Exo70 as a Link Between Vesicle Targeting and Vesicle Fusion

分泌囊泡是如何定位于质膜并与之融合的？这个问题的答案对于理解细胞在生长和分化过程中如何形成专门的质膜结构域至关重要。在真核细胞中，分泌囊泡从高尔基体网络（TGN）出芽，并沿着细胞骨架轨道运输到其与质膜融合的位点附近。囊泡运输、靶向和融合之间的联系才刚刚开始被理解。本研究将探讨Exo 70 p在这些过程中的作用。Exo 70 p是酵母外囊复合物的70千道尔顿蛋白质组分，其是在极化生长位点处囊泡对接和融合所需的。该复合物含有至少8种蛋白质组分，但对其生物化学功能知之甚少。为了更好地理解分泌囊泡靶向和融合的机制，研究人员将研究：1）Exo 70如何调节分泌囊泡靶向的特异性。2）Exo 70如何将囊泡靶向与融合机制联系起来。该研究结合了酵母遗传学与生物化学和细胞生物学方法的优势。遗传筛选将用于鉴定分泌囊泡运输和exo 70 -38突变体中囊泡靶向特异性所需的蛋白质。该项目将探索创新的想法，即分泌囊泡的运输和识别可能直接耦合到囊泡融合的Exo 70 p。为此，该项目将确定是否外囊组分蛋白可以调节分泌囊泡对接的特异性。 期望该项目将为Exo 70 p如何将囊泡运输与质膜上的囊泡对接，识别和融合联系起来提供新的见解。这将通过研究Exo 70 p在融合位点的t-SNARE聚类中的作用以及在囊泡靶向/对接至质膜期间如何区分不同类别的分泌囊泡来实现。 在极化上皮中，基底外侧囊泡靶向需要外囊复合物，并且外囊定位于发育中的神经元中的极化生长（生长锥）的位点。因此，可以预期，从酵母研究中获得的见解，如这些将适用于人类细胞中胞吐调节的理解。

How are secretory vesicles targeted to and fused with the plasma membrane? The answer to this question is fundamental to the understanding of how cells form specialized plasma membrane domains during growth and differentiation. In eukaryotic cells, secretory vesicles bud from the trans Golgi network (TGN) and are transported along cytoskeletal tracks to the vicinity of their site of fusion with the plasma membrane. The links between vesicle transport, targeting and fusion are just now beginning to be understood. This research will examine the role of Exo70p in these processes. Exo70p is the 70 kilodalton protein component of the yeast exocyst complex that is required for vesicle docking and fusion at sites of polarized growth. This complex contains at least 8 protein components, but little is known about their biochemical function. To gain a greater understanding of the mechanism of secretory vesicle targeting and fusion, the investigators will examine:1)How Exo70 regulates the specificity of secretory vesicle targeting.2)How Exo70 links vesicle targeting to the fusion machinery.The research combines the strengths of yeast genetics with biochemical and cell biological approaches. Genetic screens will be used to identify proteins that are required for secretory vesicle transport and for the specificity of vesicle targeting in an exo70-38 mutant. This project will explore the innovative idea that secretory vesicle transport and recognition may be directly coupled to vesicle fusion by Exo70p. Towards this end the project will determine whether an exocyst component protein can regulate the specificity of secretory vesicle docking. The expectations are that the project will provide new insights into how Exo70p links vesicle transport to vesicle docking, recognition and fusion at the plasma membrane. This will be accomplished by investigating the role of Exo70p in t-SNARE clustering at the site of fusion and how different classes of secretory vesicles can be discriminated during vesicle targeting/docking to the plasma membrane. In polarized epithelium, the exocyst complex is required for basolateral vesicle targeting and the exocyst is localized to the sites of polarized growth (growth cones) in developing neurons. Therefore, it is expected that insights gained from yeast studies such as these will be applicable to the understanding of the regulation ofexocytosis in human cells.