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.

项目摘要/摘要： 膜运输的一个主要功能是在正确的时间将蛋白质运送到正确的位置。然而， 对潜在机制的许多方面仍然知之甚少。这一点对于网壳蛋白来说尤其如此。 接头复合体AP-1，我们知道它对人类健康很重要，因为编码 这种复合体的亚单位至少会导致三种无关的人类疾病。我们发现了以下两个关键差距 了解AP-1的功能。首先，在体外，AP-1通过Arf1的协同作用被募集到膜上， PI4P和货物。然而，来自保守的HEATR5家族的活体蛋白对AP-1很重要 酵母、苍蝇和蠕虫中的膜缔合。为什么这些蛋白质是重要的，目前还没有被探索 任何系统。其次，准确的膜交通的一个重要方面是保持不同的路径 分开。对于使用汽车的交通来说，这一点尤其重要。因为马达可以移动整个细胞器，如果 运动活动与囊泡的形成没有正确地协调，运动很容易扰乱许多通路。 AP-1和肌球蛋白V(MyoV)依赖的流量在共同的隔间启动，但指向不同的隔间 多个单元格中的目的地。在许多情况下，AP-1功能的缺失已知会导致意外分娩 通过MyoV将AP-1货物转移到极化生长部位。是什么维持了这两条路径的分离 在任何系统中都是未知的。为了弥补这些差距，我们将使用萌芽酵母，这使得 在其他系统中不可能的水平上发现新的分子机制。在第一个目标中，我们将 探索扩大我们对酵母HEATR5蛋白Laa1的分子理解。我们已经建立了一个 监测Laa1对AP-1招募的影响的系统，并开发了生化工具来剖析其 与AP-1的相互作用以及AP-1功能的其他重要因素。在第二个目标中，我们将描述一个 我们发现在酵母中对AP-1功能起重要作用的未知蛋白质。令人惊讶的是，缺乏这一点的细胞 蛋白质表现出一种以前未被描述的表型--AP-1依赖肌球蛋白V的运动性增强 伴生细胞器。这表明依赖于AP-1和依赖于MyoV的流量不准确 在这些细胞中分离。在AIM2中，我们将定义这种新蛋白预防MyoV的机制 AP-1的依赖移动，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的一般原理 这两种机制。了解这些过程是了解基本细胞的关键一步 正常细胞和疾病细胞的生物学机制。