PROTEOLIPOSOME--MEMBRANE MODEL

蛋白脂质体--膜模型

• 批准号: 3304295
• 负责人: ANTHONY W SCOTTO
• 金额: $ 16.74万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304295
• 关键词: affinity chromatography; bacteriorhodopsins; cell free system; density gradient ultracentrifugation; laboratory rabbit; lipid bilayer membrane; liposomes; membrane fusion; membrane lipids; membrane proteins; membrane reconstitution /synthesis; phospholipids; posttranslational modifications; vesicle /vacuole

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.

我们早期发现的预先形成的囊泡的内在能力， 自发地将膜蛋白整合到脂质双层中 导致蛋白脂质体（PRL）的容易和常规形成。 我们最近发现， 细菌视紫红质不限于小单层囊泡（SUV），但 大的单层囊泡（LUV）也会自发地 这种膜蛋白。 此外，我们认为蛋白质掺入 转化为LUV或其他PRL代表了一个更有生理意义的模型， 系统比我们以前的模型基于SUV。 因此，我们建议使用这些 囊泡作为人工细胞模型，以检查细胞的几个概念， 细胞相互作用和膜生物发生。 本研究的目的是1） 探索我们的发现与膜结合的相关性， 蛋白质，如细菌视紫红质在体内，和2）研究的作用， 使用蛋白脂质体作为膜稳定性和融合中的膜脂质 一个简单的膜模型。 首先，我们将研究 在无细胞翻译系统中合成的新生细菌视蛋白， LUV和PRL。 这些研究将确定磷脂的作用 组合物对新生细菌视蛋白自发插入 已被证明接受或不接受的LUV 掺入纯化的细菌视紫红质。 第二，我们建议使用 PRL作为模型膜研究膜的稳定性和融合。 的 新生蛋白脂质体通过融合或脂质转移的生长 在不含蛋白质的情况下导致脂质含量增加 囊泡 我们计划研究磷脂转移的相对作用， 和/或蛋白脂质体"生长"中的囊泡融合。 虽然自发的 SUV与其他SUV或SUV与脂蛋白体之间的融合， 归因于裸露的高度弯曲的脂质双层的固有性质， 脂质-蛋白质膜彼此融合的程度是 不清楚 因此，蛋白脂质体与其他蛋白质的融合是不可能的。 蛋白脂质体将被检查，以确定是否脂质蛋白质 比例或磷脂组成在固有的能力发挥作用， 脂蛋白膜相互融合。 的影响 单体菌紫质的自发掺入 通过释放囊泡内或"细胞质" 该模型细胞的内容物及其与磷脂的关系 将探索囊泡的组成。