FOLDING AND ACTIVITY OF ABC PROTEINS IN YEAST

酵母中 ABC 蛋白的折叠和活性

• 批准号: 6381892
• 负责人: Susan D. Michaelis
• 金额: $ 22.01万
• 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 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381892
• 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（摘自本申请）哺乳动物CFTR和酿酒酵母交配信息素转运蛋白Ste 6p都是ATP结合盒（ABC）超家族的成员。在本项目中，我们将使用Step 6p作为研究CFTR的生物起源结构和活性的模型。由于这两种蛋白质具有共同的整体设计，它们可能需要类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Δ F508引起突变CFTR蛋白的错误折叠，导致其被ER质量控制机制识别、ER保留和随后被泛素-蛋白酶体系统降解。很少有人知道的折叠和生物合成的分泌和膜蛋白或识别时，他们没有正确折叠。该项目的一个长期目标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组活性形式的Ste 6p，以确定其生化特性。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）分离抑制子，其稳定在先前项目期间鉴定的ER保留的、快速降解的Ste 6p突变形式;这种抑制子将从遗传上精确定位参与ABC蛋白的膜插入、折叠和ER质量控制的细胞机器的组分。2)确定ER质量控制机制如何区分膜蛋白的折叠和未折叠胞质亚结构域，使用C-末端。Step 6p截断系列和一个“设计者嵌合体”。3)纯化并功能性地将His标记的Ste 6p重组到磷脂囊泡中，并检查ATP酶活性、底物特异性和转运活性。纯化的Ste 6p、CFTR和MDR的生化特性将与P. Maloney及其同事合作进行比较。4)建立第二个酵母CFTR模型（ER保留的Ycf 1 p），除Ste 6p外，还可用于剖析ER质量控制途径。我们乐观地认为，我们获得的关于酵母Step 6p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恢复CF患者中的缺陷基因产物提供坚实的基础。