Atg12-Interacting Motif Is Crucial for E2–E3 Interaction in Plant Atg8 System

Abstract

Autophagy is an intracellular degradation system regulating cellular homeostasis. The two ubiquitin-like modification systems named the Atg8 system and the Atg12 system are essential for autophagy. Atg8 and Atg12 are ubiquitin-like proteins covalently conjugated with a phosphatidylethanolamine (PE) and Atg5, respectively, via enzymatic reactions. The Atg8-PE conjugate binds to autophagic membranes and recruits various proteins through direct interaction, whereas the Atg12-Atg5 conjugate recognizes Atg3, the E2 enzyme for Atg8, and facilitates Atg8-PE conjugation by functioning as the E3 enzyme. Although structural and biochemical analyses have well established the Atg8-family interacting motif (AIM), studies on the interacting sequence for Atg12 are rare (only one example for human ATG12-ATG3), thereby making it challenging to define a binding motif. Here we determined the crystal structure of the plant ATG12b as a complex with the ATG12b-binding region of ATG3 and revealed that ATG12b recognizes the Asp-Met motif in ATG3 via a hydrophobic pocket and a basic residue, which we confirmed critical for the complex formation by mutational analysis. This recognition mode is similar to that reported between human ATG12 and ATG3, suggesting that the Asp-Met sequence is a conserved Atg12-interacting motif (AIM12). These data suggest that AIM12 mediates E2-E3 interaction during Atg8 lipidation and provide structural basis for developing chemicals that regulate autophagy by targeting Atg12-family proteins.

Linear motifs regulating protein secretion, sorting and autophagy in Leishmania parasites are diverged with respect to their host equivalents

“…As a further striking finding, neither the kinetoplastid ATG12s, nor any of the atypical ATG8-family proteins have a LIR interface compatible with the AIM (also known as AIM12) linear motif found in ATG3. The latter motif is crucial for ATG12-mediated ATG8 conjugation in the majority of known eukaryotic organisms, allowing ATG12 conjugates to act upstream of ATG8 [ 56 – 58 ]. The nearly-ubiquitous AIM12 motif, found in animals, plants and fungi, is not even conserved in kinetoplastids (although our ATG3 alignments betray that it is present in other groups within Discoba, suggesting a secondary loss, see S14 Fig ).…”

Structural view on autophagosome formation

“…The Atg12-Atg5-Atg16 complex targets autophagic membranes via interaction with Atg21, a PI-3 phosphate (PI3P) binding protein [71] (WIPI2 in the case of mammals [72]; structural studies are described below), and recruits Atg8-loaded Atg3 to the membrane via the interaction between Atg12 and the Atg12interacting motif in the FR of Atg3 (Fig. 2I) [73,74]. This process promotes the transfer of Atg8 from Atg3 to PE.…”