Dissecting the molecular functions of the ubiquitin-like Atg8 during autophagosome biogenesis in S. cerevisiae

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Autophagy, a highly conserved transport pathway, delivers cytosolic material or even whole organelles to the vacuole for degradation upon nutrient limitation. First, double membrane-layered vesicles, the so-called autophagosomes, are formed out of the preautophagosomal structure (PAS). They finally fuse with the vacuole and release a still one membrane-layered vesicle, the autophagic body, into the vacuolar lumen for breakdown and reuse of its constituents. The source of the membrane required for autophagosome biogenesis is one of the probing questions in the autophagy field. In S. cerevisiae, vesicles containing the transmembrane protein Atg9 deliver only around 2% of the required membrane surface from the non-PAS pool to the PAS. Thus additional lipid sources must be still unknown. The ubiquitin-like protein Atg8 is one of the key components of autophagy and required for autophagosome biogenesis. Similar to ubiquitin Atg8 is covalently attached to phosphatidylethanolamine and thus to membranes. The Atg8 conjugation is unique in that it catalyzes conjugation to a lipid, not to a protein. Preliminary unpublished own data pointed to the existence of a non-PAS pool of Atg8. The first aim of this proposal is the detailed analyses of this Atg8 non-PAS pool and its potential role in Atg8-mediated vesicular membrane transport to the PAS. The second aim of this proposal focuses on the function of Atg8 in membrane fusions required during autophagosome biogenesis. Atg8 consists of a N-terminal helical domain (NHD) and a ubiquitin-like domain (UBL). It differs from ubiquitin mostly by this NHD that is crucial for its function. Own published data has brought the NHD in connection with membrane fusion during autophagosome biogenesis. Thus especially the role of the Atg8-NHD will be analyzed. Elucidating the elongation and closure mechanism would lead to substantial progress in understanding autophagosome biogenesis, another challenge for the autophagic field.The data on Atg8 indicate that several Atg8 sub-complexes are required for spatially and temporally different steps of autophagosome biogenesis. The isolation of Atg8 complexes using standard methods would not allow to assign newly identified components to one of the co-existing complexes. Therefore, the different sub-complexes should be separated by native electrophoresis (blue native PAGE) to further analyze the spatial and temporal differences in complex composition. This approach should allow the suggestion of a first model for sub-complex conversion over time and space.