Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation. There is a Hot off the Press (January 2010) associated with this Scientific Report.

BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-x_L positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.

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.

Autophagy is an evolutionarily conserved degradation pathway characterized by dynamic rearrangement of membranes that sequester cytoplasm, protein aggregates, organelles, and pathogens for delivery to the vacuole and lysosome, respectively. The ability of autophagosomal membranes to act selectively toward specific cargo is dependent on the small ubiquitin-like modifier ATG8/LC3 and the LC3-interacting region (LIR) present in autophagy receptors. Here, we describe a comprehensive protein-protein interaction analysis of TBC (Tre2, Bub2, and Cdc16) domain-containing Rab GTPase-activating proteins (GAPs) as potential autophagy adaptors. We identified 14 TBC domain-containing Rab GAPs that bind directly to ATG8 modifiers and that colocalize with LC3-positive autophagy membranes in cells. Intriguingly, one of our screening hits, TBC1D5, contains two LIR motifs. The N-terminal LIR was critical for interaction with the retromer complex and transport of cargo. Direct binding of the retromer component VPS29 to TBC1D5 could be titrated out by LC3, indicating a molecular switch between endosomes and autophagy. Moreover, TBC1D5 could bridge the endosome and autophagosome via its C-terminal LIR motif. During starvation-induced autophagy, TBC1D5 was relocalized from endosomal localization to the LC3-positive autophagosomes. We propose that LC3-interacting Rab GAPs are implicated in the reprogramming of the endocytic trafficking events under starvation-induced autophagy.

Completion of meiosis in mammals depends on the formation of the synaptonemal complex, a tripartite structure that physically links homologous chromosomes during prophase I. Several components of the synaptonemal complex are known, including constituents of the cohesin core, the axial/lateral element and the transverse filaments. No protein has previously been identified as an exclusive component of the central element. Mutations in some synaptonemal-complex proteins results in impaired meiosis. In humans, cases of male infertility have been associated with failure to build the synaptonemal complex. To search for new components of the meiotic machinery, we have used data from microarray expression profiling and found two proteins localising solely to the central element of the mammalian synaptonemal complex. These new proteins, SYCE1 and CESC1, interact with the transverse filament protein SYCP1, and their localisation to the central element appears to depend on recruitment by SYCP1. This suggests a role for SYCE1 and CESC1 in synaptonemal-complex assembly, and perhaps also stability and recombination.