Fatty acid acylation at the single cysteine residue in the cytoplasmic domain of the glycoprotein of vesicular-stomatitis virus.
The cysteine residue in the cytoplasmic domain at position 489 of the sequence of the glycoprotein (G protein) isolated from vesicular-stomatitis virions is completely blocked for carboxymethylation. After release of covalently bound fatty acids by hydroxylamine at pH 6.8, this cysteine residue could be specifically labelled by iodo[14C]acetic acid. Reaction products were analysed after specific cleavage of labelled G protein at asparagine-glycine bonds by hydroxylamine at pH 9.3, which generated a C-terminal peptide of Mr 15,300 containing only the single cysteine residue. Bromelain digestion of [3H]palmitic acid-labelled membrane fractions of vesicular-stomatitis-virus-infected baby-hamster kidney cells removed almost completely the 3H radioactivity from the cytoplasmic domain of the G protein, whereas the ectodomain was completely protected by the microsomal membrane. This result indicates that the acylation site of the G protein is exposed on the cytoplasmic side of intracellular membranes. Taken together, both biochemical techniques strongly suggest that the single cysteine-489 residue, which is located six amino acid residues distal to the putative transmembrane domain, is the acylation site. The thioester bond between palmitic acid and the G protein is quite resistant to hydroxylamine treatment (0.32 M at pH 6.8 for 1 h at 37 degrees C) compared with the reactivity of the thioester linkage in palmitoyl-CoA, which is cleaved at relatively low concentrations of hydroxylamine (0.05 M).
Mack
D
D
Abteilung Molekularbiologie, Universität Hamburg, Federal Republic of Germany.
Kruppa
J
J
D000215:Acylation; D003545:Cysteine / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D006898:Hydroxylamines; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*
D005227:Fatty Acids; C028391:G protein, vesicular stomatitis virus; D006898:Hydroxylamines; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D003545:Cysteine