The Exocyst in Synthesis, Cystogenesis and Tubulogenesis

描述（由申请人提供）：囊肿和小管是上皮器官（如肾脏）的基本“构建块”，囊肿和小管形成缺陷与常染色体显性多囊肾病等疾病有关。我们的目标是了解与发育和疾病相关的膀胱发生和小管发生的生物学。使用一个良好的描述在体外胶原凝胶系统，我们已经表明，在cystogenesis和tubulogenesis的一个中心因素是外囊，一个进化上保守的八蛋白复合物参与分泌途径。分泌途径对于适当的细胞功能是必不可少的，并且已知外囊在该途径的最后阶段介导携带分泌蛋白和基底外侧蛋白的囊泡的靶向和对接。我们最近发现，外囊，特别是Sec 10组件，也有新的和意想不到的功能，特别是调节蛋白质合成，分泌途径的第一阶段，通过与Sec 61的内质网（ER）的一个组件的相互作用。在哺乳动物细胞中，蛋白质通过易位子同时翻译和易位穿过粗糙ER。我们的建议是针对这样的假设，即外囊在囊和小管形成中所起的中心作用是其对蛋白质合成的特定作用的结果。因此，我们将通过提出以下问题来建立我们的研究结果：外囊/Sec 61 a相互作用如何调节蛋白质合成？Sec 10和Sec 61 a之间的相互作用域是什么？最后，外囊/转位子如何识别并特异性调节基底外侧而非顶端的蛋白质合成？ 为了回答这些问题，我们将使用体外系统，包括无细胞测定，以测试是否外囊调节蛋白质翻译和/或易位（目标1）。然后，我们将鉴定和定位Sec 10/Sec 61相互作用结构域，并确定突变该结构域在蛋白质合成和囊肿和小管形成方面的功能后果（目的2）。最后，我们将确定序列，直接外囊/转位调控基底外侧蛋白质合成。这将使用由于基底外侧靶向序列中的突变而运输至顶膜的现有基底外侧蛋白以及由顶和基底外侧蛋白的部分组成的嵌合体来完成（Aim 3）。这些研究的完成将在细胞和分子水平上增强对囊肿和小管形成中蛋白质合成机制的理解，并为开发新的治疗方法奠定基础。

DESCRIPTION (provided by applicant): Cysts and tubules are basic "building blocks" for epithelial organs such as the kidney, and defects in cyst and tubule formation are implicated in disorders such as autosomal dominant polycystic kidney disease. Our goal is to understand the biology of cystogenesis and tubulogenesis as it relates to development and disease. Using a well-described in vitro collagen gel system, we have shown that a central factor in cystogenesis and tubulogenesis is the exocyst, an evolutionarily conserved eight-protein complex involved in the secretory pathway. The secretory pathway is essential for proper cellular function, and the exocyst is known for mediating the targeting and docking of vesicles carrying secretory and basolateral proteins during the final stage of this pathway. We recently showed that the exocyst, particularly the Sec10 component, also has the novel and unexpected function of specifically regulating protein synthesis, the first stage of the secretory pathway, by interacting with the Sec61 a component of the endoplasmic reticulum (ER) translocon. In mammalian cells, proteins are simultaneously translated and translocated across the rough ER via the translocon. Our proposal is directed toward the hypothesis that the central role played by the exocyst in cyst and tubule formation is a result of its specific effects on protein synthesis. Accordingly, we will build on our findings by asking the following questions: How does the exocyst/Sec61 a interaction regulate protein synthesis? What are the interacting domains between Secl0 and Sec61a? Finally, how does the exocyst/translocon, recognize and then specifically regulate basolateral, but not apical, protein synthesis? To answer these questions we will use in vitro systems, including cell-free assays, to test whether the exocyst regulates protein translation and/or translocation (Aim 1). We will then identify and map the SeclO/Sec61 a interacting domain, and determine the functional consequences of mutating this domain, with respect to protein synthesis and cyst and tubule formation (Aim 2). Lastly, we will identify sequences that direct exocyst/translocon regulation of basolateral protein synthesis. This will be done using existing basolateral proteins that traffic to the apical membrane, due to mutations in the basolateral targeting sequence, and chimeras composed of portions of apical and basolateral proteins (Aim 3). Completion of these studies will enhance the understanding of the mechanisms of protein synthesis in cyst and tubule formation at the cellular and molecular levels and lay the groundwork for the development of novel therapeutics.