Dynamics of Endomembrane Docking and Fusion

内膜对接和融合的动力学

基本信息

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
项目类别：
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

项目摘要

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 206.685.1792 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的基本过程生物学。它们对于分泌和内吞的运作至关重要途径和神经传递、激素分泌、脂质代谢和免疫。融合事件通常由 SNARE 蛋白催化，该蛋白在天然膜，与一系列伴侣和调节蛋白一起作用包括小 G 蛋白和多亚基束缚复合物。酵母液泡是用于理解 SNARE 介导的技术最先进的系统细胞内细胞器的融合。它提供了卓越的体内工具、无与伦比的细胞- 免费融合检测，以及完全重构的系统，允许 Rab 调节融合。在上一个资助周期中，我们广泛表征了 HOPS，一种 640 kDa 液泡融合所需的束缚复合物，我们描述了新的机制控制液泡 Rab 蛋白 Ypt7 的活性，我们研究了相互作用在 HOPS 和外套复合物 AP-3 之间，我们开发了用于第一次允许捕获和研究明确的反SNARE 全息复合物。我们现在建议将这些进步与创新的新方法相结合技术以及经典方法，以获得对导致预融合复杂组装的复杂过程及其机制这些复合物通过它启动和调节融合。在具体目标 1 中，我们使用生化和遗传学方法来剖析新发现的机制 SNARE 在体内运行的复杂质量控制，我们探索通用伴侣 Secl7 恢复融合活性的机制某些有缺陷的 trans-SNARE 复合体。在目标 2 中，我们使用新开发的光学 Rab 和 SNARE 检测可探测对接和融合的动态。在目标 3 我们使用反式 SNARE 捕获和新的 AP-3 突变体来剖析将高尔基体衍生的 AP-3 囊泡异型递送至溶酶体液泡。邮政信箱 357350 1959 NE Pacific St 西雅图, WA 93195 206.543.1660 传真 206.685.1792 bioc@u,washington.edu http。：deptsyashIngtonedu生物www；修改后的具体目标我们的目标是了解膜束缚、对接的复杂事件是如何发生的融合是在天然细胞器上执行和调节的。膜融合是其中之一细胞生物学中最基本的过程。融合和对接反应在它之前对于分泌和内吞途径的运作至关重要，脂质代谢、神经传递、营养稳态和免疫。我们以上次资助期间取得的生物和技术进步为基础循环进一步探索 SNARE 介导的对接的通用机制并对将流量引导至的特定机器有一个连贯的了解溶酶体细胞器。由于该项目的资助期限从 5 到第 4 年，并且因为第 1-4 年所请求的预算平均削减了每年 31%，我们被迫缩减具体目标。我们现在由于其费用和技术复杂性，我们消除了子目标 3C（电气来自孤立细胞器的记录），同样是由于技术复杂性的原因。两个都研究部门将这些目标确定为高风险目标，并且相对于其他目标目标，缺乏初步数据和明确的终点。修改后的目标是： 1. 确定在生活中运行的 SNARE 复杂质量控制机制细胞。我们已经获得证据表明 SNARE 复杂组件是由一个体内质量控制体系。生化和遗传策略将用于了解该质量控制体系的运作机制。我们我们还发现，通过一个明显独立的机制，普遍的 SNARE 伴侣 Secl7 (a-SNAP) 可以挽救某些有缺陷的 trans-SNARE 复合物。突变分析和生化检测将用于阐明这种新颖且意想不到的活动的潜在机制。 2. 使用光学方法探测对接动力学，SNARE-cofactor 互动、融合。我们开发了新的光学分析方法和报告基因探针对接和融合。 Rab 活性的非侵入性光学测定使我们能够在对接和融合过程中实时跟踪Rab激活状态。我们有制备的荧光 SNARE 将使我们能够同时捕获反式复杂的组装中间体并探测它们的组织。 3. 发现AP-3囊泡转运至溶酶体的分子要求液泡。在酵母菌中，从高尔基体到溶酶体液泡的直接交通需要 AP-3 货物适配器复合体。尽管几个实验室付出了巨大的努力，只有少数特定于该途径的成分是已知的。体内圈套捕获，以及卡在高尔基体上的新 AP-3 突变体，将用于识别 AP-3 途径的其他成分并了解其机制他们通过它运作。