GOLGI TRANSPORT VESICLE BINDING AND FUSION

对高尔基体内囊泡交通的体外分析发现了许多 参与运输囊泡萌发和运输过程的分子 集合。然而，这一分析方法的局限性使分析 运输囊泡更难捆绑和融合。这样做的一个目的是 建议是评估对《公约》的具体修改 体外转运试验。这些改进将产生两个阶段的反应 高尔基体内转运囊泡与受体高尔基体膜结合 没有融合，然后在添加胞质调节时融合 各种因素。改进后的化验方法将用于鉴定必要的蛋白质 用于结合和融合的具体反应步骤。之前的两个 已鉴定的胞浆活性，称为组分1-α和1-β 被发现是结合和融合试验所必需的。 这些活动将得到进一步的净化和尊重 到他们特定的行动地点。最后，一个额外的细胞质 活性，称为释放因子，将被提纯并在 确定了结合和/或融合。

In vitro analysis of intra-Golgi vesicular traffic has uncovered many of the molecules involved in the process of transport vesicle budding and assembly. However, limitations of this assay have made analysis of transport vesicle binding and fusion more difficult. One aim of this proposal is to evaluate specific modifications to the conventional in vitro transport assay. These refinements will create a two-stage reaction where intra-Golgi transport vesicles bind to acceptor Golgi membranes without fusion, then fuse upon the addition of cytosolic regulatory factors. The improved assay will be used to identify proteins necessary for the specific reaction steps of binding and fusion. Two previously identified cytosolic activities, termed fractions 1-alpha and 1-beta have been found to be necessary for the combined binding and fusion assay. These activities will be further purified and characterized with respect to their specific site of action. Finally, an additional cytosolic activity, called releasing factor, will be purified and its role in binding and/or fusion determined.