EFFECT OF SITE-DIRECTED MUTAGENESIS OF THE ARGININE RESIDUE-509 AND RESIDUE-748 ON MOUSE BAND-3 PROTEIN-MEDIATED ANION TRANSPORT

Using site-directed mutagenesis, the arginine residues 509 and 748 in mouse band 3 protein were substituted by Lys. Thr. and Cys, or by Lys and Gln, respectively. After expression in Xenopus oocytes of the cRNA s encoding wild type band 3 or any one of the band 3 mutants, chloride equilibrium exchange was measured. When the flux measurements were pe rformed two to three days after microinjection of the cRNAs, in contra st to the wild type, neither one of the mutants was able to accomplish transport, with the possible exception or the mutants R509K and R748K both of which showed some transport activity of doubtful significance . Immunoprecipitates revealed that the Arg 748 mutants were expressed similar to the wild type band 3 while no expression of the Arg 509 mut ants could be detected. When the flux measurements were performed only 3 h after microinjection of the cRNAs. transport activity was observe d in the oocytes that had received cRNAs encoding wild type band 3. In some oocytes of a population, a very slight transport activity was br ought about by cRNA encoding Arg 509 mutants. No transport activity co uld be detected after injection of the Arg 748 mutant. Immunoprecipita tion demonstrated the successful biosynthesis of wild type band 3 and of both the Arg 509 and the Arg 748 mutants. The experiments suggest t hat mutation of Arg 748 leads to biosynthesis of an inactive form of t he band 3 protein, while that of Arg 509 results in expression of an a bnormally folded, possibly functionally more or less intact form, whic h is proteolytically degraded within less than one day. (C) 1998 Elsev ier Science B.V.