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(farnesyl)-OCH3], to investigate the membrane-anchoring properties of this region of the protein and its reversible modification by S-acylation in cultured mammalian fibroblasts. The farnesylated peptide associates with lipid bilayers (large unilamellar phospholipid vesicles) with high affinity but in a rapidly reversible manner. Additional S-palmitoylation of the peptide suppresses its ability to desorb from, and hence to diffuse between, lipid bilayers on physiologically significant time scales. NBD-labeled derivatives of the farnesylated N-ras C-terminal heptapeptide, when incubated with CV-1 cells in culture, are taken up by the cells and reversibly S-acylated in a manner similar to that observed previously for the parent protein. The S-acylation process is highly specific for modification of a cysteine rather than a serine residue but tolerates replacement of the peptide-linked farnesyl moiety by other hydrophobic groups. Fluorescence microscopy reveals that in CV-1 cells the S-acylated form of the peptide is localized preferentially to the plasma membrane, as has been observed for N-ras itself. This plasma membrane localization is unaffected by either reduced temperature (15 degrees C) or exposure to brefeldin A, treatments which inhibit various trafficking steps within the secretory pathway. These results suggest that in mammalian cells the plasma membrane localization of mature N-ras is maintained by a 'kinetic trapping' mechanism based on S-acylation of the protein at the level of the plasma membrane itself.
Schroeder
H
H
Department of Biochemistry, McGill University, Montréal, Québec, Canada H3G 1Y6.
Leventis
R
R
Rex
S
S
Schelhaas
M
M
Nägele
E
E
Waldmann
H
H
Silvius
J R
JR
D009327:4-Chloro-7-nitrobenzofurazan; D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D020126:Brefeldin A; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003080:Cold Temperature; D003517:Cyclopentanes / Q000494:pharmacology; D005347:Fibroblasts; D011905:Genes, ras; D007700:Kinetics; D008081:Liposomes; D008322:Mammals; D010446:Peptide Fragments / Q000737:chemistry*; D017368:Protein Prenylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000378:metabolism*
D003517:Cyclopentanes; D008081:Liposomes; D010446:Peptide Fragments; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D016283:Proto-Oncogene Proteins p21(ras); D009327:4-Chloro-7-nitrobenzofurazan