COMPLEMENTATION STUDIES WITH COEXPRESSED FRAGMENTS OF THE HUMAN RED-CELL ANION TRANSPORTER (BAND-3, AE1) - THE ROLE OF SOME EXOFACIAL LOOPSIN ANION TRANSPORT

We constructed cDNA clones encoding fragments of band 3 in which the m embrane domain was truncated from either the N or the C terminus withi n each of the first four exofacial loops. The truncations containing t he 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 glycopho rin 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 fragment s divided in the third and fourth exofacial loops yielded transport ac tivity, but the pair separated within the second exofacial loop was no t active, We conclude that the integrity of the second exofacial loop, but not the third and fourth exofacial loops, is necessary for transp ort activity. The unusually stable association between the fragments d ivided in the second exofacial loop suggests that interactions may occ ur between polar surfaces on amphiphilic portions of the third and fif th transmembrane spans.