FOLDING AND ACTIVITY OF ABC PROTEINS IN YEAST

哺乳动物CFTR和交配信息素转运体酿酒酵母Ste6p都是三磷酸腺苷结合盒(ABC)超家族的成员。在本项目中，我们将使用Ste6P作为模型来研究CFTR的生物发生结构和活性。由于这两种蛋白质具有共同的整体设计，它们可能需要相似的细胞机制来确保它们正确的膜插入、折叠和活性。最普遍的CF等位基因Delta F508导致突变的CFTR蛋白错误折叠，导致其被ER质量控制机制识别，ER滞留，并随后被泛素-蛋白酶体系统降解。关于分泌蛋白和膜蛋白的折叠和生物发生，以及识别它们何时折叠不当的问题，人们知之甚少。这个项目的一个长期目标是利用酵母遗传学的力量来识别帮助和监测ABC蛋白质正确折叠的细胞成分。第二个长期目标是提纯并以活性形式重组Ste6P，以确定其生化性质。我们将使用遗传、分子和生化方法来实现以下特定目标：1)分离抑制物，以稳定在前一个项目期间发现的内质网保留、快速降解的Ste6p突变形式；这些抑制子将从基因上定位参与ABC蛋白质的膜插入、折叠和内质网质量控制的细胞机械的组件。2)确定ER质量控制机制如何使用C-末端区分膜蛋白的折叠和未折叠的胞浆亚域。Step6p截断系列和一个“设计师嵌合体”。3)纯化和功能重组His标记的Ste6p形成磷脂小泡，并检测ATPase活性、底物特异性和转运活性。纯化的Ste6p、CFTR和MDR的生化特性将与P.Maloney和他的同事合作进行比较。4)建立第二个酵母CFTR模型(保留内质网的Ycf1p)，该模型可替代Ste6p用于剖析内质网质量控制途径。我们乐观地认为，我们获得的许多关于酵母Ste6p的基本知识将适用于人类CFTR，并将为开发定向的化学和生理策略以重振CF患者的缺陷基因产物提供坚实的基础。

DESCRIPTION (taken from the application) Mammalian CFTR and the Saccharomyces cerevisiae a mating pheromone transporter Ste6p are both members of the ATP binding cassettes (ABC) superfamily. In this project, we will use Ste6p as a model for investigation the biogenesis structure, and activity of CFTR. Because these two proteins share a common overall design, they are likely to require similar cellular machinery to ensure their proper membrane insertion, folding, and activity. The most prevalent CF allele, delta F508, causes misfolding of the mutant CFTR protein, resulting in its recognition by the ER quality control machinery, ER retention, and subsequent degradation by the ubiquitin- proteasome system. Little is known in the folding and biogenesis of secretory and membrane proteins or that recognize when they are not properly folded. One long-term goal of this project is to utilize the power of yeast genetics to identify cellular components that assist in and monitor the proper folding of ABC proteins. A second long-term goal is to purify and reconstitute Ste6p in active form to determine its biochemical properties. We will use genetic, molecular, and biochemical approaches to accomplish the following specific aims: 1) Isolate suppressors that stabilize an ER-retained, rapidly degraded mutant form of Ste6p identified in the previous project period; such suppressors will genetically pinpoint components of the cellular machinery involved in the membrane insertion, folding, and ER quality control of an ABC protein. 2) Determine how the ER quality control machinery distinguishes folded and unfolded cytosolic subdomains of a membrane protein, suing a C-terminal. Step6p truncation series and a "designer chimera". 3) Purify and functionally reconstitute His-tagged Ste6p into phospholipid vesicles and examine ATPase activity, substrate specificity, and transport activity. The biochemical properties of purified Ste6p, CFTR, and MDR, will be compared in collaboration with P. Maloney, and colleagues. 4) To establish a second yeast CFTR model (ER-retained Ycf1p) that can be used in addition to Ste6p to dissect the ER quality control pathway. We are optimistic that much of the basic knowledge we acquired about yeast Ste6p will apply to human CFTR, and in the long-term will provide a firm foundation for developing directed chemical and physiological strategies to revitalize the defective gene product in CF patients.