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Intracellular trafficking of variant chicken kidney AE1 anion exchangers: role Of alternative NH(2) termini in polarized sorting and Golgi recycling.

Adair-Kirk TL, Cox KH, Cox JV - J. Cell Biol. (1999)

Bottom Line: Pulse-chase 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, Tennessee 38163, USA.

ABSTRACT
The variant chicken kidney AE1 anion exchangers differ only at the NH(2) 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 NH(2)-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. Pulse-chase 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.

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Localization of AE1-4 mutants in subconfluent MDCK monolayers. MDCK cells expressing AE1-4Δ21 (A), AE1-4Δ37 (B), AE1-4(Y44A) (C), AE1-4(Y47A) (D), or AE1-4(Y44A,47A) (E) were grown on coverslips in subconfluent monolayers. The cells were fixed and incubated with an AE1-specific peptide antibody, followed by incubation with DAR-IgG conjugated to lissamine (A–E) and FITC-conjugated phalloidin (F–J). Immunoreactive polypeptides and phalloidin stained microfilaments were visualized on a Zeiss Axiophot microscope. Images were collected at the basal surface of the cells. Merged images were generated in Adobe Photoshop (K–O). The arrows in L mark two of the actin-containing clusters that colocalize with AE1-4Δ37. The arrowheads in O indicate where AE1-4(Y44A,47A) colocalizes with cortical actin at sites of cell-cell contact.
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Figure 9: Localization of AE1-4 mutants in subconfluent MDCK monolayers. MDCK cells expressing AE1-4Δ21 (A), AE1-4Δ37 (B), AE1-4(Y44A) (C), AE1-4(Y47A) (D), or AE1-4(Y44A,47A) (E) were grown on coverslips in subconfluent monolayers. The cells were fixed and incubated with an AE1-specific peptide antibody, followed by incubation with DAR-IgG conjugated to lissamine (A–E) and FITC-conjugated phalloidin (F–J). Immunoreactive polypeptides and phalloidin stained microfilaments were visualized on a Zeiss Axiophot microscope. Images were collected at the basal surface of the cells. Merged images were generated in Adobe Photoshop (K–O). The arrows in L mark two of the actin-containing clusters that colocalize with AE1-4Δ37. The arrowheads in O indicate where AE1-4(Y44A,47A) colocalizes with cortical actin at sites of cell-cell contact.

Mentions: 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 AE1-4Δ21, AE1-4Δ37, 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 (Fig. 9). In addition to colocalizing with stress fibers in the basal membrane of cells, AE1-4Δ37 often accumulated in clusters in the basal membrane that were also stained by FITC-phalloidin (arrows in Fig. 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 Fig. 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.


Intracellular trafficking of variant chicken kidney AE1 anion exchangers: role Of alternative NH(2) termini in polarized sorting and Golgi recycling.

Adair-Kirk TL, Cox KH, Cox JV - J. Cell Biol. (1999)

Localization of AE1-4 mutants in subconfluent MDCK monolayers. MDCK cells expressing AE1-4Δ21 (A), AE1-4Δ37 (B), AE1-4(Y44A) (C), AE1-4(Y47A) (D), or AE1-4(Y44A,47A) (E) were grown on coverslips in subconfluent monolayers. The cells were fixed and incubated with an AE1-specific peptide antibody, followed by incubation with DAR-IgG conjugated to lissamine (A–E) and FITC-conjugated phalloidin (F–J). Immunoreactive polypeptides and phalloidin stained microfilaments were visualized on a Zeiss Axiophot microscope. Images were collected at the basal surface of the cells. Merged images were generated in Adobe Photoshop (K–O). The arrows in L mark two of the actin-containing clusters that colocalize with AE1-4Δ37. The arrowheads in O indicate where AE1-4(Y44A,47A) colocalizes with cortical actin at sites of cell-cell contact.
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Figure 9: Localization of AE1-4 mutants in subconfluent MDCK monolayers. MDCK cells expressing AE1-4Δ21 (A), AE1-4Δ37 (B), AE1-4(Y44A) (C), AE1-4(Y47A) (D), or AE1-4(Y44A,47A) (E) were grown on coverslips in subconfluent monolayers. The cells were fixed and incubated with an AE1-specific peptide antibody, followed by incubation with DAR-IgG conjugated to lissamine (A–E) and FITC-conjugated phalloidin (F–J). Immunoreactive polypeptides and phalloidin stained microfilaments were visualized on a Zeiss Axiophot microscope. Images were collected at the basal surface of the cells. Merged images were generated in Adobe Photoshop (K–O). The arrows in L mark two of the actin-containing clusters that colocalize with AE1-4Δ37. The arrowheads in O indicate where AE1-4(Y44A,47A) colocalizes with cortical actin at sites of cell-cell contact.
Mentions: 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 AE1-4Δ21, AE1-4Δ37, 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 (Fig. 9). In addition to colocalizing with stress fibers in the basal membrane of cells, AE1-4Δ37 often accumulated in clusters in the basal membrane that were also stained by FITC-phalloidin (arrows in Fig. 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 Fig. 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.

Bottom Line: Pulse-chase 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, Tennessee 38163, USA.

ABSTRACT
The variant chicken kidney AE1 anion exchangers differ only at the NH(2) 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 NH(2)-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. Pulse-chase 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.

Show MeSH
Related in: MedlinePlus