S-Acylation and Plasma Membrane Targeting of the Farnesylated Carboxyl-Terminal Peptide of N-ras in Mammalian Fibroblasts

Abstract

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.

Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

“…We and others have demonstrated that short peptide sequences are sufficient to direct the addition of both lipids and confer plasma membrane targeting (Schroeder et al, 1996(Schroeder et al, , 1997Wolven et al, 1997) (this study). Biophysical measurements of the association of lipid-modified peptides with artificial bilayers provide a rationale for the requirement of two lipid modifications in targeting proteins to membranes (Peitzsch and McLaughlin, are targeted to the plasma membrane via their association with Gpa1p, which is dually acylated.…”

Huntingtin interacting protein 14 is an oncogenic human protein: palmitoyl acyltransferase

“…The main function of protein palmitoylation is to facilitate membrane binding of the palmitate-modified proteins. Although other fatty acid modifications also increase protein hydrophobicity, those modifications result in only transient membrane association (t 1/2 p1 min) (Schroeder et al, 1997). Furthermore, proteins that undergo palmitoylation in conjunction with either myristoylation or prenylation demonstrate an even stronger membrane association with a t 1/2 >70 h (Alland et al, 1994;Shahinian and Silvius, 1995).…”

The cholesterol membrane anchor of the Hedgehog protein confers stable membrane association to lipid-modified proteins

“…To date direct determination of the membrane-binding properties of biologically functional proteins has focused on acylated and isoprenylated peptides and proteins (9)(10)(11)(12)(13)(14)(15)(16), whereas there is a gap for sterol-modified peptides and proteins.…”