PROTEOLIPOSOME--MEMBRANE MODEL

蛋白脂质体--膜模型

• 批准号: 3304296
• 负责人: ANTHONY W SCOTTO
• 金额: $ 16.26万
• 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/3304296
• 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固有的能力 自发地将完整的膜蛋白结合到脂质双层中 已经导致了蛋白脂质体(PRL)的简单和常规的形成。 我们最近发现，自发成立的 细菌视紫红质并不局限于小单层囊泡(SUV)，而是 大的单层囊泡(Luv)也会自发地合并 这种膜蛋白。此外，我们认为蛋白质的掺入 进入LUV或其他PRL代表了一种更有生理意义的模型 系统比我们之前基于SUV的模型更好。因此，我们建议使用这些 囊泡作为人造细胞模型来研究细胞的几个概念- 细胞相互作用与膜生物发生。本研究的目的是1) 探索我们的发现与膜结合的相关性 细菌视紫红质等蛋白质在体内的作用，以及2)研究 膜脂类在膜的稳定性和融合中的应用 一个简单的膜模型。首先，我们将研究成立 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 新生细菌素自发植入体内的组合物 已经被证明能接受或不能接受 纯化的细菌视紫红质的掺入。第二，我们建议使用 以PRLS膜为模型膜，研究膜的稳定性和融合性。这个 融合或脂质转移法生长新生蛋白脂质体 在不含蛋白质的情况下，导致脂肪含量增加 水泡。我们计划研究磷脂转移的相对作用。 和/或蛋白脂体“生长”过程中的囊泡融合。虽然是自发的 SUV与其他SUV或SUV与蛋白质脂质体之间的融合是 归因于裸露的高度弯曲的脂质双层的固有性质， 脂蛋白膜相互融合的程度是 不清楚。因此，蛋白质脂质体与其他 将对蛋白质脂质体进行检查，以确定脂蛋白是否 比率或磷脂成分在体内的固有能力中起作用。 脂-蛋白膜相互融合。美国政府的影响 自发掺入单体细菌视紫红质对细胞完整性的影响 以囊泡内或“胞浆”的释放来测量囊泡的数量 该模型细胞的含量及其与磷脂的关系 我们将探索囊泡的组成。