Exo70 as a Link Between Vesicle Targeting and Vesicle Fusion

分泌囊泡如何定位于质膜并与质膜融合？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特殊的质膜域是至关重要的。在真核细胞中，分泌囊泡从跨高尔基网络(TGN)萌发，并沿着细胞骨架轨道运输到它们与质膜融合的部位附近。囊泡运输、靶向和融合之间的联系现在才刚刚开始被理解。这项研究将研究Exo70p在这些过程中的作用。Exo70p是酵母胞囊复合体中70千道尔顿的蛋白质组分，在极化生长的位置，囊泡对接和融合是必需的。这种复合体包含至少8种蛋白质成分，但人们对它们的生化功能知之甚少。为了更好地了解分泌囊泡靶向和融合的机制，研究人员将研究：1)Exo70如何调节分泌囊泡靶向的特异性2)Exo70如何将囊泡靶向与融合机制联系起来。研究将酵母遗传学的优势与生化和细胞生物学方法相结合。基因筛选将用于鉴定分泌性囊泡运输所需的蛋白质，以及Exo70-38突变体中囊泡靶向的特异性。该项目将探索分泌性囊泡的运输和识别可能通过Exo70p直接偶联到囊泡融合的创新思想。为此，该项目将确定外囊成分蛋白是否可以调节分泌囊泡对接的特异性。人们的期望是，该项目将为Exo70p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Exo70p在融合部位t-SNARE聚集中的作用以及在囊泡靶向/对接到质膜过程中如何区分不同类型的分泌小泡来实现。在极化上皮中，基侧囊泡靶向需要外囊复合体，外囊定位于发育中神经元的极化生长部位(生长锥)。因此，希望从酵母研究中获得的见解将有助于理解人类细胞的胞吐作用的调节。

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.