Mechanism of autophagosome maturation and fusion with lysosomes

自噬体成熟及与溶酶体融合的机制

• 批准号: 183545120
• 负责人: Professor Dr. Christian Ungermann
• 金额: --
• 依托单位: Institut for Biomedicin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/183545120?language=en
• 关键词: Mechanism; autophagosome; maturation; fusion; lysosomes

Autophagy is an essential catabolic process to control cellular organelle and protein homeostasis. While Atg proteins have been identified and partially characterized, their interplay in the formation and closure of autophagosomes prior to autophagosome fusion with the lysosome/vacuole remains unresolved. Likewise, it remains unresolved, how autophagosomes become fusion competent (mature) and how Atg proteins may control this process.Based on multiple unpublished observations, including the initial characterization of Atg proteins and a novel autophagosome-vacuole/lysosome fusion assay, we aim to dissect key steps in the formation of fusion-competent (mature) autophagosomes using yeast as a model system. First, we will optimize our autophagosomes purification, determine their lipid- and protein composition by mass spectrometry under different starvation conditions and in different Atg knockout strains, and will analyze the ultrastructure of autophagosomes. In a second set of experiments, we will dissect the requirements of autophagosome fusion with the lysosome-like vacuoles using a novel fusion assay. We will determine the mechanism of the recruitment of the Rab7-like Ypt7 via its GEF Mon1-Ccz1, the distribution of the involved SNAREs between autophagosomes and vacuoles and their recruitment to autophagosomes. Finally, we will dissect early and late Atg proteins involved in autophagosome formation and closure. Initial assays will determine their crosstalk in solution and on membranes in the presence and absence of Atg8. Subsequent assays will take advantage of the immature autophagosomes and will determine possible in vitro maturation in the presence of missing Atg proteins. All aims will be paralleled by electron microscopy based analysis of autophagosome morphology. Where applicable, mutants will be analyzed for defects in autophagy by in vivo assays. Structural analyses of Atg proteins will complement our in vitro analyses. This mechanistic, organelle-based approach of autophagosome biogenesis will provide important new insights to understand the contribution of individual Atg proteins in this process of organelle formation and maturation.

自噬是控制细胞器和蛋白质动态平衡的重要分解代谢过程。虽然ATG蛋白已被鉴定和部分鉴定，但在自噬小体与溶酶体/空泡融合之前，它们在自噬小体形成和关闭中的相互作用仍未解决。同样，自噬小体如何变得具有融合能力(成熟)以及ATG蛋白如何控制这一过程仍未解决。基于多个未发表的观察，包括ATG蛋白质的初步特征和一种新的自噬小体-空泡/溶酶体融合实验，我们的目标是以酵母为模型系统剖析形成融合能力(成熟)自噬小体的关键步骤。首先，我们将对自噬小体的纯化进行优化，用质谱仪测定不同饥饿条件和不同ATG基因敲除菌株的自噬小体的脂和蛋白质组成，并对自噬小体的超微结构进行分析。在第二组实验中，我们将使用一种新的融合实验来剖析自噬小体与溶酶体样空泡融合的要求。我们将确定Rab7样Ypt7通过其全球环境基金Mon1-Ccz1募集的机制，所涉及的陷阱在自噬小体和空泡之间的分布以及它们募集到自噬小体。最后，我们将剖析参与自噬小体形成和关闭的早期和晚期ATG蛋白。最初的测试将确定它们在溶液中的串扰以及在存在和不存在Atg8的情况下在膜上的串扰。随后的检测将利用未成熟的自噬小体，并将确定在缺失ATG蛋白存在的情况下体外成熟的可能性。所有的AIMS都将通过基于电子显微镜的自噬小体形态分析来实现。在适用的情况下，将通过体内检测来分析突变体的自噬缺陷。ATG蛋白的结构分析将补充我们的体外分析。这种机械性的、基于细胞器的自噬小体生物发生方法将为理解单个ATG蛋白在细胞器形成和成熟过程中的作用提供重要的新见解。