La3+-induced fusion of phosphatidylserine liposomes. Close approach, intermembrane intermediates, and the electrostatic surface potential.
The fusion of large unilamellar phosphatidylserine liposomes (PS LUV) induced by La3+ has been monitored using the 1-aminoapthalene-3,6,8-trisulfonic acid/p-xylenebis(pyridinium bromide) (ANTS/DPX) fluorescence assay for the mixing of aqueous contents. The fusion event is extensive and nonleaky, with up to 95% mixing of contents in the fused liposomes. However, addition of excess EDTA leads to disruption of the fusion products in a way that implies the existence of metastable intermembrane contact sites. The maximal fusion activity occurs between 10 and 100 microM La3+ and fusion can be terminated rapidly, without loss of contents, by the addition of excess La3+, e.g., 1 mM La3+ at pH 7.4. This observation is explained by the very large intrinsic binding constant (approximately 10(5) M-1) of La3+ to the PS headgroup, as measured by microelectrophoresis. Addition of 1 mM La3+ causes charge reversal of the membrane and a large positive surface potential. La3+ binding to PS causes the release of a proton. These data can be explained if La3+ can chelate to PS at two sites, with one of the sites being the primary amino group. This binding model successfully predicts that at pH 4.5 fusion occurs up to 2 mM La3+, due to reduced La3+ binding at low pH. We conclude that the general mechanism of membrane fusion includes three kinetic steps. In addition to (a) aggregation, there is (b) the close approach of the surfaces, or thinning of the hydration layer, and (c) the formation of intermembrane intermediates which determine the extent to which membrane destabilization leads to fusion (mixing of aqueous contents), as opposed to lysis. The lifetime of these intermembrane intermediates appears to depend upon La3+ binding to both PS sites.
Bentz
J
J
Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143.
Alford
D
D
Cohen
J
J
Düzgüneş
N
N
D002118:Calcium; D007700:Kinetics; D007811:Lanthanum; D008081:Liposomes; D008564:Membrane Potentials; D008954:Models, Biological; D010718:Phosphatidylserines; D013050:Spectrometry, Fluorescence
D008081:Liposomes; D010718:Phosphatidylserines; D007811:Lanthanum; D002118:Calcium