MECHANISMS OF REGULATED EXOCYTOSIS IN TETRAHYMENA

描述：致密的核心颗粒存储激素、消化酶和 刺激偶联胞吐作用的其他蛋白质。致密核心颗粒 在许多单细胞生物体中发现，包括几个 医学上相关的寄生虫。虽然致密核的基本特征 颗粒胞吐作用在真核生物中是保守的，我们的 对这一途径中的基本步骤的理解受到 缺乏适当的实验系统，具有灵活的遗传和 分子遗传工具。分子遗传学和生物化学方法 将被用来分析参与蛋白质的功能 纤毛虫致密核颗粒的生物合成和胞吐作用 原生生物，嗜热四膜虫。颗粒组装涉及到有序的 一组始于跨高尔基体的颗粒蛋白质的凝聚 网络。颗粒蛋白质的蛋白质分解处理似乎是 调节颗粒形成。首席研究员的实验室已经克隆了 主要的钙结合颗粒蛋白，并已获得证据 在特定步骤中发生的一系列对抗性转变 颗粒分泌物。在第一个具体目标中，PI将确定 构象是否通过表达改变直接颗粒组装 主要颗粒蛋白Grl1p的变体。编码该基因的 其他主要颗粒蛋白将被干扰以确定新的和 职能重叠。随之而来的刺激胞吐作用 颗粒，新颗粒以同步的方式从头合成。 我们将使用消减文库的方法来鉴定相关基因 在颗粒合成中。这些基因产物的功能将是 通过使用表达的反义表达阻止翻译进行分析 将被用来识别其翻译抑制导致的基因 颗粒胞吐缺陷。

DESCRIPTION: Dense core granules store hormones, digestive enzymes, and other proteins for stimulation-coupled exocytosis. Dense core granules are found in number of unicellular organisms, including several medically relevant parasites. Although basic features of dense core granule exocytosis are conserved throughout eukaryotes, our understanding of fundamental steps in this pathway is limited by the paucity of appropriate experimental systems with flexible genetic and molecular genetic tools. Molecular genetic and biochemical approaches will be used to analyze the functions of proteins involved in the biosynthesis and exocytosis of dense-core granules in the ciliated protist, Tetrahymena thermophila. Granule assembly involves the ordered condensation of a set of granule proteins beginning in the trans-Golgi network. Proteolytic processing of the granule proteins appears to regulate granule formation. The principal investigator's lab has cloned the major Ca++-binding granule protein and has obtained evidence for a series of confrontational transitions that occur at specific steps in granule secretion. In the first specific aim the PI will determine whether the conformational changes direct granule assembly by expressing variants of the major granule protein Grl1p. The genes encoding the other major granule proteins will be disrupted to identify novel and overlapping functions. Following stimulated exocytosis of stored granules, new granules are synthesized de novo in a synchronous fashion. A subtractive library approach will be used to identify genes involved in granule synthesis. The function of these gene products will be analyzed by blocking translation using expressed antisense expression will be used to identify genes whose translational inhibition causes defects in granule exocytosis.