SYNTHESIS AND DISTRIBUTION OF PROTEINS IN MEMBRANES

蛋白质在膜中的合成和分布

• 批准号: 6519373
• 负责人: DAVID D SABATINI
• 金额: $ 46.17万
• 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/6519373
• 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（来自申请人摘要的逐字）：本申请是 与介导药物转运的载体囊泡的性质有关 从高尔基体网络（TGN）到上皮细胞表面的蛋白质 细胞它建议研究其形成过程， 特定蛋白质、脂质和辅因子的作用模式， 细胞质和TGN膜在此过程中起重要作用。为这些 研究中，我们使用了一种无细胞系统， 从纯化的高尔基体级分产生后高尔基体囊泡，所述纯化的高尔基体级分得自 病毒感染的MDCK细胞，其含有累积的唾液酸化VSV-G蛋白 分子。对于囊泡的产生，将高尔基体部分与 胞质蛋白质和核苷三磷酸的来源。囊泡，当 在GTP γ S存在下产生的，含有包衣体包衣。他们还 包含rab 11，它以前已经定位于TGN，并与 VSV-G通过内体室转运至质膜 高尔基体后囊泡的形成可分为两个连续的阶段， Arf依赖性包被组装/芽形成的第一种和 膜断裂我们将鉴定细胞质蛋白质和脂质， 在高尔基体后囊泡的分裂中起关键作用，并决定它们在 过程。这些包括磷脂酰肌醇转移蛋白（PITP）， 含边界未知蛋白质组成的截断因子 溶血磷脂酸（LPA），可能是溶血磷脂酸 酰基转移酶，（可能是BARS-50，高尔基体微管因子的靶点， BFA），一种脂肪酸结合蛋白（FABP）， 非活性和限制被包衣体包被的细胞分裂的假定因子 膜到包被芽的颈部。我们将测试一个模型， Arf在被膜芽的颈部启动了分裂机制， 使酰基转移酶与保持其失活的FABP解离。我们将 阐明了磷脂酶D和磷脂酶A的断裂中可能的作用。 我们还将鉴定蛋白质分子， TGN衍生物的涂层和膜有助于其靶向 到受体隔室并识别该隔室的性质。 由于囊泡含有rab 11，我们将确定囊泡是否可以 与内体对接融合。