Dynamics of Endomembrane Docking and Fusion

细胞内膜对接和融合是细胞内的基本过程 生物学。它们对内分泌和内吞的运作是必不可少的。 神经传递、激素分泌、脂类代谢和 豁免权。融合事件通常由SNARE蛋白催化，在天然情况下 膜与一系列伴侣和调节蛋白一起起作用 包括小G蛋白和多亚基连接复合体。酵母液泡 是理解陷阱中介的最先进的技术系统 胞内细胞器融合。它提供了一流的活体工具，一个无与伦比的细胞- 融合的自由分析，以及完全重组的系统，允许RAB调节 核聚变。在上一个资金周期中，我们广泛描述了啤酒花，一个640 kDa的 空泡融合所需的系链复合体，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 在啤酒花和涂层复合体AP-3之间，我们开发了用于 首次允许捕获和研究明确的跨网捕 全息复合体。我们现在建议将这些进步与创新的新技术相结合 技术以及经典方法，以获得对 导致预聚变复合体组装的复杂过程和机制 这些复合体通过这些复合体启动和调节融合。在具体目标1中，我们 使用生物化学和遗传学方法剖析一种新发现的 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 某些有缺陷的反式SNARE复合体。在目标2中，我们使用了新开发的光学系统 RAB和SNARE检测用于探测对接和融合的动力学。在……里面 目的3我们利用反式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囊泡异型递送到溶酶体空泡。 信箱357350 1959东北太平洋西雅图，华盛顿州93195 206.543.1660传真206.685.1792 Bioc@u，washington.edu http.DeptsyashIngtonedu bioww； 修改后的特定目标 我们的目标是了解膜系留、对接等复杂事件 和融合在天然细胞器上执行和调节。膜融合是一种 细胞生物学中最基本的过程。聚变与对接反应 在它之前是分泌和内吞通路运行所必需的， 脂肪代谢、神经传递、营养动态平衡和免疫。我们 在前一次供资期间取得的生物和技术进步的基础上再接再厉 循环，以进一步探索陷阱介导的对接的普遍机制，并 对引导交通进入的特定机制有一个连贯的了解 溶酶体细胞器。 因为本项目申请的资金期限从5个减少到5个 年至4年，因为第1至第4年所要求的预算平均削减了 每年31%，我们不情愿地被迫缩减具体目标。我们现在 省略了原目标1(反式SNARE络合物的质谱学)，因为它 成本和技术复杂性，我们取消了子目标3C(电气 来自孤立器官的录音)，同样是出于技术复杂性的原因。两者都有 研究科将目标确定为高风险目标，相对于其他目标 目标不明确，缺乏初步数据和明确的终点。修订后的目标是： 1.确定生活中操作的陷阱复杂质量控制的机制 细胞。我们已经获得证据表明，SNARE复合体的组装是由一个 体内质量控制体系。生化和遗传策略将被用于 了解这个质量控制系统的运作机制。我们 还发现，通过一种明显分离的机制，宇宙 诱捕伴侣Secl7(a-SNAP)可以挽救某些有缺陷的反式诱捕 复合体。将使用突变分析和生化分析来澄清 这种新奇且出乎意料的活动的潜在机制。 2.使用光学方法探测对接的动力学，陷阱-辅因 互动，融合。我们已经开发了新的光学分析和记者来 探测器对接和融合。一种非侵入性的RAB活性光学分析使我们能够 在对接和融合过程中实时跟踪RAB激活状态。我们有 准备了荧光陷阱，让我们可以同时捕获反式 复杂的装配中间体，并探索它们的组织。 3.发现AP-3囊泡转运到溶酶体的分子需求 液泡。在酵母菌中，从高尔基体到溶酶体液泡的运输 需要AP-3货物适配器复合体。尽管在几个实验室中付出了巨大的努力， 只有少数几种特定于这一途径的成分是已知的。活体诱捕 捕捉到的，以及滞留在高尔基的一个新的AP-3突变体，将被用来识别 AP-3途径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.