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.

K Okubo,N Hamasaki,K Hara,M Kageura

doi:10.1016/s0021-9258(18)55315-1

Abstract

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.

Highlights

ACYLATION OCCURSIN T H E MIDDLEOF T H E CONSENSUSSEQUENCEOFF--I-IICLAVLFOUNDIN BAND 3 PROTEIN AND G2 PROTEINOFRIFT VALLEY FEVER VIRUS*
The COOH terminus of Band 3.The fatty acidsused in Stoffel et al (1983) show by amino acidsequencing of the the modification were myristate, palmitate, oleate, andprotein that the fatty acid is bound to a threonine residue stearate, with palmitate being the major component. within a n extracellular loop
COOH terminus of human erythrocyte Band 3 is affinitylabeled with pyridoxal phosphate, and that this region constitutes a part of the active center for anion transport (HaRecently, a varietyof viral and cellular membrane proteins masaki et al, 1983; Hamasaki and Kawano, 1987; Kawano et have been reported to be modified by fatty acid acylation or al., 1988)

Summary

Wehaverecently foundthat a peptidecontainingthe

COOH terminus of human erythrocyte Band 3 is affinitylabeled with pyridoxal phosphate, and that this region constitutes a part of the active center for anion transport (HaRecently, a varietyof viral and cellular membrane proteins masaki et al, 1983; Hamasaki and Kawano, 1987; Kawano et have been reported to be modified by fatty acid acylation or al., 1988). The 10th amino acid residue of this peptide could. Determination of this cysteine residue suggested thatthe residue may be modified by lipids, and we have investigated this possibility in the present communication

MATERIALS ANDMETHODS

RESULTS

Myristate Palmitoleate Palmitate Oleate Stearate Total

Amino acid

DISCUSSION

The amino acid sequence near the acylation site of Band