Regulation of Membrane Dynamics by Sec7 Domain Arf Nucle

Sec7 域 Arf 核对膜动力学的调节

• 批准号: 6672659
• 负责人: Catherine L Jackson
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6672659
• 关键词: ADP ribosylation; Golgi apparatus; Saccharomyces cerevisiae; brefeldin A; fungal proteins; guanine nucleotide exchange factors; guanosinetriphosphatases; intermolecular interaction; intracellular transport; membrane activity; microorganism culture; protein transport

This unit studies the Sec7 domain guanine nucleotide exchange factors (GEFs) for the Arf family of small GTPases. We are interested in the roles of these proteins in membrane dynamics and protein trafficking. The Arfs and the Arf GEFs are important regulators of both organelle structure and protein transport throughout the cell. Eukaryotic cells are characterized by their internal membrane structure, which is essential for the correct spatial organization of the many biochemical reactions that take place within cells. The nucleus is separated from the surrounding cytoplasm by the nuclear envelope, a double membrane structure that is continuous with the endoplasmic reticulum (ER). Transmembrane domain proteins, proteins destined for secretion, and soluble resident proteins of organelles such as the lysosome/vacuole are translocated across the ER membrane into the lumen of this organelle. From here they are transported to their final destination via the Golgi apparatus. The ER spreads throughout cells, and gives rise to multiple Golgi elements that in mammalian cells are transported via microtubules to a region adjacent to the nucleus, thus forming the Golgi apparatus. A major function of the Golgi apparatus is the post-translational modification of proteins traveling through it, and sorting of these proteins to their correct destination in the cell. We are focusing our attention on a subfamily of Arf GEFs involved in transport through the Golgi apparatus, both in budding yeast and in mammalian cells. A central question in cell biology is how the elaborate and dynamic structures of membrane systems are maintained in the face of constant trafficking into and out of each organelle. In particular, the way organelle structure is generated and maintained, and how structure is correlated with the underlying molecular events of protein sorting and membrane remodeling are pressing questions. Evidence that the Arf GEFs play a key role in membrane dynamics and organelle structure came from studies of the cellular effects of brefeldin A (BFA), a drug that has profound effects on organelles of the secretory and endocytic pathways in a wide range of cell types. BFA causes the complete and rapid disassembly of the Golgi apparatus and its fusion with the ER, as well as fusion of the trans-Golgi network with endosomes. These experiments were the first to show the incredibly dynamic nature of the Golgi apparatus, whose elaborate structure would seem to suggest a more stable state. We demonstrated that three Arf GEFs in yeast are the major targets of the drug in the yeast secretory pathway. We also demonstrated the mechanism of action of this drug. BFA binds to a normally very short-lived reaction intermediate in the exchange reaction, an Arf-GDP-Sec7 domain complex, and forms an abortive quaternary complex that prevents the reaction from proceeding to completion. This unusual mechanism of action provides a paradigm for development of novel drugs. Instead of the usual search for drugs competing for a given substrate, screens could be designed for drugs that block reactions through stabilization of reaction intermediates. We are using a combination of techniques, including yeast genetics, molecular biology, imaging of yeast and mammalian cells and biochemistry, to elucidate the roles of the Arf GEFs in protein transport and organelle structure. An important step towards understanding the mechanisms of membrane trafficking will be to define the roles of the Arf GEFs at the molecular level, through identification of interacting partners, elucidation of membrane localization mechanisms and analysis of Arf GEF mutants in vivo. We have identified a number of interesting partners of the Arf GEFs in both budding yeast (Saccharomyces cerevisiae) and in mammalian cells, and are currently characterizing these novel partners and their roles in protein trafficking and membrane dynamics.

本单元研究小 GTP 酶 Arf 家族的 Sec7 结构域鸟嘌呤核苷酸交换因子 (GEF)。我们对这些蛋白质在膜动力学和蛋白质运输中的作用感兴趣。 Arf 和 A​​rf GEF 是整个细胞内细胞器结构和蛋白质运输的重要调节因子。真核细胞的特征在于其内部膜结构，这对于细胞内发生的许多生化反应的正确空间组织至关重要。细胞核通过核膜与周围的细胞质分开，核膜是一种与内质网 (ER) 连续的双层膜结构。跨膜结构域蛋白、用于分泌的蛋白以及细胞器（例如溶酶体/液泡）的可溶性驻留蛋白穿过内质网膜易位到该细胞器的内腔中。从这里，它们通过高尔基体被运送到最终目的地。内质网遍布整个细胞，并产生多种高尔基体元件，在哺乳动物细胞中，这些高尔基体元件通过微管转运到邻近细胞核的区域，从而形成高尔基体。高尔基体的主要功能是对通过它的蛋白质进行翻译后修饰，并将这些蛋白质分类到细胞中的正确目的地。我们将注意力集中在参与芽殖酵母和哺乳动物细胞中高尔基体运输的 Arf GEF 亚家族。细胞生物学的一个核心问题是，面对每个细胞器的不断进出，如何维持膜系统复杂和动态的结构。特别是，细胞器结构的生成和维持方式，以及结构如何与蛋白质分选和膜重塑的潜在分子事件相关，都是紧迫的问题。 Arf GEF 在膜动力学和细胞器结构中发挥关键作用的证据来自对布雷菲德菌素 A (BFA) 细胞效应的研究，布雷菲德菌素 A (BFA) 是一种对多种细胞类型的分泌和内吞途径的细胞器具有深远影响的药物。 BFA 导致高尔基体完全快速分解并与内质网融合，以及跨高尔基体网络与内体的融合。这些实验首次展示了高尔基体令人难以置信的动态性质，其复杂的结构似乎表明了一种更稳定的状态。我们证明酵母中的三个 Arf GEF 是酵母分泌途径中药物的主要靶标。我们还展示了这种药物的作用机制。 BFA 与交换反应中通常寿命非常短的反应中间体（Arf-GDP-Sec7 结构域复合物）结合，并形成无效的四元复合物，阻止反应进行至完成。这种不寻常的作用机制为新药的开发提供了范例。可以设计筛选通过稳定反应中间体来阻断反应的药物，而不是通常寻找与给定底物竞争的药物。 我们正在结合使用酵母遗传学、分子生物学、酵母和哺乳动物细胞成像以及生物化学等技术来阐明 Arf GEF 在蛋白质运输和细胞器结构中的作用。了解膜运输机制的一个重要步骤是通过鉴定相互作用伙伴、阐明膜定位机制和分析体内 Arf GEF 突变体，在分子水平上定义 Arf GEF 的作用。我们已经在芽殖酵母（酿酒酵母）和哺乳动物细胞中鉴定出许多有趣的 Arf GEF 伙伴，目前正在表征这些新伙伴及其在蛋白质运输和膜动力学中的作用。