ATPASE PROTEINS OF THE YEAST 26S PROTEIN

酵母 26S 蛋白的ATP酶蛋白

• 批准号: 2392102
• 负责人: Daniel J Finley
• 金额: $ 28.21万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392102
• 关键词: Saccharomyces cerevisiae; adenosinetriphosphatase; chemical association; chemical cleavage; chemical conjugate; conformation; covalent bond; enzyme activity; exopeptidase; fungal genetics; fusion gene; gene mutation; hydrolysis; immunoelectron microscopy; molecular chaperones; mutant; nucleic acid sequence; peptidases; protein degradation; protein structure function; ubiquitin

Modification by ubiquitin has been implicated in the turnover of many proteins, such as cyclins, p53, and various oncoproteins. This grant focuses on the role of ATPases in the degradation of ubiquitin-conjugates. A family of 5 related but functionally distinct ATPases appears to reside within a multisubunit complex known as the 26S protease, which is essential for degradation of ubiquitin-conjugates. The importance of these ATPases is suggested by the strict ATP-dependence of ubiquitin-conjugate degradation, the requirement for all 5 genes for viability in yeast, and effects of point mutations in these genes on proteolysis. However, nothing is known about the mechanism by which these ATPases participate in protein degradation. We propose a molecular chaperone model for 26S ATPase function, namely that the these proteins use ATP hydrolysis to drive cycles of association and dissociation of the proteolytic substrate. We further propose that functional distinctions among 26S ATPases are at least partly attributable to their having distinct substrate binding specificities. The multiplicity of ATPases in the 26S protease, and their extreme evolutionary conservation, suggests that the role of ATP in 26S function is of fundamental importance but also poses difficulties in addressing these roles, since adding ATP to the complex may alter the functioning of not one but many of its components. In this proposal, we describe the use of yeast genetics to overcome this problem and to dissect systematically the role of ATP in proteolysis. We have constructed synoptic sets of mutations in the predicted ATP-binding regions of the 5 ATPase genes. Surprisingly, the mutations are for the most part nonlethal, although they result in pleiotropic phenotypes and dramatic protein stabilization effects. Using these mutations, the detailed in vivo functions of each ATPase activity can be determined. In particular, we will determine the role of each ATPase in the degradation of a variety of available in vivo substrates. We have developed a purification of the yeast 26S protease, which will allow us to study the ATPase mutants in vitro as well. Biochemical experiments will address specific mechanistic models for the individual role of each ATPase in the proteolytic process. For example, our hypothesis that these ATPases bind proteolytic substrates in an ATP- dependent manner will be examined by searching for peptide ligands, which may be used to study Yam-substrate interactions and their possible role in proteolysis. Finally, each ATPase will be localized within the architecture of the 26S protease by high-resolution immunoelectron microscopy. In summary, the powerful combination of biochemistry and genetics proposed below will provide insights into a novel ATPase particle, and allow us to test the molecular chaperone model of 26S ATPase function.

泛素修改已与许多人的营业额有关 蛋白质，例如细胞周期蛋白，p53和各种癌蛋白。这笔赠款 着重于ATPases在泛素结合物降解中的作用。 一个相关但功能上不同ATPases的家族似乎存在 在称为26S蛋白酶的多育种复合物中， 泛素 - 偶联物的降解至关重要。这些的重要性 泛素 - 偶联物的严格依赖性ATP依赖性建议ATPases 降解，对酵母生存能力的所有5个基因的要求，以及 这些基因中点突变对蛋白水解的影响。但是，什么都没有 了解这些ATP酶参与蛋白质的机制 降解。我们提出了26S ATPase的分子伴侣模型 功能，即这些蛋白质使用ATP水解驱动 蛋白水解底物的缔合和解离。我们 进一步提出，26S ATPases之间的功能区别是 至少部分归因于它们具有独特的底物结合 特殊性。 26S蛋白酶中的ATPases多样性及其极端 进化保护表明ATP在26S功能中的作用 至关重要，但在解决问题方面也很困难 这些角色，因为将ATP添加到复合物中可能会改变 不是一个，而是许多组成部分。在此提案中，我们描述了使用 酵母遗传学以克服这个问题并系统解剖 ATP在蛋白水解中的作用。我们已经构建了概要集 5个ATPase基因的预测ATP结合区域中的突变。 令人惊讶的是，这些突变在大多数情况下是非致命的，尽管它们 导致多效性表型和戏剧性的蛋白质稳定 效果。 使用这些突变，每个ATPase的详细体内函数 可以确定活动。特别是，我们将确定 每个ATPase在体内可用的各种可用的降解中 基材。我们已经开发了酵母26S蛋白酶的纯化， 这将使我们也可以在体外研究ATPase突变体。 生化实验将解决特定机械模型 每个ATPase在蛋白水解过程中的个体作用。例如， 我们的假设是这些ATP酶结合了ATP中的蛋白水解底物 依赖方式将通过搜索肽配体来检查 可用于研究山毛基底相互作用及其在 蛋白水解。最后，每个ATPase将位于 高分辨率免疫电子的26S蛋白酶结构 显微镜。总而言之，生物化学和 下面提出的遗传学将为新的ATPase提供见解 粒子，允许我们测试26S ATPase的分子伴侣模型 功能。