Cell surface expression and internalization of the murine erythroid AE1 anion exchanger tagged with an extracellular FLAG epitope
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Anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells andis essential for maintaining red cell mechanical stability. However, the mechanism for theassembly of AE1 into the membrane skeletal network remains unknown. Several mutants ofmurine AE1 tagged with an N-terminal enhanced green fluorescent protein (EGFP) and/oran extracellular FLAG epitope inserted adjacent to the N-glycosylation site were prepared,and their expression was analyzed in HEK293 or COS-1 cells by immunofluorescencemicroscopy, biotinylation, and deglycosylation. The EGFP- and FLAG-tagged AE1 mutant,as well as the wild-type AE1, exhibited cell surface expression in transfected cells andshowed a rapid internalization that appeared to occur through the early endosome into theGolgi apparatus. Interestingly, the form of the protein with an endoglycosidase H (endoH)-sensitive N-glycan was the major component of EGFP-tagged and wild-type AE1. Bycontrast, the polypeptide with an endo H-resistant oligosaccharide was the predominantform of FLAG-tagged AE1. These data demonstrate that the processing of N-glycan is not aprerequisite to cell surface expression of AE1 and suggest that the FLAG tag insertionaltered the accessibility of the N-glycan to enzymes in the Golgi which facilitate processingof oligosaccharides. Although whether this structural alteration would affect the structuraland functional properties of AE1 remains unknown, cell surface expression and endocyticinternalization of FLAG-tagged AE1 mutants indicate that these mutants are suitable forstudying the mechanisms of the assembly and plasma membrane insertion of AE1.
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