The Exocyst in Synthesis, Cystogenesis and Tubulogenesis

描述(申请人提供)：囊和肾小管是肾脏等上皮器官的基本“积木”，囊和肾小管形成缺陷与常染色体显性遗传性多囊肾病等疾病有关。我们的目标是了解与发育和疾病有关的囊性发生和小管发生的生物学。利用一个已被广泛描述的体外胶原凝胶系统，我们已经证明了囊泡和小管发生的一个中心因素是外囊，它是一种进化上保守的八蛋白复合体，参与了分泌途径。分泌途径对于正常的细胞功能是必不可少的，而在该途径的最后阶段，外囊被认为是介导携带分泌蛋白和基侧蛋白的囊泡的靶向和对接。我们最近发现，外囊，特别是Sec10组分，通过与内质网(ER)转位蛋白Sec61的相互作用，还具有新的和意想不到的功能，特异性地调节蛋白质的合成，这是分泌途径的第一阶段。在哺乳动物细胞中，蛋白质通过转运子同时翻译和跨粗略内质网转运。我们的建议是针对这样一种假设，即外囊在囊和小管形成中所起的中心作用是由于它对蛋白质合成的特殊影响。因此，我们将以我们的发现为基础，提出以下问题：胞囊/Sec61a相互作用是如何调节蛋白质合成的？Secl0和Sec61a之间有哪些相互作用的结构域？最后，外囊/转位蛋白是如何识别并特异性地调节基侧而不是顶端的蛋白质合成的？ 为了回答这些问题，我们将使用体外系统，包括无细胞检测，以测试外囊是否调节蛋白质翻译和/或易位(目标1)。然后，我们将识别和定位Sec10/Sec61的一个相互作用的结构域，并确定该结构域突变在蛋白质合成和囊和小管形成方面的功能后果(目标2)。最后，我们将确定指导基侧蛋白合成的胞外/转位调控的序列。这将使用现有的基边蛋白，由于基边靶向序列的突变而运输到顶膜，以及由部分顶端和基边蛋白组成的嵌合体(目标3)。这些研究的完成将在细胞和分子水平上加深对囊和小管形成过程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.