title: Clathrin-mediated endocytosis of mammalian erythroid AE1 anion exchanger facilitated by a YXXΦ or a noncanonical YXXXΦ motif in the N-terminal stretch.
abstract: To explore the roles of the conserved YXXΦ-type motif in the erythroid-specific N-terminal stretch of anion exchanger 1 (AE1), cell surface expression and internalization of various mutants derived from murine erythroid AE1 tagged with an N-terminal enhanced green fluorescent protein and an extracellular FLAG (EGFP-mAE1Flag) were explored in K562 and HEK293 cells. EGFP-mAE1Flag showed rapid internalization, in association with the internalizations of transferrin and the endogenous AE1 chaperone-like protein glycophorin A in K562 cells. Disruption of the conserved Y72VEL sequence markedly reduced the internalization and increased the relative abundance of cell-surface AE1, whereas substitution of the N-terminal region from bovine AE1 that lacks the relevant motif for the corresponding region had less of an effect on internalization. Deletion or substitution mutations of the Y7EDQL sequence in the bovine N-terminal stretch resulted in the decreased internalization of the AE1 proteins. Cell surface biotinylation and deglycosylation studies showed that approximately 30% of the cell-surface EGFP-mAE1Flag and several other mutants was sorted to the plasma membrane without N-glycan maturation in the Golgi apparatus. These findings indicate that the conserved YXXΦ sequence or a noncanonical YXXXΦ sequence in the N-terminal region facilitates the endocytic recycling of erythroid AE1 through a clathrin-mediated pathway.
authors.affiliation: Laboratory of Molecular Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
authors.forename: Chen-Chi
authors.initials: CC
authors.lastname: Wang
authors.forename: Kota
authors.initials: K
authors.lastname: Sato
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Otsuka
authors.forename: Wataru
authors.initials: W
authors.lastname: Otsu
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Inaba
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology; D049452:Green Fluorescent Proteins; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D051379:Mice
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D002966:Clathrin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins