SYNTHESIS AND DISTRIBUTION OF PROTEINS IN MEMBRANES

我们建议研究载体囊泡的形成过程， 介导蛋白质从高尔基体网络（TGN）转运到 上皮细胞的细胞表面，并建立的作用方式， 特异性蛋白质、脂质和辅因子， 膜，在这个过程中发挥重要作用。对于这些研究，我们将 使用我们开发的无细胞系统， 从获得的纯化的高尔基体级分产生后高尔基体囊泡 来自病毒感染的MDCK细胞。有了这个系统， 高尔基体囊泡可以在以下两个连续阶段中受到影响：I）ArfGTP 依赖的外壳组装/芽形成和，2）囊泡断裂。的 分裂期需要TGN膜中的PKC样分子，但不需要。 其磷酸化活性，以及几种胞质蛋白， 包括存在于高浓度中的膜断裂促进活性（MSPA）， 含有NEM敏感组分的分子量复合物， 我们已经初步鉴定为磷脂酰肌醇转移蛋白 （PITP）。利用生物化学遗传学和电子显微镜 我们将： l）通过以下鉴定TGN衍生的囊泡的外壳和膜组分： 在存在下产生的纯化囊泡的包衣的分析 GTPPDAS，它防止囊泡脱壳，并通过组装 囊泡与纯化的胞质亚组分含有外壳亚单位。 2)纯化并阐明胞质内NEM敏感的 膜断裂促进因子（MSPA），我们已经确定， 是产生囊泡所必需的，当与其他 组分，导致未涂覆的TGN膜的不受控制的囊泡化。 3)测试一个模型，在该模型中，囊泡断裂依赖于一个调节因子， 涉及膜相关PKC样蛋白的作用的机制 分子，与Arf-GTP一致，是一种细胞溶质PITP， PtdIns（4，5）P2，可能还有RhoA，导致一个 磷脂酶D（PLD），在TGN膜中，它将是最终的效应物 囊泡破裂的过程在这些研究中，我们将确定PKC样 分子，我们已经暗示在囊泡的产生和确定 它是否直接与高尔基体相关的PLD相互作用并激活它。 我们还将阐明PITP的作用，并确定磷酸肌醇 它有助于分裂过程。

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.