Novel Topology in C-terminal Region of the Human Plasma Membrane Anion Exchanger, AE1*

Human AE1 performs electroneutral exchange of Cl⁻ for HCO${}_3^{-}$ across the erythrocyte membrane. We examined the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Eighty individual cysteine residues, introduced into an otherwise cysteine-less mutant between Phe⁸⁰⁶ and Cys⁸⁸⁵, were expressed by transient transfection of HEK293 cells. Topology of the region was determined by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent biotin maleimide, with or without prior labeling with the membrane-impermeant reagents, bromotrimethylammoniumbimane bromide (qBBr) and lucifer yellow iodoacetamide (LYIA). Phe⁸⁰⁶–Leu⁸³⁵, Ser⁸⁵²–Ala⁸⁵⁵, and Ile⁸⁷²–Cys⁸⁸⁵ were labeled by biotin maleimide, suggesting their location in an aqueous environment. In contrast, Phe⁸³⁶–Lys⁸⁵¹ and Ser⁸⁵⁶–Arg⁸⁷¹ were not labeled by biotin maleimide and therefore localize to the plane of the bilayer, as transmembrane segments (TM). Labeling by qBBr revealed that Pro⁸¹⁵–Lys⁸²⁹ and Ser⁸⁵²–Ala⁸⁵⁵ are accessible to the extracellular medium. Pro⁸¹⁵–Lys⁸²⁹ mutants were also labeled with LYIA. Mutants Ile⁸⁷²–Cys⁸⁸⁵ were inaccessible to the extracellular medium and thus localized to the intracellular surface of AE1. Functional assays revealed that one face of each of two AE1 TMs was sensitive to mutation. Based on these results, we propose a topology model for the C-terminal region of the membrane domain of human AE1.