Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells

Tracy L. Adair-Kirk*, Frank C. Dorsey and John V. Cox{ddagger}

Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, Tennessee 38163, USA
* Present address: Department of Medicine, Washington University School of Medicine at Barnes-Jewish Hospital, St Louis, MO 63110, USA

{ddagger} Author for correspondence (e-mail: jcox{at}utmem.edu)

Accepted 6 November 2002


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 Materials and Methods
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 References
 
AE1/Fc receptor chimeras have been used to define the sequences that direct the basolateral sorting, recycling and cytoskeletal association of the chicken AE1-4 anion exchanger in MDCK cells. These analyses revealed that amino acids 1-63 of AE1-4 were sufficient to redirect a cytoplasmic tailless murine IgG FcRII B2 receptor from the apical to the basolateral membrane of MDCK cells, where Fc1-63 associated with elements of the actin cytoskeleton. In contrast to Fc1-63, chimeras containing amino acids 1-37 (Fc1-37) or 38-63 (Fc38-63) of AE1-4 accumulated in intracellular membrane compartments that overlapped late endosomes and the trans-Golgi network (TGN), respectively. Internalization assays indicated that the patterns of localization observed for Fc1-37 and Fc38-63 resulted from the recycling of these chimeras from the cell surface. These assays further indicated that Fc1-37 and Fc38-63 each possess a basolateral sorting activity. Mutagenesis studies revealed that the endocytic and basolateral sorting activities in Fc1-37 are dependent upon serine 25, which is located in a sequence similar to a sorting signal in the polymeric immunoglobulin receptor. In addition, the sorting activities associated with Fc38-63 were dependent upon tyrosine 47 and leucine 50. These residues resided within the sequence, YVEL, which matches the YXX{Phi} motif (where X is any amino acid and {Phi} is a hydrophobic residue) that functions as an endocytic and TGN recycling signal for other membrane proteins. Our data indicate that amino acids 1-63 of AE1-4 contain sorting and cytoskeletal binding activities that account for most of the properties previously associated with AE1-4 in MDCK cells. Furthermore, the alternative localization patterns exhibited by chimeras containing various combinations of these activities suggest that interplay between these cytoplasmic activities is critical for specifying AE1-4 localization in epithelial cells.

Key words: Sorting signal, Actin, Recycling, TGN, Late endosomes


    Introduction
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 Introduction
 Materials and Methods
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 References
 
Biochemical studies have revealed novel trafficking pathways for variant chicken AE1 anion exchangers in chicken embryonic erythroid cells (Ghosh et al., 1999Go) and in transfected Madin Darby canine kidney (MDCK) cells (Adair-Kirk et al., 1999Go). In these cell types, newly synthesized AE1 anion exchangers with simple N-linked sugar modifications are delivered to the plasma membrane and subsequently undergo recycling to the Golgi where they receive complex N-linked sugars (Adair-Kirk et al., 1999Go; Ghosh et al., 1999Go). A similar non-conventional trafficking pathway has recently been described for a newly synthesized cystic fibrosis transmembrane conductance regulator, which passes through late endosomes prior to the acquisition of mature N-linked sugars (Yoo et al., 2002Go). These observations suggest that specific subsets of membrane proteins can either be sequestered from Golgi-modifying enzymes in their initial passage through the secretory pathway or alternatively these proteins initially bypass the Golgi en route to later compartments in the secretory pathway.

Although the precise sorting pathway followed by newly synthesized chicken AE1 anion exchangers has not been defined, previous studies have shown that the variant chicken kidney AE1-4 anion exchanger has the capacity to undergo recycling to the Golgi following delivery to the plasma membrane. By contrast, the AE1-3 variant, which lacks the N-terminal 63 amino acids of AE1-4, does not recycle to the Golgi (Adair-Kirk et al., 1999Go). Mutagenesis studies have further shown that a tyrosine-dependent sorting signal within the N-terminal 63 amino acids of AE1-4 is necessary both for Golgi recycling and for efficient basolateral sorting of this variant anion exchanger in transfected MDCK cells (Adair-Kirk et al., 1999Go). The tyrosines at amino acids 44 and 47 of AE1-4 were critical for these sorting activities and for the association of AE1-4 with elements of the actin cytoskeleton. Tyrosine 47 of AE1-4 resides within the sequence YVEL, which is conserved among all characterized AE1 anion exchangers except for chicken AE1-3 (Adair-Kirk et al., 1999Go) and the mammalian kidney AE1 variants (Brosius et al., 1989Go; Kollert-Jons et al., 1993Go; Kudrycki and Shull, 1993Go). This peptide matches the sequence motif, YXX{Phi}, where X is any amino acid and {Phi} is a hydrophobic residue. This motif associates with adaptor complexes (Ohno et al., 1996Go; Dell'Angelica et al., 1997Go; Aguilar et al., 2001Go; Boehm and Bonifacino, 2001Go) and functions as an endocytic (Collawn et al., 1990Go) and trans-Golgi network (TGN) recycling (Wong and Hong, 1993Go) signal for several membrane proteins. The AE1-3 variant and AE1-4 mutants that lack this tyrosine-dependent sorting signal are rapidly degraded in MDCK cells, suggesting that the trafficking events directed by this signal are necessary for the stable accumulation of AE1 in this epithelial cell type.

To further define the sequence requirements for the sorting and cytoskeletal binding activities associated with the AE1-4 variant in transfected MDCK cells, we have fused various portions of the N-terminal cytoplasmic tail of AE1-4 to a cytoplasmic tailless version of the murine IgG FcRII B2 receptor. Studies with these chimeras revealed that multiple sorting activities reside within amino acids 1-63 of AE1-4. Amino acids 38-63 of AE1-4 were sufficient to direct efficient basolateral sorting of a chimeric receptor in MDCK cells. This region of AE1-4 was also sufficient to direct chimeric receptors from the basolateral membrane to the TGN. Another activity, which targeted chimeric receptors from the cell surface to late endosomes, was characterized within amino acids 1-37 of AE1-4. This region of AE1-4 was also capable of directing basolateral sorting. In addition to these sorting signals, amino acids 38-63 of AE1-4 were sufficient to mediate the association of chimeric receptors with the actin cytoskeleton. The alternative localization patterns exhibited by AE1/Fc receptor chimeras containing various combinations of these cytoplasmic activities suggest that interplay between these activities is critical for specifying the intracellular distribution of AE1-4 in epithelial cells.


    Materials and Methods
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 Materials and Methods
 Results
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 References
 
Construction of FcR/AE1 chimeras
A cytoplasmic tailless version of the murine IgG FcRII B2 receptor was generated using the site-directed mutagenesis kit Altered Sites II (Promega). The mutagenic oligonucleotide used for this analysis contained an AflII restriction site, which maintains a lysine at position 208 of the Fc receptor, followed by a stop codon (Fc- in Fig. 1).



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Fig. 1. Sequence of AE1/Fc receptor chimeras. The sequence of the AE1/Fc receptor chimeras is illustrated. The putative boundary between the cytoplasmic domain and the transmembrane domain of these proteins is shown. The numbering at the top refers to the amino-acid sequence at the N-terminus of AE1-4. The sequences within amino acids 1-63 of AE1-4 that are homologous to known sorting signals are underlined. Residues within this region of AE1-4 that are identical to amino acids in the poly Ig receptor sorting signal are marked with a plus. The spacing between the arginine and serine in this poly Ig-like sorting signal is the same as that in the poly Ig receptor. The asterisk at the end of each sequence corresponds to a stop codon.

 

Various portions of the N-terminal cytoplasmic tail of AE1-4 were amplified by the polymerase chain reaction (PCR) and fused to the cytoplasmic tailless Fc receptor that was cloned in the pcDNA3 mammalian expression vector (Invitrogen). Sense oligonucleotides corresponding to nucleotides 26-40 or 137-151 of the AE1-4 anion exchanger cDNA (Adair-Kirk et al., 1999Go) were generated. These oligonucleotides were flanked at their 5' ends by an AflII restriction site. Antisense oligonucleotides corresponding to nucleotides 122-136 or 200-214 that were flanked at their 3' ends by a stop codon followed by a XbaI site were also generated. These oligonucleotides were used in various combinations to amplify the regions of AE1-4 illustrated in the chimeras in Fig. 1. cDNAs encoding wild-type AE1-4, AE1-4Y44A, AE1-4Y47A or AE1-4Y44AY47A (Adair-Kirk et al., 1999Go) were used as templates for these PCR reactions. For certain reactions, antisense oligonucleotides that introduced an alanine for serine 25 or an alanine for leucine 50 were used for the amplifications. The PCR reactions were performed using Pfu polymerase (Stratagene), and the amplification products were digested with AflII and XbaI restriction enzymes (Gibco) and ligated to the cytoplasmic tailless Fc receptor utilizing the AflII site in Fc- and an XbaI site in the polylinker of pcDNA3. All chimeras were confirmed by DNA sequence analysis.

Cell culture and stable transfection
MDCK cells were maintained in Dulbecco's modified Eagle's media (DMEM) 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. Some analyses were performed with MDCK cells transiently expressing AE1/Fc receptor chimeras that were introduced into the cells using the lipid-based transfection reagent, Effectene (Qiagen). Alternatively, cell lines stably expressing AE1/Fc receptor chimeras were established by growing MDCK cells that were transfected by the calcium-phosphate method in the presence of 600 µM G418. In all instances, similar results were observed in stably and transiently transfected cells.

Immunolocalization analysis
Cells grown either on coverslips or on Transwell filters (Costar) were washed in phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde in PBS and permeabilized by incubation in acetone. Following permeabilization, the cells were washed with PBS and incubated with the rat 2.4G2 anti-Fc receptor monoclonal antibody (Matter et al., 1992Go). After washing, the cells were incubated with donkey anti-rat IgG conjugated to lissamine (Jackson Immunoresearch) and phalloidin conjugated to fluorescein isothiocyanate (FITC). Alternatively, cells were incubated with the rat anti-Fc receptor monoclonal antibody and either a rabbit polyclonal antibody directed against furin (Affinity Bioreagents) or a mouse monoclonal antibody directed against the cation-independent mannose 6-phosphate receptor (Affinity Bioreagents). Following washing, the cells were incubated with donkey anti-rat IgG conjugated to lissamine, and either donkey anti-rabbit IgG conjugated to FITC (Jackson Immunoresearch) or goat anti-mouse IgG conjugated to FITC (Jackson Immunoresearch). In each instance, the cells were washed in PBS and the localization of fluorescently labeled proteins was visualized using either a Zeiss LSM 510 laser scanning microscope or a Zeiss Axiophot epifluorescent microscope.

Cell-surface binding and internalization assays
MDCK cells stably expressing AE1/Fc receptor chimeras were grown on coverslips or Transwell filters. In each instance, the cells were incubated with the anti-Fc receptor antibody, which recognizes an extracellular epitope of the receptor, for 1 hour at 4°C. Following extensive washing with cold DMEM to remove unbound antibody, pre-warmed media containing 5% fetal calf serum was added to the cells and they were incubated for various times at 37°C. At each time point, the cells were fixed by incubation in PBS containing 3% paraformaldehyde and permeabilized with PBS containing 0.5% Triton X-100 (PBST). The cells were then incubated with the rabbit anti-furin or the mouse anti-mannose 6-phosphate receptor antibodies in PBST. The cells were again washed and incubated with donkey anti-rat IgG conjugated to lissamine and either donkey anti-rabbit IgG conjugated to FITC or goat anti-mouse IgG conjugated to FITC. Following washing, immunoreactive polypeptides were visualized on a Zeiss LSM 510 laser-scanning microscope.

Experiments were performed to control for the possibility that the immunofluorescence profiles observed in internalization assays resulted from the dissociation of the Fc receptor antibody from the AE1/Fc chimeras following endocytosis. MDCK cells expressing the chimeras were incubated with the Fc receptor antibody as described above and shifted to 37°C for 45 minutes. The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch). Following washing, the cells were incubated with the Fc receptor antibody directly conjugated to Alexa 488 (Molecular Probes) to label the entire cellular pool of the chimera. The cells were again washed and immunoreactive polypeptides were visualized by confocal microscopy. This analysis revealed that the fluorescence derived from surface-labeled chimeras only accumulated in intracellular compartments that were also labeled by the directly conjugated Fc receptor antibody (data not shown), strongly suggesting that surface-bound antibodies did not dissociate from the chimeras following internalization into the cell.


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 Introduction
 Materials and Methods
 Results
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 References
 
The N-terminal 63 amino acids of AE1-4 are sufficient to direct basolateral sorting
The variant chicken AE1-4 anion exchanger primarily accumulates in the basolateral membrane of transfected MDCK cells, whereas the AE1-3 variant accumulates in the apical membrane and in an undefined intracellular compartment (Adair-Kirk et al., 1999Go). This result indicates that the N-terminal 63 amino acids of AE1-4, which are absent in AE1-3, are necessary for the basolateral accumulation of AE1-4 in this polarized epithelial cell type. Additional studies have shown that the N-terminal 63 amino acids of AE1-4 are necessary for the efficient recycling of this variant transporter from the plasma membrane to the Golgi and for its association with the actin cytoskeleton of MDCK cells (Adair-Kirk et al., 1999Go). To determine whether sequences at the N-terminus of AE1-4 are both necessary and sufficient to direct the intracellular trafficking and cytoskeletal association of this variant transporter, we have fused various portions of its N-terminal cytoplasmic tail to a cytoplasmic tailless version of the murine IgG FcRII B2 receptor (Fig. 1). The cytoplasmic tailless Fc receptor, Fc-, was generated by introducing a stop codon after lysine 208 of the polypeptide (Fig. 1). As shown previously by other investigators (Matter et al., 1992Go), a Fc receptor truncated after this residue is sorted to the apical membrane of MDCK cells (Fig. 2A).



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Fig. 2. Amino acids 1-63 of AE1-4 can direct a cytoplasmic tailless mutant of the Fc receptor to the basolateral membrane of MDCK cells. Confluent MDCK cells stably expressing Fc- (A), Fc1-63 (B), Fc1-63Y44A (C), Fc1-63Y47A (D) or Fc1-63Y44A, Y47A (E) were fixed and incubated with the rat monoclonal antibody specific for the Fc receptor and phalloidin-FITC. The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine, and the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center (B, C and D) or near the apical surface (A and E) of the cells. Regions that are yellow indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image of the transfected cells. The bar in each xy image is 10 µm.

 

MDCK cells expressing a chimera that fused amino acids 1-63 of AE1-4 to this cytoplasmic tailless Fc receptor (FcR1-63, Fig. 2B) were fixed and double stained with a Fc-receptor-specific antibody and phalloidin. Confocal analysis revealed that this chimera primarily accumulated in the basolateral membrane and to a lesser extent in an intracellular membrane compartment of polarized MDCK cells. This result indicated that amino acids between 1 and 63 of AE1-4 are sufficient to direct basolateral sorting in this epithelial cell type.

