Doubly-lipid-modified protein sequence motifs exhibit long-lived anchorage to lipid bilayer membranes

Abstract

To understand better the potential functional importance of the dual-lipid modifications found in a number of intracellular proteins of eukaryotes, we have examined how "tenaciously" various doubly-lipid-modified peptides, with sequences and lipid modifications reflecting those found in intracellular proteins, are anchored to lipid bilayer membranes. Fluorescent-labeled peptides bearing dual-lipid modifications were incorporated into large unilamellar egg phosphatidylcholine/phosphatidylglycerol vesicles, and the kinetics of spontaneous intervesicle transfer of the lipopeptides were monitored by a fluorescence-dequenching assay. Lipopeptides incorporating the stable "dual-anchor" motif -C(geranylgeranyl)XC(geranylgeranyl)-OMe found in several rab and homologous proteins exhibit very slow rates of interbilayer transfer (t1/2 > 50 h), as do lipopeptides incorporating myristoyl-GC(palmitoyl)X- and -C(palmitoyl)XC(farnesyl)-OMe motifs found in various src-related intracellular tyrosine kinases and G-protein alpha-subunits and in p21H-ras, respectively. Lipopeptides terminating in an unmethylated -C(geranylgeranyl)C(geranylgeranyl)-OH motif show somewhat greater but still very slow rates of spontaneous interbilayer transfer (t1/2 = ca. 10 h). Extrapolating from these results, we estimate that the rate of spontaneous desorption of the corresponding doubly-anchored proteins from membranes should be much slower than that of regulated, protein-mediated release (effected by binding to an "escort" protein or by de-S-acylation). As a result the intracellular distributions of these species (and particularly their targeting to specific intracellular membranes) are likely to be governed (and regulated) primarily by kinetic rather than thermodynamic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…11, March 20001993; Silvius and l'Heureux, 1994;Shahinian and Silvius, 1995). A C15 farnesyl isoprenoid or an N-myristoyl group does not provide sufficient hydrophobicity to anchor a peptide stably to membranes.…”

“…However, the addition of a second lipid modification dramatically slows the rate of interbilayer transfer such that the peptide is essentially permanently anchored. Shahinian and Silvius (1995) suggested the kinetic bilayer-trapping model as a mechanism for targeting proteins to a specific membrane. The model proposes that a farnesylated or N-myristoylated protein diffuses through the cytosol transiently associating with all membranes until it encounters the site where the second lipid is added.…”

Organic Synthesis and Biological Signal Transduction

“…In a series of biophysical investigations of the thermodynamics of insertion of lipid-modified model peptides in vesicles, functioning as model membranes, the contribution of the lipids to lipopeptide membrane affinity was determined. [45][46][47] This showed that an Nmyristoyl or an S-farnesyl group alone cannot contribute enough hydrophobic character to maintain stable membrane insertion of peptides, and therefore also of proteins.…”

“…Biophysical model experiments on the kinetics of transfer of lipidmodified peptides from one model membrane to another [49,50] showed that peptides and proteins that only carry a single lipid modification are rapidly inserted into the membrane (within seconds) yet they are also rapidly exchanged between two different membranes. The half time for transfer of a singly farnesylated or palmitoylated peptide is in the region of seconds.…”

Emerging roles for protein S-palmitoylation in Toxoplasma biology

“…In the kinetic trapping model (Peitzsch and McLaughlin, 1993;Shahinian and Silvius, 1995), palmitoylation acts in concert with two other types of acylation to ensure a stable interaction within membrane leaflets (Fig. 1B).…”