Mechanisms of Polytopic Protein Biogenesis in the ER

描述(改编自申请人的摘要)：该项目的长期目标 建议建立真核细胞多面性的分子基础 膜蛋白整合、折叠和组装在细胞的脂双层中。 内质网(ER)。水通道蛋白代表多面体的一个原型类 含有六个跨膜(TM)片段并形成选择性的蛋白质 细胞膜中的透水通道。至少有六个水通道蛋白 在哺乳动物肾脏中表达，在液体和尿液中起关键作用 电解质动态平衡。而内质网中水通道蛋白组装的基本步骤有 最近被描述的是，对细胞机械如何 介导特定的转位、膜整合和折叠事件 需要建立AQP拓扑。这是我们的 了解正常的肾脏生理，特别是病理状态 水通道蛋白折叠被破坏的地方，例如肾源性尿崩症。 我们实验室最近的研究，现在第一次提供了一种方法 定义导致不同和新的多面体的分子相互作用 蛋白质折叠途径。因为它们在正常生活中的重要作用 生理学，它们相对简单的结构，以及它们不寻常的生物发生 水通道蛋白是这类研究的理想候选者。 具体的目标是：i)表征不同的分子途径 内质网细胞膜中的水孔组装，II)定义初级结构 决定因素在这些折叠途径中产生变异，以及iii)到 在ER中确定专业化认证所需的新组件 水通道蛋白生物发生的一些方面。拟议的实验将使用无细胞 翻译系统在以下位置结合光活性交联剂 天然、突变和嵌合水通道蛋白中的工程位点。这些 实验将定义新生多肽之间的分子相互作用 和内质网易位机制，介导蛋白质折叠并决定如何 序列中的细微变化影响正常的生物发生事件和拓扑结构 结果。最后，水通道蛋白成熟所需的新因素将是 通过易位的分级和异源重组来鉴定 在水通道蛋白中表现出明显差异的有活性的ER膜 成熟。总之，这些研究将为我们的 多聚体蛋白生物发生和相关分子事件的知识 将为理解遗传突变是如何破坏 人类疾病的生物发生学。

DESCRIPTION (Adapted from the Applicant's Abstract): The long term goal of this proposal is to establish the molecular basis by which eukaryotic polytopic membrane proteins integrate, fold and assemble in the lipid bilayer of the endoplasmic reticulum (ER). Aquaporins represent a prototype class of polytopic proteins that contain six transmembrane (TM) segments and form selective water-permeable channels in cell membranes. At least six aquaporins are expressed in the mammalian kidney where they play critical roles in fluId and electrolyte homeostasis. While the basic steps of aquaporin assembly in ER have recently been described, very little is known about how cellular machinery mediates specific translocation, membrane integration, and folding events required to establish AQP topology. This is a major limitation in our understanding of normal renal physiology and in particular, pathologic states where aquaporin folding is disrupted, e.g. nephrogenic diabetes insipidus. Recent studies from our laboratory, now provide for the first time, a means to define the molecular interactions responsible for different and novel polytopic protein folding pathways. Because of their significant role in normal physiology, their relatively simple architecture, and their unusual biogenesis mechanisms, aquaporins represent ideal candidates for such a study. The specific aims are: i) to characterize different molecular pathways of aquaporm assembly in the ER membrane, ii) to define how primary structural determinants generate variations in these folding pathways, and iii) to identify novel components within the ER that are required for specialized aspects of aquaporin biogenesis. Proposed experiments will use cell free translation systems to incorporate photoactive crosslinking probes at engineered sites in native, mutant and chimeric aquaporin proteins. These experiments will define molecular interactions between the nascent polypeptide and ER translocation machinery that mediate protein folding and determine how subtle variations in sequence influence normal biogenesis events and topologic outcome. Finally novel factors required for aquaporin maturation will be identified by fractionation and heterologous reconstitution of translocation competent ER membranes that exhibit distinct differences in aquaponn maturation. Together these studies will provide a major advance in our knowledge of the molecular events involved in polytopic protein biogenesis and will establish a foundation for understanding how inherited mutations disrupt biogenesis in human disease.