Efficient basolateral sorting of AE1-4 in MDCK cells is dependent upon the cytoplasmic tyrosine residues at amino acids 44 and 47 (Adair-Kirk et al., 1999Go). To investigate the role of these residues in the basolateral sorting of Fc1-63, point mutants were generated that substituted an alanine for each of the tyrosine residues in this chimera separately or together (Fig. 1). Substituting an alanine for tyrosine 44, Fc1-63Y44A, resulted in a chimera that still primarily accumulated in the basolateral membrane of transfected cells (Fig. 2C). By contrast, substituting an alanine for tyrosine 47, Fc1-63Y47A, resulted in a polypeptide that accumulated both in the basolateral and apical membrane (Fig. 2D), whereas the double mutant, Fc1-63Y44AY47A, accumulated exclusively in the apical membrane of transfected cells (Fig. 2E). These data indicated that tyrosines 44 and 47 were necessary for the basolateral sorting activity that resides within amino acids 1-63 of AE1-4. Furthermore, the results suggested that tyrosine 47, which is located within the sequence YVEL (underlined in Fig. 1), was critical for efficient basolateral sorting of this chimera. Experiments described below will further address the activities associated with the YVEL tetrapeptide.

The FcR1-37 and FcR38-63 chimeras are delivered to the plasma membrane and recycled to distinct intracellular membrane compartments
The acquisition of mature N-linked sugars by newly synthesized AE1-4 anion exchangers is dependent upon recycling of these transporters from the plasma membrane to the Golgi (Adair-Kirk et al., 1999Go). The mutant anion exchangers, AE1-4{Delta}37 and AE1-4Y47A, are partially defective in this recycling process (Adair-Kirk et al., 1999Go), suggesting that the sequence between amino acids 1-37 and tyrosine 47 of AE1-4 are both necessary for efficient internalization from the plasma membrane and subsequent Golgi recycling. Interestingly, chimeric receptors containing amino acids 1-37, Fc1-37, or 38-63, Fc38-63 (Fig. 1), of AE1-4 accumulated in intracellular membrane compartments in transfected MDCK cells (Fig. 3). Confocal analyses revealed that Fc1-37 accumulated in membrane vesicles that partially overlapped the distribution of the cation-independent mannose 6-phosphate receptor, which primarily accumulates in late endosomes (Fig. 3A-C). By contrast, Fc38-63 was restricted to a perinuclear membrane compartment that substantially overlapped the distribution of the TGN marker, furin (Fig. 3D-F). Additional experiments revealed that there was no overlap in the localization profiles of Fc1-37 and furin, nor was there any significant overlap in the localization profiles of Fc38-63 and the mannose 6-phosphate receptor (data not shown). The observed accumulation of Fc1-37 and Fc38-63 in intracellular membrane compartments may have been the result of their specific retention in these compartments. Alternatively, amino acids 1-37 and 38-63 of AE1-4 may each be able to direct the surface delivery and subsequent recycling of these chimeric proteins to these intracellular compartments.



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Fig. 3. Amino acids 1-37 and 38-63 of AE1-4 target a cytoplasmic tailless Fc receptor to distinct intracellular membrane compartments. MDCK cells stably expressing Fc1-37 (A-C) or Fc38-63 (D-F) were fixed and incubated with rat monoclonal antibodies specific for the Fc receptor (A and D), a mouse monoclonal antibody specific for the mannose 6-phosphate receptor (B) or a rabbit polyclonal antibody specific for furin (E). The cells were washed and incubated with donkey anti-rat IgG conjugated to lissamine (A and D), goat anti-mouse IgG conjugated to FITC (B) or donkey anti-rabbit IgG conjugated to FITC (E). The localization of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The merged images illustrate significant overlap in the distribution of Fc 1-37 and the mannose 6-phosphate receptor (C) and Fc38-63 and furin (F). Bar in A and D, 10 µm.

 

To distinguish between these possibilities, internalization assays were performed. Subconfluent cells expressing the chimeras were incubated with the Fc receptor antibodies, which recognize an extracellular epitope on the receptor. The incubation was carried out at 4°C to prevent endocytosis. The cells were then shifted to 37°C, and the fate of surface-labeled chimeras was followed over time by confocal microscopy. Prior to shifting to 37°C, labeled Fc1-37 and Fc38-63 were present on the cell surface where they did not overlap markers for intracellular membrane compartments (data not shown). Although some of the surface-labeled Fc1-37 was internalized from the plasma membrane 15 minutes after the shift to 37°C, a substantial fraction of Fc1-37 still resided on the cell surface at this time point (arrows in Fig. 4A). By 45 minutes, however, the majority of surface-labeled Fc1-37 had internalized and accumulated in a compartment that significantly overlapped the distribution of the mannose 6-phosphate receptor (Fig. 4). Unlike Fc1-37, the bulk of surface-labeled Fc38-63 had internalized 15 minutes after the shift to 37°C and its localization partially overlapped the distribution of the TGN marker, furin (Fig. 5). Forty-five minutes after the shift, the localization profiles of Fc38-63 and furin were very similar (Fig. 5). These data indicated that sequences between amino acids 1-37 and 38-63 of AE1-4 can independently direct the internalization of chimeric proteins from the cell surface. These results may account for the observation that AE1-4{Delta}37 and AE1-4Y47A are only partially defective in recycling, since one internalization signal is presumably functional in each.



