The effects of human red cell glycophorin A (GPA) on the translocation to the plasma membrane and anion transport activity of the human erythrocyte anion transporter (band 3; AE1) have been examined using the Xenopus oocyte expression system. We show that band 3 accumulates steadily at the oocyte surface with time in the presence or absence of GPA, but this occurs more quickly when GPA is coexpressed. The amount of band 3 at the surface is determined by the concentrations of band 3 and GPA cRNA that are injected, with a higher proportion of total band 3 being translocated to the surface in the presence of GPA cRNA. The increased expression of DNDS-sensitive chloride transport is highly specific to GPA, and is not observed when the cRNA to the putative glycophorin E or a very high concentration of the cRNA to glycophorin C are coexpressed with band 3 in oocytes.

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.

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.

We constructed cDNA clones encoding N-terminal, C-terminal and internal polypeptide fragments of the human red cell anion exchanger (band 3; AE1). The internal fragments comprised between one and seven putative transmemhrane spans with two or more spans deleted from both termini of the membrane domain of band 3. Sets of three, four or five complementary fragments, which together represented the complete amino acid sequence of the membrane domain, were co-expressed in Xenopus oocytes. Stilbene disulphonate-sensitive chloride uptake assays revealed that all six of the three-fragment combinations and two of the four-fragment combinations reassembled functionally in vivo. Unexpectedly, co-expression of a non-complementary pair of fragments comprising the first five and last seven putative transmemhrane spans (i.e. entirely lacking spans six and seven) was also found to be sufficient to generate stilbene disulphonate-sensitive chloride uptake.

South-East Asian ovalocytosis (SAO) is caused by the heterozygous presence of a variant form of the human erythrocyte anion transporter (band 3; AEl). The expression of band 3 SAO has been studied in Xenopus oocytes. Band 3 SAO is not functional as an anion transporter but is inserted stably into the plasma membrane of oocytes. Band 3 SAO translocation to the cell surface does not require co-expression of normal band 3. Co-expression of glycophorin A @PA) increases the rate of translocation of band 3 SAO to the oocyte membrane but is not essential for this process.We suggest that the increased tendency of band 3 SAO to form oligomers may facilitate its translocation to the cell surface.