Lipid-modified, cysteinyl-containing peptides of diverse structures are efficiently S-acylated at the plasma membrane of mammalian cells.

Abstract

Abstract. A variety of cysteine-containing, lipidmodified peptides are found to be S-acylated by cultured mammalian cells. The acylation reaction is highly specific for cysteinyl over serinyl residues and for lipid-modified peptides over hydrophilic peptides. The S-acylation process appears by various criteria to be enzymatic and resembles the S-acylation of plasma membrane-associated proteins in various characteristics, including inhibition by tunicamycin. The substrate range of the S-acylation reaction encompasses, but is not limited to, lipopeptides incorporating the motifs myristoylGC-and -CXC(farnesyl)-OCH3, which are reversibly S-acylated in various intracellular proteins. Mass-spectrometric analysis indicates that palmitoyl residues constitute the predominant but not the only type of S-acyl group coupled to a lipopeptide carrying the myristoylGC-motif, with smaller amounts of S-stearoyl and S-oleoyl substituents also detectable. Fluorescence microscopy using NBD-labeled cysteinyl lipopeptides reveals that the products of lipopeptide S-acylation, which cannot diffuse between membranes, are in almost all cases localized preferentially to the plasma membrane. This preferential localization is found even at reduced temperatures where vesicular transport from the Golgi complex to the plasma membrane is suppressed, strongly suggesting that the plasma membrane itself is the preferred site of S-acylation of these species. Uniquely among the lipopeptides studied, species incorporating an unphysiological N-myristoylcysteinyl-motif also show substantial formation of S-acylated products in a second, intracellular compartment identified as the Golgi complex by its labeling with a fluorescent ceramide. Our results suggest that distinct S-acyltransferases exist in the Golgi complex and plasma membrane compartments and that S-acylation of motifs such as myristoylGC-occurs specifically at the plasma membrane, affording efficient targeting of cellular proteins bearing such motifs to this membrane compartment.VARIETY of integral membrane proteins and reversibly membrane-associated proteins in eukaryotic cells exhibits posttranslational acylation on one or more cysteine residues, a modification that for a number of such proteins appears to be dynamic (6,39,42,45,81,82,84) and, in some cases, is modulated by physiological or pharmacological stimuli (15,31,45,46,63,86). Integral membrane proteins may be S-acylated either on cysteine residues near the cytoplasmic termini of transmembrane helixes (3,14,28,30,32,68,74) or on cytoplasmic cysteine residues more distant from a transmembrane helix (9,18,21,88). Among the reversibly membrane-associated proteins that undergo S-acylation are found a number of srchomologous nonreceptor tyrosine kinases, heterotrimeric G protein a subunits and monomeric G proteins (for reviews see 10,43,44,61,65,75). S-acylation has been shown to enhance the membrane association of a variety Address all correspondence to Dr. John R. Silvius, Department of Biochemistry, McGill University, Montrral, Qu6bec,...

Organic Synthesis and Biological Signal Transduction

“…The results of the biophysical and cell biology investigations support a model [51] for specific localization of proteins by myristoylation/ palmitoylation or farnesylation/palmitoylation. According to this model, the specific localization is not only determined by the lipid groups introduced in the course of the biosynthesis (in the case of Ras proteins, the farnesyl residue).…”

“…This hypothesis was tested by in vivo studies with fluorescently labeled N-myristoylated [51] and S-farnesylated peptides. [43,50,51] Peptides 30 and 31 (Fig.…”

“…[43,50,51] Peptides 30 and 31 (Fig. 4), which represent forms of the N-terminus of the human non-receptor tyrosine kinase Lck and the C-terminus of human N-Ras protein with a single lipid modification, were S-palmitoylated in fibroblast cells.…”

Association of Prenylated Proteins with the Plasma Membrane and the Inner Nuclear Membrane Is Mediated by the Same Membrane-targeting Motifs

“…S-Acylation of H-and N-Ras occurs in an early compartment of the exocytotic pathway (Choy et al, 1999;Magee and Marshall, 1999;Apolloni et al, 2000). Studies with lipid-modified peptides have shown that efficient S-acylation can occur directly at the PM (Schroeder et al, 1996) and that doubly lipidated peptides are bound to membrane vesicles by kinetic trapping (Shahinian and Silvius, 1995). Whether K-Ras exists in a dynamic equilibrium and can rapidly switch between a PM-bound and a cytosolic form (Yokoe and Meyer, 1996) or shows fast lateral diffusion at the PM (Niv et al, 1999) is still a matter of debate.…”

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.…”