SYNTHESIS AND DISTRIBUTION OF PROTEINS IN MEMBRANES

蛋白质在膜中的合成和分布

• 批准号: 6636001
• 负责人: DAVID D SABATINI
• 金额: $ 47.53万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636001
• 关键词: Golgi apparatus; MDCK cell; acyltransferase; cell free system; enzyme activity; fatty acid binding protein; immunoelectron microscopy; intracellular membranes; intracellular transport; laboratory mouse; laboratory rabbit; laboratory rat; lysophospholipids; membrane activity; membrane biogenesis; membrane proteins; phosphatidylinositols; phospholipase D; protein biosynthesis; protein localization; protein purification; protein transport; transport proteins; vesicle /vacuole

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.

描述（申请人摘要的逐字记录）：本申请是 与介导转运的载体囊泡的性质有关 从跨高尔基体网络 (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 特定蛋白质、脂质和辅因子的作用方式 细胞质和 TGN 膜在此过程中发挥着重要作用。对于这些 研究中我们使用我们开发的无细胞系统在体外重现 从纯化的高尔基体级分中产生后高尔基体囊泡 病毒感染的 MDCK 细胞，含有积累的唾液酸化 VSV-G 蛋白 分子。为了产生囊泡，将高尔基体部分与 胞浆蛋白和三磷酸核苷的来源。囊泡，当 在 GTPgammaS 存在下产生，含有涂层。他们还 包含 rab11，它之前已定位于 TGN 并与 VSV-G 通过内体隔室转运至质膜 高尔基体后囊泡的形成可以通过两个连续的阶段来实现， 第一个是 Arf 依赖性外套组装/芽形成，第二个是 膜破裂。我们将确定发挥作用的胞质蛋白和脂质 在高尔基体后囊泡的分裂中起关键作用并确定它们的作用 的过程。其中包括磷脂酰肌醇转移蛋白 (PITP)，一种 含有伯恩的未知蛋白质成分的断裂因子 溶血磷脂酸 (LPA) 并且可能是溶血磷脂酸 酰基转移酶（可能是 BARS-50，高尔基体管状物质的靶标， BFA），一种保持断裂因子的脂肪酸结合蛋白（FABP） 不活跃并且是限制涂层异构体涂层裂解的假定因素 将膜覆盖到被包芽的颈部。我们将测试一个模型，其中激活 Arf 通过以下方式在包覆芽的颈部启动切割机械： 将酰基转移酶从 FABP 上解离，使其保持失活。我们将 阐明磷脂酶 D 和磷脂酶 D 分裂中的可能作用 调节它的类似 PKC 的分子。我们还将鉴定蛋白质分子 TGN 衍生的外壳和膜有助于其靶向 到受体室并识别该室的性质。 由于囊泡含有rab11，我们将确定囊泡是否可以 与内体对接并融合。