title: Co-expressed complementary fragments of the human red cell anion exchanger (band 3, AE1) generate stilbene disulfonate-sensitive anion transport.
abstract: We have constructed cDNA clones encoding various portions of the human red cell anion transporter (band 3), a well characterized integral membrane protein with up to 14 transmembrane segments. The biosynthesis, stability, cell surface expression, and functionality of these band 3 fragments were investigated by expression from the cRNAs into microsomal membranes using the reticulocyte cell-free translation system and in Xenopus oocytes. Co-expression of the pairs of recombinants encoding the first 8 and last 6 transmembrane spans (8 + 6) or the first 12 and last 2 spans (12 + 2) of band 3 generated stilbene disulfonate-sensitive anion transport in oocytes. When the pairs of fragments 8 + 6 or 12 + 2 were co-expressed with glycophorin A (GPA), translocation to the plasma membrane of the fragment corresponding to the first 12 or the first 8 transmembrane spans was greater than in the absence of GPA. Only the fragment encoding the first 12 transmembrane spans showed GPA-dependent translocation when expressed in the absence of its complementary fragment. A truncated form of band 3 encoding all 14 transmembrane spans but lacking the carboxyl-terminal 30 amino acids of the cytoplasmic tail did not induce anion transport activity in oocytes and was not translocated to the plasma membrane but appeared to be degraded in oocytes. Our results suggest that there is no single signal for the insertion of the different transmembrane spans of band 3 into membranes and that the integrity of the loops between transmembrane spans 8-9 or 12-13 is not essential for anion transport function. Our data also suggest that a region of transmembrane spans 9-12 of band 3 is involved in the process by which GPA facilitates the translocation of band 3 to the surface.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom.
authors.forename: J D
authors.initials: JD
authors.lastname: Groves
authors.forename: M J
authors.initials: MJ
authors.lastname: Tanner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002712:Chlorides / Q000378:metabolism*; D018076:DNA, Complementary / Q000302:isolation & purification; D006021:Glycophorins / Q000032:analysis; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D013267:Stilbenes / Q000494:pharmacology*; D014357:Trypsin / Q000494:pharmacology; D014981:Xenopus
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D018076:DNA, Complementary; D006021:Glycophorins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013267:Stilbenes; D014357:Trypsin