TARGETING OF VESICLES FROM THE TRANS GOLGI NETWORK

所有细胞都依赖于特定蛋白质的有效靶向 正确的细胞内位置。在分泌途径中，这是被携带的 通过囊泡运输出来的。人们对膜成分知之甚少。 参与囊泡运输的主要原因是 剖析和重组这样一个复杂的过程。最近，在体外 膜转运步骤的重建使人们能够第一次洞察到 转化为高尔基体之间运输所需的胞质组分 水池。本提案中描述的研究的目标是 进一步发展无细胞系统，以重组分选 分子与反高尔基体运输囊泡的形成 以及它们对质膜的正确靶向。具体来说， 参与分选、囊泡形成和 囊泡靶向将被识别和表征。 我们引入的无细胞系统来研究囊泡的运输 跨高尔基体网络始于一个孤立的细胞器，一个堆叠的高尔基体 通过一种固定在磁珠表面上的组份 抗原/抗体相互作用(免疫隔离)。运输囊泡是 在以下情况下从跨高尔基网络生成 细胞器不存在，供体细胞器可以快速移除 因为它被固定在磁性固体支撑体上。市场中的中间商 交通事件，运输小泡，将与萌芽的 使用特定抗体的部分。在随后的一系列实验中， 将添加受体膜，并研究融合过程。 生化解剖和免疫试剂将应用于 分析两个亚细胞隔间在向 细胞表面：跨高尔基体网络和运输小泡。两者都有 参与运输过程的细胞质和细胞膜蛋白将 寻求并研究他们之间的相互作用。对其特异性的验证 这些蛋白质在运输过程中将通过以下方式完成 将它们重新引入我们的无细胞系统。

All cells depend on efficient targeting of specific proteins to their correct intracellular locations. In the secretory pathway this is carried out by vesicular transport. Little is known about the membrane components involved in vesicular transport primarily due to the difficulty of dissecting and reconstituting such a complex process. Recently, in vitro reconstitution of membrane transport steps have allowed the first insights into cytosolic components which are required for transport between Golgi cisternae. The objective of the research described in this proposal is the further development of cell-free systems to reconstitute the sorting of molecules and the formation of transport vesicles from the trans Golgi network and their correct targeting to the plasma membrane. Specifically, the components that are involved in the sorting, vesicle formation and vesicle targeting will be identified and characterized. The cell-free system we introduce to study vesicular transport from the trans Golgi network starts with an isolated organelle, a stacked Golgi fraction which is immobilized on the surface of a magnetic bead by an antigen/antibody interaction (immunoisolation). Transport vesicles are generated from the trans Golgi network under conditions where the recipient organelles are not present and the donor organelle can be rapidly removed since it is bound to a magnetic solid support. The intermediates in the traffic event, the transport vesicles, will be isolated from the budded fraction using specific antibodies. In a consequent set of experiments, recipient membranes will be added and the fusion process will be studied. Biochemical dissection and immunological reagents will be applied to analyze the two sub-cellular compartments involved in the transport to the cell surface: the trans Golgi network and the transport vesicles. Both cytosolic and membrane proteins involved in the traffic process will be sought and their interactions studied. Verification of the specificity of these proteins in the transport process will be accomplished by reintroducing them into our cell-free systems.