Polylysine induces pH-dependent fusion of acidic phospholipid vesicles: a model for polycation-induced fusion.
Polylysine induced aggregation and fusion of negatively charged small unilamellar phosphatidylcholine vesicles containing at least 10% anionic lipid. Aggregation was followed by absorbance changes and fusion was assayed both by electron microscopy and by fluorescence energy transfer between lipid probes. A method for preparing asymmetric vesicles, where the fluorescent probes were present only in the inner monolayer of the vesicle membrane, was developed. These vesicles were used to distinguish the inner and outer monolayer when measuring lipid mixing between vesicles. Since polylysine induced lipid mixing of both monolayers equally, fusion of these vesicles did occur. The extent of fusion was dependent on the charge ratio between bound polylysine and phosphatidylserine (PS) in the outer monolayer and was optimal at a ratio of about 1:1. Excess polylysine inhibited fusion. At a given concentration of polypeptide, fusion increased as the pH was lowered toward 3 with an apparent pKa near 4. Since this value is close to the pKa of the PS-carboxyl groups and far from the pKa of the lysine epsilon-amino groups, the pH dependence observed for fusion resides in the lipids rather than in the peptide. Fusion was dependent on the available lysine and not the size or molarity of the polypeptide. The data indicate that there must be sufficient sites on the vesicles and sufficient polypeptide to achieve effective aggregation. For fusion to occur after aggregation, charges on the vesicles must be neutralized either by polypeptide-PS interaction or by protonation of the PS carboxyl groups. Optimal conditions for fusion occur when charge neutralization is possible without completely covering the vesicles with polypeptide. The results are consistent with the notion that the polypeptide is necessary for fusion because of requirements for crosslinking, but limits fusion by steric inhibition.
D002413:Cations, Divalent / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000032:analysis; D005614:Freeze Fracturing; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008560:Membrane Fluidity / Q000187:drug effects; D008561:Membrane Fusion / Q000187:drug effects*; D008563:Membrane Lipids / Q000032:analysis*; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011107:Polylysine / Q000494:pharmacology*
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D011107:Polylysine