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

基本信息

批准号：
10458493
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
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10458493
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 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

项目摘要

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：膜流量的主要功能是在正确的时间将蛋白质传递到正确的位置。然而，基本机制的许多方面仍然鲜为人知。对于甲板蛋白而言尤其如此适配器复合物AP-1，我们知道这对人类健康很重要，因为编码基因的突变这种复杂的亚基至少三种无关的人类疾病。我们确定了我们的两个关键差距了解AP-1功能。首先，通过ARF1的合作作用招募了体外AP-1， PI4P和货物。但是，来自保守Heatr5家族的体内蛋白对于AP-1很重要酵母，苍蝇和蠕虫中的膜关联。为什么这些蛋白质当前很重要任何系统。第二，准确的膜流量的一个重要方面是保持不同的途径分离。这对于使用电动机的流量特别重要。因为电动机可以移动整个细胞器，如果运动活动与囊泡的形成未正确协调，电动机很容易破坏许多途径。 AP-1和肌球蛋白V（MYOV）依赖性流量是在一个共同的隔间中启动的，但针对不同许多细胞的目的地。在许多情况下，已知缺乏AP-1功能会导致无意的交付 AP-1货物到MOYOV的两极分化生长地点。是什么保持这两种途径的分离在正常细胞中，在任何系统中均不清楚。为了解决这些差距，我们将使用萌芽的酵母，这允许在其他系统中不可能的水平发现新的分子机制。在第一个目标中，我们将探索我们对酵母Heatr5蛋白LAA1的分子理解的扩展。我们已经建立了监视LAA1对AP-1招聘的影响的系统，并开发了生化工具以剖析其与AP-1的相互作用和AP-1功能重要的其他因素。在第二个目标中，我们将表征一个我们认为对酵母中AP-1功能很重要的未表征蛋白质。令人惊讶的是，细胞缺乏这个蛋白质表现出先前未描述的表型 - 增强的肌球蛋白V依赖性运动性AP-1 相关细胞器。这表明AP-1依赖性和MYOV依赖性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阻止MYOV的机制 AP-1的依赖运动并导致正常流量。这些研究的成功结果将是关于细胞如何控制AP-1将正确蛋白定位在正确位置的新的一般原则这两个机制。了解这些过程是理解基本细胞的重要一步正常细胞和患病细胞中的生物学机制。