title: Cell surface expression and internalization of the murine erythroid AE1 anion exchanger tagged with an extracellular FLAG epitope.
abstract: Anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is essential for maintaining red cell mechanical stability. However, the mechanism for the assembly of AE1 into the membrane skeletal network remains unknown. Several mutants of murine AE1 tagged with an N-terminal enhanced green fluorescent protein (EGFP) and/or an extracellular FLAG epitope inserted adjacent to the N-glycosylation site were prepared, and their expression was analyzed in HEK293 or COS-1 cells by immunofluorescence microscopy, biotinylation, and deglycosylation. The EGFP- and FLAG-tagged AE1 mutant, as well as the wild-type AE1, exhibited cell surface expression in transfected cells and showed a rapid internalization that appeared to occur through the early endosome into the Golgi apparatus. Interestingly, the form of the protein with an endoglycosidase H (endo H)-sensitive N-glycan was the major component of EGFP-tagged and wild-type AE1. By contrast, the polypeptide with an endo H-resistant oligosaccharide was the predominant form of FLAG-tagged AE1. These data demonstrate that the processing of N-glycan is not a prerequisite to cell surface expression of AE1 and suggest that the FLAG tag insertion altered the accessibility of the N-glycan to enzymes in the Golgi which facilitate processing of oligosaccharides. Although whether this structural alteration would affect the structural and functional properties of AE1 remains unknown, cell surface expression and endocytic internalization of FLAG-tagged AE1 mutants indicate that these mutants are suitable for studying the mechanisms of the assembly and plasma membrane insertion of AE1.
authors.affiliation: Laboratory of Molecular Medicine, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, 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: Mutsumi
authors.initials: M
authors.lastname: Inaba
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D000939:Epitopes; D005786:Gene Expression Regulation / Q000502:physiology*; D051379:Mice; D009154:Mutation; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry*
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide