MECHANISMS OF REGULATED EXOCYTOSIS IN TETRAHYMENA

描述：致密的核心颗粒储存激素，消化酶， 其他蛋白质刺激偶联胞吐作用。 致密核心颗粒 在许多单细胞生物中发现， 医学相关寄生虫 虽然致密核的基本特征 颗粒胞吐作用在整个真核生物中是保守的，我们 对这一途径基本步骤的理解受到以下因素的限制： 缺乏适当的实验系统， 分子遗传学工具 分子遗传学和生物化学方法 将用于分析蛋白质的功能， 纤毛虫致密核心颗粒的生物合成和胞吐 原生生物，嗜热四膜虫Tetrahymena thermophila。 颗粒组装涉及有序的 一组颗粒蛋白质开始于反式高尔基体的浓缩 网络 颗粒蛋白的蛋白水解加工似乎 调节颗粒形成。 首席研究员的实验室克隆了 主要的Ca++结合颗粒蛋白，并已获得证据， 一系列对抗性的过渡，发生在特定的步骤， 颗粒分泌 在第一个具体目标中，PI将确定 构象变化是否通过表达 主要颗粒蛋白Grl1p的变体。 编码基因 其它主要颗粒蛋白将被破坏以鉴定新的和 职能重叠。 刺激胞吐后， 颗粒，新的颗粒以同步的方式从头合成。 一个消减文库的方法将用于确定基因参与 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断翻译进行分析 将被用来确定基因的翻译抑制导致 颗粒胞吐作用的缺陷。

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.