, most of the AE1-4(Y47A) polypeptides that associated with the detergent insoluble fraction possessed only simple N-linked sugars suggesting that AE1-4(Y47A) may acquire detergent insolubility by a mechanism distinct from that used by AE1-4. Finally, AE1-4
53 failed to acquire complex N-linked sugars and was entirely detergent soluble (Figure 10). A similar phenotype was observed for AE1-4(Y44A,Y47A) suggesting that Golgi recycling and the acquisition of detergent insolubility, like efficient basolateral targeting, requires at least one of the tyrosines at amino acids 44 and 47 of AE1-4.
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Pulsechase analyses have examined whether mutations that alter the posttranslational processing of AE1-4 affect the kinetics with which these polypeptides acquire complex N-linked sugars as well as their stability. These studies revealed that each mutant acquired N-linked modifications that were sensitive to endo H during a 15-min pulse (Figure 11). During the chase period, AE1-437 and AE1-4(Y47A) acquired additional sugar modifications that were resistant to endo H. However, the percentage of newly synthesized AE1-4
37 and AE1-4(Y47A) polypeptides that acquired complex N-linked sugars by 4 h of chase was substantially lower than that observed for AE1-4 (compare Figure 4 and Figure 11). These data indicate that tyrosine 47 and sequences in the NH2-terminal 37 amino acids of AE1-4 are both necessary for efficient Golgi recycling of AE1-4. The inefficient recycling of AE1-4
37 and AE1-4(Y47A) may account for the reduced ability of these mutant transporters to associate with the detergent insoluble fraction of MDCK cells. Similar analyses with AE1-4
53 and AE1-4(Y44A,Y47A) indicated that these mutants acquired no additional modifications after the pulse, and like AE1-3, they were almost completely turned over by 4 h of chase (Figure 11). The pulsechase and immunoblotting studies indicate that Golgi recycling requires at least one of the tyrosine residues at amino acids 44 and 47 of AE1-4. Furthermore, these data suggest that the stable accumulation of newly synthesized AE1 in this kidney epithelial cell type is completely dependent upon its ability to undergo recycling from the plasma membrane to the Golgi.
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Discussion |
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The alternative NH2-terminal sequences of the variant chicken kidney AE1 anion exchangers direct the intracellular trafficking of these membrane transporters in transfected MDCK cells. The AE1-4 variant accumulates in the basolateral membrane of transfected cells, while the AE1-3 variant, which lacks the 63 NH2-terminal cytoplasmic amino acids of AE1-4, primarily accumulates in the apical membrane. In addition to targeting AE1-4 to the basolateral membrane of transfected cells, the NH2-terminal 63 amino acids of AE1-4 are also required for recycling of this membrane transporter from the plasma membrane to the Golgi. Previous studies have shown that the association of chicken erythroid AE1 anion exchangers with cytoskeletal ankyrin occurs during recycling of newly synthesized polypeptides from the plasma membrane to the Golgi (
The sorting of type I membrane proteins to the basolateral membrane of kidney epithelial cells has been shown to be dependent upon dominant cytoplasmic sorting signals that direct the vectorial transport of newly synthesized proteins from the TGN to the basolateral membrane (
Either of the tyrosine residues at amino acids 44 and 47 of AE1-4 is sufficient to direct the basolateral accumulation of this type III membrane protein. However, there are differential requirements for these tyrosine residues for Golgi recycling. Substitution of an alanine for tyrosine 47 reduces the efficiency of Golgi recycling of AE1-4 by ~50%, while a similar substitution at tyrosine 44 has no significant effect on recycling. Tyrosine 47 resides within the sequence, YVEL, that is conserved among all characterized AE1 anion exchangers except for chicken AE1-3 (, where X is any amino acid and
is a hydrophobic residue. This sequence motif has been shown to associate with the µ subunits of the AP1, AP2, and AP3 adaptor complexes (
motif is critical for the ability of this sequence to serve as a sorting signal (
The molecular basis of detergent insolubility of AE1 in MDCK cells varies as a function of polarity. Immunolocalization analyses coupled with studies using the actin-depolymerizing drug, latrunculin B, have indicated that the detergent insolubility of AE1-4 in subconfluent, nonpolarized MDCK cells is almost entirely dependent upon an intact actin cytoskeleton. Although the exact role of the actin cytoskeleton in directing the intracellular trafficking of AE1-4 is unclear, the ability of mutant AE1 constructs to accumulate in the basolateral membrane of this cell type correlated with their ability to colocalize with phalloidin-stained stress fibers. Immunolocalization studies with AE1-437 have further suggested that the organization of the actin cytoskeleton is modified as a consequence of associating with this AE1-4 mutant. Filamentous actin colocalizes with AE1-4
37 in large clusters in the basal membrane of subconfluent MDCK cells. This reorganization of actin, which was not observed with wild-type AE1-4 or the other mutant constructs, may contribute to the slow rate at which AE1-4
37 recycles to the Golgi and acquires mature N-linked sugars.
As cells establish a more polarized epithelial phenotype, the maintenance of detergent insolubility of AE1-4 is only partially dependent upon the actin cytoskeleton. The alternative interactions responsible for the maintenance of AE1-4 insolubility in polarized MDCK cells are not known. However, the fact that chicken erythroid AE1 anion exchangers acquire detergent insolubility in part through their association with cytoskeletal ankyrin (
Other investigators have hypothesized that phosphorylation of the tyrosine residue in the YVEL tetrapeptide in the cytoplasmic domain of skate erythroid AE1 may be involved in regulating its association with elements of the cytoskeleton, such as ankyrin (
Our analyses have suggested that a subset of the newly synthesized AE1-3 anion exchanger is directed to the apical membrane of transfected MDCK cells. However, the rapid turnover of this polypeptide in this epithelial cell type prevents high levels of this variant transporter from accumulating in the apical membrane domain. This rapid rate of turnover may account for the fact that polypeptides with the predicted molecular mass of AE1-3 are not detected in chicken kidney membrane preparations by immunoblotting analysis (
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Footnotes |
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1 Abbreviations used in this paper: BFA, brefeldin A; endo H, endoglycosidase H.
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Acknowledgements |
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This research was supported by a grant from the National Chapter of the American Heart Association (96-008610). We thank S. Ghosh and F.C. Dorsey for their critical evaluation of the manuscript. We thank Dr. S. Gluck for generously providing monoclonal antibodies directed against the H+-ATPase.
Submitted: 19 August 1999
Revised: 11 October 1999
Accepted: 6 November 1999
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