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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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Steady state fractionation properties of the transfected AE1-3 and AE1-4 anion exchangers. MDCK cells transiently expressing the AE1-3 and AE1-4 anion exchangers, or a point mutant of AE1-4 that eliminates the single N-linked glycosylation site, AE1-4N638T, were detergent lysed and separated into soluble (S) and insoluble (I) fractions by centrifugation. Equivalent amounts of the soluble and insoluble fractions were separated on a 6% SDS polyacrylamide gel, transferred to nitrocellulose, and probed with an AE1-specific antibody. After washing, the blot was incubated with GAR-IgG conjugated to horseradish peroxidase, and immunoreactive species were detected by enhanced chemiluminescence. Molecular mass markers to the left correspond to phosphorylase b (97 kD), and fructose-6-P-kinase (84 kD).
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Figure 3: Steady state fractionation properties of the transfected AE1-3 and AE1-4 anion exchangers. MDCK cells transiently expressing the AE1-3 and AE1-4 anion exchangers, or a point mutant of AE1-4 that eliminates the single N-linked glycosylation site, AE1-4N638T, were detergent lysed and separated into soluble (S) and insoluble (I) fractions by centrifugation. Equivalent amounts of the soluble and insoluble fractions were separated on a 6% SDS polyacrylamide gel, transferred to nitrocellulose, and probed with an AE1-specific antibody. After washing, the blot was incubated with GAR-IgG conjugated to horseradish peroxidase, and immunoreactive species were detected by enhanced chemiluminescence. Molecular mass markers to the left correspond to phosphorylase b (97 kD), and fructose-6-P-kinase (84 kD).

Mentions: 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 (Fig. 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 (Fig. 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 (Cox and Cox 1995). Quantitation of several fractionation experiments identical to those described above has indicated that ∼5% of AE1-3 is detergent insoluble, while ∼45% of AE1-4 is detergent insoluble. Although AE1-4N638T exhibited fractionation properties similar to AE1-4, a single species of ∼95 kD accumulated in cells transfected with this construct (Fig. 3). This suggests the complex array of polypeptides observed in AE1-4 transfected cells is due to heterogeneity in the N-linked modifications acquired by this AE1 variant. The size of the polypeptides detected for both AE1-3 and AE1-4 in this analysis is smaller than their predicted molecular masses. The basis for this discrepancy is currently unknown.


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)

Steady state fractionation properties of the transfected AE1-3 and AE1-4 anion exchangers. MDCK cells transiently expressing the AE1-3 and AE1-4 anion exchangers, or a point mutant of AE1-4 that eliminates the single N-linked glycosylation site, AE1-4N638T, were detergent lysed and separated into soluble (S) and insoluble (I) fractions by centrifugation. Equivalent amounts of the soluble and insoluble fractions were separated on a 6% SDS polyacrylamide gel, transferred to nitrocellulose, and probed with an AE1-specific antibody. After washing, the blot was incubated with GAR-IgG conjugated to horseradish peroxidase, and immunoreactive species were detected by enhanced chemiluminescence. Molecular mass markers to the left correspond to phosphorylase b (97 kD), and fructose-6-P-kinase (84 kD).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168086&req=5

Figure 3: Steady state fractionation properties of the transfected AE1-3 and AE1-4 anion exchangers. MDCK cells transiently expressing the AE1-3 and AE1-4 anion exchangers, or a point mutant of AE1-4 that eliminates the single N-linked glycosylation site, AE1-4N638T, were detergent lysed and separated into soluble (S) and insoluble (I) fractions by centrifugation. Equivalent amounts of the soluble and insoluble fractions were separated on a 6% SDS polyacrylamide gel, transferred to nitrocellulose, and probed with an AE1-specific antibody. After washing, the blot was incubated with GAR-IgG conjugated to horseradish peroxidase, and immunoreactive species were detected by enhanced chemiluminescence. Molecular mass markers to the left correspond to phosphorylase b (97 kD), and fructose-6-P-kinase (84 kD).
Mentions: 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 (Fig. 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 (Fig. 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 (Cox and Cox 1995). Quantitation of several fractionation experiments identical to those described above has indicated that ∼5% of AE1-3 is detergent insoluble, while ∼45% of AE1-4 is detergent insoluble. Although AE1-4N638T exhibited fractionation properties similar to AE1-4, a single species of ∼95 kD accumulated in cells transfected with this construct (Fig. 3). This suggests the complex array of polypeptides observed in AE1-4 transfected cells is due to heterogeneity in the N-linked modifications acquired by this AE1 variant. The size of the polypeptides detected for both AE1-3 and AE1-4 in this analysis is smaller than their predicted molecular masses. The basis for this discrepancy is currently unknown.

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