Dynamics of Endomembrane Docking and Fusion

内膜对接和融合的动力学

• 批准号: 8235481
• 负责人: Alexey Jarrell Merz
• 金额: $ 34.17万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8235481
• 关键词: Amino Acids; Architecture; Back; Biochemical; Biochemical Genetics; Biochemistry; Biological; Biological Assay; Boxing; Budgets; Cells; Cellular biology; Coated vesicle; Complex; Cytoplasm; Data; Docking; Electrophysiology (science); Embryonic Development; Endocrine; Event; Evolution; Funding; Glucose; Goals; Golgi Apparatus; Homeostasis; Hormones; Immunity; Individual; Intracellular Membranes; Life; Lipids; Location; Lysosomes; Mass Spectrum Analysis; Mediating; Membrane; Membrane Fusion; Methods; Modification; Molecular; Molecular Chaperones; Monitor; Monomeric GTP-Binding Proteins; Neurons; Nutrient; Operating System; Optical Methods; Optics; Organelles; Pathway interactions; Post-Translational Protein Processing; Process; Proteins; Quality Control; Reaction; Relative (related person); Reporter; Role; Rupture; S-nitro-N-acetylpenicillamine; SNAP receptor; Saccharomyces; Study Section; System; Telefacsimile; Time; Transport Vesicles; Universities; Vacuole; Vesicle; Washington; Yeasts; advanced system; cofactor; genetic regulatory protein; high risk; in vivo; innovation; isotope incorporation; lipid metabolism; lysosome membrane; millisecond; mutant; neurotransmission; new technology; novel; operation; professor; programs; protein complex; reconstitution; stable isotope; tool; trafficking

Intracellular membrane docking and fusion are fundamental processes in cell biology. They are essential for the operation of the secretory and endocytic pathways and for neurotransmission, hormone secretion, lipid metabolism and immunity. Fusion events are usually catalyzed by SNARE proteins that, on native membranes, act together with an array of chaperones and regulatory proteins including small G proteins and multisubunit tethering complexes. The yeast vacuole is the most technically advanced system for understanding the SNARE-mediated fusion of intracellular organelles. It offers superb in vivo tools, an unsurpassed cell- free assay of fusion, and a fully reconstituted system that allows Rab-regulated fusion. In the previous funding cycle we extensively characterized HOPS, a 640 kDa tethering complex required for vacuole fusion, we delineated new mechanisms that control the activity of the vacuolar Rab protein Ypt7, we studied interactions between HOPS and a coat complex, AP-3, and we developed methods that for the first time allow the capture and study of unambiguous trans-SNARE holocomplexes. We now propose to combine these advances with innovative new technologies as well as classical approaches, to obtain an integrated view of the complex processes leading to pre-fusion complex assembly, and the mechanisms through which these complexes initiate and regulate fusion. In Specific Aim 1 we use biochemical and genetic approaches to dissect a newly discovered mechanism of SNARE complex quality control that operates in vivo, and we explore the mechanism by which the universal chaperone Secl7 restores fusion activity to certain defective trans-SNARE complexes. In Aim 2 we use newly developed optical assays of Rab and SNARE function to probe the dynamics of docking and fusion. In Aim 3 we use trans-SNARE capture and a new AP-3 mutant to dissect the heterotypic delivery of Golgi-derived AP-3 vesicles to the lysosomal vacuole. Box 357350 1959 NE Pacific St Seattle, WA 93195 206.543.1660 fax [phone] bioc@u,washington.edu http. :deptsyashIngtonedu biowww; Modified Specific Aims Our goal is to understand how the complex events of membrane tethering, docking and fusion are executed and regulated on native organelles. Membrane fusion is one of the most fundamental processes in cell biology. Fusion and the docking reactions preceding it are essential for the operation of the secretory and endocytic pathways, lipid metabolism, neurotransmission, nutrient homeostasis, and immunity. We build on biological and technical advances achieved during the previous funding cycle to further explore universal mechanisms of SNARE-mediated docking and to obtain a coherent understanding of the specific machinery that directs traffic into lysosomal organelles. Because the requested funding period for this Project was reduced from 5 years to 4, and because the requested budget over years 1-4 was cut by an average of 31% per year, we are reluctantly compelled to scale back the Specific Aims. We now omit the original Aim 1 (mass spectrometry of trans-SNARE complexes) due to its expense and technical complexity, and we eliminate sub-Aim 3C (electrical recordings from isolated organelles), again for reasons of technical complexity. Both Aims were identified by the Study Section as high-risk and, relative to the other Aims, lacking in preliminary data and clear end-points. The Modified Aims are to: 1. Identify mechanisms of SNARE complex quality control that operate in living cells. We have obtained evidence that SNARE complex assembly is monitored by a quality control system in vivo. Biochemical and genetic strategies will be used to understand the mechanisms through which this quality control system operates. We have also discovered that, through an apparently separate mechanism, the universal SNARE chaperone Secl7 (a-SNAP) can rescue certain defective trans-SNARE complexes. Mutational analyses and biochemical assays will be used to clarify the underlying mechanism of this novel and unexpected activity. 2. Use optical methods to probe the dynamics of docking, SNARE-cofactor interaction, and fusion. We have developed new optical assays and reporters to probe docking and fusion. A noninvasive optical assay of Rab activity allows us to follow Rab activation status in real time during docking and fusion. We have prepared fluorescent SNAREs that will allow us to simultaneously capture trans complex assembly intermediates and probe their organization. 3. Discover the molecular requirements for AP-3 vesicle transport to the lysosomal vacuole. In Saccharomyces, direct traffic from the Golgi to the lysosomal vacuole requires the AP-3 cargo adaptor complex. Despite enormous efforts in several labs, only a few of the components specific to this pathway are known. In vivo SNARE capture, and a new AP-3 mutant that is stuck at the Golgi, will be used to identify additional components of the AP-3 pathway and to understand the mechanisms through which they operate.

