FOLDING AND TRAFFICKING OF STE 6 AND CFTR IN YEAST

CFTR是一种蛋白质，其缺陷会导致囊性纤维化(CF)，是一种 结构相关的膜蛋白超家族的成员 命名为ATP结合盒(ABC)蛋白。ABC蛋白质是 由两个同源的一半组成，每个一半包含六个预测的 膜跨段和ATP核苷酸结合折叠(NBF) 域。ABC超家族的另一个膜是STE6蛋白 酿酒酵母，一种调节啤酒出口的转运蛋白 交配信息素是一个重要因素。在本项目中，我们将使用STE6作为模型 用于研究CFTR的生物发生和结构；类似的 对这两种蛋白质的总体设计表明，它们可能需要 相似的细胞成分，以确保其适当的生物发生，即 膜的插入、折叠和运输到细胞表面。 最普遍的CF等位基因deltaF508似乎导致错误折叠 Cftr，导致细胞内运输和降解 突变的蛋白质。人们对帮助这些因素的了解很少 复杂多跨膜蛋白的生物发生。一个 该项目的主要目标是利用酵母遗传学的力量 识别编码细胞成分的基因，以确保适当的 ABC蛋白的折叠和进展，特别是STE6和CFTR， 从它们的合成部位到细胞表面。第二个目标是 探测STE6(和STE6-CFTR嵌合体)中的分子内相互作用 作为一种确定这些蛋白质中哪些区域相互作用的手段 以促进分子的正确构象。第三个目标是 在酵母中产生高水平的正确折叠的CFTR。我们将使用 遗传、分子和生物化学方法来实现 以下特定目标：1)分离STE6内导致的突变 在其错误本地化或快速降解中，2)确定抑制因素 在Aim 1中获得了ste6突变体；这样的抑制子将从基因上 精确定位与膜有关的细胞机械的组件 插入、折叠、质量控制和贩运STE6，3)用途 STE6-CFTR嵌合体用于识别细胞的其他成分 折叠和质量控制机械，4)正确折叠的快递CFTR 在酿酒酵母中的高水平，以及5)分子内的解剖 管理STE6组装的相互作用，特别是关注 带电残基在跨膜跨度中的作用。 这些研究将使我们能够确定候选的细胞成分 这有助于CFTR的生物发生，并将提供更详细的视图 CFTR的结构和折叠。这些信息可以作为 为制定振兴缺陷基因产品的战略奠定基础 在某些CF患者中。

CFTR, the protein whose defect results in cystic fibrosis (CF), is a member of a superfamily of structurally related membrane proteins designated the ATP binding cassette (ABC) proteins. ABC proteins are comprised of two homologous halves, each half containing six predicted membrane spanning segments and an ATP nucleotide binding fold (NBF) domain. Another membrane of the ABC superfamily is the STE6 protein of Saccharomyces cerevisiae, a transporter which mediates export of the mating pheromone a-factor. In this project, we will use STE6 as a model for investigating the biogenesis and structure of CFTR; the similar overall design of the two proteins suggests they are likely to require similar cellular components to ensure their proper biogenesis, i.e. membrane insertion, folding, and trafficking to the cell surface. The most prevalent CF allele, deltaF508, appears to cause misfolding of CFTR, resulting in aberrant intracellular trafficking and degradation of the mutant protein. Little is known about factors that aid the biogenesis of complex multispanning membrane proteins such as CFTR. A major goal of this project is to utilize the power of yeast genetics to identify genes encoding cellular components that ensure the proper folding and progression of ABC proteins, in particular STE6 and CFTR, from their site of synthesis to the cell surface. A second goal is to probe intramolecular interactions within STE6 (and STE6-CFTR chimeras) as a means of determining which regions within these proteins interact to promote the proper conformation of the molecule. At third goal is to produce high levels of properly folded CFTR in yeast. We will use genetic, molecular, and biochemical approaches to accomplish the following specific aims: 1) Isolate mutations within STE6 that result in its mislocalization or rapid degradation, 2) identify suppressors of ste6 mutants obtained in aim 1; such suppressors will genetically pinpoint components of the cellular machinery involved in membrane insertion, folding, quality control, and trafficking of STE6, 3) Use STE6-CFTR chimeras to identify additional components of the cellular folding and quality control machinery, 4) Express properly folded CFTR at high levels in S. cerevisiae, and 5) Dissect the intramolecular interactions that govern assembly of STE6, particularly focusing on the role of charged residues in the transmembrane spans. These studies will allow us to identify candidate cellular components that assist CFTR in its biogenesis, and will provide a more detailed view of the structure and folding of CFTR. This information could serve as a basis for devising strategies to revitalize the defective gene product in certain CF patients.