Effect of site-directed mutagenesis of the arginine residues 509 and 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 cRNAs encoding wild type band 3 or any one of the band 3 mutants, chloride equilibrium exchange was measured. When the flux measurements were performed two to three days after microinjection of the cRNAs, in contrast to the wild type, neither one of the mutants was able to accomplish transport, with the possible exception of 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 mutants could be detected. When the flux measurements were performed only 3 h after microinjection of the cRNAs, transport activity was observed in the oocytes that had received cRNAs encoding wild type band 3. In some oocytes of a population, a very slight transport activity was brought about by cRNA encoding Arg 509 mutants. No transport activity could be detected after injection of the Arg 748 mutant. Immunoprecipitation demonstrated the successful biosynthesis of wild type band 3 and of both the Arg 509 and the Arg 748 mutants. The experiments suggest that mutation of Arg 748 leads to biosynthesis of an inactive form of the band 3 protein, while that of Arg 509 results in expression of an abnormally folded, possibly functionally more or less intact form, which is proteolytically degraded within less than one day.
Karbach
D
D
Max Planck Institut für Biophysik, Frankfurt am Main, Germany.
Staub
M
M
Wood
P G
PG
Passow
H
H
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000235:genetics / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D017136:Ion Transport; D051379:Mice; D008845:Microinjections; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism*; D018075:RNA, Complementary / Q000235:genetics; D014981:Xenopus
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D018075:RNA, Complementary; D001120:Arginine