COMPLEMENTATION STUDIES WITH COEXPRESSED FRAGMENTS OF HUMAN RED-CELL BAND-3 (AE1) - THE ASSEMBLY OF THE ANION-TRANSPORT DOMAIN IN XENOPUS OOCYTES AND A CELL-FREE TRANSLATION SYSTEM

We examined the assembly of the membrane domain of the human red cell anion transporter (band 3; AE1) by co-expression of recombinant N- and C-terminal fragments in Xenopus oocytes and in cell-free translation with canine pancreatic microsomes. Co-immunoprecipitation was performe d in non-denaturing detergent solutions using antibodies directed agai nst the N- and C-termini of the membrane domain. Eleven of the twelve fragments were expressed stably in oocytes in the presence or absence of their respective partners. However, the fragment containing from pu tative span nine to the C-terminus could be detected in oocytes only w hen co-expressed with its complementary partner containing the first e ight spans. Go-expression of pairs of fragments divided in the first, second, third and fourth exofacial loops and in the fourth cytoplasmic loop resulted in a concentration-dependent association, but a pair of fragments divided in the sixth cytoplasmic loop did not co-immunoprec ipitate. When two complementary fragments were translated separately i n the cell-free system and the purified microsomes were then mixed, co immunoprecipitation was observed only if the membranes were first fuse d using polyethylene glycol. This shows that co-immunoprecipitation re sults from specific interactions within the membrane and is not an art efact of detergent solubilization or immunoprecipitation. We demonstra te that band 3 assembly can occur within the membrane after translatio n, insertion and initial folding of the individual fragments have been completed. We conclude that most band 3 fragments contain the necessa ry information to fold in the membrane and adopt a structure that is s ufficiently similar to the native protein that it permits correct asse mbly with its complementary partner.