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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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Posttranslational modification and turnover of the chicken AE1-3 and AE1-4 anion exchangers. MDCK cells stably expressing the AE1-3 or AE1-4 anion exchanger were pulsed with 35S-Translabel™ in methionine-free DME for 15 min, and chased in DME containing 5% FCS for times ranging from 0–4 h. At each time point, immunoprecipitates were prepared from total cell lysates using polyclonal antibodies directed against the cytoplasmic domain of chicken AE1-4. Immunoprecipitates were either undigested, digested with endo H, or digested with N-glycosidase. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.
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Figure 4: Posttranslational modification and turnover of the chicken AE1-3 and AE1-4 anion exchangers. MDCK cells stably expressing the AE1-3 or AE1-4 anion exchanger were pulsed with 35S-Translabel™ in methionine-free DME for 15 min, and chased in DME containing 5% FCS for times ranging from 0–4 h. At each time point, immunoprecipitates were prepared from total cell lysates using polyclonal antibodies directed against the cytoplasmic domain of chicken AE1-4. Immunoprecipitates were either undigested, digested with endo H, or digested with N-glycosidase. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.

Mentions: Pulse–chase analyses have investigated the mechanisms involved in generating the steady state profile of anion exchangers detected in AE1-3 and AE1-4 transfected cells. MDCK cells stably transfected with AE1-3 or AE1-4 were pulsed with 35S-Translabel™ for 15 min, and chased for 1 or 4 h. At each time point, the cells were lysed and immunoprecipitates prepared using AE1-specific peptide antibodies were either undigested, digested with endo H, or digested with N-glycosidase. These studies revealed an AE1-3 polypeptide of ∼80 kD accumulated in MDCK cells at the end of a 15-min pulse (Fig. 4). This species was susceptible to digestion with endo H and N-glycosidase yielding a polypeptide of ∼78 kD. The ∼80-kD AE1-3 polypeptide underwent no further modification, and was completely turned over by the end of a 4-h chase. Analysis of AE1-4 transfected cells revealed a discrete AE1-4 species of ∼97 kD accumulated in MDCK cells after the 15-min pulse (Fig. 4). This polypeptide was susceptible to digestion with endo H and N-glycosidase yielding a polypeptide of ∼95 kD. Quantitation of three independent experiments has shown that ∼50% of the newly synthesized AE1-4 polypeptides are turned over during a 4-h chase. The remaining AE1-4 polypeptides acquired additional modifications that eventually resulted in a diffuse array of polypeptides ranging in size from ∼105 to ∼112 kD (Fig. 4). The ∼105- to ∼112-kD polypeptides were still sensitive to digestion with N-glycosidase yielding a polypeptide of ∼95 kD. However, the N-linked sugar modifications of the ∼105- to ∼112-kD polypeptides were insensitive to digestion with endo H (Fig. 4). This suggests that AE1-4 initially receives high mannose or biantennary hybrid sugars on its single N-linked site. By 1 h after synthesis, ∼10% of these modifications are converted to endo H–resistant complex sugars, and by 4 h after synthesis the bulk of the newly synthesized AE1-4 polypeptides have received these more complex endo H-resistant sugar modifications. The addition of these complex sugars to AE1-4 requires the activity of α-mannosidase II and N-acetylglucosamine transferase, markers of the medial compartment of the Golgi. This suggests that AE1-4 passes through the medial compartment of the Golgi between 1 and 4 h after synthesis.


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)

Posttranslational modification and turnover of the chicken AE1-3 and AE1-4 anion exchangers. MDCK cells stably expressing the AE1-3 or AE1-4 anion exchanger were pulsed with 35S-Translabel™ in methionine-free DME for 15 min, and chased in DME containing 5% FCS for times ranging from 0–4 h. At each time point, immunoprecipitates were prepared from total cell lysates using polyclonal antibodies directed against the cytoplasmic domain of chicken AE1-4. Immunoprecipitates were either undigested, digested with endo H, or digested with N-glycosidase. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168086&req=5

Figure 4: Posttranslational modification and turnover of the chicken AE1-3 and AE1-4 anion exchangers. MDCK cells stably expressing the AE1-3 or AE1-4 anion exchanger were pulsed with 35S-Translabel™ in methionine-free DME for 15 min, and chased in DME containing 5% FCS for times ranging from 0–4 h. At each time point, immunoprecipitates were prepared from total cell lysates using polyclonal antibodies directed against the cytoplasmic domain of chicken AE1-4. Immunoprecipitates were either undigested, digested with endo H, or digested with N-glycosidase. Immune complexes were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.
Mentions: Pulse–chase analyses have investigated the mechanisms involved in generating the steady state profile of anion exchangers detected in AE1-3 and AE1-4 transfected cells. MDCK cells stably transfected with AE1-3 or AE1-4 were pulsed with 35S-Translabel™ for 15 min, and chased for 1 or 4 h. At each time point, the cells were lysed and immunoprecipitates prepared using AE1-specific peptide antibodies were either undigested, digested with endo H, or digested with N-glycosidase. These studies revealed an AE1-3 polypeptide of ∼80 kD accumulated in MDCK cells at the end of a 15-min pulse (Fig. 4). This species was susceptible to digestion with endo H and N-glycosidase yielding a polypeptide of ∼78 kD. The ∼80-kD AE1-3 polypeptide underwent no further modification, and was completely turned over by the end of a 4-h chase. Analysis of AE1-4 transfected cells revealed a discrete AE1-4 species of ∼97 kD accumulated in MDCK cells after the 15-min pulse (Fig. 4). This polypeptide was susceptible to digestion with endo H and N-glycosidase yielding a polypeptide of ∼95 kD. Quantitation of three independent experiments has shown that ∼50% of the newly synthesized AE1-4 polypeptides are turned over during a 4-h chase. The remaining AE1-4 polypeptides acquired additional modifications that eventually resulted in a diffuse array of polypeptides ranging in size from ∼105 to ∼112 kD (Fig. 4). The ∼105- to ∼112-kD polypeptides were still sensitive to digestion with N-glycosidase yielding a polypeptide of ∼95 kD. However, the N-linked sugar modifications of the ∼105- to ∼112-kD polypeptides were insensitive to digestion with endo H (Fig. 4). This suggests that AE1-4 initially receives high mannose or biantennary hybrid sugars on its single N-linked site. By 1 h after synthesis, ∼10% of these modifications are converted to endo H–resistant complex sugars, and by 4 h after synthesis the bulk of the newly synthesized AE1-4 polypeptides have received these more complex endo H-resistant sugar modifications. The addition of these complex sugars to AE1-4 requires the activity of α-mannosidase II and N-acetylglucosamine transferase, markers of the medial compartment of the Golgi. This suggests that AE1-4 passes through the medial compartment of the Golgi between 1 and 4 h after synthesis.

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