4 Alternative macroautophagic pathways Beclin 1-independent Atg5/Atg7- independent Degradation of peptidase-resistant peptides Atg5 Atg7 Ulk1/2 LC3 Beclin 1 Vps34 Ulk1 Fip200 LC3 (but not essential) Beclin 1 (Vps34) Atg5 Atg7 Atg9 Atg12 Atg16 LC3 Atg5 WIPI-1 p62 Tsg101 Vps24 Table 1: Types of alternative macroautophagic pathways. Macroautopagic molecules involved Macroautopagic molecules not involved Cell type Reference Induction Resveratrol Staurosporine Etoposide MK801 H2O2 MPP+ As2O3 Etoposide Staurosporine Starvation Resistance against cytoplasmic peptidases International Journal of Cell Biology MCF-7 (breast cancer cells) primary cortical neurons RAW 264.7 (macrophage cells) SH-SY5Y (neuroblastoma cells) primary dopaminergic neurons ovarian cells Atg5-/- MEF Atg7-/- MEF wt MEF HeLa Atg5-/- MEF wt MEF [50] [51] [52] [53] [55] [49] [43] protein degradation via this pathway was inhibited by the PI3K inhibitor 3-MA. Furthermore, silencing of components of the Ulk1 complex, a mammalian serine/threonine protein kinase that plays a key role in the initial stages of autophagy, decreased autophagic vacuoles, suggesting that the Ulk1 complex is needed for Atg5/Atg7-independent autophagy [49]. Apoptosis-induced stress, for example, by staurosporine, resveratrol, or H2O2 can also induce the so-called non- canonical autophagy pathway, where autophagosomes can be formed independent of Beclin 1 or Vps34 and with an insensitivity to 3-MA [50–52]. However, this speciﬁc pathway still requires Atg7-activity for LC3-I lipidation and is, therefore, diﬀerent from the Atg5/Atg7-independent pathway described above [49]. Furthermore, Scarlatti et al. have shown that resveratrol inhibits the mTOR activation by a direct inhibitory eﬀect on the upstream class 1A PI3K [50]. Similarly, a Beclin 1-independent pathway has been reported in neuronal cells treated with the neurotoxin 1-methyl-4- phenylpyridinium (MPP+) [53] and in other cellular systems in response to various drugs [54, 55]. These studies have shown that several agents stimulate autophagic cell death through Beclin 1 in canonical autophagy pathways [56]. Recently, evidence emerged that autophagy and cell death are induced independent of Beclin 1 and Vps34. In breast cancer cells, resveratrol induces autophagic cell death in a Beclin 1-independent manner [50]. Silencing of Atg7 impairs the cellular death elicited by resveratrol. In dopaminergic neuronal cells, the neutotoxin MPP+ induces Beclin 1- independent autophagy and cell death [53]. As most studies on the noncanonical pathway used compounds to induce cell death, it is tempting to link the noncanonical autophagy pathway to a death execution mechanism or cell survival. However, it has also been suggested that the independency of the noncanonical autophagy pathway may provide an adaptation to loss of Beclin 1, for example, in various tumors where Beclin 1 is deleted, in immune cell development, and may even be an evolutionary way to circumvent inhibition of Beclin 1 by various viruses in order to prevent autophagy [57–59]. None of these alternative autophagy pathways seem to correspond to the traﬃcking we observed for the peptidase- resistant peptides, as the Atg5/Atg7-independent pathway is still 3-MA sensitive (in contrast to the peptide targeted to lysosomes), while the noncanonical pathway (Beclin 1- independent) is 3-MA insensitive but still depends on LC3. Thus, lysosomal degradation of peptidase-resistant peptides and proteins, as we and others have demonstrated [35, 41– 45], deﬁnes a novel authophagy route independent of known regulators of the constitutive macroautophagic pathway like Beclin 1, Atg5 or LC3. A better understanding of the role of these alternative autophagic pathways and their molecular regulators raise to two crucial questions: (1) What is the origin of the autophagic membrane in the diﬀerent autophagic routes, and (2) Which stimuli trigger the diﬀerent autophagic pathways? In mammalian macroautophagy, various sources for the origin of the autophagosome membrane have been proposed including the ER, the Golgi complex, the plasma membrane, and the mitochondria [17, 60–66]. Alternatively, de novo synthesis of a nucleating structure, the phagophore, is proposed to elongate by the addition of lipids via the integral