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

Quansheng Zhu,Diana W.K Lee,Joseph R Casey

doi:10.1074/jbc.m207797200

Abstract

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 Phe(806) and Cys(885), 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). Phe(806)-Leu(835), Ser(852)-Ala(855), and Ile(872)-Cys(885) were labeled by biotin maleimide, suggesting their location in an aqueous environment. In contrast, Phe(836)-Lys(851) and Ser(856)-Arg(871) were not labeled by biotin maleimide and therefore localize to the plane of the bilayer, as transmembrane segments (TM). Labeling by qBBr revealed that Pro(815)-Lys(829) and Ser(852)-Ala(855) are accessible to the extracellular medium. Pro(815)-Lys(829) mutants were also labeled with LYIA. Mutants Ile(872)-Cys(885) 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.

Highlights

Human AE1, called Band 3, is the most abundant integral membrane protein of the erythrocyte membrane (50% membrane protein, 1.2 ϫ 106 copies per cell) [1]
Y555C was used as the extracellular surface control, since it is adjacent to the two chymotryptic cleavage sites in intact erythrocytes [33]; K892C was used as intracellular surface control because it is located in the C-terminal tail of the protein, which previously mapped to the cytoplasm [20, 34]
Labeling of an introduced cysteine mutant with biotin maleimide indicates that the residue is accessible to the aqueous environment

Summary

EXPERIMENTAL PROCEDURES

Materials—Restriction endonucleases were from New England Biolabs. Pwo DNA polymerase was from Roche Molecular Diagnostics. Transfected HEK cells were washed with 5 ml of PBS (140 mM NaCl, 3 mM KCl, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) and allowed to detach in 2 ml of PBS for 10 min, at room temperature. The qBBr was removed by aspiration, and cells were washed once with PBS Both cell samples were incubated with biotin maleimide (prepared in Me2SO, final concentration 0.2 mM), for 20 min at room temperature. Anion Exchange Assays—HEK cells were grown on polylysine-coated glass coverslips in 60-mm tissue culture dishes and transfected as described. Two days post-transfection, cells were rinsed with serumfree DMEM (Invitrogen) and incubated with in 4 ml of serum-free DMEM medium containing 2 ␮M BCECF-AM (37 °C, 30 min). Statistical analysis—Standard errors were calculated with Kaleidagraph 3.5 software (Synergy Software)

RESULTS

Biotin maleimide labeling

DISCUSSION