OUTER LEAFLET-PACKING DEFECTS PROMOTE POLY(ETHYLENE GLYCOL)-MEDIATED FUSION OF LARGE UNILAMELLAR VESICLES

Poly(ethylene glycol)-induced fusion of two different vesicle systems has been examined: dipalmitoylphosphatidylcholine (DPPC) large unilame llar vesicles (LUV) and cardiolipin (CL)/dioleoylphosphatidylcholine ( DOPC) (1:10) LUVs. A slight perturbation was established in the outer leaflets of DPPC LUVs by hydrolyzing 0.8% of the outer leaflet lipid w ith phospholipase A(2) to produce lysophosphatidylcholine and palmitat e which were then removed by bovine serum albumin. Similarly, 5 mM Ca2 + was added to the external compartment of CL/DOPC LUVs to alter the s hape of the CL molecule and thereby create a perturbation in the outer leaflet packing of these vesicles. Contents mixing assays showed that both vesicle systems fused only when the outer leaflets of both conta cting vesicles were perturbed as described. Two fluorescent probes (C- 6-NBD-PC and TMA-DPH) were used to detect changes in outer leaflet mol ecular packing between nonfusing and fusing systems. The steady-state fluorescence intensity of C-6-NBD-PC added externally to either fusing system was enhanced relative to that of nonfusing vesicles. Phase-res olved measurements of probe lifetime showed that this was due mainly t o enhanced partitioning of probe from a micellar state into fusing ver sus nonfusing membranes. Similarly, TMA-DPH was found to undergo more rapid motion when incorporated into fusing as opposed to nonfusing ves icles. The effects of deuterium exchange on probe lifetime also indica ted that C-6-NBD-PC and TMA-DPH penetrated more deeply into fusing tha n into nonfusing membranes. These results suggest that the fusogenic p erturbations produced in these two very different lipid systems took t he form of altered outer leaflet packing. We conclude that, for the tw o model lipid bilayers examined, small perturbations in lipid packing within contacting bilayer leaflets are necessary and probably sufficie nt to promote membrane fusion.