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First published online 18 December 2002
doi: 10.1242/jcs.00260

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Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells

Tracy L. Adair-Kirk^*, Frank C. Dorsey and John V. Cox

Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, Tennessee 38163, USA
^* Present address: Department of Medicine, Washington University School of Medicine at Barnes-Jewish Hospital, St Louis, MO 63110, USA

View larger version (28K): [in a new window]   Fig. 1. Sequence of AE1/Fc receptor chimeras. The sequence of the AE1/Fc receptor chimeras is illustrated. The putative boundary between the cytoplasmic domain and the transmembrane domain of these proteins is shown. The numbering at the top refers to the amino-acid sequence at the N-terminus of AE1-4. The sequences within amino acids 1-63 of AE1-4 that are homologous to known sorting signals are underlined. Residues within this region of AE1-4 that are identical to amino acids in the poly Ig receptor sorting signal are marked with a plus. The spacing between the arginine and serine in this poly Ig-like sorting signal is the same as that in the poly Ig receptor. The asterisk at the end of each sequence corresponds to a stop codon.

View larger version (128K): [in a new window]   Fig. 2. Amino acids 1-63 of AE1-4 can direct a cytoplasmic tailless mutant of the Fc receptor to the basolateral membrane of MDCK cells. Confluent MDCK cells stably expressing Fc- (A), Fc1-63 (B), Fc1-63Y44A (C), Fc1-63Y47A (D) or Fc1-63Y44A, Y47A (E) were fixed and incubated with the rat monoclonal antibody specific for the Fc receptor and phalloidin-FITC. The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine, and the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center (B, C and D) or near the apical surface (A and E) of the cells. Regions that are yellow indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image of the transfected cells. The bar in each xy image is 10 µm.

View larger version (76K): [in a new window]   Fig. 3. Amino acids 1-37 and 38-63 of AE1-4 target a cytoplasmic tailless Fc receptor to distinct intracellular membrane compartments. MDCK cells stably expressing Fc1-37 (A-C) or Fc38-63 (D-F) were fixed and incubated with rat monoclonal antibodies specific for the Fc receptor (A and D), a mouse monoclonal antibody specific for the mannose 6-phosphate receptor (B) or a rabbit polyclonal antibody specific for furin (E). The cells were washed and incubated with donkey anti-rat IgG conjugated to lissamine (A and D), goat anti-mouse IgG conjugated to FITC (B) or donkey anti-rabbit IgG conjugated to FITC (E). The localization of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The merged images illustrate significant overlap in the distribution of Fc 1-37 and the mannose 6-phosphate receptor (C) and Fc38-63 and furin (F). Bar in A and D, 10 µm.

View larger version (80K): [in a new window]   Fig. 4. Fc1-37 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of the mannose 6-P receptor. MDCK cells stably expressing Fc1-37 were incubated with the anti-Fc receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a mouse monoclonal directed against the mannose 6-phosphate receptor (M-6-P). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and goat anti-mouse IgG conjugated to FITC. Following washing, the localization of Fc1-37 (A,B) and the mannose 6-phosphate receptor (C,D) was visualized on a Zeiss LSM 510 laser-scanning microscope. The merged images showing the overlap of Fc1-37 and the mannose 6-phosphate receptor are shown in E and F. The arrows in A indicate the Fc1-37 chimeras that still reside on the cell surface following a 15 minute incubation at 37°C. Bar (A,B), 10 µm.

View larger version (92K): [in a new window]   Fig. 5. Fc38-63 recycles from the plasma membrane to a membrane compartment that overlaps the distribution of furin. MDCK cells stably expressing Fc38-63 were incubated with the anti-Fc receptor antibody for 1 hour at 4°C. Following washing with cold DMEM, the cells were incubated for 15 minutes (A,C,E) or 45 minutes (B,D,F) at 37°C. At each time point, the cells were fixed, permeabilized and incubated with a rabbit polyclonal directed against furin. The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine and donkey anti-rabbit IgG conjugated to FITC. Following washing, the localization of Fc38-63 (A,B) and furin (C,D) was visualized on a Zeiss LSM 510 laser scanning microscope. The merged images showing the overlap of Fc38-63 and furin are shown in E and F. Bar (A,B), 10 µm.

View larger version (111K): [in a new window]   Fig. 6. The surface distribution and steady-state localization of wild-type and mutant Fc38-63 constructs in polarized MDCK cells. Polarized MDCK cells stably expressing Fc38-63 (A,B) or Fc38-63Y47A (C) or transiently expressing Fc38-63Y47L (D) or Fc38-63L50A (E) were grown on Transwell filters. The intact cells were either incubated with the Fc-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the Fc-receptor-specific antibody (B-E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-E) and phalloidin conjugated to FITC (B-E). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is near the center of the cells. Regions that are yellow in B-E indicate significant overlap in the distribution of the chimera and actin. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.

View larger version (64K): [in a new window]   Fig. 7. The surface distribution and steady-state localization of wild-type and mutant Fc1-37 constructs in polarized MDCK cells. Polarized MDCK cells transiently (A,C) or stably (B) expressing Fc1-37 (A and B) or Fc1-37S25A (C) were grown on Transwell filters. The intact cells were either incubated with the Fc-receptor-specific antibody for 1 hour at 4°C, washed and fixed in 3% paraformaldehyde (A) or the cells were fixed in 3% paraformaldehyde, permeabilized by incubation in PBST and incubated with the Fc-receptor-specific antibody (B,C). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine (A-C) and phalloidin conjugated to FITC (B and C). Following washing, the distribution of fluorescently labeled proteins was visualized using a Zeiss LSM510 confocal microscope. The 0.5 µm xy image in each panel is either near the center (A,B) or near the apical surface (C) of the cells. The surface population of Fc1-37 could be detected in a longer exposure of the image in B. The black arrowhead next to each panel marks the position of the basal membrane in the xz image. Bars, 10 µm.

View larger version (130K): [in a new window]   Fig. 8. Fc1-63 and Fc38-63Y47A colocalize with phalloidin-stained stress fibers in subconfluent MDCK cells. Subconfluent MDCK cells stably expressing Fc1-63 (A-C) or Fc38-63Y47A (D-F) were fixed, permeabilized and incubated with the Fc-receptor-specific antibody (A,D) and phalloidin conjugated to FITC (B,E). The cells were then washed and incubated with donkey anti-rat IgG conjugated to lissamine. Following washing, the localization of fluorescently labeled polypeptides was visualized on a Zeiss Axiophot microscope. The merged images showing the overlap of these chimeras with phalloidin-stained microfilaments are shown in C and F. Bars, 10 µm.

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