Localization of the Palmitoylation Site in the Transmembrane Protein p12E of Friend Murine Leukaemia Virus

Friend murine leukaemia virus complex was propagated on murine cells in the presence of [9,10‐³H]palmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size‐exclusion chromatography and purified by narrowbore reverse‐phase HPLC. Analysis of the purified product by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI‐TOF‐MS or fractionated by microbore reverse‐phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the C‐terminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid.