Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells

Autophagy is mediated by a unique organelle, the autophagosome. Autophagosome formation involves a number of autophagy-related (ATG) proteins and complicated membrane dynamics. Although the hierarchical relationships of ATG proteins have been investigated, how individual ATG proteins or their complexes contribute to the organization of the autophagic membrane remains largely unknown. Here, systematic ultrastructural analysis of mouse embryonic fibroblasts (MEFs) and HeLa cells deficient in various ATG proteins reveals that the emergence of the isolation membrane (phagophore) requires FIP200 (also known as RB1CC1), ATG9A and phosphatidylinositol (PtdIns) 3-kinase activity. By contrast, small premature isolation-membrane-like and autophagosome-like structures were generated in cells lacking VMP1 and both ATG2A and ATG2B, respectively. The isolation membranes could elongate in cells lacking ATG5, but did not mature into autophagosomes. We also found that ferritin clusters accumulated at the autophagosome formation site together with p62 (also known as SQSTM1) in autophagy-deficient cells. These results reveal the specific functions of these representative ATG proteins in autophagic membrane organization and ATG-independent recruitment of ferritin to the site of autophagosome formation.