SYNTHESIS AND DISTRIBUTION OF PROTEINS IN MEMBRANES

我们建议研究载体囊泡的形成过程 介导蛋白质从反式高尔基体网络(TGN)到 细胞表面上皮细胞，并建立其作用模式 胞浆和TGN中的特定蛋白质、脂类和辅因子 膜在这一过程中起着至关重要的作用。对于这些研究，我们将 使用我们开发的无细胞系统在体外重建 从纯化的高尔基体组分中产生高尔基体后囊泡 来自病毒感染的MDCK细胞。有了这一制度，岗位的形成 高尔基体小泡可在以下两个相继阶段起作用：i)ArfGTP 从属的被膜组装/芽形成和(2)囊泡破裂。这个 裂解相需要在TGN膜上有一个类似PKC的分子，但不需要 它的磷酸化活性，以及几种胞浆蛋白， 包括一种膜分裂促进活性(MSPA)，存在于高水平 含有NEM敏感组分的分子量复合体，它 我们已初步鉴定为磷脂酰肌醇转移蛋白 (PITP)。采用生化、遗传学和电子显微镜相结合的方法 我们将采用以下方法： L)通过以下方法鉴定TGN来源的囊泡的包膜和膜成分 水提纯囊泡的包衣分析 GTPlambdaS，防止囊泡脱落，并通过组装 含有含有被衣亚单位的纯化胞浆亚组分的囊泡。 2)纯化和阐明一种胞质NEM敏感蛋白的调节 我们已经确定的膜分裂促进因子(MSPA)，它 是水泡生成所必需的，并且当与其他 成分，导致未涂覆的TGN膜不受控制的囊泡。 3)测试一个模型，在该模型中，囊泡的断裂依赖于 涉及膜相关的PKC样蛋白作用的机制 与Arf-GTP结合的一种胞质PITP分子，对 PtdIns(4，5)P2，可能还有RhoA，导致一种 磷脂酶D(PLD)，在TGN膜中，它将是最终的效应者 囊泡破裂的可能性。在这些研究中，我们将确定PKC样蛋白 我们已经发现的与囊泡生成有关的分子，并确定 它是否直接与高尔基体关联的PLD相互作用并激活。 我们还将阐明PITP的作用并鉴定磷脂酰肌醇 它在分裂过程中做出了贡献。

We propose to study the process of formation of the carrier vesicles that mediate the transport of proteins from the trans Golgi network (TGN) to the cell surface of epithelial cells and to establish the mode of action of the specific proteins, lipids and cofactors that, in the cytosol and TGN membranes, play essential roles in this process. For these studies, we will use a cell-free system we have developed that recreates in vitro the generation of post Golgi vesicles from a purified Golgi fraction obtained from virus-infected MDCK cells. With this system, the formation of post Golgi vesicles can be effected in two sequential phases of: I) ArfGTP dependent coat assembly/bud formation and, 2) vesicle scission. The scission phase requires a PKC-like molecule in the TGN membrane, but not its phosphorylating activity, as well as several cytosolic proteins, including a membrane scission promoting activity (MSPA) present in a high molecular weight complex that contains an NEM-sensitive component, which we have tentatively identified as the phosphatidylinositol transfer protein (PITP). Employing a combined biochemical, genetic, and electron microscopic approach we will: l) Identify the coat and membrane components of TGN-derived vesicles by analysis of the coats of purified vesicles produced in the presence of GTPlambdaS, which prevents vesicle uncoating, and by assembling the vesicles with purified cytosolic subfractions containing coat subunits. 2) Purify and elucidate the regulation of a cytosolic NEM-sensitive membrane scission promoting factor (MSPA) that we have identified, which is required for vesicle generation and, when separated from other components, causes the uncontrolled vesiculation of uncoated TGN membranes. 3) Test a model in which vesicle scission is dependent on a regulatory mechanism that involves the action of a membrane-associated PKC-like molecule that, in concert with Arf-GTP, a cytosolic PITP that contributes PtdIns(4,5)P2, and possibly RhoA, leads to the activation of a phospholipase D (PLD), which, in TGN membranes, would be the final effector of vesicle scission. In these studies, we will identify the PKC-like molecule that we have implicated in vesicle generation and determine whether it interacts directly with and activates a Golgi associated PLD. We will also clarify the role of PITP and identify the phosphoinositides that it contributes to the scission process.