SYNTHESIS & DISTRIBUTION OF PROTEINS IN MEMBRANES

合成

• 批准号: 3302678
• 负责人: DAVID D SABATINI
• 金额: $ 31.98万
• 依托单位: NEW YORK UNIVERSITY
• 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 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302678
• 关键词: Golgi apparatus; actins; basolateral membrane; cell free system; cell membrane; cellular polarity; crosslink; cytoplasm; endocytosis; endoplasmic reticulum; epithelium; glycoproteins; guanine nucleotide binding protein; immunoelectron microscopy; immunofluorescence technique; kidney cell; laboratory rabbit; membrane activity; membrane biogenesis; membrane permeability; membrane proteins; membrane structure; protein biosynthesis; protein reconstitution; protein sequence; protein transport; radioimmunoassay; radiotracer; spectrin; vesicle /vacuole; virus protein

The overall goal of this proposal is to elucidate the sorting mechanisms that serve to establish the distinct protein compositions of the two plasma membrane domains of polarized epithelial cells. We wish to determine: i) how newly synthesized membrane proteins emerging from the trans Golgi network (TGN) are incorporated into specific vesicles, and, ii) how these vesicles are directed to one aspect of the cell surface. We propose to test a model in which certain proteins (Class I) sort by themselves by interaction in the TGN with adaptor molecules that recognize their cytoplasmic segments and lead to the incorporation of the proteins into vesicles directed to the plasma membrane. Other proteins, such as the HA of influenza and G of VSV, would be recognized by sorting receptors, which are Class I molecules that interact with the luminal domains of the proteins to be sorted and transport them to the cell surface. Polarized cell monolayers, perforated in one or the other surface, and a cell-free system, will be employed to identify, and ultimately purify, cellular components, including adaptor proteins and GTP-binding proteins and their cognate docking proteins, that participate in the formation and targeting of the transport vesicles. Complexes between the viral glycoproteins and their putative sorting receptors and the corresponding adaptors will be searched for in total cell extracts and subcellular fractions, including purified post Golgi vesicles. To investigate the role in sorting of the cytoplasmic segments of proteins that sort by themselves, peptides corresponding to the cytoplasmic tails of various receptors will be used in permeabilized cells and in the cell-free system, in attempts to specifically inhibit transport to one or the other aspect of the cell surface. The specific association of the cytoplasmic segments with adaptor-like molecules will be directly investigated by cross-linking and label-transfer techniques and by attempts to purify the adaptors using the cytoplasmic segments as affinity ligands. The perforated cell system will be used to examine the requirements for the formation of endocytic vesicles from each surface and for the transport of these vesicles within the cell that leads to their fusion with endosomes. The role of specific cytoskeletal elements, such as actin and fodrin, in endocytosis at each surface will be examined.

该提案的总体目标是阐明排序机制 用于确定两种血浆的不同蛋白质组成 极化上皮细胞的膜域。 我们希望确定：i) 新合成的膜蛋白如何从反式高尔基体中出现 网络（TGN）被整合到特定的囊泡中，以及，ii）这些如何 囊泡指向细胞表面的一方面。 我们建议 测试一个模型，其中某些蛋白质（I 类）通过自身排序 TGN 中与识别其自身的接头分子的相互作用 细胞质片段并导致蛋白质掺入 囊泡指向质膜。 其他蛋白质，例如 HA 流感的病毒和 VSV 的 G，将被分选受体识别， 是与管腔域相互作用的 I 类分子 蛋白质被分类并将它们运输到细胞表面。 偏振 细胞单层，在一个或另一个表面穿孔，以及无细胞 系统，将用于识别并最终纯化细胞 成分，包括接头蛋白和 GTP 结合蛋白及其 同源对接蛋白，参与形成和靶向 的运输囊泡。 病毒糖蛋白和病毒糖蛋白之间的复合物 他们假定的分选受体和相应的适配器将是 在总细胞提取物和亚细胞部分中搜索，包括 纯化的高尔基体后囊泡。 研究在排序中的作用 自行排序的蛋白质的细胞质片段，肽 对应于各种受体的细胞质尾部将被用于 透化细胞和无细胞系统中，试图 特异性抑制向细胞的一个或另一个方面的转运 表面。 细胞质片段的特定关联 类似接头的分子将通过交联和 标签转移技术并尝试使用 细胞质片段作为亲和配体。 穿孔细胞系统将 用于检查内吞囊泡形成的要求 来自每个表面并用于细胞内这些囊泡的运输 这导致它们与内体融合。 具体作用 细胞骨架元件，例如肌动蛋白和胞嘧啶，在每个细胞的内吞作用中 表面将被检查。