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Shown are the execution stages of autophagy, controlled by an upstream signaling cascade centered around the Ser/Thr kinase Tor, a metabolic regulator of autophagy, and a cascade of Atg factors, starting with Atg1. 1. Initiation. Upstream signaling brings about complex changes in autophagic initiation machinery leading to the formation of membranous precursors of autophagosome. (a) Signaling: nutrient or growth factor starvation signals via Ser/Thr kinase Tor; immunological stimuli (e.g. TLR activation with PAMPs) act through protein complexes containing MyD88 and Beclin 1 (Atg6), a key regulator of autophagy. (b) Beclin 1 is in an inhibitory complex with Bcl-2. Following stimulation, JNK-1 phosphorylates Bcl-2 (Bcl 2-P_i) releasing Beclin 1 from the inhibitory complex with Bcl-2. (c) Activated Beclin 1, in a tri-partite complex with type III phosphatidylinositol 3-kinase hVPS34 and Atg14, cooperates with other Atg factors to initiate autophagosome formation. (d) Atg16L1 (a genetic risk locus for inflammatory Crohn's disease) is in a noncovalent complex with the Atg5–Atg12 protein conjugate Atg16L1 marks the site of phosphatidylinositol 3-phosphate (PI3P) -dependent initiation of autophagosome formation and sets off phases e, f and g (shown only in elongation phase for clarity). Autophagic isolation membrane (phagophore) wraps around the cytoplasmic target (cytosol, protein aggregates, mitochondria, peroxisomes, microbes, etc.). 2. Elongation. Phagophore enlarges and closes to form a double membrane organelle termed the autophagosome. (e,f) Atg16L1/Atg5–Atg12 (e) acts as an E3 enzyme to stimulates conversion of LC3 I into the LC3 II form (f) via a protein-protein and protein-lipid conjugation cascade (not shown) LC3 I has a free C-terminal Gly residue, whereas LC3 II C-terminal Gly is lipidated with phosphatidylethanolamine (PE) leading to membrane association. This stage is pre-proteolytic and the lumen is not yet acidified. (g) Atg4 reverses LC3 II into LC3 I by removing PE. Atg4 is sensitive to reactive oxygen species (ROS) released from mitochondria and may be one of the signals promoting initiation+elongation stages. 3. Maturation. Autophagosome matures into autolysosome, where the captured cargo is degraded. (h) The maturation phase is controlled by another Beclin 1-interacting factor, UVRAG. A second tri-partite complex with Beclin 1 and hVPS34 has been postulated (indicated by parentheses), containing UVRAG (Vps38) as a subunit in place of the initiation-specific factor Atg14; however, published evidence suggests that UVRAG acts independent of Beclin 1 at this stage. UVRAG interacts with HOPS which acts as a tethering and Rab gunanine nucleotide exchange factor (GEF). (i) HOPS with Vps39 stimulates activation of Rab7 by loading Rab7 with GTP via the Rab7 Vps39 (GEF) associated with HOPS complexes. Rab7 is a small GTPase controlling trafficking and identity of late endosomal/lysosomal compartments. (j) Maturation occurs through fusion with late endosomal/lysosomal organelles or delivery of trafficking intermediates carrying H⁺ ATPase components and lysosomal hydrolytic enzymes. The maturing autophagosome becomes acidified and is converted into a degradative organelle (autolysosome) with single delimiting membrane (the inner of the two membranes is dissolved) containing degraded material including internal membranes originating form the captured cytoplasmic material.