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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 the variant chicken AE1-3 and AE1-4 anion exchangers in transfected MDCK cells. MDCK cells transfected with the AE1-3 (A and C) or AE1-4 (B and D) anion exchangers were grown to confluency on polycarbonate filters. The cells were then fixed and incubated with an AE1-specific peptide antibody. Immunoreactive polypeptides were detected with DAR-IgG conjugated to lissamine and visualized on a BioRad laser scanning confocal microscope. The 0.5-μm x-y confocal images were collected at the apical surface (A) or near the center (B) of the cell monolayer. The corresponding x-z images are shown in C and D. The arrows in A indicate the boundary between adjacent cells.
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Figure 2: Localization of the variant chicken AE1-3 and AE1-4 anion exchangers in transfected MDCK cells. MDCK cells transfected with the AE1-3 (A and C) or AE1-4 (B and D) anion exchangers were grown to confluency on polycarbonate filters. The cells were then fixed and incubated with an AE1-specific peptide antibody. Immunoreactive polypeptides were detected with DAR-IgG conjugated to lissamine and visualized on a BioRad laser scanning confocal microscope. The 0.5-μm x-y confocal images were collected at the apical surface (A) or near the center (B) of the cell monolayer. The corresponding x-z images are shown in C and D. The arrows in A indicate the boundary between adjacent cells.

Mentions: 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 (Fig. 2B and Fig. D). In contrast, AE1-3 accumulates in or near the apical membrane where it exhibits a diffuse pattern of localization (Fig. 2A and Fig. C). Although AE1-3 is primarily apical in the cells shown in Fig. 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.


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 the variant chicken AE1-3 and AE1-4 anion exchangers in transfected MDCK cells. MDCK cells transfected with the AE1-3 (A and C) or AE1-4 (B and D) anion exchangers were grown to confluency on polycarbonate filters. The cells were then fixed and incubated with an AE1-specific peptide antibody. Immunoreactive polypeptides were detected with DAR-IgG conjugated to lissamine and visualized on a BioRad laser scanning confocal microscope. The 0.5-μm x-y confocal images were collected at the apical surface (A) or near the center (B) of the cell monolayer. The corresponding x-z images are shown in C and D. The arrows in A indicate the boundary between adjacent cells.
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Related In: Results  -  Collection

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Figure 2: Localization of the variant chicken AE1-3 and AE1-4 anion exchangers in transfected MDCK cells. MDCK cells transfected with the AE1-3 (A and C) or AE1-4 (B and D) anion exchangers were grown to confluency on polycarbonate filters. The cells were then fixed and incubated with an AE1-specific peptide antibody. Immunoreactive polypeptides were detected with DAR-IgG conjugated to lissamine and visualized on a BioRad laser scanning confocal microscope. The 0.5-μm x-y confocal images were collected at the apical surface (A) or near the center (B) of the cell monolayer. The corresponding x-z images are shown in C and D. The arrows in A indicate the boundary between adjacent cells.
Mentions: 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 (Fig. 2B and Fig. D). In contrast, AE1-3 accumulates in or near the apical membrane where it exhibits a diffuse pattern of localization (Fig. 2A and Fig. C). Although AE1-3 is primarily apical in the cells shown in Fig. 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.

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