Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy

Abstract

The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase-like activity by which the lipidation of members of the LC3 family is facilitated. The crystal structure of the human ATG12~ATG5 conjugate bound to the amino-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous patch. Mutagenesis data support the importance of both the ATG12–ATG5 interface and the continuous patch for E3 activity. The ATG12~ATG5 conjugate interacts with the E2 enzyme ATG3 with high-affinity through another surface location that is exclusive to ATG12, suggesting a different role of the continuous patch in E3 activity. These findings provide a foundation for understanding the mechanism of LC3 lipidation.

Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

“…The possibility that ATG5 masks a destabilizing region in ATG12 seems unlikely given that disruption of the ATG12 and ATG5 binding interface does not appear to destabilize the ATG12-ATG5 conjugate. 16,23 Importantly, ATG12 proteasomal degradation neither requires autophagy nor an exposed C-terminal glycine (that conjugates to ATG5 or ATG3) since ATG12 instability persisted in various autophagy-deficient settings or following mutation of the ATG12 C-terminal glycine to an alanine (a modification that inhibits ATG12-ATG5 conjugation). 15 Free ATG12 has recently been shown to promote mitochondrial-dependent apoptosis.…”

“…16,17 Therefore, we addressed whether the ability of free ATG12 to promote cell death was modulated by autophagy. For this purpose, we generated U2OS cells stably expressing a dominant-negative ATG4B mutant (C74A).…”

The machinery of macroautophagy

“…The structural data of the ATG12-ATG5-ATG16L1N (containing the N-terminal part of ATG16L1) complex reveal that ATG16L1N binds to ATG5 at the opposite side to where ATG12 binds; no interaction is detected between ATG16L1N and ATG12 [170]. Human ATG12 can interact with ATG3 through its Phe108 residue [162].…”

Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4

“…The assembled ATG5 complex appears to play a role as an E3-like enzyme for the lipidation of ATG8-family members, such as the LC3 mammalian orthologs of yeast Atg8. 13 The conserved Gly of the processed ATG8 is implicated in adduct formation with PE after the other ubiquitin-like conjugation reaction by ATG7, E2-like ATG3, and ATG12-ATG5-ATG16L1. 9,10,13 While yeast has single Atg4 and Atg8 proteins, genes in higher eukaryotes such as animals and plants contain large families of ATG4 and ATG8, suggesting their diverse functions.…”

“…13 The conserved Gly of the processed ATG8 is implicated in adduct formation with PE after the other ubiquitin-like conjugation reaction by ATG7, E2-like ATG3, and ATG12-ATG5-ATG16L1. 9,10,13 While yeast has single Atg4 and Atg8 proteins, genes in higher eukaryotes such as animals and plants contain large families of ATG4 and ATG8, suggesting their diverse functions. 3,[14][15][16][17][18][19][20] In humans, 4 ATG4s (HsATG4s) and 7 ATG8s (HsATG8s) have been identified based on sequence similarity to the yeast ATG4 (ScATG4) and ATG8 (ScATG8), respectively.…”