Palmitoylation of cysteine 69 from the COOH-terminal of band 3 protein in the human erythrocyte membrane. Acylation occurs in the middle of the consensus sequence of F--I-IICLAVL found in band 3 protein and G2 protein of Rift Valley fever virus.
One of the major physiologic functions of erythrocytes is the mediation of chloride-bicarbonate exchange in the transport of carbon dioxide from the tissues to the lungs. The anion exchange is mediated by a typical polytopic transmembrane protein in the cell membrane, designated Band 3. A carboxyl-terminal peptide of Band 3 was affinity-labeled with pyridoxal phosphate, a substrate for the anion transport system, and then sequenced (Kawano, Y., Okubo, K., Tokunaga, F., Miyata, T., Iwanaga, S., and Hamasaki, N. (1988) J. Biol. Chem. 263, 8232-8238). The 10th amino acid residue of the peptide could not be determined, suggesting post-translational modification of the residue. In the present communication, we have investigated the molecular structure of human Band 3 and the COOH-terminal 8500-dalton peptide using gas-liquid chromatography-mass spectrometry. Band 3 was modified covalently by fatty acids and these acids were released from Band 3 by hydroxylamine treatment at either pH 7 or 11, indicating that the linkage between Band 3 and the fatty acid is a thio ester bond. 1 mol of Band 3 interacted with 1 mol of fatty acid at a cysteine residue located 69 residues from the COOH terminus of Band 3. The fatty acids used in the modification were myristate, palmitate, oleate, and stearate, with palmitate being the major component. The esterified site is close to the site affinity-labeled with pyridoxal phosphate (Kawano, Y., Okubo, K., Tokunaga, F., Miyata, T., Iwanaga, S., and Hamasaki, N. (1988) J. Biol. Chem. 263, 8232-8238). The amino acid sequence including the acylation site was Phe-Thr-Gly-Ile-Gln-Ile-Ile-Cys-Leu-Ala-Val-Leu, which is conserved in the G2 protein of Rift Valley fever virus as Phe-Ser-Ser-Ile-Ala-Ile-Ile-Cys-Leu-Ala-Val-Leu. The G2 protein, like Band 3, is a polytopic transmembrane protein. Although acylation of the cysteine residue of G2 protein has not been examined, the Phe-X-X-Ile-X-Ile-Ile-Cys-Leu-Ala-Val-Leu sequence could be a common motif for fatty acylation of certain membrane proteins.
Okubo
K
K
Department of Clinical Chemistry, University School of Medicine, Fukuoka, Japan.
Hamasaki
N
N
Hara
K
K
Kageura
M
M
D000215:Acylation; D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D016384:Consensus Sequence; D003545:Cysteine / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D010169:Palmitic Acids / Q000378:metabolism*; D012296:Rift Valley fever virus / Q000378:metabolism*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D005227:Fatty Acids; D006898:Hydroxylamines; D010169:Palmitic Acids; D014764:Viral Proteins; D019811:Hydroxylamine; D003545:Cysteine