Biogenesis, maturation and fusion of autophagosomes

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Autophagy is of central importance in the catabolism and quality control of cells, which can be tuned to the metabolic needs of the cells. This becomes particularly obvious during nitrogen starvation, when cells sequester part of their cytosol and its organelles into newly formed autophagosomes. Autophagosome form de novo, starting with the fusion of small vesicles, which grow by lipid transfer into a double membrane structure, followed by closure and finally fusion with the vacuole. Within this project, we will focus on two main aspects of this process, using yeast as model organism: (i) How is the coordinated formation of the phagophore achieved (by dissecting the interplay of the key proteins Atg2, Atg18 and Atg9)? This will be addressed in Aim 1. (ii) How do the lipid phosphatase Ymr1 and the Mon1-Ccz1 guanine nucleotide exchange factor (GEF) as a Rab7/Ypt7 activator control steps in autophagosome maturation and membrane fusion? This will be the focus of Aim 2. In Aim 1, we will analyze how lipid transfer to the growing phagophore is mediated and regulated. Based on our previous work on the in vitro reconstitution of the Atg2-Atg18 mediated lipid transfer and the Atg9-dependent lipid scrambling, we will combine structural approaches by cryo-electron microscopy with functional analyses of lipid transfer and scrambling to understand the crosstalk of these three proteins and the role of PI3P. This will include the comparative analysis of Atg2 with the Atg2-Atg18 complex, and the detailed dissection of the Atg2-Atg9 interface to identify critical residues involved in lipid gating. We postulate that the Atg2 binding controls the ability of Atg9 to accept and scramble lipids, thus promoting a continuous and efficient transfer of lipids. We will further ask, how the kinase Atg1 controls this process by identifying phosphosites and their role in controlling the proteins’ activity. All relevant sites will be analyzed by in vivo assays with respective mutants to validate the in vitro findings. In Aim 2, we will ask how distinct key proteins promote autophagosome maturation and fusion. At the center of our analysis will be a) the control of the PI3P content by the lipid phosphatase Ymr1, and b) the mechanism of Mon1-Ccz1 complex activation and its crosstalk with Atg8 to decorate autophagosomes with Ypt7. For both subprojects, purified proteins and assays have been established to monitor the respective ligands. We will determine in particular the role of the Atg1 kinase complex in the control of Ymr1 and Mon1-Ccz1. Any point mutant, derived from a phosphoproteomic screen, controlling their activity will be validated by in vivo autophagy assays.