Role of Anionic Lipids on Peg-Mediated Model Membrane Fusion

Abstract

Poly(ethylene glycol)- (PEG) mediated fusion of 25 nm vesicles composed of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), bovine brain sphingomyelin (SM), cholesterol (CH) and phosphatidylserine (PS) was examined in order to investigate the effects of phosphatidylserine (PS) on the fusion mechanism. Lipid mixing (LM), content mixing (CM) and content leakage (L) measurements were carried out with vesicles containing from 0 to 10 mol% PS and similar amounts of phosphatidylglycerol (PG) as controls. Fitting these time courses globally to a 3-state (aggregate, intermediate, pore) sequential model established the rate constants for each step as well as probabilities for the occurrence of LM, CM, or L in each state. Charged lipids inhibited rates of intermediate and pore formation, with inhibition of intermediate formation being directly proportional to negative surface potential for PG but greater for PS. Inhibition of pore formation was limited up to 4% PG or PS but increased dramatically above that. Even low PG content inhibited the rapid rate of PEG-induced aggregation (detected by turbidity) and led to smaller aggregates (detected by DLS), while a slower component of turbidity increase roughly tracked the dependence on PG content of the rate of pore formation. PS or PG content above 6% also inhibited lipid mixing in the initial intermediate. We conclude that, aside from an expected effect on the rate and extent of PEG-induced aggregation, PS at physiological membrane contents alters the nature of the initial intermediate such that lipid mixing between joined monolayers is considerably reduced prior to formation of a stable fusion pore. Supported by NIH grant GM32707 to BRL.