Mechanism of autophagosome maturation and fusion with lysosomes

自噬是控制细胞器和蛋白质稳态的重要分解代谢过程。虽然Atg蛋白已被鉴定并部分表征，但在自噬体与溶酶体/液泡融合之前，它们在自噬体形成和关闭中的相互作用仍未得到解决。同样，自噬体如何成为融合能力（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结果，包括Atg蛋白的初步表征和一种新的自噬体-液泡/溶酶体融合实验，我们的目标是以酵母为模型系统，解剖融合能力（成熟）自噬体形成的关键步骤。首先，我们将优化我们的自噬体纯化，用质谱法测定不同饥饿条件下和不同Atg敲除菌株下自噬体的脂质和蛋白质组成，并分析自噬体的超微结构。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体与溶酶体样液泡融合的要求。我们将通过其GEF Mon1-Ccz1来确定rab7样Ypt7的募集机制，相关SNAREs在自噬体和空泡之间的分布以及它们向自噬体的募集。最后，我们将剖析参与自噬体形成和关闭的早期和晚期Atg蛋白。初始分析将确定它们在存在和不存在at8的情况下在溶液和膜上的串扰。随后的检测将利用未成熟的自噬体，并确定在缺失Atg蛋白的情况下体外成熟的可能性。所有目的都将通过基于自噬体形态的电子显微镜分析来实现。在适用的情况下，突变体将通过体内实验分析自噬缺陷。Atg蛋白的结构分析将补充我们的体外分析。这种机制的、基于细胞器的自噬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。

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how autophagosomes become fusion competent (mature) and how Atg proteins may control this process.Based on multiple unpublished observations, including the initial characterization of Atg proteins and a novel autophagosome-vacuole/lysosome fusion assay, we aim to dissect key steps in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.