Lipid-binding characteristics of the polybasic carboxy-terminal sequence of K-ras4B.
We have examined the association with lipid vesicles of fluorescent lipidated peptides based on the farnesylated, polybasic carboxy-terminal region of mature K-ras4B, which functions physiologically as an autonomous plasma membrane-targeting motif. While the peptides bind to neutral lipid (phosphatidylcholine/phosphatidylethanolamine) vesicles with relatively low affinity, the vesicle-binding affinity increases exponentially as increasing amounts of anionic lipids are incorporated into the vesicle bilayers. Competitive vesicle-binding experiments reveal that the K-ras4B carboxy-terminal sequence accordingly discriminates strongly between lipid surfaces of differing surface charge, such that two lipid bilayers differing in anionic lipid content by 10 mol % will show a 45-fold preferential accumulation of the lipidated peptide in the more negatively charged surface. At the same time, the carboxyl-terminal region of K-ras4B exhibits no preferential binding to particular anionic lipids, including the polyanionic species phosphatidylinositol-4'-phosphate and phosphatidylinositol-4',5'-bisphosphate, beyond that predicted on the basis of surface-charge effects. The K-ras4B carboxyl-terminal sequence dissociates rapidly (with half-times of seconds or less) from lipid bilayers containing up to 40 mol % anionic lipid. These results suggest that the targeting of the mature K-ras4B carboxy-terminus to the plasma membrane, if it is based on interactions with plasma membrane lipids, is not mediated by a kinetic-trapping mechanism or by specific binding to particular anionic lipids but may rest on the sensitive surface potential-sensing function of this region of the protein.
Leventis
R
R
Department of Biochemistry, McGill University, Montréal, Québec, Canada.
Silvius
J R
JR
D000818:Animals; D000838:Anions; D002784:Cholesterol / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D050356:Lipid Metabolism; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D011485:Protein Binding; D017368:Protein Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry*; D055672:Static Electricity; D014982:Xenopus laevis
D000838:Anions; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010455:Peptides; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; C042511:1,2-dioleoylphosphatidylserine; D002784:Cholesterol; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)