Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3‐interacting region (LIR) at the C‐terminus of the protein and a novel motif at the N‐terminus. Although both sites are important for Atg4–Atg8 interaction in vivo, only the new N‐terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE‐bound Atg8.
Atg4 proteases are essential for the processing of Atg8/LC3 and autophagic flux. This study shows that yeast Atg4 is recruited to autophagosomal membranes via two conserved motifs, one of which specifically recognizes lipidated Atg8 (Atg8‐PE).
Atg4 is recruited on autophagosomal membranes through direct interaction with Atg8 via two conserved sites.
A C‐terminal LIR motif is involved in Atg4 binding to both unlipidated and lipidated Atg8.
A novel N‐terminal motif in close vicinity to the catalytic site allows Atg4 to specifically recognize lipidated Atg8.
- Received August 1, 2016.
- Revision received February 12, 2017.
- Accepted February 20, 2017.
- © 2017 The Authors
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