Anatomy of autophagy: from the beginning to the end

Abstract

Autophagy is a highly regulated process in eukaryotes to maintain homeostasis and manage stress responses. Understanding the regulatory mechanisms and key players involved in autophagy will provide critical insights into disease-related pathogenesis and potential clinical treatments. In this review, we describe the hallmark events involved in autophagy, from its initiation, to the final destruction of engulfed targets. Furthermore, based on structural and biochemical data, we evaluate the roles of key players in these processes and provide rationale as to how they control autophagic events in a highly ordered manner.

Keywords: ATG; Autophagosome; Lysosome; SNARE; Vps34.

Fig. 1

Schematic depiction of the autophagic process. The autophagic process is directed by green arrowheads. The whole process can be roughly divided into six steps: (1) initiation of autophagy; (2) biogenesis of phagophore; (3) expansion of phagophore; (4) formation of autophagosome; (5) fusion with lysosome, and (6) reformation of lysosome. The red arrows indicate the potential membrane sources for autophagosomes, including cytoplasm, ER (endoplasmic reticulum), mitochondria, and Golgi. The blue arrow indicates that degraded materials are transported back to the cytosol for reuse via lysosomal transporters. PAS phagophore assembly site

Fig. 2

Structural analysis of the Atg1 complex. a Signaling pathways that transmit stress signals to regulate TORC1 and autophagy. b The Atg1/Atg13 complex structure. The domain organization schemes of Atg1 and Atg13 are shown above. Below left is the kinase domain structure of Atg1 in complex with its inhibitor (yellow) (4WNP), where the activation loop is highlighted in red. Below middle is the domain complex structure of Atg1 MIT (green)/Atg13 MIM (cyan) (4P1N). Below right is the HORMA domain complex structure of Atg13 (cyan)/Atg101 (grey) (4YK8), where Atg13 is folded in the closed state, compared to Atg101’s open state conformation. c The Atg13–Atg17–Atg31–Atg29 complex structure. Atg17 (yellow) forms a dimer with a curvature radius close to 10 nm and appears as a letter “S”. Atg29 (blue) and Atg31 (magenta) are located at the concave face of Atg17. Atg13 interacts with the N-terminus and C-terminus via 17BR (red) and 17LR (orange), respectively. The ability of Atg13 to bind to different Atg17 molecules simultaneously allows the supramolecular molecular assembly. 5JHF is used as a reference here but 4HPQ and 4P1W should also be noted

Fig. 3

Structural analysis of the Vps34 complex. a The helical and kinase domain structure of Vps34. The domain organization scheme of Vps34 is shown above. Below is the structure of Vps34 helical and catalytic domains (2X6H). The different colors of C-terminal helix indicate a potential conformational switch from its cytosolic state (magenta) to its membrane-associated state (cyan). The activation loop is highlighted in blue and the catalytic loop in black, when the inhibitor is shown in red. b The complex structure of Vps30–Vps38–Vps15–Vps34-nb. The domain organization schemes of individual subunits are shown above. Below is the complex structure (5DFZ) that appears as a letter “Y”. One arm of “Y” is formed by intertwined Vps15 (orange) and Vps34 (green), where Vps15 contacts the activation loop of Vps34 to regulate its activity. The other arm is formed by intertwined Vps30 (yellow) and Vps38 (blue), which embraces the WD40 domain of Vps15 and the C2 domain of Vps34. nb (nanobody) is used as a crystallization chaperone

Fig. 4

Structural analysis of the Atg8 conjugating system. a The complex structure of Atg8–Atg4. The tail of Atg8 (blue) is inserted into the catalytic center of Atg4 (yellow) (2ZOD). C74 (red) stands for the catalytic residue of Atg4. W142 (red) and the regulatory loop (G257-A263, magenta) undergo a large conformational change, compared to Atg4’s apo structure (grey, 2CY7). b The complex structure of Atg8–Atg7. The tail of Atg8 (blue) is inserted into the catalytic center of Atg7 (pink) (3RUI). C507 (red) stands for the catalytic residue of Atg7, which is located in the proximity of the last amino acid glycine of Atg8. c The complex structure of Atg7–Atg3. Two Atg7 molecules (Atg7′ and Atg7″ labeled here) form a dimer via their CTDs and coordinate in Atg3 binding and catalysis (4GSL). Atg7’ NTD (purple) (Atg7″ NTD is not shown) pushes the backside of Atg3 (orange) to force its front side (containing the catalytic residue C234 in black) facing the catalytic center of Atg7″ CTD (pink) (containing the catalytic residue C507 in red). The juxtaposition of active sites of Atg7″ and Atg3 facilitates the transfer of Atg8 (modeled here) between two enzymes, as indicated by the arrow. d The complex structure of Atg3–Atg12–Atg5–Atg16. The Atg12–Atg5–Atg16 complex is present as a tetramer due to Atg16 dimerization (3A7O), and anchored to the target membrane via the association of Atg16 and Atg5 with the membrane, as indicated by the arrows. From the interaction between Atg3 (orange) and Atg12 (green) (4NAW), it is speculated that Atg8-conjugated Atg3 is recruited to the target membrane by Atg12. Then, Atg8 is transferred from Atg3 to the membrane substrate PE, as indicated by the arrow. e The factors involved in the autophagosomal fusion. Stx17, SNAP29, and VAMP8 form a SNARE complex that mediates the membrane tethering and fusion (4WY4). The regulatory factors are listed, which play either a positive or negative role in this process