Mechanism of autophagosome maturation and fusion with lysosomes

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how autophagosomes become fusion competent (mature) and how Atg proteins may control this process.Based on multiple unpublished observations, including the initial characterization of Atg proteins and a novel autophagosome-vacuole/lysosome fusion assay, we aim to dissect key steps in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