Regulation of Membrane Dynamics by Sec7 Domain Arf Nucle

Sec7 域 Arf 核对膜动力学的调节

• 批准号: 7209898
• 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/7209898
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; biochemistry; brefeldin A; cell component structure /function; endoplasmic reticulum; fungal genetics; fungal proteins; guanine nucleotide exchange factors; intracellular transport; membrane structure; molecular biology; protein localization; protein structure function; 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.

该单位研究Arf家族小GTP酶的sec7结构域的鸟嘌呤核苷酸交换因子(GEF)。我们感兴趣的是这些蛋白质在膜动力学和蛋白质运输中的作用。ARF和Arf GEF是细胞器结构和细胞内蛋白质运输的重要调节因子。真核细胞以其内部膜结构为特征，这对于细胞内发生的许多生化反应的正确空间组织是必不可少的。细胞核被核膜与周围的细胞质隔开，核膜是一个与内质网相连的双层膜结构。跨膜结构域蛋白、分泌蛋白和细胞器中的可溶性驻留蛋白，如溶酶体/液泡，通过内质网膜转运到这个细胞器的管腔。从这里，它们通过高尔基仪器被运送到最终目的地。内质网扩散到整个细胞，并产生多种高尔基体，在哺乳动物细胞中，这些高尔基体通过微管运输到邻近细胞核的区域，从而形成高尔基体。高尔基体的一个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它们在细胞中的正确目的地。我们将注意力集中在Arf GEF的一个亚家族上，该家族参与了通过高尔基体的运输，无论是在发芽酵母中还是在哺乳动物细胞中。细胞生物学中的一个中心问题是，面对不断进出每个细胞器的运输，如何保持膜系统的精细和动态结构。特别是，细胞器结构的产生和维持方式，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，是迫切需要解决的问题。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尔丁A(BFA)的细胞效应的研究，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导致高尔基体的完全和快速解体及其与内质网的融合，以及跨高尔基体网络与内小体的融合。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。我们证明了酵母中的三个Arf GEF是该药物在酵母分泌途径中的主要靶点。我们还对该药的作用机制进行了论证。BFA与交换反应中通常非常短暂的反应中间体Arf-GDP-Sec7结构域复合体结合，并形成阻止反应进行到完成的流产的四元复合体。这种不同寻常的作用机制为新药的开发提供了一个范例。与通常寻找与特定底物竞争的药物不同，筛查可以设计成通过稳定反应中间产物来阻止反应的药物。我们正在利用酵母遗传学、分子生物学、酵母和哺乳动物细胞成像以及生物化学等多种技术来阐明Arf GEF在蛋白质运输和细胞器结构中的作用。了解膜转运机制的一个重要步骤是在分子水平上确定Arf GEF的作用，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。我们已经在萌芽酵母(酿酒酵母)和哺乳动物细胞中发现了一些有趣的Arf GEF伙伴，目前正在表征这些新的伙伴及其在蛋白质运输和膜动力学中的作用。