title: Topology of the anion exchange protein AE1: the controversial sidedness of lysine 743.
abstract: The topology of the band 3 (AE1) polypeptide of the erythrocyte membrane is not fully established despite extensive study. Residues near lysine 743 (K743) have been reported to be extracellular in some studies and cytoplasmic in others. In the work presented here, we have attempted to establish the sidedness of K743 using in situ proteolysis. Trypsin, papain, and proteinase K do not cleave band 3 at or near K743 in intact red cells, even under conditions that cause cleavage on the C-terminal side of the glycosylation site (N642) in extracellular loop 4. In contrast, trypsin sealed inside red cell ghosts cleaves at K743, as does trypsin treatment of inside-out vesicles (IOVs). The transport inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H(2)DIDS), acting from the extracellular side, blocks trypsin cleavage at K743 in unsealed membranes by inducing a protease-resistant conformation. H(2)DIDS added to IOVs does not prevent cleavage at K743; therefore, trypsin cleavage at K743 in IOVs is not a consequence of cleavage of right-side-out or leaky vesicles. Finally, microsomes were prepared from HEK293 cells expressing the membrane domain of AE1 lacking the normal glycosylation site. This polypeptide does not traffic to the surface membrane; trypsin treatment of microsomes containing this polypeptide produces the 20 kDa fragment, providing further evidence that K743 is exposed at the cytoplasmic surface. Therefore, the actions of trypsin on intact cells, resealed ghosts, unsealed ghosts, inside-out vesicles, and microsomes from HEK293 cells all indicate that K743 is cytoplasmic and not extracellular.
authors.affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kuma
authors.forename: Anjali A
authors.initials: AA
authors.lastname: Shinde
authors.forename: Todd R
authors.initials: TR
authors.lastname: Howren
authors.forename: Michael L
authors.initials: ML
authors.lastname: Jennings
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010206:Papain / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D014357:Trypsin; D019286:Endopeptidase K; D010206:Papain; D008239:Lysine