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Fig. 4. Fc1-37 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of the mannose 6-P receptor. MDCK cells stably expressing Fc1-37 were incubated with the anti-Fc receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a mouse monoclonal directed against the mannose 6-phosphate receptor (M-6-P). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and goat anti-mouse IgG conjugated to FITC. Following washing, the localization of Fc1-37 (A,B) and the mannose 6-phosphate receptor (C,D) was visualized on a Zeiss LSM 510 laser-scanning microscope. The merged images showing the overlap of Fc1-37 and the mannose 6-phosphate receptor are shown in E and F. The arrows in A indicate the Fc1-37 chimeras that still reside on the cell surface following a 15 minute incubation at 37°C. Bar (A,B), 10 µm.

 


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Fig. 5. Fc38-63 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of furin. MDCK cells stably expressing Fc38-63 were incubated with the anti-Fc receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a rabbit polyclonal directed against furin. The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and donkey anti-rabbit IgG conjugated to FITC. Following washing, the localization of Fc38-63 (A,B) and furin (C,D) was visualized on a Zeiss LSM 510 laser scanning microscope. The merged images showing the overlap of Fc38-63 and furin are shown in E and F. Bar (A,B), 10 µm.

 

The internalization of Fc1-37 and Fc38-63 from the cell surface was not induced by antibody binding to the chimeras, since studies with Fc1-63 revealed that this chimera remained on the cell surface throughout the time course of similar assays (data not shown). Although surface labeled Fc1-63 did not internalize, it did undergo redistribution on the cell surface to sites of cell-cell contact. This redistribution of Fc1-63 mimicked the redistribution of phalloidin-stained microfilaments in these cells, suggesting a critical role for the actin cytoskeleton in maintaining the cell-surface localization of this chimeric receptor.

Identification of amino acids necessary for the sorting activities associated with amino acids 1-37 and 38-63 of AE1-4
Internalization assays performed with polarized MDCK cells expressing Fc38-63 yielded results essentially identical to those shown in Fig. 5 (data not shown). However, incubation of the Fc receptor antibody at 4°C with the apical and basolateral surfaces of cells grown on permeable supports revealed that the surface population of Fc38-63 accumulated exclusively in the basolateral membrane of this polarized epithelial cell type (Fig. 6A). This result indicates that sequences within amino acids 38-63 of AE1-4 are not only sufficient to direct internalization from the plasma membrane and subsequent transport to the TGN, but are also sufficient to direct basolateral sorting in MDCK cells. The fact that tyrosines 44 and 47 of AE1-4 are necessary for both the basolateral sorting and Golgi recycling activities of this transporter in MDCK cells (Adair-Kirk et al., 1999Go) prompted us to examine the role of these residues in the trafficking of Fc38-63. Mutants were generated that substituted an alanine for each of the tyrosines in this chimera. MDCK cells expressing the mutant chimeras were fixed and stained with the Fc receptor antibody and phalloidin. This analysis revealed that Fc38-63Y44A (data not shown) exhibited a steady-state pattern of localization indistinguishable from Fc38-63 (Fig. 6B). Longer exposure of the image in Fig. 6B again revealed that the surface population of Fc38-63 was exclusively basolateral (data not shown). By contrast, Fc38-63Y47A accumulated in both the basolateral and apical membranes of polarized MDCK cells and to a lesser extent in an intracellular membrane compartment (Fig. 6C). This result suggested that tyrosine 47 was critical both for efficient basolateral sorting and for efficient endocytosis of Fc38-63 following cell-surface delivery. To directly test whether tyrosine 47 was critical for endocytosis, internalization assays identical to those described above were performed with Fc38-63Y47A. This analysis revealed that surface-labeled Fc38-63Y47A was almost entirely retained in the plasma membrane throughout the time course of the assay (data not shown), illustrating the importance of this residue for efficient endocytosis. This result provides additional evidence that the uptake of surface-labeled chimeras in our internalization assay does not simply occur as a consequence of antibody binding. Furthermore, the very slow rate of internalization of Fc38-63Y47A suggests that the surface distribution of this chimera primarily resulted from sorting events that occurred at the level of the TGN.



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Fig. 6. The surface distribution and steady-state localization of wild-type and mutant Fc38-63 constructs in polarized MDCK cells. Polarized MDCK cells stably expressing Fc38-63 (A,B) or Fc38-63Y47A (C) or transiently expressing Fc38-63Y47L (D) or Fc38-63L50A (E) were grown on Transwell filters. The intact cells were either incubated with the Fc-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the Fc-receptor-specific antibody (B-E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-E) and phalloidin conjugated to FITC (B-E). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center of the cells. Regions that are yellow in B-E indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.

