Dissecting the functions of yeast COPI - Resubmission 01

描述（由申请人提供）：在分泌途径中，外壳蛋白I （COPI）复合物对膜运输和室室组织很重要，但COPI的生物学作用仍然非常不确定。尽管COPI被认为是分泌和高尔基池成熟所必需的，但酵母COPI温度敏感（ts）突变体表现出令人惊讶的轻微缺陷：分泌交通仅被某些货物蛋白阻断，池成熟减慢但不停止。在其他研究中，COPI从膜上的位移改变了高尔基结构，但潜在的机制尚不清楚。我们建议阐明COPI在酿酒酵母中的作用。我们的主要方法是“锚移”方法的改进版本。雷帕霉素的加入导致被标记的蛋白被隔离在锚位点，从而在功能上使被标记的蛋白失活。初步数据证实，COPI可在几分钟内被雷帕霉素隔离和灭活。此外，我们还对酵母的相关荧光和电镜检测方法进行了优化。目前的假设是：(a) COPI对高尔基体内部交通至关重要，因为COPI囊泡驱动池成熟；(b) COPI负调控高尔基体早期的同型融合。具体目的1是测试COPI在高尔基池成熟中的作用。假设是COPI囊泡将驻留的高尔基蛋白从老年池循环到年轻池，从而推动池成熟。通过结合锚移法和我们建立的视频显微镜技术来观察酵母高尔基体动力学，我们将测试池成熟是否需要COPI。预测是，当COPI失活时，池的成熟将“冻结”。具体目标#2是通过分泌路径测试COPI在流量中的作用。假设是所有通过分泌途径的蛋白质运输都需要COPI。秘书流量将通过脉冲追踪分析进行分析。预测是，COPI的失活将通过阻止关键贩运成分（如SNAREs和ER输出受体）的再循环，完全阻断分泌交通。具体目的#3是测试COPI在高尔基同型融合中的作用。假设除了促进囊泡形成外，COPI还抑制早期高尔基膜的同型融合。在这种情况下，COPI可能负性调节高尔基体池大小和高尔基体开孔。我们将使用荧光和电子显微镜来测试这些想法。预测是控制高尔基相关的COPI的减少将加强同型融合。十多年来，COPI的功能一直是分泌领域的核心谜团之一。新的酵母工具将使我们能够在这个问题上取得重大进展。

DESCRIPTION (provided by applicant): The coat protein I (COPI) complex is important for membrane traffic and compartmental organization in the secretory pathway, but the biological roles of COPI are still remarkably uncertain. Even though COPI is postulated to be essential for secretion and for Golgi cisternal maturation, yeast COPI temperature- sensitive (ts) mutants show surprisingly mild defects: secretory traffic is blocked only for some cargo proteins, and cisternal maturation is slowed but not arrested. In other studies, displacement of COPI from membranes alters Golgi architecture, but the underlying mechanism is unknown. We propose to clarify the action of COPI in Saccharomyces cerevisiae. Our main approach is a refined version of the "anchor-away" method. Addition of rapamycin causes a tagged protein to be sequestered at an anchor site, thereby functionally inactivating the tagged protein. Preliminary data confirm that COPI can be sequestered and inactivated by rapamycin within minutes. In addition, we are optimizing a method for correlative fluorescence and electron microscopy of yeast. The working hypothesis is that (a) COPI is essential for intra-Golgi traffic because COPI vesicles drive cisternal maturation, and (b) COPI negatively regulates homotypic fusion at the early Golgi. Specific Aim #1 is to test the role of COPI in Golgi cisternal maturation. The hypothesis is that COPI vesicles recycle resident Golgi proteins from older to younger cisternae, thereby driving cisternal maturation. By combining the anchor-away method with our established video microscopy technique for visualizing yeast Golgi dynamics, we will test whether cisternal maturation requires COPI. The prediction is that when COPI is inactivated, cisternal maturation will "freeze". Specific Aim #2 is to test the role of COPI in traffic through the secretory pathwa. The hypothesis is that all protein traffic through the secretory pathway requires COPI. Secretory traffic will be assayed by pulse-chase analysis. The prediction is that inactivation of COPI will completely block secretory traffic by preventing the recycling of key trafficking components such as SNAREs and ER export receptors. Specific Aim #3 is to test the role of COPI in Golgi homotypic fusion. The hypothesis is that in addition to promoting vesicle formation, COPI restrains the homotypic fusion of early Golgi membranes. In this context, COPI may negatively regulate Golgi cisternal size and Golgi fenestration. We will test these ideas using fluorescence and electron microscopy. The prediction is that a controlled reduction of Golgi-associated COPI will enhance homotypic fusion. For over a decade, the function of COPI has been one of the central mysteries in the secretion field. New yeast tools will enable us to make major inroads into this problem.