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）：外壳蛋白I（COPI）复合物对于分泌途径中的膜运输和隔室组织非常重要，但COPI的生物学作用仍然非常不确定。即使COPI被假定为对于分泌和对于高尔基体池成熟是必需的，酵母COPI温度敏感性（ts）突变体显示出令人惊讶的轻微缺陷：分泌运输仅对于一些货物蛋白被阻断，并且池成熟被减慢但不被阻止。在其他研究中，COPI从膜上的位移改变了高尔基体的结构，但潜在的机制尚不清楚。我们建议澄清COPI在酿酒酵母中的作用。我们的主要方法是“锚定”方法的改进版本。添加雷帕霉素导致标记蛋白被隔离在锚位点，从而功能性地失活标记蛋白。初步数据证实，COPI可以在几分钟内被雷帕霉素隔离和灭活。此外，我们正在优化酵母相关荧光和电子显微镜的方法。工作假设是：（a）COPI对于高尔基体内交通是必不可少的，因为COPI囊泡驱动池成熟，和（B）COPI负调节早期高尔基体的同型融合。具体目标#1是测试COPI在高尔基体池成熟中的作用。假设是COPI囊泡将常驻高尔基体蛋白从较老的脑池回收到较年轻的脑池，从而驱动脑池成熟。通过结合锚的方法与我们建立的视频显微镜技术可视化酵母高尔基体动态，我们将测试是否池成熟需要COPI。预测是当COPI失活时，脑池成熟将“冻结”。具体目标#2是测试COPI在通过分泌途径的交通中的作用。假设所有通过分泌途径的蛋白质运输都需要COPI。将通过脉冲追踪分析测定分泌交通。预测是COPI的失活将通过阻止关键运输组分如SNARE和ER输出受体的再循环来完全阻断分泌运输。具体目标#3是测试COPI在高尔基体同型融合中的作用。假设除了促进囊泡形成，COPI抑制早期高尔基体膜的同型融合。在这种情况下，COPI可能负调节高尔基体池的大小和高尔基体开窗。我们将使用荧光和电子显微镜来检验这些想法。预测是，高尔基体相关的COPI的受控减少将增强同型融合。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。