MECHANISMS OF AP-3 FUNCTION IN VESICLE FORMATION AND GOLGI MATURATION

AP-3 在囊泡形成和高尔基体成熟中的功能机制

• 批准号: 10017291
• 负责人: Alexey Jarrell Merz
• 金额: $ 47万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-12 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017291
• 关键词: Animal Model; Architecture; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biological; Biological Models; Biological Process; Bypass; Capsid; Cell physiology; Cells; Coated vesicle; Communicable Diseases; Complex; Data; Defect; Destinations; Disease; Early Endosome; Electron Microscopy; Endocytosis; Endosomes; Epilepsy; Eukaryotic Cell; Event; Fluorescence Microscopy; Foundations; Functional disorder; Genetic; Genetic Diseases; Goals; Golgi Apparatus; HIV; HIV-1; Hermanski-Pudlak Syndrome; Human; Impairment; Lesion; Link; Lipids; Location; Lysosomes; Membrane; Microscopic; Mission; Modeling; Molecular; Morphology; Multivesicular Body; National Institute of General Medical Sciences; Organelles; Outcome; Pathway interactions; Pharmacology; Play; Prevention; Process; Proteins; Proteomics; Public Health; Publishing; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Site; Sorting - Cell Movement; Structure; Surveys; Syndrome; System; TFAP2A gene; Testing; Transcription Factor AP-1; Ubiquitination; Vacuole; Vesicle; Viral; Virus Assembly; Work; Yeasts; base; cofactor; disease diagnosis; follow-up; functional genomics; genetic approach; imaging approach; innovation; late endosome; novel; prevent; programs; spatiotemporal; trafficking; trans-Golgi Network; ubiquitin ligase; ubiquitin-protein ligase; vesicle transport; yeast genetics

PROJECT SUMMARY The secretory pathway folds, assembles, and sorts membrane and secreted proteins and targets them to a variety of locations inside and outside the cell. The `Grand Central Station' of the cell is the trans Golgi network (TGN), an incredibly dynamic and complex structure that packages cargo into separate containers and dispatches them to their terminal destinations. The AP-3 trafficking complex sorts cargo from endosomes or the Golgi into containers bound for endolysosomal compartments, bypassing the endosomal multivesicular body and ESCRT pathway. AP-3 is needed for productive assembly of HIV and other viral capsids. Genetic lesions impairing AP-3 function cause multiple diseases, including Hermansky-Pudlak Syndrome type 2 and an epilepsy-like syndrome. Despite its importance, remarkably little is known about the mechanisms of AP-3 function. In this collaborative Project, we exploit published and unpublished genetic, cell biological, and biochemical advances, and employ state of the art imaging approaches, to dissect the mechanisms of AP-3 budding and the consequences of AP-3 function for the overall dynamics of the TGN, in the model organism Saccharomyces cerevisiae. In Aim 1, we build on proteomic surveys, functional genomic surveys, and follow-up analyses, wich identify new candidate AP-3 cofactors including a ubiquitin ligase. We will use these advances to test hypotheses about the assembly, maturation, and dispatch of AP-3 vesicles from the TGN. In Aim 2, we build on preliminary findings showing that disruption of AP-3 activity causes striking and unexpected global perturbations of Golgi architecture. We hypothesize, based on published and unpublished data, that distinct carriers exit the TGN in a programmed, sequential manner, with AP-3 carriers departing early. We will further test this model and dissect the molecular mechanisms though which the sequence of events is programmed. Together, these studies should establish new paradigms for AP-3 vesicle formation and for organelle maturation dynamics in the Golgi-endolysosomal system.

项目总结 分泌途径折叠、组装和分类膜和分泌的蛋白质，并以它们为靶点。 牢房内外的各种位置。细胞的“中央车站”是跨高尔基网络 (TGN)，这是一种令人难以置信的动态和复杂的结构，将货物包装在不同的集装箱中，并将它们发送到各自的码头目的地。AP-3运输复合体将货物从内体或 高尔基体进入容器前往内溶体隔室，绕过内体多囊体小体 和ESCRT途径。AP-3是艾滋病毒和其他病毒衣壳的有效组装所必需的。遗传性损害 AP-3功能受损会导致多种疾病，包括Hermansky-Pudlak综合征2型和癫痫样综合征。尽管AP-3很重要，但人们对AP-3的作用机制知之甚少。 在这个合作项目中，我们利用已发表和未发表的遗传学、细胞生物学和生化进展，并使用最先进的成像方法，剖析AP-3发芽和 模式生物酵母菌中AP-3功能对TGN整体动力学的影响 酿酒。在目标1中，我们建立在蛋白质组调查、功能基因组调查和后续分析的基础上， 确定新的候选AP-3辅因子，包括泛素连接酶。我们将使用这些进展来测试关于从TGN组装、成熟和发送AP-3囊泡的假设。在《目标2》中，我们建立在 初步研究结果表明，AP-3活动的中断会导致高尔基体结构的显著和意想不到的全球扰动。我们根据已公布和未公布的数据推测，不同的运营商 以程序、顺序的方式离开TGN，AP-3航母提前离开。我们将进一步测试这一点 模拟和剖析分子机制，通过这些机制对事件序列进行编程。一起， 这些研究将为AP-3囊泡的形成和高尔基体-内溶酶体系统中细胞器成熟动力学建立新的范式。