Mechanisms of Polytopic Protein Biogenesis in the ER

描述（改编自申请人的摘要）：本发明的长期目标是： 建议是建立真核多位体的分子基础 膜蛋白整合，折叠和组装在脂质双分子层中， 内质网（ER）。水通道蛋白是一类典型的多胞体 包含六个跨膜（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，在肾脏中它们在体液中起关键作用， 电解质稳态虽然ER中水通道蛋白组装的基本步骤 最近被描述，很少有人知道细胞机器是如何 介导特异性易位、膜整合和折叠事件 需要建立AQP拓扑。这是我们的一个主要限制。 了解正常肾脏生理学，特别是病理状态 其中水通道蛋白折叠被破坏，例如肾源性尿崩症。 我们实验室最近的研究首次提供了一种方法， 定义负责不同的和新的多面体的分子相互作用 蛋白质折叠途径由于其在正常情况下的重要作用 它们的生理结构相对简单， 因此，水通道蛋白是这种研究的理想候选者。 具体目标是：i）表征不同的分子途径， ii）确定初级结构如何在ER膜中组装， 决定簇在这些折叠途径中产生变化，和iii） 确定ER中的新组件， 水通道蛋白生物发生的方面。拟议的实验将使用无细胞 翻译系统结合光活性交联探针. 天然、突变和嵌合水通道蛋白中的工程化位点。这些 实验将确定新生多肽之间的分子相互作用， 和ER易位机制，介导蛋白质折叠，并决定如何 层序的细微变化影响正常的生物成因事件和拓扑 结果。最后，水通道蛋白成熟所需的新因子将被 通过分离和易位的异源重建鉴定 表现出明显差异的感受态内质网膜， 成熟这些研究将为我们的研究提供重大进展。 多位蛋白生物发生中涉及的分子事件的知识， 将为理解遗传突变如何破坏 人类疾病的生物成因

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.