Complementation studies with Co-expressed fragments of the human red cell anion transporter (Band 3; AE1). The role of some exofacial loops in anion transport.
We constructed cDNA clones encoding fragments of band 3 in which the membrane domain was truncated from either the N or the C terminus within each of the first four exofacial loops. The truncations containing the C terminus of the protein were fused with the cleavable N-terminal signal sequence of glycophorin A to facilitate the correct orientation of the most N-terminal band 3 membrane span. Cleavage of the glycophorin A signal sequence was observed, except when the truncation was in the first exofacial loop where the signal peptidase cleavage site was probably too close to the membrane. The anion transport activity of co-expressed complementary pairs of truncations which together contained the entire band 3 membrane domain was examined. The pairs of fragments divided in the third and fourth exofacial loops yielded transport activity, but the pair separated within the second exofacial loop was not active. We conclude that the integrity of the second exofacial loop, but not the third and fourth exofacial loops, is necessary for transport activity. The unusually stable association between the fragments divided in the second exofacial loop suggests that interactions may occur between polar surfaces on amphiphilic portions of the third and fifth transmembrane spans.
Wang
L
L
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Groves
J D
JD
Mawby
W J
WJ
Tanner
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002712:Chlorides / Q000378:metabolism; D017931:DNA Primers; D005260:Female; D005816:Genetic Complementation Test; D005914:Globins / Q000096:biosynthesis; D006021:Glycophorins / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014982:Xenopus laevis
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D017931:DNA Primers; D006021:Glycophorins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D005914:Globins