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

AE1/F_c receptor chimeras have been used to define the sequences that direct the basolateral sorting, recycling and cytoskeletal association of the chicken AE1-4 anion exchanger in MDCK cells. These analyses revealed that amino acids 1-63 of AE1-4 were sufficient to redirect a cytoplasmic tailless murine IgG F_cRII B2 receptor from the apical to the basolateral membrane of MDCK cells, where F_c1-63 associated with elements of the actin cytoskeleton. In contrast to F_c1-63, chimeras containing amino acids 1-37 (F_c1-37) or 38-63 (F_c38-63) of AE1-4 accumulated in intracellular membrane compartments that overlapped late endosomes and the trans-Golgi network (TGN), respectively. Internalization assays indicated that the patterns of localization observed for F_c1-37 and F_c38-63 resulted from the recycling of these chimeras from the cell surface. These assays further indicated that F_c1-37 and F_c38-63 each possess a basolateral sorting activity. Mutagenesis studies revealed that the endocytic and basolateral sorting activities in F_c1-37 are dependent upon serine 25, which is located in a sequence similar to a sorting signal in the polymeric immunoglobulin receptor. In addition, the sorting activities associated with F_c38-63 were dependent upon tyrosine 47 and leucine 50. These residues resided within the sequence, YVEL, which matches the YXX motif (where X is any amino acid and is a hydrophobic residue) that functions as an endocytic and TGN recycling signal for other membrane proteins. Our data indicate that amino acids 1-63 of AE1-4 contain sorting and cytoskeletal binding activities that account for most of the properties previously associated with AE1-4 in MDCK cells. Furthermore, the alternative localization patterns exhibited by chimeras containing various combinations of these activities suggest that interplay between these cytoplasmic activities is critical for specifying AE1-4 localization in epithelial cells.