细胞内膜对接和融合是细胞中的基本过程 生物学。它们对于分泌和内吞作用至关重要 途径和神经传递，激素分泌，脂质代谢和 免疫。融合事件通常是由圈套蛋白催化的，在本地上 膜，与一系列伴侣和调节蛋白一起起作用 包括小的G蛋白和多生育束缚复合物。酵母液泡 是最先进的系统，用于理解工资介导的 细胞内细胞器的融合。它提供了极佳的体内工具，这是一种无与伦比的细胞 - 融合的免费测定和完全重构的系统，允许RAB调节 融合。在上一个融资周期中，我们广泛表征了啤酒花，一个640 kDa 液泡融合所需的绑扎复合物，我们描述了新机制 控制液泡Rab蛋白YPT7的活性，我们研究了相互作用 在啤酒花和外套复合物之间，AP-3，我们开发了用于 第一次允许捕获和研究明确的跨弹 全旋转。我们现在建议将这些进步与创新的新型相结合 技术和经典方法，以获取对 复杂的过程导致融合前复合物组装和机制 这些复合物通过其引发和调节融合。在特定的目标1中我们 使用生化和遗传方法剖析新发现的机制 在体内运行的圈套复杂质量控制，我们探索 通用伴侣secl7将融合活性恢复到的机制 某些有缺陷的跨弹复合物。在AIM 2中，我们使用新开发的光学 RAB和SNARE功能的测定方法，以探测对接和融合的动力学。在 AIM 3我们使用跨环捕获和新的AP-3突变体进行剖析 Golgi衍生的AP-3囊泡的异型递送到溶酶体液泡。 Box 357350 1959 NE Pacific St Seattle，WA 93195 206.543.1660传真206.685.1792 bioc@u，washington.edu http。 ：deptsyashingtonu biowww; 修改了特定目标 我们的目标是了解膜束缚的复杂事件如何停靠 融合受到本地细胞器的执行和调节。膜融合是一个 细胞生物学中最基本的过程。融合和对接反应 在它之前，它对于分泌和内吞途径的操作至关重要， 脂质代谢，神经传递，营养稳态和免疫力。我们 基于以前的资金中实现的生物和技术进步 循环进一步探索圈套介导的对接的通用机制和 获得对将流量引导到的特定机械的连贯理解 溶酶体细胞器。 因为该项目的要求的资金期限从5个缩短 年限到4岁，并且因为1 - 4年的要求预算平均削减 每年31％，我们不愿迫使我们缩减具体目标。我们现在 省略原始目标1（跨弹头复合物的质谱） 费用和技术复杂性，我们消除了Sub-Aim 3C（电气 出于技术复杂性的原因，来自孤立的细胞器的录音。两个都 目的是通过研究部分确定为高风险，相对于另一个 目的，缺乏初步数据和明确的终点。修改后的目的是： 1。确定在生活中运作的军鼓复杂质量控制的机制 细胞。我们已经获得了证据表明，军鼓复合组装由 体内质量控制系统。生化和遗传策略将用于 了解该质量控制系统运行的机制。我们 还发现，通过明显分开的机制，通用机制 SNARE Chaperone Secl7（A-SNAP）可以挽救某些有缺陷的跨诺 复合物。突变分析和生化测定将用于澄清 这种新颖和意外活动的基本机制。 2。使用光学方法来探测对接的动力学，圈圈式伴随器 相互作用和融合。我们已经开发了新的光学测定法和记者 探针对接和融合。 RAB活动的无创光学测定使我们能够 在对接和融合过程中实时遵循RAB激活状态。我们有 准备的荧光贪食片将使我们同时捕获Trans 复杂的集会中间体并调查其组织。 3。发现AP-3囊泡传输到溶酶体的分子要求 液泡。在糖含量中，直接从高尔基人到溶酶体液泡的交通 需要AP-3货物适配器复合物。尽管在多个实验室做出了巨大的努力，但 仅知道该途径的少数组件。体内军鼓 捕获，以及卡在高尔基的新的AP-3突变体，将用于识别 AP-3途径的其他组件，并了解机制 他们通过的操作。