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 and HeLa cells deficient in various ATG proteins revealed that the emergence of the isolation membrane (phagophore) requires FIP200/RB1CC1, ATG9A, and PtdIns 3-kinase activity. By contrast, small premature isolation membrane- and autophagosome-like structures were generated in cells lacking VMP1 and ATG2A/B, respectively. The isolation membranes could elongate in cells lacking ATG5, but these did not mature into autophagosomes. We also found that ferritin clusters accumulated at the autophagosome formation site together with p62/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 autophagosome formation site.