SYNTHESIS & DISTRIBUTION OF PROTEINS IN MEMBRANES

合成

• 批准号: 3302676
• 负责人: DAVID D SABATINI
• 金额: $ 33.47万
• 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/3302676
• 关键词: Golgi apparatus; actins; basolateral membrane; cell free system; cell membrane; crosslink; cytoplasm; endocytosis; endoplasmic reticulum; epithelium; glycoproteins; guanine nucleotide binding protein; immunoelectron microscopy; immunofluorescence technique; kidney cell; laboratory rabbit; membrane activity; membrane permeability; membrane proteins; membrane reconstitution /synthesis; 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.

这项提议的总体目标是阐明分类机制 这有助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) 反式高尔基体如何产生新合成的膜蛋白 网络(TGN)被结合到特定的囊泡中，以及，ii)这些 小泡被定向到细胞表面的一个方面。我们建议 测试一个模型，在该模型中，某些蛋白质(I类)按其自身进行排序 TGN与识别其受体的接头分子的相互作用 胞质片段，并导致蛋白质掺入 小泡直接进入质膜。其他蛋白质，如HA 流感的G和VSV的G，将通过分类受体识别，这 是与光域相互作用的I类分子 蛋白质被分类，并将它们输送到细胞表面。极化 细胞单层，在一个或另一个表面上穿孔，以及无细胞 系统，将被用来识别并最终净化细胞 组分，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 运输囊泡。病毒糖蛋白和糖蛋白的复合体 它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进行搜索，包括 纯化后高尔基体小泡。要调查在分类中的角色 蛋白质的细胞质片段，它们自己分类，多肽 相应于细胞质尾部的各种受体将被用于 渗透性细胞和在无细胞系统中，试图 特别是抑制运输到细胞的一个或另一个方面 浮出水面。胞质片段与细胞的特异性联系 接头类分子将直接通过交联剂和 标记转移技术，并尝试使用 胞质片段作为亲和配基。穿孔细胞系统将 用来检查形成内吞小泡的要求 从每个表面和这些小泡在细胞内的运输 这导致了它们与内小体的融合。具体的作用 胞吞作用中的细胞骨架元素，如肌动蛋白和纤维蛋白 将对表面进行检查。