Studies of the generation and function of phospholipid asymmetry

It is well established that biological membranes maintain an asymmetric transbilayer distribution of component molecules, including lipids. The mechanisms by which this lipid asymmetry is established and maintained are not well understood. In addition, little is known concerning the biological significance of lipid asymmetry. This thesis employs large unilamellar vesicle (LUV) model membrane systems to examine the ability of transmembrane pH gradients (ΔpH) to generate lipid asymmetry and investigate the consequences of lipid asymmetry in membrane fusion phenomena. The first area of investigation demonstrates that transmembrane pH gradients can influence the inter-vesicular exchange of stearylamine and oleic acid. Vesicles containing stearylamine are shown to aggregate immediately with vesicles containing phosphatidylserine and disaggregation occurs as stearylamine equilibrates between the two vesicle populations. Despite visible flocculation during the aggregation phase, vesicle integrity is maintained. It is also shown that stearylamine is the only lipid to exchange, fusion does not occur and vesicles are able to maintain a pH gradient. When stearylamine is sequestered to the inner monolayer in response to a transmembrane pH gradient (inside acidic) aggregation is not observed and diffusion of stearylamine to acceptor vesicles is greatly reduced. The ability of ΔpH-dependent lipid asymmetry to modulate lipid exchange is also demonstrated for fatty acids. Oleic acid can be induced to transfer from one population of vesicles to another by maintaining a basic interior pH in the acceptor vesicles. It is also shown that the same acceptor vesicles can deplete serum albumin of bound fatty acid. The second area of investigation concerns asymmetric transbilayer distributions of dioleoylphosphatidic acid (DOPA) induced by transmembrane pH gradients. A fluorescent assay is developed employing 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS) as a probe of lipid asymmetry. The kinetics of DOPA transport are shown to be consistent with the transport of the uncharged (protonated) form. Transport of the neutral species can be rapid, exhibiting half-times for transbilayer transport of approximately 25 s at 45°C. These studies also indicate that the transport of DOPA is associated with a large activation energy (28 Kcal/mol). The third area builds on the ability to generate LUVs with an asymmetric distribution of DOPA and concerns studies on the ability of lipid asymmetry to regulate Ca2+ stimulated fusion of LUV systems. It is shown that for LUVs composed of DOPC:DOPE:PI:DOPA (25:60:5:10 mol/mol) rapid and essentially complete fusion is observed by fluorescent resonance energy transfer techniques when Ca2+ is added. Alternatively, for LUVs with the same lipid composition but when DOPA has been sequestered to the inner monolayer, due to the presence of a pH gradient (interior basic), little or no fusion is observed upon addition of Ca2+ It is demonstrated that the extent of Ca2+induced fusion correlates with the amount of exterior DOPA. It is also shown that LUVs containing only 2.5 mol% DOPA, but when all the DOPA is in the outer monolayer, can be induced to fuse to the same extent and with the same initial rate as LUVs containing 5 mol% DOPA. These results strongly support a regulatory role for lipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipid bilayers are largely determined by the properties of one monolayer.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat