PROTEOLIPOSOME--MEMBRANE MODEL

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Our earlier discovery of the inherent ability of preformed vesicles to spontaneously incorporate integral membrane proteins into the lipid bilayer has resulted in the facile and routine formation of proteoliposomes (PRLs). We have recently found that the spontaneous incorporation of bacteriorhodopsin is not limited to small unilamellar vesicles (SUVs), but that large unilamellar vesicles (LUVs) will also spontaneously incorporate this membrane protein. Furthermore, we believe that protein incorporation into LUVs or other PRLs represents a more physiologically significant model system than our prior model based on SUVs. Thus, we propose to use these vesicles as an artificial cell model to examine several concepts of cell- cell interaction and membrane biogenesis. The purpose of this study is 1) to explore the relevance of our findings to the incorporation of membrane proteins such as bacteriorhodopsin in vivo, and 2) to study the role of membrane lipids in membrane stability and fusion using a proteoliposome as a simple membrane model. First, we will examine the incorporation of nascent bacterio-opsin synthesized in a cell-free translation system into LUVs and PRLs. These studies will determine the effect of phospholipid composition on the spontaneous insertion of nascent bacterio-opsin into LUVs which have been shown to be receptive or non-receptive to the incorporation of purified bacteriorhodopsin. Second, we propose to use the PRLs as a model membrane to study membrane stability and fusion. The growth of nascent proteoliposomes by either fusion or lipid transfer results in an increase in lipid content in the presence of protein-free vesicles. We plan to examine the relative role of phospholipid transfer and/or vesicle fusion in proteoliposome "growth". Although the spontaneous fusion of SUVs with other SUVs or between SUVs and proteoliposomes is attributed to an inherent property of naked highly curved lipid bilayers, the extent of fusion of lipid-protein membranes with each other is unclear. Therefore, the fusion of proteoliposomes with other proteoliposomes will be examined to determine whether the lipid-to-protein ratio or phospholipid composition play a role in the inherent ability of lipid-protein membranes to fuse with each other. The effect of the spontaneous incorporation of monomeric bacteriorhodopsin on the integrity of the vesicles measured by the release of intravesicular or "cytoplasmic" contents of this model cell, and its relationship to the phospholipid composition of the vesicle will be explored.