 

The hydrophobic residue in the YXX{Phi} motif is critical for the ability of this motif to serve as a sorting signal (Collawn et al., 1990Go; Wong and Hong, 1993Go) and for its ability to associate with adaptins (Ohno et al., 1996Go; Dell'Angelica et al., 1997Go; Aguilar et al., 2001Go; Boehm and Bonifacino, 2001Go). To assess the contribution of the leucine residue in the YVEL50 sequence of AE1-4 (Fig. 1) to the sorting activities contained within amino acids 38-63, we mutated this residue to an alanine. This amino-acid substitution did not alter the basolateral sorting activity of Fc38-63, as the surface population of Fc38-63L50A primarily accumulated in the basolateral membrane of polarized MDCK cells (Fig. 6E). Although this chimera could still be detected in a sub-apical compartment of transfected cells, the majority of Fc38-63L50A resided on the cell surface. The fact that efficient basolateral sorting was unaffected by substituting an alanine for leucine 50, whereas the accumulation of this chimera in intracellular compartments was substantially reduced, suggested that there were different sequence requirements for the basolateral sorting and endocytic activities of Fc38-63. However, it must be pointed out that the reduced intracellular accumulation of Fc38-63L50A could be due to a reduced rate of endocytosis as well as an increased rate of recycling to the basolateral membrane following internalization. Whether one or both of these possibilities contributes to the localization of this chimera in MDCK cells is not known.

The data described above suggested that the YVEL peptide of AE1-4 comprises a YXX{Phi} motif that is critical for the sorting activities associated with this transporter. The fact that alanine is a relatively hydrophobic residue in many of the algorithms used to predict hydrophobicity further suggested the possibility that the residual basolateral sorting activity associated with Fc38-63Y47A (Fig. 6C) resulted from the creation of a YXX{Phi} sequence surrounding tyrosine 44. To test whether a more hydrophobic residue at amino acid 47 would fully suppress the sorting defects associated with Fc38-63Y47A, we replaced tyrosine 47 with a leucine. Confocal analysis revealed that this mutant, Fc38-63Y47L (Fig. 6D), exhibited a localization profile very similar to Fc38-63Y47A (Fig. 6C). The failure of this mutant to undergo efficient basolateral sorting and subsequent internalization from the plasma membrane demonstrates that the sequence surrounding the tyrosine and leucine residues in the YXXL tetrapeptide of AE1-4 is critical for determining its sorting activity.

As was observed for Fc38-63, internalization assays with polarized MDCK cells expressing Fc1-37 yielded results very similar to those shown in Fig. 4 (data not shown). Surprisingly, incubation of the Fc receptor antibody at 4°C with the apical and basolateral surfaces of cells revealed that a significant percentage of the surface population of Fc1-37 accumulated in the basolateral membrane of polarized cells (Fig. 7A). This suggested that amino acids 1-37 of AE1-4 contained an inefficient basolateral sorting signal, as well as sequences sufficient to direct the internalization of chimeric receptors from the cell surface to late endosomes. A comparison of the sequence within this region to other known sorting signals revealed that amino acids 17-25 (underlined in Fig. 1) are similar to a cytoplasmic sorting signal that is involved in both the basolateral sorting and transcytosis of the poly Ig receptor (Casanova et al., 1990Go; Casanova et al., 1991Go). The residues in Fig. 1 that are marked with a plus are identical to amino acids in this poly Ig receptor signal. The serine in the poly Ig receptor signal is required for the transcytosis of the receptor from the basolateral to the apical membrane of transfected MDCK cells (Casanova et al., 1990Go). To determine whether the serine in the putative sorting signal in Fc1-37 was involved in directing the intracellular trafficking of this chimera, a mutant was generated that substituted an alanine for serine, Fc1-37S25A. Immunolocalization analyses revealed that unlike the steady-state localization profile of Fc1-37 (Fig. 7B), Fc1-37S25A accumulated exclusively in the apical membrane of transfected cells (Fig. 7C). This pattern of localization suggested that serine 25 was necessary for both the basolateral sorting and endocytic activities that resided within amino acids 1-37. Again, to directly test the role of this residue in endocytosis, internalization assays were performed with Fc1-37S25A. This analysis indicated that the surface population of this chimera was retained in the apical membrane during the time course of the assay (data not shown), demonstrating that serine 25 is critical for efficient endocytosis.



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Fig. 7. The surface distribution and steady-state localization of wild-type and mutant Fc1-37 constructs in polarized MDCK cells. Polarized MDCK cells transiently (A,C) or stably (B) expressing Fc1-37 (A and B) or Fc1-37S25A (C) were grown on Transwell filters. The intact cells were either incubated with the Fc-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the Fc-receptor-specific antibody (B,C). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-C) and phalloidin conjugated to FITC (B and C). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is either near the center (A,B) or near the apical surface (C) of the cells. The surface population of Fc1-37 could be detected in a longer exposure of the image in B. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.

 

Similar sequence requirements for the basolateral sorting and the cytoskeletal association of AE1-4 in MDCK cells
Previous analyses had shown that AE1-4 colocalizes both with actin stress fibers underlying the basal membrane of subconfluent MDCK cells and with cortical actin at sites of cell-cell contact (Adair-Kirk et al., 1999Go). To determine whether the AE1/Fc chimeras that accumulate in the basolateral membrane of polarized MDCK cells exhibit a similar capacity to colocalize with elements of the actin cytoskeleton, subconfluent MDCK cells expressing Fc1-63 (Fig. 8A-C) were double stained with phalloidin and the Fc-receptor-specific antibody. This analysis revealed that Fc1-63 colocalized both with actin stress fibers and with cortical actin at sites of cell-cell contact. Similar analyses with Fc38-63Y47A, which inefficiently sorts to the basolateral membrane of MDCK cells (Fig. 6C), revealed that this chimera also colocalized with stress fibers and with cortical actin (Fig. 8D-F). This result indicated that sequences between amino acids 38 and 63 of AE1-4 are not only sufficient to direct basolateral sorting, they can also mediate the association of a chimeric receptor with various elements of the actin cytoskeleton. Whether this interaction represents a functional association with actin that is involved in the basolateral sorting of AE1-4 in MDCK cells is not known.



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Fig. 8. Fc1-63 and Fc38-63Y47A colocalize with phalloidin-stained stress fibers in subconfluent MDCK cells. Subconfluent MDCK cells stably expressing Fc1-63 (A-C) or Fc38-63Y47A (D-F) were fixed, permeabilized and incubated with the Fc-receptor-specific antibody (A,D) and phalloidin conjugated to FITC (B,E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine. Following washing, the localization of fluorescently labeled polypeptides was visualized on a Zeiss Axiophot microscope. The merged images showing the overlap of these chimeras with phalloidin-stained microfilaments are shown in C and F. Bars, 10 µm.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our analyses revealed that the N-terminal 63 amino acids of AE1-4 contained sequences that accounted for essentially all of the sorting and cytoskeletal binding activities that have been attributed to full-length AE1-4 in MDCK cells (Adair-Kirk et al., 1999Go). Two independent endocytic activities were present at the N-terminus of AE1-4. One of these activities resided within amino acids 1-37 of AE1-4, whereas the second activity resided within amino acids 38-63. The residues nearer the N-terminus targeted internalized chimeras to a compartment that substantially overlapped late endosomes, whereas amino acids 38-63 targeted internalized chimeras to the TGN. Although the regions between amino acids 1-37 and 38-63 of AE1-4 could both direct the basolateral sorting of chimeric receptors, the signal within amino acids 38-63 was much more efficient in directing the basolateral accumulation of an AE1/Fc chimera. In addition to its efficient basolateral sorting signal, the region between amino acids 38-63 of AE1-4 was also sufficient to mediate the association of chimeras with the actin cytoskeleton. Fc1-63 contains all of the above-mentioned activities, and like AE1-4 it primarily accumulates in the basolateral membrane of transfected MDCK cells. Other investigators have postulated that the stable accumulation of the Na+,K+-ATPase in the basolateral membrane of MDCK cells is the result of its ability to associate with the spectrinbased membrane cytoskeleton in this cell type (Nelson and Hammerton, 1989Go). Whether the retention of Fc1-63 in the basolateral membrane of MDCK cells reflects its ability to associate with the actin cytoskeleton in this membrane domain is not known. However, the fact that the wild-type and mutant Fc1-63 constructs all primarily accumulate on the cell surface (Fig. 2) suggests that their ability to associate with the actin cytoskeleton may stabilize their surface distribution regardless of whether they are delivered to the basolateral or apical membrane of this polarized epithelial cell.

Sequences within amino acids 38-63 of AE1-4 are sufficient to mediate the association of a chimeric receptor with actin stress fibers in subconfluent MDCK cells. Yet Fc38-63, which is sorted to the basolateral membrane of polarized cells, is rapidly internalized and delivered to the TGN. If indeed the surface retention of Fc1-63 is due to its interaction with actin, the observation that Fc38-63 is rapidly internalized following surface delivery suggests that sequences within amino acids 1-37 of AE1-4 are necessary for a stable association with actin. This idea is supported by the observation that some of the surface population of Fc1-37 colocalizes with actin stress fibers in the basal membrane of polarized MDCK cells (data not shown). In the absence of this cytoskeletal binding activity within amino acids 1-37, the endocytic activity of the Y47VEL peptide is dominant and it directs the rapid internalization of Fc38-63. Mutations in the YVEL peptide that reduce its endocytic activity, such as the tyrosine 47 to alanine substitution, results in a chimera that is primarily retained on the cell surface where it colocalizes with actin.

Other investigators have shown that certain membrane proteins, including TGN38 (Wong and Hong, 1993Go) and furin (Schafer et al., 1995Go), recycle from the plasma membrane to the TGN. Each of these proteins contains a YXXL tetrapeptide in their cytoplasmic domain, and in the case of TGN38, this sequence is necessary to direct recycling to the TGN (Wong and Hong, 1993Go). Furthermore, both the tyrosine and leucine residues are necessary for the recycling activity of TGN38. Our chimera studies have revealed a similar role for the YVEL peptide of AE1-4 in TGN recycling. Mutation of either the tyrosine or leucine residue in this sequence to an alanine dramatically increases the percentage of the Fc38-63 chimera that resides on the cell surface. By contrast, the basolateral sorting activity of Fc38-63 is substantially inhibited by mutation of the tyrosine residue and unaffected by mutation of the leucine. The differential effect of these mutations on basolateral sorting and endocytosis suggest distinct requirements for the recognition of this tyrosine-based signal by the sorting machinery at the TGN and plasma membrane.

