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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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Surface delivery and recycling of newly synthesized AE1-4 anion exchangers. MDCK cells stably expressing AE1-4 were pulsed with 35S-Translabel™ for 15 min, followed by 15 min of chase (A, lane 1). After 15 min of chase, the cells were chased an additional 45 min in the absence (A, lane 3) or presence (A, lane 4) of 1.5 mg/ml chymotrypsin. Cells were also incubated continuously with 1.5 mg/ml chymotrypsin during a 15-min preincubation in methionine-free DME, a 15-min pulse with 35S-Translabel™, and a 15-min chase (A, lane 2). Alternatively, cells were pulsed for 15 min and chased for 1 h at 37°C. The cells were then shifted to 4°C, and incubated an additional 45 min in the presence of 1.5 mg/ml chymotrypsin (A, lane 5, marked with asterisk). For each labeling scheme, AE1 immunoprecipitates were prepared from total cell lysates using a polyclonal antibody directed against the cytoplasmic domain of AE1-4. A fraction of the total cell lysate was also analyzed by immunoblotting using a polyclonal antibody that recognizes α-fodrin (A) or a chicken AE1-specific peptide antibody (A). Additional studies examined the effect of various reagents on the posttranslational processing of AE1-4. MDCK cells stably expressing AE1-4 were pulsed with 35S-Translabel™ for 15 min, and chased for 1 h (B, lane 1), or 4 h (B, lanes 2–5). For some of the cells, 25 mM ammonium chloride (A, lane 3), 10 μg/ml BFA (B, lane 4), or 0.4 M sucrose (B, lane 5) was added to media after 45 min of chase, and was present for the remainder of the chase period. At each time point, AE1 immunoprecipitates were prepared from total cell lysates. Immunoprecipitates in A and B were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.
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Figure 5: Surface delivery and recycling of newly synthesized AE1-4 anion exchangers. MDCK cells stably expressing AE1-4 were pulsed with 35S-Translabel™ for 15 min, followed by 15 min of chase (A, lane 1). After 15 min of chase, the cells were chased an additional 45 min in the absence (A, lane 3) or presence (A, lane 4) of 1.5 mg/ml chymotrypsin. Cells were also incubated continuously with 1.5 mg/ml chymotrypsin during a 15-min preincubation in methionine-free DME, a 15-min pulse with 35S-Translabel™, and a 15-min chase (A, lane 2). Alternatively, cells were pulsed for 15 min and chased for 1 h at 37°C. The cells were then shifted to 4°C, and incubated an additional 45 min in the presence of 1.5 mg/ml chymotrypsin (A, lane 5, marked with asterisk). For each labeling scheme, AE1 immunoprecipitates were prepared from total cell lysates using a polyclonal antibody directed against the cytoplasmic domain of AE1-4. A fraction of the total cell lysate was also analyzed by immunoblotting using a polyclonal antibody that recognizes α-fodrin (A) or a chicken AE1-specific peptide antibody (A). Additional studies examined the effect of various reagents on the posttranslational processing of AE1-4. MDCK cells stably expressing AE1-4 were pulsed with 35S-Translabel™ for 15 min, and chased for 1 h (B, lane 1), or 4 h (B, lanes 2–5). For some of the cells, 25 mM ammonium chloride (A, lane 3), 10 μg/ml BFA (B, lane 4), or 0.4 M sucrose (B, lane 5) was added to media after 45 min of chase, and was present for the remainder of the chase period. At each time point, AE1 immunoprecipitates were prepared from total cell lysates. Immunoprecipitates in A and B were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.

Mentions: The acquisition of mature N-linked sugars by chicken erythroid AE1 anion exchangers occurs via recycling of newly synthesized polypeptides from the plasma membrane to the Golgi (Ghosh et al. 1999). To determine whether a similar mechanism is responsible for the slow acquisition of endo H-resistant sugars by AE1-4, pulse–chase studies have examined the time after synthesis that newly synthesized AE1-4 becomes susceptible to digestion with extracellular chymotrypsin. This analysis revealed that the immunoprecipitable AE1-4 species of ∼97 kD was not susceptible to digestion with extracellular chymotrypsin that was present in the media during the pulse and for the first 15 min of chase (Fig. 5 A, lane 2). However, when chymotrypsin was included in the media from 15 min to 60 min of chase, all of the immature AE1-4 species of ∼97 kD was susceptible to digestion (Fig. 5 A, lane 4). Immunoblotting analysis revealed that the steady state population of AE1-4 was digested to the same extent regardless of the time when chymotrypsin was added (Fig. 5 A). Additional controls revealed that α-fodrin was not susceptible to chymotrypsin digestion at either of the time points (Fig. 5 A) indicating the susceptibility of newly synthesized AE1-4 to chymotrypsin was not due to the cells becoming leaky during digestion. These results indicate that essentially all of the newly synthesized polypeptides arrive at the plasma membrane by 1 h after synthesis. Furthermore, these data suggest that the acquisition of endo H–resistant N-linked sugars by AE1-4 between 1 and 4 h of chase primarily occurs via recycling of cell surface polypeptides to the Golgi. Interestingly, when cells were shifted to 4°C after 1 h of chase to block vesicular trafficking and incubated with chymotrypsin for an additional 45 min at 4°C, newly synthesized AE1-4 was resistant to chymotrypsin digestion (Fig. 5 A, lane 5). This result together with the fact that newly synthesized polypeptides have undergone cell surface delivery by 1 h of chase suggests that newly synthesized AE1-4 is rapidly internalized after surface delivery.


