Dissection of autophagosome formation using Apg5-deficient mouse embryonicstem cells

In macroautophagy, cytoplasmic components are delivered to lysosomes for de gradation via autophagosomes that are formed by closure of cup-shaped isola tion membranes. However, how the isolation membranes are formed is poorly u nderstood. We recently found in yeast that a novel ubiquitin-like system, t he Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mou se embryonic stem cells. Using green fluorescent protein-tagged Apg5, we re vealed that the cup-shaped isolation membrane is developed from a small cre scent-shaped compartment. Apg5 localizes on the isolation membrane througho ut its elongation process. To examine the role of Apg5, we generated Apg5-d eficient embryonic stem cells, which showed defects in autophagosome format ion. The covalent modification of Apg5 with Apg12 is not required for its m embrane targeting, but is essential for involvement of Apg5 in elongation o f the isolation membranes. We also show that Apg12-Apg5 is required for tar geting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.