LIPID-MODIFIED, CYSTEINYL-CONTAINING PEPTIDES OF DIVERSE STRUCTURES ARE EFFICIENTLY S-ACYLATED AT THE PLASMA-MEMBRANE OF MAMMALIAN-CELLS

A variety of cysteine-containing, lipid-modified 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-modi fied peptides over hydrophilic peptides. The S-acylation process appea rs by various criteria to be enzymatic and resembles the S-acylation o f plasma membrane-associated proteins in various characteristics, incl uding inhibition by tunicamycin. The substrate range of the S-acylatio n reaction encompasses, but is not limited to, lipopeptides incorporat ing the motifs myristoylGC- and -CXC(farnesyl)-OCH3, which are reversi bly S-acylated in various intracellular proteins. Mass-spectrometric a nalysis indicates that palmitoyl residues constitute the predominant b ut not the only type of S-acyl group coupled to a lipopeptide carrying the myristoylGC- motif, with smaller amounts of S-stearoyl and S-oleo yl substituents also detectable. Fluorescence microscopy using NBD-lab eled 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 preferenti al 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 sec ond, intracellular compartment identified as the Golgi complex by its labeling with a fluorescent ceramide. Our results suggest that distinc t S-acyltransferases exist in the Golgi complex and plasma membrane co mpartments 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.