FOLDING AND ACTIVITY OF ABC PROTEINS IN YEAST

酵母中 ABC 蛋白的折叠和活性

• 批准号: 6178512
• 负责人: Susan D. Michaelis
• 金额: $ 21.43万
• 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/6178512
• 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（取自应用程序）哺乳动物CFTR和酿酒酵母A配合信息素转运蛋白转运蛋白ste6p都是ATP结合盒（ABC）超级家族的成员。在这个项目中，我们将使用Ste6P作为研究生物发生结构和CFTR活性的模型。由于这两种蛋白质具有共同的总体设计，因此它们可能需要类似的细胞机械，以确保其适当的膜插入，折叠和活性。最普遍的CF等位基因Delta F508会导致突变CFTR蛋白的错误折叠，从而导致其通过ER质量控制机制，ER保留率以及随后的泛素蛋白酶体系统的降解。在分泌和膜蛋白的折叠和生物发生中，几乎不知道它们何时未正确折叠。该项目的一个长期目标是利用酵母遗传学的能力来识别有助于和监测ABC蛋白适当折叠的细胞成分。第二个长期目标是以主动形式净化和重构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特定目的：1）稳定稳定ER的抑制剂，该抑制剂稳定了上一个项目期间确定的STE6P的迅速降解的突变体形式；这样的抑制剂将从遗传上查明参与ABC蛋白的膜插入，折叠和ER质量控制的细胞机械的成分。 2）确定ER质量控制机制如何区分膜蛋白的折叠和展开的胞质亚域，并起诉C末端。 STEP6P截断系列和“设计师嵌合体”。 3）纯化并在功能上将其标记的Ste6p重新构成磷脂囊泡，并检查ATPase活性，底物特异性和运输活性。将与P. Maloney和同事合作比较纯化的Ste6p，CFTR和MDR的生化特性。 4）建立第二种酵母CFTR模型（ER保留的YCF1P），该模型可用于剖析ER质量控制途径。我们乐观的是，我们获得的有关酵母菌Ste6P的许多基本知识将适用于人类CFTR，从长远来看，将为开发有导化的化学和生理策略提供坚定的基础，以振兴CF患者中有缺陷的基因产物。