COEXPRESSED COMPLEMENTARY FRAGMENTS OF THE HUMAN RED-CELL ANION-EXCHANGER (BAND-3, AE1) GENERATE STILBENE DISULFONATE-SENSITIVE ANION TRANSPORT

We have constructed cDNA clones encoding various portions of the human red cell anion transporter (band 3), a well characterized integral me mbrane 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 microso mal membranes using the reticulocyte cell-free translation system and in Xenopus oocytes, Co expression of the pairs of recombinants encodin g 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-sensitiv e anion transport in oocytes. When the pairs of fragments 8+6 or 12+2 were co-expressed with glycophorin A (GPA), translocation to the plasm a 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-dependen t translocation when expressed in the absence of its complementary fra gment, A truncated form of band 3 encoding all 14 transmembrane spans but lacking the carboxyl-terminal 30 amino acids of the cytoplasmic ta il did not induce anion transport activity in oocytes and was not tran slocated 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 tha t the integrity of the loops between transmembrane spans 8-9 or 12-13 is not essential for anion transport function. Our data also suggest t hat 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 su rface.