Abstract
The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.
Highlights
Autophagy, a highly conserved cellular pathway responsible for recycling and degradation of cellular components and organelles, is essential for differentiation, development and homeostasis[1]
The same behavior is common to its homolog Bnip[3], which serves as a mitochondrial autophagy receptor[14,18]
Interaction between autophagy receptors and ATG8/LC3/GABARAP proteins through specific LIR motifs has been shown to be important for proper activation of selective autophagy
Summary
A highly conserved cellular pathway responsible for recycling and degradation of cellular components and organelles, is essential for differentiation, development and homeostasis[1]. Selective autophagy is a precisely regulated process aimed to remove unwanted components (cargo) like protein aggregates, damaged organelles, etc, from the cytosol It is based on specific cargo recognition by direct interaction of cargo receptors with members of the Atg[8] protein family[3,4,5,6,7,8,9]. Recent data on Optineurin (Optn), an autophagy receptor for selective recognition and removal of intracellular bacteria (Salmonella) and protein aggregates, revealed that phosphorylation of serine residues juxtaposed to the LIR sequence is a major contributor to the specific interaction with Atg[8] proteins[5]. Biochemical and biophysical analyses, including a crystal structure of the LC3B:Nix-LIR complex, revealed that the LIR phosphorylation status is essential for the fine-tuning of the LC3B:Nix interaction and crucial for the initiation of Nix-mediated mitophagy
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