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 (t(1/2) 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 p21(H-ras), respectively. Lipopeptides terminating in an ummethylated -C(geranylgeranyl)C(geranylgeranyl)-OH motif show somewhat greater but still very slow rates of spontaneous interbilayer transfer (t(1/2) = 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. In parallel vesicle-binding measurements, peptides modified with S-palmitoyl groups were found to associate with lipid bilayers even more avidly than comparable geranylgeranylated peptides, explaining the 'tenacious' membrane-binding properties of dual-anchor motifs incorporating an S-palmitoyl residue.