Our studies with Fc1-37 have shown that amino acids 1-37 of AE1-4 possess an endocytic activity as well as a weak basolateral sorting signal. The fact that Fc1-63Y44A, Y47A accumulates exclusively in the apical membrane of polarized MDCK cells (Fig. 2E) suggests that the basolateral sorting activity within amino acids 1-37 is masked or non-functional in this context. Although the basis for this is not understood, previous analyses have shown that mutation of the tyrosine-dependent basolateral sorting signal of the full-length AE1-4 variant, AE1-4Y44A, Y47A, does not completely abrogate the basolateral sorting of this membrane transporter (Adair-Kirk et al., 1999Go). At this time it is not clear whether the residual basolateral accumulation of AE1-4Y44A, Y47A is dependent upon the signal within amino acids 1-37.

Amino acids 17-25 of AE1-4 (Fig. 1) share sequence similarity with the membrane proximal cytoplasmic sorting signal of the poly Ig receptor. Other investigators have shown that the serine residue in the poly Ig receptor signal, which is homologous to serine 25 in AE1-4, is necessary for the transcytosis of the receptor (Casanova et al., 1990Go). In addition, the histidine and arginine in the poly Ig receptor signal, which are homologous to amino acids 17 and 18, respectively, in AE1-4, are necessary for basolateral sorting of the receptor (Casanova et al., 1991Go). Our mutagenesis studies with AE1/Fc chimeras have indicated that serine 25 is necessary for both the endocytic and basolateral sorting activities that reside within amino acids 1-37 of AE1-4. It is not known whether histidine 17 and arginine 18 of AE1-4 are necessary for either of the sorting activities associated with amino acids 1-37. However, additional studies have shown that AE1-4{Delta}21Y47A accumulates in the apical membrane of MDCK cells (data not shown), whereas AE1-4Y47A accumulates in the basolateral membrane (Adair-Kirk et al., 1999Go). This suggests that residues in the first 21 amino acids of AE1-4 contribute to the basolateral sorting of this electroneutral transporter in this epithelial cell type.

Previous pulse-chase studies have shown that deletion of the N-terminal 37 amino acids of AE1-4 dramatically slows the rate at which this polypeptide recycles to the Golgi for the acquisition of mature N-linked sugars. Yet at steady state, the ratio of AE1-4{Delta}37 with mature to immature N-linked sugars is not significantly different from the wild-type AE1-4 (Adair-Kirk et al., 1999Go). By contrast, even though AE1-4Y47A recycles much more rapidly than AE1-4{Delta}37, the majority of AE1-4Y47A in the cell possesses immature N-linked sugars (Adair-Kirk et al., 1999Go). One explanation that could account for these observations is that the late endosomal targeting signal that we have characterized within amino acids 1-37 is involved in regulating the constitutive turn over of AE1-4 in this epithelial cell type. In the absence of this sorting signal, AE1-4{Delta}37 turns over slowly. As a consequence of this slower turnover, newly synthesized polypeptides have more time to recycle to the Golgi and acquire mature N-linked sugars. This would result in a steady-state profile for AE1-4{Delta}37 that is similar to wild-type AE1-4 even though the two proteins recycle at very different rates. The observation that AE1-4Y47A with mature N-linked sugars does not accumulate in the cell may be a result of the sorting signal within amino acids 1-37 directing a more rapid rate of turnover of this polypeptide in the absence of a wild-type tyrosine-dependent signal. Future studies will address how the interplay between the sorting signals we have characterized at the N-terminus of AE1-4 controls the Golgi recycling and stability of this variant transporter in MDCK cells.

Studies of investigators have shown that the actin cytoskeleton is involved in regulating the vesicular transport (Rozelle et al., 2000Go; Brown and Song, 2001Go; Kanzaki et al., 2001Go; Valderrama et al., 2001Go) and endocytosis (Fujimoto et al., 2000Go; Pol et al., 2000Go; Jiang et al., 2002Go) of some membrane proteins. Our data presented here as well as previous analyses (Adair-Kirk et al., 1999Go) suggest that the actin cytoskeleton plays a critical role in directing the localization of AE1 in polarized MDCK cells. Although it is unclear whether the ability to associate with actin is directly involved in the Golgi recycling or turnover of AE1-4, mechanisms must exist to regulate the availability of the sorting signals at the N-terminus of AE1-4 to elements of the cellular sorting machinery. In the case of the poly Ig receptor, it has been proposed that the phosphorylation of the serine residue within its cytoplasmic sorting signal is required for the transcytosis of the receptor (Casanova et al., 1990Go). It is tempting to speculate that similar phosphorylation events may be involved in determining how the various activities at the N-terminus of AE1-4 are coordinated to regulate its localization and stability. Along these lines it is interesting to note that the tyrosine residue in skate erythroid AE1 that is homologous to tyrosine 47 in chicken AE1-4 is phosphorylated (Musch et al., 1999Go). In addition, the phosphorylation of this residue is stimulated when skate erythroid cells are grown in hypertonic medium. Whether tyrosine 47 or other residues at the N-terminus of AE1-4 are phosphorylated and the potential role of these phosphorylation events in regulating AE1 localization in epithelial cells will be the subject of future investigations.


    Acknowledgments
 
This research was supported by grants from the National Chapter (96-008610) and the Southeast Affiliate (0050997B) of the American Heart Association. We thank M. Whitt for providing the murine IgG FcRII B2 receptor cDNA. In addition, we thank K. H. Cox and P.Ryan for their critical evaluation of the manuscript.


    References
 Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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