The traditional view is that autophagosome formation starts at phagophore assembly sites (PAS). This concept is derived from studies in yeast which observed that a number of the key proteins involved in autophagosome formation colocalise at a single site in the cell. This operational definition led to statements in the literature that describe the autophagosome formation as an event that occurred ‘de novo’. In mammalian cells, there are multiple PAS at any one time. The formation of phagophores requires the class III phosphoinositide 3-kinase (PI3K) Vps34, which acts in a large macromolecular complex, along with Beclin-1 (mammalian Atg6), Atg14 and Vps15 (previously known as p150), to form PI3-phosphate (PI(3)P). The activity of this complex is dependent on upstream autophagy regulators, including the mammalian Atg1 orthologues ULK1 and ULK2, Atg13 and focal adhesion kinase (FAK)-family interacting protein of 200 kDa (FIP200).
The elongation of membranes that evolve into autophagosomes is regulated by two ubiquitination-like reactions. First, the ubiquitin-like molecule Atg12 is conjugated to Atg5 by Atg7, which acts like an E1 ubiquitin-activating enzyme, and by Atg10, which is similar to an E2 ubiquitin-conjugating enzyme. The Atg5–Atg12 complex then interacts non-covalently with Atg16L1 and this resulting ternary complex associates with phagophores but dissociates from completed autophagosomes.
The second of the ubiquitin-like reactions involves the conjugation of ubiquitin-like molecules of the Atg8 family, which comprises the three subfamilies LC3, GABARAP and GATE-16, to the lipid phosphatidylethanolamine. Microtubule-associated protein 1 light chain 3 (MAP-LC3/Atg8/ LC3), the most well-characterised member of the Atg8 protein family, is conjugated to phosphatidylethanolamine by Atg7 (E1-like) and Atg3 (E2-like), resulting in autophagosome-associated LC3-II. The Atg5–Atg12 complex may be able to enhance LC3 conjugation to phosphatidylethanolamine by acting in an E3-like fashion. In this way, the Atg5– Atg12–Atg16L complex may determine the sites of autophagosome synthesis by regulating the targeting of LC3 to Atg5–Atg12-associated membranes.
Although the Atg5–Atg12–Atg16L1 complex localizes to phagophores and pre-phagophore structures and dissociates from fully formed (completed) autophagosomes, LC3-II remains associated with autophagosomes until after their fusion with lysosomes. The LC3-II inside the autolysosomes is degraded, while the LC3-II on the cytoplasmic surface can be delipidated and recycled. Thus, Atg5–Atg12–Atg16L1-positive LC3-negative vesicles represent pre-autophagosomal structures (pre-phagophores and possibly early phagophores), Atg5–Atg12–Atg16L1-positive LC3-positive structures can be considered to be phagophores, and Atg5–Atg12–Atg16L1-negative LC3-positive vesicles can be regarded as completed autophagosomes. Note that these definitions do not consider intermediate structures and therefore may end up being imprecise as our understanding of the structure, shape and protein composition of autophagy-related membranes evolves. Also, the PAS, at least in mammalian cells, may include some of these distinct structures.
Autophagosome biogenesis is the key event associated with the stress-responsive autophagic pathway, allowing the capture of specific cargoes and their delivery to the lysosomal degradative compartment. Although the endoplasmatic reticulum (ER) appears to be central for the assembly of autophagosomal membranes, it is also involved in several events regulating trafficking and local signaling, e.g., the establishment of contact sites with other organelles, the vesicular transport to the Golgi apparatus, and the biogenesis and turnover of lipid droplets.
The mechanisms involved in autophagosome biogenesis
Once activated, ULK1 is able to phosphorylate several substrates. Among them, there are two initial complexes, the ULK1 complex itself and the PI3KC3 complex 1 (PI3KC3-C1). In the first complex, ULK1 phosphorylates to itself (Thr180/1046, Ser1042), and the other members of the complex, Atg13 (Ser318/203), FIP200 (Ser943/986/1323) and ATG101 (Ser11/203). In the second complex, ULK1 potentiates the PI3K activity of the catalytic subunit Vps34, by the phosphorylation of two members of the complex, BECN1 (Ser14) and ATG14L (Ser29), resulting in the increment of PI3P production. Following to ULK1 complex activation, the transmembrane protein VMP1 interacts with the BH3 domain of BECN1 through its ATG domain, recruiting the PI3KC3-C1 to the autophagosomal membrane.
There are two main PI3KC3 complexes in autophagosome biogenesis. The complex 1 is composed by BECN1, ATG14L, Vps15 and Vps34, which is a key component in autophagosome initiation. The other complex, PI3KC3-C2, is related to autophagosome maturation and endosomal trafficking and is composed by the same members except for the regulatory protein ATG14L which is replaced by UVRAG. Structurally, the PI3KC3-C1 is stabilized in pairs, BECN1/ATG14L and Vps15/Vps34. Upon autophagy induction, BECN1 recruitment induces the complex assembly, through the adaptor ATG14L, where the WD domain of Vps15 organizes the proteins into the complex allowing the activity of Vps34. Moreover, the KAP1-mediated SUMOylation of Vps34 enhances the interaction of this protein with the rest of the complex. As it was commented before, ULK1-mediated phosphorylation of BECN1, ATG14L and Vps34 potentiates PI3K activity in this complex. The tumor suppressor DAPK, a calcium/calmodulin serine/threonine kinase, also contributes to the PI3KC3-C1 recruitment to the autophagosome membrane. This kinase phosphorylates BECN1 on its BH3 domain interfering with the BECN1-Bcl-xL association and releasing BECN1. This effect is reaffirmed by TRAF6 which ubiquitinates BECN1 on the same region. Recently, it has been proposed that Vps34 activity may be switched on/off by an EP300-dependent acetylation/deacetylation on K771, as another regulation of the PI3KC3-C1.
The cascade of subsequent activations of ULK1 and PI3KC3-C1 complex members is limited by a series of degradative processes. The deubiquitinase A20 (DUB A20) controls BECN1 participation on autophagosome formation by elimination of poly ubiquitin chain in the BH3 domain placed by ATF6 E3 ligase. Beyond that regulation, the E3 ligases NEDD4 and NEDD4L induce degradation of key members in ULK1, and Vps34 complexes respectively. BECN1 is poly ubiquitinated with K11-linked ubiquitin chain by NEDD4 to be eliminated in the proteasome. Similar activity is carried out by NEDD4L on ULK1 targeting this protein with K27- and K29-linked ubiquitin chains. In both cases, the proteasome-mediated elimination of those proteins causes the destabilization of its respective complexes. In a redundant way of labeling for degradation, the poly ubiquitination with K48-linked ubiquitin chains on ULK1, BECN1, and Vps34 is catalyzed by the complex CUL3-KHLH20.
1. Mercer, Thomas J.; et al. Initial steps in mammalian autophagosome biogenesis. Journal of Biological Chemistry, 2018, 293(15):jbc.R117.810366.
2. David C. Rubinsztein.; et al. Mechanisms of autophagosome biogenesis. Current Biology, 2012, 22(1): R29-34.