Dissecting the functions of yeast COPI - Resubmission 01

剖析酵母 COPI 的功能 - 重新提交 01

• 批准号: 8868127
• 负责人: BENJAMIN S GLICK
• 金额: $ 34.33万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8868127
• 关键词: Architecture; Automobile Driving; Biological; Biological Assay; Capsid Proteins; Cell Culture Techniques; Cells; Coat Protein Complex I; Culture Media; Data; Defect; Disease; Electron Microscopy; Fluorescence Microscopy; Freezing; Genetic study; Golgi Apparatus; Image; Immunoprecipitation; Individual; Label; Malignant Neoplasms; Mammalian Cell; Membrane; Membrane Protein Traffic; Membrane Proteins; Methods; Microscopy; Monomeric GTP-Binding Proteins; Pathway interactions; Phenotype; Physiologic pulse; Process; Proteins; Recruitment Activity; Recycling; Regulation; Role; Saccharomyces cerevisiae; Sirolimus; Site; Staging; System; Techniques; Temperature; Testing; Variant; Vesicle; Video Microscopy; Work; Yeasts; developmental disease; mutant; operation; prevent; protein transport; receptor; secretory protein; temperature sensitive mutant; tool; trafficking

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.

描述（由申请人提供）：外套I I（COPI）复合物对于分泌途径中的膜交通和隔室组织很重要，但是COPI的生物学作用仍然非常不确定。即使假定COPI对于分泌和高尔基体的成熟是必不可少的，但酵母COPI温度敏感（TS）突变体表现出令人惊讶的轻度缺陷：仅针对某些货物蛋白而被阻塞，并且蓄水组织成熟却被放大但没有被逮捕。在其他研究中，膜上的COPI位移会改变高尔基体的结构，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“锚定”方法的精制版本。雷帕霉素的添加会导致标记的蛋白在锚固位点隔离，从而在功能上灭活标记的蛋白质。初步数据证实，COPI可以在几分钟之内被雷帕霉素隔离并灭活。此外，我们正在优化一种相关荧光和酵母电子显微镜的方法。工作假设是（a）COPI对于高尔基体的交通是必不可少的，因为Copi囊泡驱动了蓄水池成熟，并且（b）COPI在高尔基体早期对同型融合进行负调节。具体目的1是测试COPI在高尔基体蓄积成熟中的作用。假设是，铜囊泡从较大的水库中回收了驻留的高尔基蛋白，从而驱动蓄水池成熟。通过将锚定方法与我们已建立的视频显微镜技术相结合，以可视化酵母高尔基动力学，我们将测试脑海中的成熟是否需要Copi。预测是，当COPI灭活时，脑海成熟将“冻结”。特定目标是通过分泌Pathwa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流量都需要COPI。分泌流量将通过脉冲练习分析测定。预测，COPI的灭活将通过防止关键贩运组件（例如SNARES和ER出口受体）的回收来完全阻止分泌流量。特定目的＃3是测试COPI在Golgi同型融合中的作用。假设是，除了促进囊泡形成外，COPI还限制了早期高尔基膜的同型融合。在这种情况下，COPI可能会负调节高尔基体的大小和高尔基体。我们将使用荧光和电子显微镜测试这些想法。预测是，受控的高尔基相关COPI的减少将增强同质融合。十多年来，COPI的功能一直是分泌领域的中心奥秘之一。新的酵母工具将使我们能够重大介绍这个问题。