Alternative primary structures in the transmembrane domain of the chicken erythroid anion transporter.

Abstract

Isolation and characterization of the chicken erythroid anion transporter (band 3) cDNA clone, pCHB3-1, revealed that the chicken erythroid band 3 polypeptide is 844 amino acids in length with a predicted mass of 109,000 daltons. This polypeptide is composed of a hydrophilic N-terminal cytoplasmic domain and a hydrophobic C-terminal transmembrane domain. The -90 N-terminal amino acids of the human and murine erythroid band 3 polypeptides are absent in the predicted sequence of the chicken erythroid band 3 polypeptide. The absence of this very acidic N-terminal region is consistent with the lack of binding of glyceraldehyde-3-phosphate dehydrogenase to chicken erythroid band 3, as weH as the relatively basic isoelectric point observed for this molecule. The remainder of the cytoplasmic domain shows little similarity to the cytoplasmic domain of the murine and human erythroid band 3, with the exception of the putative ankyrin-binding site, which is highly conserved. In contrast, the transmembrane domain of the chicken band 3 polypeptide is very similar to that of the murine erythroid and human nonerythroid band 3 polypeptides. The transmembrane domain contains 10 hydrophobic regions that could potentially traverse the membrane 12 to 14 times. In addition, a variant of chicken erythroid band 3, pCHB3-2, was cloned in which one of the hydrophobic regions of pCHB3-1 is lacking. The transcript complementary to pCHB3-2 accumulated in chicken erythroid ceUs in a similar manner as the transcript complementary to pCHB3-1 during embryonic development. This is the first example of a transporter protein or ion channel with alternative primary structures in its membrane-spanning segments.

Two different mRNAs are transcribed from a single genomic locus encoding the chicken erythrocyte anion transport proteins (band 3).

“…This result is clearly in disagreement with the chicken erythroid anion transport protein cDNA sequence and polypeptide sequence (predicted by conceptual translation of the cDNA clone pCHB3-1) previously described by Cox and Lazarides (9). The band 3 protein which these authors deduce from pCHB3-1 DNA sequence begins translation at amino acid 79 of the pBIIIC1 sequence ( Fig.…”

“…Recombinants pBIIIC1 through pBIIIC3 all contain this exon by direct DNA sequence analysis (data not shown). Since the experiment reporting the missing exon was technically flawed (only protection fragments corresponding to either full-length deletion recombinant probe or the band 3 sequence reported here were visualized in that experiment [9]), we cannot comment on the possibility that such an mRNA does or does not exist in chicken RBC mRNA, although we find no evidence for the existence of that species of band 3 mRNA in the experiments reported here.…”

“…The band 3 protein which these authors deduce from pCHB3-1 DNA sequence begins translation at amino acid 79 of the pBIIIC1 sequence ( Fig. 2; 3-1), predicting a polypeptide of 844 amino acids with a molecular mass of 93,784 (erroneously reported as 109,000 [9]) and a pI of 9.83; both the molecular weight and pl of this predicted protein substantially differ from values in the established literature (17). Although internal sequences within cDNA clone pCHB3-1 match the DNA sequence of pBlIICl with long regions of identity (including, however, a number of base sequence and several frameshift discrepancies), sequences 5' to position 267 of pBIICl are not related to pCHB3-1 (Fig.…”

“…We have not examined pBIIIC1-C9 to specifically address the question of whether or not these recombinants contain a band 3 mRNA which is reported to lack one membranespanning exon (9). Recombinants pBIIIC1 through pBIIIC3 all contain this exon by direct DNA sequence analysis (data not shown).…”

“…8,1988 TWO mRNAs FROM CHICKEN RBC ANION TRANSPORT GENE 4419 10 20 30 40 50 60 4 Cl GGCGGCGGCG TCCCCAGAGC GGGGCATGGT GCCGCCAAGA TGAGCGCCGG CACGCAGCGG GGGGGTGAGT 70 C1 Cl GGAGCCX3 AGGGGCCCGG CCAGGACACC GAGGACGCGC TACGCAGGAG CCTGGACCCC GAGGGCTACG 140 C2 TGAG 1 ---------------------------- ) is shown, in comparison to the 5' ends of recombinants pBIIIC2 and pCHB3-1 (9). The AUG corresponding to translation initiation (boxed) indicates the predicted translational start site for band 3 polypeptides corresponding to cDNA clones pBIIIC1 (nt 77 to 79), pBIIIC2 (nt 176 to 178), and pCHB3-1 (nt 311 to 313).…”