DOUBLY-LIPID-MODIFIED PROTEIN-SEQUENCE MOTIFS EXHIBIT LONG-LIVED ANCHORAGE TO LIPID BILAYER-MEMBRANES

To understand better the potential functional importance of the dual-l ipid modifications found in a number of intracellular proteins of euka ryotes, we have examined how ''tenaciously'' various doubly-lipid-modi fied peptides, with sequences and lipid modifications reflecting those found in intracellular proteins, are anchored to lipid bilayer membra nes. Fluorescent-labeled peptides bearing dual-lipid modifications wer e incorporated into large unilamellar egg phosphatidylcholine/phosphat idylglycerol vesicles, and the kinetics of spontaneous intervesicle tr ansfer of the lipopeptides were monitored by a fluorescence-dequenchin g assay. Lipopeptides incorporating the stable ''dual-anchor'' motif - C(geranylgeranyl)XC(geranylgeranyl)-OMe found in several rab and homol ogous proteins exhibit very slow rates of interbilayer transfer (t(1/2 ) 50 h), as do lipopeptides incorporating myristoyl-GC(palmitoyl)X- an d -C(palmitoyl)XC(farnesyl)-OMe motifs found in various src-related in tracellular tyrosine kinases and G-protein alpha-subunits and in p21(H -ras), respectively. Lipopeptides terminating in an ummethylated -C(ge ranylgeranyl)C(geranylgeranyl)-OH motif show somewhat greater but stil l very slow rates of spontaneous interbilayer transfer (t(1/2) = ca. 1 0 h). Extrapolating from these results, we estimate that the rate of s pontaneous desorption of the corresponding doubly-anchored proteins fr om membranes should be much slower than that Of regulated, protein-med iated release (effected by binding to an ''escort'' protein Or by de-S -acylation), As a result the intracellular distributions of these spec ies (and particularly their targeting to specific intracellular membra nes) are likely to be governed (and regulated) primarily by kinetic ra ther than thermodynamic factors. In parallel vesicle-binding measureme nts, peptides modified with S-palmitoyl groups were found to associate with lipid bilayers even more avidly than comparable geranylgeranylat ed peptides, explaining the 'tenacious' membrane-binding properties of dual-anchor motifs incorporating an S-palmitoyl residue.