Trafficking pathways to the cell surface in yeast

描述（由申请人提供）：我们拟研究两种调节质膜结构和功能的主要机制：内质网质量控制和脂质筏。我们利用编码酵母质膜H+ atp酶的PMA1突变体作为工具来了解正常的膜生物发生以及内质网和质膜调控的分子机制。显性阴性Pma1-D378N突变体被er相关降解（ERAD）途径识别并靶向破坏，而温度敏感的Pma1-10突变体不能与脂筏相关，并从质膜上去除以进行空泡降解。每个pma1突变体都被用作遗传选择的基础，从而鉴定出Eps1和Yvh1，前者是ERAD途径的新组分，后者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。本研究将结合生物化学、细胞生物学和遗传学的方法来研究这些新蛋白的分子机制。特异性Aim I解决了Eps1在ERAD过程中底物识别中的作用，Eps1是蛋白质二硫异构酶家族的成员。此外，我们将详细分析ERAD底物Pma1-D378N，因为它是研究ERAD的主要资源。特异性Aim II使用pma1-10突变体及其抑制因子yvh1来研究蛋白质磷酸化、泛素化、与脂筏的关联以及蛋白质稳定性之间的关系。我们的研究对于理解由重要分子的错误折叠和ERAD引起的人类疾病，包括囊性纤维化，具有重要意义。我们关于脂筏的研究结果将对理解免疫反应（信号在脂筏中发生）、阿尔茨海默病（蛋白质加工以脂筏为基础）和脂质储存病（脂筏异常积聚在溶酶体中）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).