REGULATION OF PHOSPHOLIPID TRANSFER BETWEEN MEMBRANES

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The experiments presented in this proposal are directed towards two major goals. The first is to continue to study how lipid transfer proteins enhance lipid movement between membranes, and the second is to study the extent of and mechanism for phospholipid surface transfer between HDL and fibroblasts--that is, phospholipid transfer that occurs in addition to transfer of intact, HDL particles. These studies focus on the nonspecific lipid transfer protein (nsLTP) because it has been studied extensively and is well characterized, yet its mechanism of action and physiological function are not known. Although it is isolated from liver cytosol and has the unique ability to stimulate the intermembrane transfer of a wide range of lipid molecules, it is not essential to normal intracellular lipid metabolism, and therefore its function may be to transfer lipids in the plasma. A new technique developed in our laboratory using fluorescent- labeled phospholipids has demonstrated for the first time phospholipid binding to nsLTP. Studies of the binding kinetics suggest a bind-and-release carrier model for the mechanism of nsLTP-stimulated intermembrane transfer, in which nsLTP collides with the donor membrane surface, binds a phospholipid molecule, and removes it from the membrane. The bound phospholipid rapidly equilibrates with the water phase and either the free lipid or protein-bound lipid reassociates with the acceptor membrane. This hypothesis will be tested and the membrane and lipid properties that determine nsLTP effectiveness will be characterized. The second goal of this proposal is to study the surface transfer of phospholipids between HDL particles and human fibroblasts. Although cholesterol movement between HDL and cells has been studied extensively, little is known about phospholipid transfer. Yet phosphatidylcholine transfer to HDL is a requirement for cholesterol removal from peripheral cells since the majority of cholesterol is carried by HDL in the form of cholesterol esters which requires the enzymatic transfer of a phosphatidylcholine acyl chain to cholesterol. The source of this HDL phospholipid is not known. In this light, the rate and mechanism of transfer of fluorescent-labeled phospholipids between fibroblasts and HDL will be investigated to determine whether spontaneous or nsLTP- stimulated phospholipid transfer may provide a physiologically important source of HDL phospholipid.