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: 10226217
• 负责人: Alexey Jarrell Merz
• 金额: $ 46.65万
• 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/10226217
• 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.

项目摘要 分泌途径折叠，组装和分类膜和分泌蛋白，并将它们靶向到细胞膜上。 细胞内外的各种位置。细胞的“大中央站”是高尔基体网络 (TGN)这是一个令人难以置信的动态和复杂的结构，将货物包装到单独的集装箱中，并将其发送到终端目的地。AP-3运输复合物从核内体或 高尔基体进入为内溶酶体隔室绑定的容器，绕过内体多泡体 和ESCRT途径。AP-3是HIV和其他病毒衣壳的生产性组装所必需的。遗传损伤 损害AP-3功能引起多种疾病，包括2型Hermansky-Pudlak综合征和癫痫样综合征。尽管其重要性，但对AP-3功能的机制知之甚少。 在这个合作项目中，我们利用已发表和未发表的遗传学、细胞生物学和生物化学进展，并采用最先进的成像方法，来剖析AP-3出芽的机制和细胞凋亡的机制。 在模式生物酵母中，AP-3功能对TGN整体动力学的影响 啤酒。在目标1中，我们建立在蛋白质组学调查、功能基因组学调查和随访分析的基础上， 鉴定新候选AP-3辅因子，包括泛素连接酶。我们将利用这些进展来检验关于AP-3囊泡从TGN的组装、成熟和调度的假设。在目标2中， 初步研究结果表明AP-3活性的破坏导致高尔基体结构的显著和意想不到的全局扰动。根据已发表和未发表的数据，我们假设不同的携带者 以编程的顺序方式退出TGN，AP-3载波提前离开。我们将进一步对此进行测试 模型和剖析的分子机制，通过该序列的事件是编程。在一起， 这些研究应该建立AP-3囊泡形成和高尔基体-内溶酶体系统中细胞器成熟动力学的新范例。