LOCALIZATION OF THE PALMITOYLATION SITE IN THE TRANSMEMBRANE PROTEIN P12E OF FRIEND MURINE LEUKEMIA-VIRUS

Friend murine leukaemia virus complex was propagated on murine cells i n the presence of [9,10-H-3]palmitic acid. Virus particles were harves ted from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size-exc lusion chromatography and purified by narrowbore reverse-phase HPLC. A nalysis of the purified product by matrix-assisted laser desorption/io nization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The rad iolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For alloc ation of the acylation site, p12E was digested with trypsin. The resul ting peptides were either directly subjected to MALDI-TOF-MS or fracti onated by microbore reverse-phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylat ed at a cysteine residue situated at the C-terminal side of the putati ve transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusivel y palmitic acid.