Trafficking pathways to the cell surface in yeast

描述(申请人提供)：我们建议研究调节质膜结构和功能的两个主要机制：内质网质量控制和脂筏。我们已经使用编码酵母质膜H+ATPase的PMA1突变体作为工具来理解正常的膜生物发生以及在内质网和质膜上调节的分子机制。显性的负性突变体Pma1-D378N被识别并被ER相关降解(ERAD)途径破坏，而温度敏感的Pma1-10突变体无法与脂筏结合，而被从质膜上移除以进行空泡降解。每个pma1突变体都被用作遗传选择的基础，导致鉴定出EPS1和Yvh1，EPS1是ERAD途径的新组成部分，Yvh1是一种双特异性磷酸酶，可能在细胞表面的质量控制中发挥作用。这一建议结合了生化、细胞生物学和遗传学的方法来研究这些新蛋白质的分子机制。特定目的I阐述了EPS1，蛋白质二硫键异构酶家族的成员，在ERAD过程中在底物识别中的作用。此外，还将详细分析ERAD衬底Pma1-D378N，因为它是研究ERAD的主要资源。特异性目的II利用pma1-10突变体及其抑制子yvh1来研究蛋白质磷酸化、泛素化、与脂筏的结合以及蛋白质稳定性之间的关系。我们的研究对理解包括囊性纤维化在内的重要分子的错误折叠和ERAD引起的人类疾病具有重要意义。我们在脂筏上的结果将对理解免疫反应(其间信号发生在脂筏上)、艾兹海默氏病(其中蛋白质处理是基于脂筏)和脂类储存疾病(脂筏脂质在溶酶体中异常积累)具有重要意义。

DESCRIPTION (provided by applicant): We propose to study two major mechanisms that regulate the structure and function of the plasma membrane: ER quality control and lipid rafts. We have used mutants of PMA1 encoding the yeast plasma membrane H+ATPase as tools to understand normal membrane biogenesis as well as the molecular mechanisms of regulation at the ER and at the plasma membrane. The dominant negative Pma1-D378N mutant is recognized and targeted for destruction by the ER-associated degradation (ERAD) pathway, whereas the temperature-sensitive Pma1-10 mutant fails to associate with lipid rafts and is removed from the plasma membrane for vacuolar degradation. Each pma1 mutant has been used as the basis for genetic selection leading to identification of Eps1, a novel component of the ERAD pathway, and Yvh1, a dual-specificity phosphatase, which may play a role in quality control at the cell surface. This proposal integrates biochemical, cell biological and genetic approaches to study the molecular mechanisms of these novel proteins. Specific Aim I addresses a proposed role for Eps1, a member of the protein disulfide isomerase family, in substrate recognition during ERAD. In addition, the ERAD substrate, Pma1-D378N, will be analyzed in detail because it represents a major resource for studying ERAD. Specific Aim II uses the pma1-10 mutant and its suppressor yvh1 to study the relationship between protein phosphorylation, ubiquitination, association with lipid rafts, and protein stability. Our studies have important implications for understanding human diseases caused by misfolding and ERAD of important molecules, including cystic fibrosis. Our results on lipid rafts will have significance for understanding the immune response (during which signaling occurs in rafts), AIzheimer's disease (in which protein processing is raft-based), and lipid storage diseases (in which raft lipids are abnormally accumulated in lysosomes).