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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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Acquisition and maintenance of detergent insolubility by AE1-4. MDCK cells stably expressing the AE1-4 anion exchanger were pulsed with 35S-Translabel™ for 15 min, and chased for times ranging from 0–48 h. At each time point, cells were detergent lysed and separated into soluble and insoluble fractions by centrifugation. Immunoprecipitates were prepared from each fraction using antibodies generated against the cytoplasmic domain of AE1-4. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography (A). Confluent or 50% confluent monolayers of MDCK cells expressing AE1-4 were incubated in the absence or presence of 25 μg/ml latrunculin B for 1 h. Cells were then detergent lysed, and the detergent soluble and insoluble fractions were analyzed by immunoblotting analysis using AE1-specific peptide antibodies (B).
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Figure 7: Acquisition and maintenance of detergent insolubility by AE1-4. MDCK cells stably expressing the AE1-4 anion exchanger were pulsed with 35S-Translabel™ for 15 min, and chased for times ranging from 0–48 h. At each time point, cells were detergent lysed and separated into soluble and insoluble fractions by centrifugation. Immunoprecipitates were prepared from each fraction using antibodies generated against the cytoplasmic domain of AE1-4. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography (A). Confluent or 50% confluent monolayers of MDCK cells expressing AE1-4 were incubated in the absence or presence of 25 μg/ml latrunculin B for 1 h. Cells were then detergent lysed, and the detergent soluble and insoluble fractions were analyzed by immunoblotting analysis using AE1-specific peptide antibodies (B).

Mentions: Pulse–chase 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 (Fig. 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 Fig. 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 pulse–chase 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 cell–cell 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.


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)

Acquisition and maintenance of detergent insolubility by AE1-4. MDCK cells stably expressing the AE1-4 anion exchanger were pulsed with 35S-Translabel™ for 15 min, and chased for times ranging from 0–48 h. At each time point, cells were detergent lysed and separated into soluble and insoluble fractions by centrifugation. Immunoprecipitates were prepared from each fraction using antibodies generated against the cytoplasmic domain of AE1-4. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography (A). Confluent or 50% confluent monolayers of MDCK cells expressing AE1-4 were incubated in the absence or presence of 25 μg/ml latrunculin B for 1 h. Cells were then detergent lysed, and the detergent soluble and insoluble fractions were analyzed by immunoblotting analysis using AE1-specific peptide antibodies (B).
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Related In: Results  -  Collection

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Figure 7: Acquisition and maintenance of detergent insolubility by AE1-4. MDCK cells stably expressing the AE1-4 anion exchanger were pulsed with 35S-Translabel™ for 15 min, and chased for times ranging from 0–48 h. At each time point, cells were detergent lysed and separated into soluble and insoluble fractions by centrifugation. Immunoprecipitates were prepared from each fraction using antibodies generated against the cytoplasmic domain of AE1-4. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography (A). Confluent or 50% confluent monolayers of MDCK cells expressing AE1-4 were incubated in the absence or presence of 25 μg/ml latrunculin B for 1 h. Cells were then detergent lysed, and the detergent soluble and insoluble fractions were analyzed by immunoblotting analysis using AE1-specific peptide antibodies (B).
Mentions: Pulse–chase 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 (Fig. 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 Fig. 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 pulse–chase 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 cell–cell 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.

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