FOLDING AND ACTIVITY OF ABC PROTEINS IN YEAST

酵母中 ABC 蛋白的折叠和活性

• 批准号: 6157648
• 负责人: Susan D. Michaelis
• 金额: $ 20.81万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2002-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6157648
• 关键词: Saccharomyces cerevisiae; adenosinetriphosphatase; cadmium; cystic fibrosis; disease /disorder model; endoplasmic reticulum; enzyme activity; fungal genetics; fungal proteins; liposomes; membrane transport proteins; mutant; phospholipids; protein folding; protein purification; protein reconstitution; secretory protein

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.

描述（摘自申请）哺乳动物 CFTR 和酿酒酵母交配信息素转运蛋白 Ste6p 都是 ATP 结合盒 (ABC) 超家族的成员。在这个项目中，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，因此它们可能需要类似的细胞机制来确保其正确的膜插入、折叠和活性。最常见的 CF 等位基因 delta F508 会导致突变 CFTR 蛋白错误折叠，从而导致其被 ER 质量控制机制识别、ER 保留以及随后被泛素蛋白酶体系统降解。人们对分泌蛋白和膜蛋白的折叠和生物发生知之甚少，也对它们何时未正确折叠的识别知之甚少。该项目的长期目标之一是利用酵母遗传学的力量来识别有助于和监测 ABC 蛋白正确折叠的细胞成分。第二个长期目标是纯化和重构活性形式的 Ste6p，以确定其生化特性。我们将使用遗传、分子和生化方法来实现以下具体目标：1）分离抑制子，以稳定上一个项目期间鉴定的 ER 保留的、快速降解的 Ste6p 突变体形式；这些抑制因子将从基因上精确定位参与 ABC 蛋白的膜插入、折叠和 ER 质量控制的细胞机制的组成部分。 2) 确定 ER 质量控制机制如何区分膜蛋白的折叠和未折叠的胞质子结构域（使用 C 端）。 Step6p 截断系列和“设计师嵌合体”。 3) 纯化带有 His 标签的 Ste6p，并在功能上将其重构为磷脂囊泡，并检查 ATP 酶活性、底物特异性和转运活性。我们将与 P. Maloney 及其同事合作比较纯化的 Ste6p、CFTR 和 MDR 的生化特性。 4) 建立第二个酵母 CFTR 模型（ER 保留的 Ycf1p），除了 Ste6p 之外，还可以使用该模型来剖析 ER 质量控制途径。我们乐观地认为，我们获得的关于酵母 Ste6p 的大部分基本知识将适用于人类 CFTR，并且从长远来看，将为开发定向化学和生理策略以振兴 CF 患者的缺陷基因产物提供坚实的基础。