Chemical identification of cysteine as palmitoylation site in a transmembrane protein (Semliki Forest virus E1).
The palmitoylation site of the membrane glycoprotein E1 of Semliki Forest virus (SFV) has been identified by chemical analysis of an acylpeptide. 3H-Palmitoylated E1 isolated from SFV grown in baby hamster kidney cells was digested with chymotrypsin and the resulting peptides subjected to high performance liquid chromatography on a wide-pore column. The 3H-acylated peptide fraction peaked at above 60% 2-propanol in the eluent, indicating its hydrophobic character. Polyacrylamide gel electrophoresis analysis revealed a molecular weight of about Mr = 6000 for the radiolabeled peptide. Manual sequencing of this material by the 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate procedure on solid phase revealed the amino-terminal sequence Ala-Ala-Ser-His-Ser-Asn-Val-Val-Phe-Pro. The same peptide also labels with [35S]cysteine. Comparison with the deduced amino acid sequence of E1 revealed that the palmitoylated peptide contains at least 43 amino acid residues, and thus includes the membrane spanning region down to the only cysteine residue five positions up from the carboxyl terminus of E1. Since [3H]palmitic acid was cleaved from E1 with thiol reagents, and since the peptide labels with [14C]iodoacetamide only after the release of fatty acids by hydroxylamine treatment, cysteine in position 433 represents the palmitoylation site in SFV E1.
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003545:Cysteine / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D017879:Isothiocyanates; D008565:Membrane Proteins / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000032:analysis*; D012672:Semliki forest virus / Q000032:analysis*; D013861:Thiocyanates; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D017879:Isothiocyanates; D008565:Membrane Proteins; D010169:Palmitic Acids; D013861:Thiocyanates; D019308:Palmitic Acid; C012926:4-(N,N-dimethylaminoazobenzene)-4'-isothiocyanate; D004124:p-Dimethylaminoazobenzene; D003545:Cysteine