Dynamics of Endomembrane Docking and Fusion

细胞内膜对接和融合是细胞生物学的基本过程 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 免疫力融合事件通常由SNARE蛋白催化， 膜，与一系列伴侣蛋白和调节蛋白一起起作用 包括小G蛋白和多亚基拴系复合物。酵母泡 是了解SNARE介导的 细胞内细胞器的融合。它提供了极好的体内工具，一个无与伦比的细胞- 融合的自由测定，以及允许Rab-regulated 核聚变在上一个融资周期中，我们广泛描述了HOPS，一个640 kDa的 我们描绘了新的机制， 控制液泡Rab蛋白Ypt 7的活性，我们研究了它们之间的相互作用。 HOPS和外套复合物AP-3之间的联系，我们开发了一种方法， 首次允许捕获和研究明确的trans-SNARE 全复合物我们现在建议将这些进步与创新的新技术相结合， 技术以及经典的方法，以获得一个综合的看法， 复杂的工艺导致预融合复杂的组装， 这些复合物通过其启动和调节融合。具体目标1， 利用生物化学和遗传学方法来剖析一种新发现的 SNARE复杂的质量控制，在体内运作，我们探讨了 通用伴侣Secl 7恢复融合活性的机制， 某些有缺陷的反式陷阱复合物。在目标2中，我们使用新开发的光学 Rab和SNARE功能的测定以探测对接和融合的动力学。在 目的3我们使用反式SNARE捕获和一个新的AP-3突变体来解剖 高尔基体衍生的AP-3囊泡向溶酶体空泡的异型递送。 美国华盛顿州西雅图1959 NE Pacific St，邮编93195 206.543.1660传真：206.685.1792 bioc@u，washington.edu http.：deptsyashIngtonedu biowwww; 修改后的具体目标 我们的目标是了解膜系留、对接等复杂事件 和融合是在天然细胞器上执行和调节的。膜融合是一种 细胞生物学中最基本的过程。聚变和对接反应 在其之前的蛋白质对于分泌和内吞途径的运作是必需的， 脂质代谢、神经传递、营养平衡和免疫。我们 在以前资助期间取得的生物和技术进步的基础上， 循环，以进一步探索SNARE介导的对接的通用机制， 获得对引导交通进入的特定机制的连贯理解 溶酶体细胞器 由于该项目申请的供资期限从5年减少到10年， 由于第1-4年的预算要求平均削减了 每年31%，我们不得不被迫缩减具体目标。我们现在 省略了最初的目标1（反式陷阱复合物的质谱分析），因为其 成本和技术复杂性，我们消除了子目标3C（电气 从分离的细胞器的记录），再次由于技术复杂性的原因。两 研究科将目标确定为高风险， 缺乏初步数据和明确的终点。修改后的目标是： 1.识别在生活中运作的SNARE复杂质量控制机制 细胞我们已经获得的证据表明，SNARE复杂的组装是由一个 体内质量控制系统。生物化学和遗传学策略将被用于 了解质量控制体系的运作机制。我们 他们还发现，通过一个明显独立的机制， SNARE分子伴侣Sec 17（a-SNAP）可以拯救某些缺陷的反式SNARE 配合物将使用突变分析和生化测定来阐明 这一新的和意想不到的活动的潜在机制。 2.使用光学方法探测对接的动力学，SNARE-辅因子 互动和融合。我们已经开发了新的光学检测和报告， 探测器对接和聚变。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真实的实时跟踪Rab激活状态。我们有 制备荧光陷阱，使我们能够同时捕获反式 复杂的组装中间体和探测它们的组织。 3.发现AP-3囊泡运输到溶酶体的分子要求 空泡在酵母属中，从高尔基体到溶酶体空泡的直接运输 需要AP-3货物适配器尽管几个实验室付出了巨大的努力， 只有少数对该途径特异的组分是已知的。体内圈套 捕获，和一个新的AP-3突变体，是停留在高尔基体，将用于识别 AP-3通路的其他成分，并了解其机制 他们通过它来运作。

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.