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.
Hensel
J
J
Biochemisches Institut am Klinikum der Justus-Liebig-Universität, Giessen, Germany.
Hintz
M
M
Karas
M
M
Linder
D
D
Stahl
B
B
Geyer
R
R
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005227:Fatty Acids / Q000737:chemistry; D005622:Friend murine leukemia virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015686:Gene Products, env / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005227:Fatty Acids; D015686:Gene Products, env; D010169:Palmitic Acids; D010446:Peptide Fragments; D019308:Palmitic Acid