Characterizing new proteins that determine AP-1 recruitment and distribution

表征决定 AP-1 募集和分布的新蛋白质

• 批准号: 10004142
• 负责人: Mara C Duncan
• 金额: $ 30.1万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004142
• 关键词: Address; Ankyrins; Binding; Biochemical; Bioinformatics; Biological Models; Blood Coagulation Factor; Capsid Proteins; Cell Adhesion; Cell Adhesion Molecules; Cell Polarity; Cell model; Cells; Cellular biology; Clathrin Adaptors; Complex; Data; Defect; Destinations; Disease; Ensure; Exhibits; Family; Fission Yeast; Foundations; Functional disorder; Genes; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Health; Human; Immune System Diseases; In Vitro; Investigation; Link; Location; Mediating; Membrane; Membrane Protein Traffic; Molecular; Monitor; Motor; Motor Activity; Movement; Mutation; Myocardial Infarction; Myosin ATPase; Myosin Type V; Nematoda; Neurodegenerative Disorders; Normal Cell; Organelles; Outcome Study; Pathway interactions; Phenotype; Phosphatidylinositols; Physiological; Play; Point Mutation; Positioning Attribute; Process; Protein Family; Protein Sortings; Proteins; Role; Saccharomycetales; Secretory Vesicles; Signal Transduction; Signaling Molecule; Site; Sorting - Cell Movement; Stroke; Structure; System; Testing; Time; To specify; Transcription Factor AP-1; Vesicle; Work; Yeasts; afadin; base; biochemical tools; cell motility; cofactor; fly; human disease; in vivo; inositol 4-phosphate; insight; mimicry; novel; polarized cell; prevent; public health relevance; recruit; trans-Golgi Network

Project summary/Abstract: A main function of membrane traffic is to deliver proteins to the correct locations at the correct time. However, many facets of the underlying mechanisms remain poorly understood. This is particularly true for the clathrin adaptor complex AP-1, which we know is important for human health because mutations in genes that encode subunits of this complex cause at least three unrelated human diseases. We identified two key gaps in our understanding of AP-1 function. First, in vitro AP-1 is recruited to membrane by the cooperative action of Arf1, PI4P and cargo. However, in vivo proteins from the conserved HEATR5 family are important for AP-1 membrane association in yeast, flies, and worms. Why these proteins are important is currently unexplored in any system. Second, an important aspect of accurate membrane traffic is keeping the different pathways separate. This is particular important for traffic that uses motors. Because motors can move entire organelles, if motor activity is not correctly coordinated with vesicle formation, a motor could easily disrupt many pathways. AP-1 and Myosin V (MyoV) dependent traffic are initiated at a common compartment, but directed to different destination in many cells. In many cases, the absence of AP-1 function is known to cause inadvertent delivery of AP-1 cargo to the sites of polarized growth by MyoV. What maintains the separation of these two pathways in normal cells is unknown in any system. To address these gaps, we will use budding yeast, which allows the discovery of novel molecular mechanisms at a level not possible in other systems. In the first aim, we will explore the extend our molecular understanding of the yeast HEATR5 protein, Laa1. We have established a system to monitor the effects Laa1 on AP-1 recruitment and have developed biochemical tools to dissect its interactions with AP-1 and other factors important of AP-1 function. In the second aim, we will characterize an uncharacterized protein that we identified as important for AP-1 function in yeast. Strikingly, cells lacking this protein exhibit a previously undescribed phenotype-the enhanced Myosin V dependent motility of AP-1 associated organelles. This suggests that AP-1 dependent and MyoV-dependent traffic are not accurately separated in these cells. In Aim2, we will define the mechanism by which this new protein prevents MyoV dependent movement of AP-1 and contributes to normal traffic. The successful outcome of these studies will be new general principles about of how the cell controls AP-1 to position the right protein in the right place via these two mechanisms. Understanding these processes is an essential step toward understanding basic cell biology mechanisms in normal and diseased cells.

项目概要/摘要： 膜运输的主要功能是在正确的时间将蛋白质递送到正确的位置。然而， 其根本机制的许多方面仍然知之甚少。对于网格蛋白来说尤其如此 接头复合物 AP-1，我们知道它对人类健康很重要，因为编码基因的突变 该复合物的亚基会导致至少三种不相关的人类疾病。我们发现了我们的两个关键差距 了解 AP-1 功能。首先，体外AP-1通过Arf1的协同作用被招募到膜上， PI4P和货物。然而，来自保守 HEATR5 家族的体内蛋白质对于 AP-1 很重要 酵母、果蝇和蠕虫中的膜结合。为什么这些蛋白质如此重要目前尚未被探索 任何系统。其次，准确的膜流量的一个重要方面是保持不同的路径 分离。这对于使用电机的交通尤其重要。因为马达可以移动整个细胞器，如果 由于运动活动与囊泡形成没有正确协调，运动很容易破坏许多通路。 AP-1 和肌球蛋白 V (MyoV) 相关流量在公共隔间启动，但定向到不同的 许多细胞中的目的地。在许多情况下，AP-1 功能的缺失会导致意外分娩 AP-1 货物通过 MyoV 到达极化生长位点。是什么维持了这两条路径的分离 在正常细胞中，在任何系统中都是未知的。为了解决这些差距，我们将使用芽殖酵母，它可以 在其他系统中不可能达到的水平上发现新的分子机制。在第一个目标中，我们将 探索扩展我们对酵母 HEATR5 蛋白 Laa1 的分子理解。我们已经建立了一个 系统监测 Laa1 对 AP-1 招募的影响，并开发了生化工具来剖析其 与 AP-1 和其他对 AP-1 功能重要的因素的相互作用。在第二个目标中，我们将描述 我们确定对酵母中 AP-1 功能很重要的未表征蛋白质。引人注目的是，细胞缺乏这种 蛋白质表现出先前未描述的表型 - AP-1 增强的肌球蛋白 V 依赖性运动 相关细胞器。这表明 AP-1 相关和 MyoV 相关流量并不准确 分离在这些细胞中。在 Aim2 中，我们将定义这种新蛋白预防 MyoV 的机制 AP-1 的相关移动并有助于正常流量。这些研究的成功结果将是 关于细胞如何控制 AP-1 将正确的蛋白质定位在正确的位置的新一般原理 这两种机制。了解这些过程是了解基本细胞的重要一步 正常和患病细胞的生物学机制。