IDENTIFICATION OF THE PALMITOYLATION SITE IN RAT MYELIN P-0 GLYCOPROTEIN

P-0 glycoprotein, the major protein of PNS myelin, contains approximat ely 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P-0 was labeled in rat sciatic nerve slices with [H-3]palmitic acid and subsequently tre ated with various reagents. The protein-bound palmitate was released b y incubation with the reducing agents dithiothreitol and 2-mercaptoeth anol, and with 1 M hydroxylamine at pH 7.5. In addition, P-0 was deacy lated by treatment with 10 mM NaBH4 with the concomitant production of [H-3]hexadecanol, indicating that the fatty acid is bound in a thioes ter linkage. This conclusion was supported further by the fact that de acylation with hydroxylamine generated free thiol groups, which were t itrated with [C-14]- iodoacetamide. To identify the cysteine residue i nvolved in the thioester linkage, [C-14]carboxyamidomethylated P-0 was digested with trypsin and the resulting peptides analyzed by reversed -phase HPLC. Identification of the radioactive protein fragments by am ino acid analysis and amino-terminal peptide sequencing revealed that Cys(153) in rat P-0 glycoprotein is the acylation site. The acylated c ysteine is located at the junction of the putative transmembrane and c ytoplasmic domains. This residue is also present in the P-0 glycoprote in of other species, including human, bovine, mice, and chicken.