Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1).
We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.
Groves
J D
JD
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
Tanner
M J
MJ
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D017920:Antiporters / Q000737:chemistry*; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism; D019281:Dimerization; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D009865:Oocytes; D010446:Peptide Fragments / Q000737:chemistry; D011233:Precipitin Tests; D013267:Stilbenes / Q000494:pharmacology; D014981:Xenopus
D001457:Anion Exchange Protein 1, Erythrocyte; D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D008565:Membrane Proteins; D010446:Peptide Fragments; D013267:Stilbenes; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid