S-ACYLATION AND PLASMA-MEMBRANE TARGETING OF THE FARNESYLATED CARBOXYL-TERMINAL PEPTIDE OF N-RAS IN MAMMALIAN FIBROBLASTS

We have used a series of fluorescent lipid-modified peptides, based on the farnesylated C-terminal sequence of mature N-ras [-GCMGLPC(farnes yl)-OCH3], to investigate the membrane-anchoring properties of this re gion of the protein and its reversible modification by S-acylation in cultured mammalian fibroblasts. The farnesylated peptide associates wi th lipid bilayers (large unilamellar phospholipid vesicles) with high affinity but in a rapidly reversible manner. Additional S-palmitoylati on of the peptide suppresses its ability to desorb from, and hence to diffuse between, lipid bilayers on physiologically significant time sc ales. NBD-labeled derivatives of the farnesylated N-ras C-terminal hep tapeptide, when incubated with CV-I cells in culture, are taken up by the cells and reversibly S-acylated in a manner similar to that observ ed previously for the parent protein. The S-acylation process is highl y specific for modification of a cysteine rather than a serine residue but tolerates replacement of the peptide-linked farnesyl moiety by ot her hydrophobic groups. Fluorescence microscopy reveals that in CV-1 c ells the S-acylated form of the peptide is localized preferentially to the plasma membrane, as has been observed for N-ras itself. This plas ma membrane localization is unaffected by either reduced temperature ( 15 degrees C) or exposure to brefeldin A, treatments which inhibit var ious trafficking steps within the secretory pathway. These results sug gest that in mammalian cells the plasma membrane localization of matur e N-ras is maintained by a 'kinetic trapping' mechanism based on S-acy lation of the protein at the level of the plasma membrane itself.