QUALITY CONTROL--SELECTIVE PROTEIN DEGRADATION OF THE ER

质量控制--ER的选择性蛋白质降解

• 批准号: 2468920
• 负责人: ANTONY A COOPER
• 金额: $ 10.86万
• 依托单位: UNIVERSITY OF MISSOURI KANSAS CITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2468920
• 关键词: Saccharomyces cerevisiae; endoplasmic reticulum; membrane proteins; molecular chaperones; proteasome; protein degradation; protein folding; ubiquitin

A quality control system is associated with the endoplasmic reticulum (ER) which retains and selectively degrades both soluble and membrane proteins which have failed to fold, oligomerize or assemble correctly. This process assures that only fully functional proteins exit the ER and prevents delivery of a mutant protein or partially assembled receptor complex to the cell surface where it may have deleterious effects. The quality control system affects a number of proteins, including the cystic fibrosis conductance transmembrane regulator, the T cell receptor, MHC class I heavy chain, alpha-1 proteinase inhibitor, apolipoprotein B and acetyl-cholinesterase. In addition, pathogens including the human immunodeficiency virus utilize the degradative machinery to promote and conceal their infection. The overall goal of this research is to develop a detailed molecular understanding of the quality control machinery associated with ER degradation. Although the proteins destined for degradation are initially associated with the ER, the site of degradation and the machinery responsible for proteolysis remain to be identified. This proposal focuses on the degradation of an unassembled membrane spanning protein, Vph1p, in the ER of the yeast S. cervisiae. In the ER of wildtype cells, Vph1p is assembled with other subunits into an ATPase complex destined for the vacuole. However, in the absence of certain assembly proteins Vph1p is rapidly degraded in a manner that is independent of vacuolar (lysosomal equivalent) proteases and occurs in the absence of transport to the Golgi. The turnover of Vph1p exemplifies regulated degradation of membrane proteins retained in the ER and provides powerful tools with which to analyze this important phenomenon. The specific aims are: 1) to determine the precise subcellular location of Vph1p degradation using both immuno-localization and subcellular fractionation techniques; 2) to ascertain if domains of Vph1p on both sides of the ER membrane are degraded, and if so, establish whether such degradation is co-ordinated; 3) to utilize yeast strains that contain mutations in molecular chaperones, ubiquitin and/or subunits of the proteasome complex, to determine the influence of these proteins on Vph1p degradation; 4) to employ genetic approaches to isolate and characterize novel mutants that stabilize Vph1p. The identification, localization and characterization of these mutants and corresponding gene products should complement the biochemical approaches and offer insights into the process of quality control through ER associated protein degradation.

质量控制系统与内质网相关 (ER) 保留并选择性降解可溶性物质和膜物质 未能正确折叠、寡聚或组装的蛋白质。 这个过程确保只有功能齐全的蛋白质才能离开内质网 阻止突变蛋白或部分组装受体的传递 与细胞表面形成复合物，可能产生有害影响。 这 质量控制系统影响许多蛋白质，包括 囊性纤维化电导跨膜调节因子，T 细胞 受体、MHC I 类重链、α-1 蛋白酶抑制剂、 载脂蛋白 B 和乙酰胆碱酯酶。 此外，病原体 包括人类免疫缺陷病毒利用降解 促进和掩盖其感染的机制。 本研究的总体目标是开发详细的分子 了解与 ER 相关的质量控制机制 降解。 尽管注定要降解的蛋白质是 最初与 ER、降解位点和 负责蛋白水解的机制仍有待确定。 这 提案的重点是未组装的跨膜的降解 酿酒酵母内质网中的蛋白质 Vph1p。 在急诊室 野生型细胞中，Vph1p 与其他亚基组装成 ATP 酶 复合物注定要进入液泡。 然而，在缺乏一定的 组装蛋白 Vph1p 会以以下方式快速降解： 独立于液泡（溶酶体等价物）蛋白酶，并发生在 缺乏通往高尔基体的运输。 Vph1p的营业额 举例说明了保留在细胞中的膜蛋白的调节降解 ER 并提供了强大的工具来分析这一重要的 现象。 具体目标是：1）确定精确的亚细胞位置 使用免疫定位和亚细胞研究 Vph1p 降解 分馏技术； 2) 确定 Vph1p 的域是否在两者上 ER 膜的两侧被降解，如果是这样，确定是否存在这种情况 降解是协调的； 3) 利用含有以下成分的酵母菌株 分子伴侣、泛素和/或亚基的突变 蛋白酶体复合物，以确定这些蛋白质对 Vph1p 退化； 4）采用遗传方法来分离和 表征稳定 Vph1p 的新型突变体。 身份证明， 这些突变体的定位和表征以及相应的 基因产品应补充生化方法并提供 通过 ER 关联深入了解质量控制流程 蛋白质降解。