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)

Surface delivery and recycling of newly synthesized AE1-4 anion exchangers. MDCK cells stably expressing AE1-4 were pulsed with 35S-Translabel™ for 15 min, followed by 15 min of chase (A, lane 1). After 15 min of chase, the cells were chased an additional 45 min in the absence (A, lane 3) or presence (A, lane 4) of 1.5 mg/ml chymotrypsin. Cells were also incubated continuously with 1.5 mg/ml chymotrypsin during a 15-min preincubation in methionine-free DME, a 15-min pulse with 35S-Translabel™, and a 15-min chase (A, lane 2). Alternatively, cells were pulsed for 15 min and chased for 1 h at 37°C. The cells were then shifted to 4°C, and incubated an additional 45 min in the presence of 1.5 mg/ml chymotrypsin (A, lane 5, marked with asterisk). For each labeling scheme, AE1 immunoprecipitates were prepared from total cell lysates using a polyclonal antibody directed against the cytoplasmic domain of AE1-4. A fraction of the total cell lysate was also analyzed by immunoblotting using a polyclonal antibody that recognizes α-fodrin (A) or a chicken AE1-specific peptide antibody (A). Additional studies examined the effect of various reagents on the posttranslational processing of AE1-4. MDCK cells stably expressing AE1-4 were pulsed with 35S-Translabel™ for 15 min, and chased for 1 h (B, lane 1), or 4 h (B, lanes 2–5). For some of the cells, 25 mM ammonium chloride (A, lane 3), 10 μg/ml BFA (B, lane 4), or 0.4 M sucrose (B, lane 5) was added to media after 45 min of chase, and was present for the remainder of the chase period. At each time point, AE1 immunoprecipitates were prepared from total cell lysates. Immunoprecipitates in A and B were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.
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Figure 5: Surface delivery and recycling of newly synthesized AE1-4 anion exchangers. MDCK cells stably expressing AE1-4 were pulsed with 35S-Translabel™ for 15 min, followed by 15 min of chase (A, lane 1). After 15 min of chase, the cells were chased an additional 45 min in the absence (A, lane 3) or presence (A, lane 4) of 1.5 mg/ml chymotrypsin. Cells were also incubated continuously with 1.5 mg/ml chymotrypsin during a 15-min preincubation in methionine-free DME, a 15-min pulse with 35S-Translabel™, and a 15-min chase (A, lane 2). Alternatively, cells were pulsed for 15 min and chased for 1 h at 37°C. The cells were then shifted to 4°C, and incubated an additional 45 min in the presence of 1.5 mg/ml chymotrypsin (A, lane 5, marked with asterisk). For each labeling scheme, AE1 immunoprecipitates were prepared from total cell lysates using a polyclonal antibody directed against the cytoplasmic domain of AE1-4. A fraction of the total cell lysate was also analyzed by immunoblotting using a polyclonal antibody that recognizes α-fodrin (A) or a chicken AE1-specific peptide antibody (A). Additional studies examined the effect of various reagents on the posttranslational processing of AE1-4. MDCK cells stably expressing AE1-4 were pulsed with 35S-Translabel™ for 15 min, and chased for 1 h (B, lane 1), or 4 h (B, lanes 2–5). For some of the cells, 25 mM ammonium chloride (A, lane 3), 10 μg/ml BFA (B, lane 4), or 0.4 M sucrose (B, lane 5) was added to media after 45 min of chase, and was present for the remainder of the chase period. At each time point, AE1 immunoprecipitates were prepared from total cell lysates. Immunoprecipitates in A and B were analyzed on a 6% SDS polyacrylamide gel, and labeled anion exchangers were detected by fluorography.
Mentions: The acquisition of mature N-linked sugars by chicken erythroid AE1 anion exchangers occurs via recycling of newly synthesized polypeptides from the plasma membrane to the Golgi (Ghosh et al. 1999). To determine whether a similar mechanism is responsible for the slow acquisition of endo H-resistant sugars by AE1-4, pulse–chase studies have examined the time after synthesis that newly synthesized AE1-4 becomes susceptible to digestion with extracellular chymotrypsin. This analysis revealed that the immunoprecipitable AE1-4 species of ∼97 kD was not susceptible to digestion with extracellular chymotrypsin that was present in the media during the pulse and for the first 15 min of chase (Fig. 5 A, lane 2). However, when chymotrypsin was included in the media from 15 min to 60 min of chase, all of the immature AE1-4 species of ∼97 kD was susceptible to digestion (Fig. 5 A, lane 4). Immunoblotting analysis revealed that the steady state population of AE1-4 was digested to the same extent regardless of the time when chymotrypsin was added (Fig. 5 A). Additional controls revealed that α-fodrin was not susceptible to chymotrypsin digestion at either of the time points (Fig. 5 A) indicating the susceptibility of newly synthesized AE1-4 to chymotrypsin was not due to the cells becoming leaky during digestion. These results indicate that essentially all of the newly synthesized polypeptides arrive at the plasma membrane by 1 h after synthesis. Furthermore, these data suggest that the acquisition of endo H–resistant N-linked sugars by AE1-4 between 1 and 4 h of chase primarily occurs via recycling of cell surface polypeptides to the Golgi. Interestingly, when cells were shifted to 4°C after 1 h of chase to block vesicular trafficking and incubated with chymotrypsin for an additional 45 min at 4°C, newly synthesized AE1-4 was resistant to chymotrypsin digestion (Fig. 5 A, lane 5). This result together with the fact that newly synthesized polypeptides have undergone cell surface delivery by 1 h of chase suggests that newly synthesized AE1-4 is rapidly internalized after surface delivery.

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