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: 10312028
• 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/10312028
• 关键词: 3-Dimensional; Architecture; Area; Autophagocytosis; Autophagosome; Binding; Biogenesis; Cell Line; Cells; Characteristics; Complement; Cytosol; 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; Resolution; Role; Seeds; Site; Source; Starvation; Structure; System; Tomogram; Toxin; Ubiquitin Like Proteins; Vesicle; Yeasts; biological adaptation to stress; chronic infection; delivery vehicle; established cell line; falls; flexibility; microorganism; pathogen; progenitor; protein aggregation; reconstitution; recruit; response; stressor; unilamellar vesicle; 3-Dimensional; Architecture; Area; Autophagocytosis; Autophagosome; Binding; Biogenesis; Cell Line; Cells; Characteristics; Complement; Cytosol; 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; Resolution; Role; Seeds; Site; Source; Starvation; Structure; System; Tomogram; Toxin; Ubiquitin Like Proteins; Vesicle; Yeasts; biological adaptation to stress; chronic infection; delivery vehicle; established cell line; falls; flexibility; microorganism; pathogen; progenitor; protein aggregation; reconstitution; recruit; response; stressor; 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.

宏大学是细胞内应力响应途径，细胞包装部分的部分 用于输送到溶酶体的细胞质。这种“包装”是由从头形成进行的 一种称为自噬体的新细胞器，它生长并封装了胞质材料以最终 溶酶体降解。自噬体如何形成，尤其包括膜如何形成 协调捕获胞质毒素的膨胀和闭合是一个强烈的区域 学习。在货物捕获和自噬体动力学中实施的一个因素是类似泛素的因素 蛋白质，ATG8。在自噬期间，ATG8与磷脂酰乙醇胺（PE）共价结合 在牙科膜膜上，并通过成熟过程保持 自噬体。 我们的初步结果表明，ATG8-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 预示自噬体本身的事件增长。这些活动取决于 ATG8-PE将单个蔬菜绑在基础支持中。至关重要的是，ATG8必须保持关联 在自噬体的整个生长过程中，膜的膜。我们现在的结果也描述了如何 这种ATG8-PE池受到保护，免受回收保护酶，否则将其发挥作用 在其他站点删除ATG8。两个重要的技术使我们的发现成为可能 进步。首先，我们开发了各种体外重构方法来研究ATG8- PE和其他自噬蛋白会影响膜变形和结构。第二，我们现在是 能够在三个维度上以超级分辨率在超级分辨率上成像自噬体中间结构 在微米深电子断层图中识别相同的结构，揭示了最早的关键特征 ATG8装饰的膜。 通过此建议，我们希望准确地证明ATG8-PE蛋白如何组织蛋白质和 支持自噬体膜扩展的机制。