Regulation of Autophagosome Membrane Dynamics by the Atg8 Family of Proteins

Atg8 蛋白家族对自噬体膜动力学的调节

• 批准号: 10544093
• 负责人: Thomas James Melia
• 金额: $ 49.75万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10544093
• 关键词: 3-Dimensional; Architecture; Area; Autophagocytosis; Autophagosome; Binding; Biogenesis; Cell Line; Cells; Characteristics; Complement; Cytoplasm; Cytosol; Dedications; Electrons; Encapsulated; Event; Excision; Exhibits; Family; Family member; Goals; Growth; Image; In Vitro; Individual; Invaded; Knock-out; Lipids; Lumen of the Lysosome; Lysosomes; Mediating; Membrane; Morphology; Nerve Degeneration; Nutrient; Organelles; Pathology; Pathway interactions; Peptide Hydrolases; Phase; Phosphatidylethanolamine; Phosphorylation; Phosphoserine; Physiologic pulse; Play; Process; Protein Family; Proteins; Recycling; Regulation; Resistance; Role; Site; Source; Starvation; Structure; System; Technology; Tomogram; Toxin; Ubiquitin Like Proteins; Vesicle; Yeasts; biological adaptation to stress; chronic infection; established cell line; falls; flexibility; lipid transfer protein; membrane reconstitution; microorganism; pathogen; progenitor; protein aggregation; protein expression; reconstitution; recruit; response; stressor; ultra high resolution; unilamellar vesicle

Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This “packaging” is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane coordinates the capture of cytosolic toxins with its own expansion and closure is an area of intense study. One factor implicated in both cargo-capture and autophagosome dynamics is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the preautophagosomal membrane and remains bound through the maturation process of the autophagosome. Our preliminary results suggest that Atg8-PE decorates the earliest autophagosome membrane progenitor structure and plays an integral role in organizing the accumulation of membranes that presages the eventual growth of the autophagosome itself. These activities depend upon the ability of Atg8-PE to tether individual vesicles into an underlying support. Critically, Atg8 must remain associated with the membranes throughout the growth of the autophagosome. Our results now also describe how this pool of Atg8-PE is protected from recycling proteases that otherwise function to constitutively remove Atg8 at other sites. Our discoveries are made possible by two important technological advances. First, we have developed a variety of in vitro reconstitution approaches to study how Atg8- PE and other autophagy proteins influence membrane deformation and structure. Second, we are now able to image autophagosome intermediate structures at super resolution in three dimensions and identify the same structures in micron deep electron tomograms revealing key features of the earliest Atg8-decorated membranes. With this proposal, we expect to demonstrate exactly how Atg8-PE proteins organize the proteins and membranes that support autophagosome membrane expansion.

大自噬是细胞内应激反应途径，细胞通过该途径包装部分 细胞溶质用于递送到溶酶体中。这种“包装”是通过重新形成 一种叫做自噬体的新细胞器， 溶酶体降解自噬体是如何形成的，特别是细胞膜是如何 协调细胞溶质毒素的捕获与其自身的扩张和关闭是一个强烈的领域， study.与货物捕获和自噬体动力学有关的一个因素是泛素样蛋白， 蛋白质，Atg 8.在自噬过程中，Atg 8与磷脂酰乙醇胺（PE）共价结合。 在前自噬体膜上，并在自噬体的成熟过程中保持结合。 自噬体 我们的初步结果表明，Atg 8-PE装饰最早的自噬体膜 祖细胞结构，并在组织膜的积累中起着不可或缺的作用， 预示着自噬体本身的最终生长。这些活动取决于 Atg 8-PE将单个囊泡拴系到下面的支持物中。至关重要的是，Atg 8必须与 在自噬体的整个生长过程中与细胞膜相连。我们的结果现在也描述了 该Atg 8-PE库被保护免于再循环蛋白酶，否则所述蛋白酶的功能是组成性地 删除其他站点的Atg 8。我们的发现是由两个重要的技术 预付款。首先，我们已经开发了多种体外重建方法来研究Atg 8- PE和其他自噬蛋白影响膜的变形和结构。第二，我们现在 能够以超分辨率三维成像自噬体中间结构， 在微米深的电子断层扫描图中识别出相同的结构， Atg 8修饰的膜。 有了这个提议，我们希望确切地展示Atg 8-PE蛋白如何组织蛋白质， 支持自噬体膜扩张的细胞膜。