SYNTHESIS AND DISTRIBUTION OF PROTEINS IN MEMBRANES

描述(逐字摘自申请者摘要)：本申请书为 与介导药物转运的载体囊泡的性质有关的 从反高尔基网络(TGN)到上皮细胞表面的蛋白质 细胞。它建议研究它们的形成过程，并建立 特定的蛋白质、脂类和辅因子的作用方式 胞浆和TGN膜在这一过程中起着至关重要的作用。为了这些 研究中我们使用的是我们开发的无细胞系统，该系统在体外重建 从纯化的高尔基体组分中产生高尔基体后囊泡 含积聚唾液酸化VSV-G蛋白的病毒感染MDCK细胞 分子。对于囊泡的产生，高尔基体部分与 胞浆蛋白和核苷三磷酸的来源。水泡，当 在GTP-Gamma存在的情况下生产的，含有椰壳涂层。他们也 包含Rab11，它之前已本地化到TGN并牵涉到 VSV-G经内膜室向质膜的转运 高尔基体后囊泡的形成可以分两个阶段进行，一个是 第一个依赖于Arf的涂层组件/芽形成，以及第二个 膜断裂。我们将鉴定起作用的胞浆蛋白和脂类 在高尔基体后囊泡断裂中的关键作用并确定它们在 这一过程。其中包括磷脂酰肌醇转移蛋白(PITP)，一种 含有边界的未知蛋白质组成的裂解因子 溶血磷脂酸(LPA)，很可能是溶血磷脂酸 酰基转移酶，(可能是BARS-50，高尔基体管状剂的靶标， BFA)，一种保持裂解因子的脂肪酸结合蛋白(FABP) 非活性和限制辅酶A涂层切割的假定因素 膜到被覆的花蕾的脖子上。我们将测试一个模型，其中激活了 ARF通过以下方式启动包衣花蕾颈部的裂开机械 使酰基转移酶与FABP解离，使其处于不活跃状态。我们会 阐明磷脂酶D和磷脂酶A断裂的可能作用 调节它的类似PKC的分子。我们还将确定蛋白质分子在 TGN来源的有助于其靶标的外衣和膜 以识别该隔室的性质。 由于囊泡含有Rab11，我们将确定囊泡是否可以 对接并与内体融合。

DESCRIPTION (Verbatim from the applicant's abstract): This application is concerned with the nature of the carrier vesicles that mediate the transport of proteins from the trans-Golgi network (TGN) to the cell surface of epithelial cells. It proposes to study the process of their formation 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 use a cell free system that we developed that recreates in vitro the generation of post-Golgi vesicles from a purified Golgi fraction obtained from virus-infected MDCK cells that contains accumulated sialylated VSV-G protein molecules. For vesicle generation, the Golgi fraction is incubated with cytosolic proteins and a source of nucleoside triphosphates. The vesicles, when produced in the presence of GTPgammaS, contain a coatomer coat. They also contain rab11, which has been previously localized to the TGN and implicated in transport of VSV-G to the plasma membrane through the endosomal compartment The formation of post-Golgi vesicles can be effected in two sequential phases, a first one of Arf-dependent coat assembly/bud formation and a second one of membrane scission. We will identify the cytosolic proteins and lipid that play a key role in the scission of post-Golgi vesicles and determine their role in the process. These include the phospatidylinositol transfer protein (PITP), a scission factor of unknown protein composition that contains bourn lysophospatidic acid (LPA) and is likely to be a lysophosphatidic acid acyltransferase, (possibly BARS-50, the target of the Golgi tubulating agent, BFA), a fatty acid binding protein (FABP) that keeps the scission factor inactive and a postulated factor that restricts cleavage of coatomer-coated membranes to the necks of coated buds. We will test a model in which activated Arf sets the scission machinery in motion at the neck of a coated bud by dissociating the acyltransferase from the FABP that keeps it inactive. We will elucidate the possible role in scission of a phospholipase D and that of a PKC-like molecule that regulates it. We will also identify protein molecules in the coat and membranes of the TGN-derived that contribute to their targetting to an acceptor compartment and to identify the nature of that compartment. Since the vesicles contain rab11, we will determine whether the vesicle can dock and fuse with endosomes.