The first crucial event in autophagy is the induction or nucleation of the membrane that will become an autophagosome. This is also the least well understood step (Tooze & Yoshimuri 2010). Following PI3P enrichment of the membrane, the associated membrane is distinct from its precursor and considered to be a new cellular structure, called a phagophore or an isolation membrane. Though the origins of this membrane are not unequivocally established, recent studies using mammalian cells indicate a strong relationship between autophagosome formation sites and the ER. The PI3P binding protein DFCP1 is localized to the ER. In starved cells it forms dot-like structures on the ER. LC3-positive membranes were observed to emerge from these structures, and named omegasomes as they resembled the Greek letter omega (Axe et al. 2008). 3D tomographic imaging of isolation membranes have shown cup-shaped isolation membranes sandwiched between two sheets of ER, connected by a narrow membrane tube (Hayashi-Nishino et al. 2009, Yla-Anttila et al. 2009) suggesting that isolation membrane formation and elongation may be guided by the adjacent ER sheets (Shibutani & Yoshimuri 2014).
ATG9 is a multi transmembrane spanning protein that may directly or indirectly participate in the formation of phagophore curvature, for example, by wedging of the membrane. It is also possible that ATG9 could function as a lipid transfer protein (Carlsson & Simonsen 2015). In nutrient rich conditions ATG9 localizes to the trans Golgi network and endosomes. Under starvation conditions it localizes to autophagosomes, in a process dependent on ULK1 (Young et al. 2006, Orsi et al. 2012). In yeast, the induction of autophagy leads to Atg9 rich Golgi derived vesicles 30 60nm in diameter (Mari et al. 2010, Yamamoto et al. 2012). These vesicles accumulate at the PAS in an Atg1 dependent manner, where Atg1 mediated phosphorylation of Atg9 facilitates the recruitment of Atg8 and Atg18 and subsequent phagophore expansion (Papinski et al. 2014). Vesicular or tubular trafficking from recycling endosomes might is thought to feed ATG9 and ATG16L1 positive membrane onto the growing phagophore in a process that appears to be regulated by the PX BAR protein SNX18 and by the RAB11 effector protein TBC1D14 in an opposite manner (Knævelsrud et al. 2013, Longatti et al. 2012, Puri et al. 2013).