Anion transport in oocytes of Xenopus laevis induced by expression of mouse erythroid band 3 protein--encoding cRNA and of a cRNA derivative obtained by site-directed mutagenesis at the stilbene disulfonate binding site.
A vector was constructed containing a cDNA for mouse band 3 obtained from Demuth et al. (1986, EMBO J., 5, 1205-1214), a synthetic linker (containing 5'-non-translated region, start codon and a coding region for the first 12 N-terminal amino acids), and RNA polymerase promoters suitable for in vitro transcription of cRNA. After injection of the cRNA into the cytoplasm of Xenopus oocytes and incubation for 16 h, expression of mouse band 3 was demonstrated by immunoprecipitation, immunohistochemical methods and influx or efflux measurements with 36Cl-. Antisense cRNA inhibits the expression. Lysines 558 and 561 were replaced by asparagines using oligonucleotide-directed mutagenesis. Like the original band 3, the mutant shows stilbene disulfonate-inhibitable anion exchange. However, in contrast to the original band 3, inhibition by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate (H2DIDS) is no longer irreversible. This indicates that thiourea bond formation between H2DIDS and band 3 involves one of the two modified lysine residues. It also shows that the two lysine residues are not essential for the execution of the anion transport function of band 3. The results described suggest that the cDNA clone of Demuth et al. (1986) encodes a protein with properties that are representative for the properties of the bulk of the band 3 protein in the plasma membrane of the red cell of the mouse.
Bartel
D
D
Max-Planck-Institut für Biophysik, Frankfurt am Main, FRG.
Lepke
S
S
Layh-Schmitt
G
G
Legrum
B
B
Passow
H
H
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000378:metabolism*; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000096:biosynthesis* / Q000235:genetics; D001665:Binding Sites; D001692:Biological Transport; D002474:Cell-Free System; D002712:Chlorides / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009865:Oocytes / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics*; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000378:metabolism; D013267:Stilbenes / Q000378:metabolism*; D014158:Transcription, Genetic; D014982:Xenopus laevis
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D018075:RNA, Complementary; D012333:RNA, Messenger; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; D012313:RNA; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid