GOLGI TRANSPORT VESICLE BINDING AND FUSION

对高尔基体内囊泡运输的体外分析揭示了许多 参与运输囊泡出芽过程的分子， 组装件.然而，该测定的局限性使得分析 运输囊泡的结合和融合更加困难。这样做的目的之一 建议是评估对常规的具体修改， 体外转运试验这些改进将产生两阶段反应 其中高尔基体内转运囊泡与受体高尔基体膜结合 不融合，然后在加入胞质调节因子后融合。 因素改进的检测方法将用于鉴定所需的蛋白质 用于结合和融合的特定反应步骤。两个先前 已鉴定的胞质活性，称为组分1-α和1-β， 已经发现对于结合和融合的组合测定是必需的。 这些活动将进一步净化和特点， 到它们的特定作用部位。最后，一个额外的细胞溶质 活性，称为释放因子，将被纯化，其作用， 确定结合和/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.