International Journal of Cell Biology 5 Conventional macroautophagy Atg5/Atg7-independent autophagy ER Atg5 LC3 ? LC3 Stress for example, staurosporine resveratrol H2O2 Noncanonical autophagy 3-MA Lysosome 3-MA Rab9 LE Golgi Atg5 LC3 Peptidase-resistant peptides Figure 1: Alternative macroautophagic pathways lead to lysosomal degradation. At least four autophagic pathways can be distinguished that all show double-membrane autophagic structures and end in lysosomal degradation of cytoplasmic cargo. Conventional macroautophagy is hallmarked by the recruitment of lipidated LC3 to autophagosomal membranes that may origin from the endoplasmatic reticulum (ER). This process is dependent on Atg5 and Beclin 1 and can be inhibited by 3-methyladenine (3-MA). In contrast, the observed Atg5/Atg7- independent autophagy pathway forms Rab9-positive double-membrane vesicles derived from the trans-Golgi network and late endosomes (LE), and while it can be inhibited by 3-MA and is dependent on Beclin 1, the process is independent of Atg5 and LC3. Almost similar, the degradation of accumulated peptidase-resistant peptides is independent of Atg5 and LC3 and is also insensitive to 3-MA treatment. Finally, the noncanonical autophagy pathway induced by diﬀerent stress factors is dependent on Atg5 and LC3 and independent of Beclin 1 but cannot be impaired by 3-MA. membrane protein Atg9 [67–70]. Atg9 seems to be a key reg- ulator in regulating the formation and expansion of nascent autophagosomes. Unfortunately, the identity of proteins that partition to the autophagosomal membrane remains largely unknown. Therefore, attempting to determine the origin of the autophagosomal membrane based on the associated proteins remains a challenge [71]. Alternatively, others at- tempted to determine the source of the autophagosomal membrane by inspecting its thickness and lipid composition [15]. Several studies reported that the autophagosomal membrane can be classiﬁed as of a thin type (6–8 nm), sim- ilar to membranes of the ER and mitochondria [60, 72–75]. Furthermore, lipid structures enriched in PI3P (known as omegasomes) were formed in the vicinity of ER membranes after amino acid starvation, suggesting that these omega- somes originate from the ER [76–79]. As the omegasomes carry autophagosomal proteins like Atg5 and LC3, they may represent the source of isolated membranes required for autophagosome expansion. In contrast, in the Atg5/Atg7- independent autophagic pathway, autophagosomes with membranes of the thick type (9-10 nm) were observed, sim- ilar to membranes of lysosomes and the trans-Golgi network [49]. Intriguingly, unlike the conventional pathway the alter- native Atg5/Atg7-independent form of autophagy is blocked by brefeldin A, indicating that autophagosomes are derived from the Golgi-apparatus. Etoposide-induced Atg5/Atg7- independent autophagy is accompanied by colocalization of markers of the trans-Golgi and late endosomes (such as the mannose 6-phosphate receptor, TGN38, and Rab9) with Lamp-2-positive autolysosomes, further pointing to the requirement of the trans-Golgi or late endosomes in this alternative form of autophagy. Indeed, silencing of Rab9 or expression of a Rab9 dominant negative mutant established an essential role for Rab9 in membrane expansion from isolated membranes and led to an accumulation of isolated membranes after silencing of Rab9 but not upon inhibition of Ulk1 or Beclin 1. Since the Atg5/Atg7-independent type of alternative autophagy is activated by starvation and the stress-inducing reagent etoposide, but not by rapamycin, this suggests that a speciﬁc stimulus for induction of autophagy activates nonconventional macroautophagy with diﬀerent lipid structures compared to conventional macroautophagy. To the best of our knowledge, there is no clear data on the source of membrane for the Beclin 1-independent non- canonical autophagy pathway. So far, several sources have been proposed to provide the putative moiety of autophagosomal membranes. However, autophagosomal membranes could derive from multiple membrane sources and the origin of lipids may vary depen- dent on the cell type, the stimulus that triggers the degra- dation, and the type of cargo for autophagic destruction (proteins, aggregates or even whole organelles). As shown in Figure 1, there are now at least three alternative pathways that target cytosolic content to lysosomes, which can be discrim- inated by their dependence on Atg5 and 3-MA (Figure 1). The identiﬁcation of key players and the origin of membrane