SYNTHESIS & DISTRIBUTION OF PROTEINS IN MEMBRANES

本提案的总体目标是阐明分类机制 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） 从高尔基体中新合成的膜蛋白 网络（TGN）被纳入特定的囊泡，以及，ii）如何这些 囊泡指向细胞表面的一个方面。 我们建议 测试一个模型，其中某些蛋白质（I类）通过以下方式自行排序： 在TGN中与识别它们的接头分子的相互作用 细胞质片段，并导致蛋白质掺入到 囊泡直接到达质膜。 其他蛋白质，如HA 流感病毒的G和VSV的G，将被分选受体识别， 是I类分子，其与细胞的管腔结构域相互作用， 将待分选的蛋白质运输到细胞表面。 极化 细胞单层，在一个或另一个表面穿孔，和无细胞的 系统，将用于鉴定，并最终纯化， 组分，包括衔接蛋白和GTP结合蛋白及其 同源对接蛋白，参与形成和靶向 运输囊泡的结构 病毒糖蛋白和 它们假定的分选受体和相应的衔接子将 在总细胞提取物和亚细胞组分中搜索，包括 纯化后的高尔基体囊泡。 为了研究在分类中的作用， 蛋白质的细胞质片段， 对应于各种受体的细胞质尾区， 在透化细胞和无细胞系统中，试图 特异性地抑制向细胞的一个或另一个方面的转运 面 细胞质片段的特异性结合， 接头样分子将通过交联直接研究， 标记转移技术，并尝试使用 细胞质片段作为亲和配体。 穿孔单元系统将 用于检查内吞囊泡形成的要求 以及这些囊泡在细胞内的运输 导致它们与核内体融合 具体的作用 细胞骨架元件，如肌动蛋白和胞衬蛋白，在每个细胞的内吞作用中， 表面将被检查。

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.