Regulation of ER to Golgi Transport byLuminal Calcium

腔内钙对 ER 至高尔基体运输的调节

• 批准号: 10114882
• 负责人: JESSE C HAY
• 金额: $ 42.2万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10114882
• 关键词: Acute; Affect; Agonist; Apoptosis; Apoptotic; Binding; Biogenesis; Biological Assay; Blood Coagulation Disorders; Buffers; COPII-Coated Vesicles; Calcium; Calcium Channel; Cell Line; Cell Survival; Cell physiology; Cells; Coagulation Factor Deficiency; Communication; Complex; Cytoplasm; Degradation Pathway; Disease; Docking; EF Hand Motifs; Estrogen receptor positive; Event; Exposure to; Factor V; Factor VIII; Failure; Familial benign pemphigus; Golgi Apparatus; Histamine; Homeostasis; Human; ITPR1 gene; In Vitro; Inositol; Kinetics; Learning; Mammalian Cell; Mammals; Measurement; Mediating; Membrane; Mental Depression; Microscopy; Microtubules; Nerve Degeneration; Neuropathy; Organelles; Outcome; Output; Pathology; Pathway interactions; Pentas; Phospholipase C; Physiological; Process; Protein Dynamics; Protein Isoforms; Proteins; Quality Control; Reaction; Regulation; Role; SNAP receptor; Signal Transduction; Site; Sorting - Cell Movement; Stress; System; Testing; Time; Tubular formation; Vesicle; Work; cell growth; cell type; endoplasmic reticulum stress; in vivo; novel; protein expression; receptor; reconstitution; response; secretory protein; trafficking; vesicle transport

PROJECT SUMMARY The secretory pathway employs vesicle transport to provide a linear pathway for export of cellular products and distribution of membrane and organelle components throughout the cytoplasm. Many diseases, including neurodegeneration, involve disruption of the biosynthetic secretory pathway and unresolved secretory stress--making it essential to understand how secretion is up- and down-regulated under different physiological conditions. While the basic engines of vesicle budding, docking and fusion have been identified, little is known of how they are tuned to respond to physiological conditions and stresses. In mammals, ER-to-Golgi transport, which represents the rate-limiting step in the secretory pathway and the step most relevant to transport-related diseases, has been extensively characterized in vivo and reconstituted in vitro. In broad terms, ER-to-Golgi transport is initiated at ER exit sites (ERES) and has been shown to comprise: 1) cargo sorting and vesicle budding mediated by the COPII vesicle coat; 2) homotypic COPII vesicle tethering and fusion mediated by tethers and SNAREs to form pre-Golgi organelles called vesicular tubular clusters (VTCs); and 3) VTC-mediated cargo sorting and transport along microtubules leading to fusion with the Golgi. Little is known about how these processes are adjusted dynamically to match secretory output rates with the needs to enforce secretory quality control, avoid ER stress, and keep pace with secretory protein biogenesis and cell growth. One key aspect to regulation of ER-to-Golgi transport that has become apparent in recent years is the role of ER luminal calcium. Calcium, when released from the ER by channel proteins appears to interact with penta-EF hand proteins (PEFs) in the cytoplasm that bind to the COPII coat at ER exit sites and modulate its assembly and the rate of cargo egress from the ER. However, the mechanisms of these proteins to produce different secretory outcomes and how they are integrated with ER calcium homeostasis and calcium channels are not understood. This project will employ kinetic assays of ER-to-Golgi transport in intact mammalian cell lines, live-cell calcium measurements, and microscopy of protein dynamics at ER exit sites to learn how calcium channels, PEF proteins, and COPII components are integrated to dynamically regulate secretion rate. These studies will have wide significance because proper regulation of secretion is fundamental to cell function and cell survival during physiological stresses.

项目摘要 分泌途径采用囊泡传输来提供导出细胞产物的线性途径 整个细胞质中的膜和细胞器成分的分布。许多疾病， 包括神经变性，涉及生物合成分泌途径的破坏和未解决 分泌的压力 - 制造分泌物必须了解分泌如何上调和下调。 生理条件。虽然囊泡萌芽的基本引擎，所以对接和融合已经 鉴定出来的是，他们如何调整如何对生理条件和压力做出反应。 在哺乳动物中，ER到高尔基运输，代表分泌途径的限速步骤 与运输相关疾病最相关的步骤已在体内广泛表征 在体外重构。从广义上讲，ER到高尔基的运输是在ER出口站点（ERES）启动的，并且具有 已显示出：1）由复菌囊泡涂层介导的货物分选和囊泡萌芽； 2） 同型复印囊泡的束缚和融合，由系tethers和sners介导，形成高尔基体前细胞器 称为囊泡管状簇（VTC）； 3）VTC介导的货物分类和运输 微管导致与高尔基体融合。关于如何调整这些过程的知之甚少 动态以将分泌的产出率与执行分泌质量控制的需求相匹配，避免ER 压力，并与分泌蛋白的生物发生和细胞生长保持同步。 近年来已经显而易见的ER到高尔基运输调节的一个关键方面是 ER腔钙。钙，当通过通道蛋白从ER释放时似乎与 细胞质中的Penta-Fef手蛋白（PEF）与ER出口位点结合并调节 它的组装和从ER的货物出口速率。但是，这些蛋白质的机制 产生不同的分泌结果，以及如何与ER钙稳态整合在一起 钙通道尚不清楚。该项目将采用ER到高尔基运输的动力学测定 完整的哺乳动物细胞系，活细胞钙测量和ER出口蛋白质动力学的显微镜 了解钙通道，PEF蛋白和COPII成分的网站如何动态集成到 调节分泌率。这些研究将具有广泛的意义，因为对分泌的适当调节是 生理胁迫期间细胞功能和细胞存活的基础。