Nix is a selective autophagy receptor for mitochondrial clearance

Abstract

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.

Figure 1

Nix interacts with Atg8/LC3/GABARAP proteins. (A) Yeast clones harbouring empty bait vector (pYTH9, mock) or those encoding ScAtg8, LC3B, Atg12 and SUMO1 were transformed with empty prey vector (pACT2, mock) or those encoding fragments of ATG4A, ATG4B, NBR1, p62 and full-length Nix. Interaction was assessed by yeast growth on the SD-W/-L/-H medium with a β-gal assay. (B) GST pulldown assays using cell extracts of Flag-Nix-expressing COS7 cells and immobilized GST or the indicated GST fusions. Coprecipitated Nix was detected with Flag antibodies. (C) Purified GST-tagged NixΔTM was cleaved from the GST moiety by thrombin, purified and used for precipitation by GST fusion proteins. Precipitated proteins were analysed by western blotting (WB) with Nix antibodies. Ponceau S staining was used to visualize GST-fusion proteins. (D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β-gal, β-galactosidase; BafA1, bafilomycin-A1; CCCP, carbonyl cyanide m-chlorophenyl hidrazone; GST, glutathione-S-transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin-like modifier 1; TCL, total cell lysate; TM, transmembrane domain.

Figure 2

Identification of Nix-LIRs. (A) Amino-acid sequences of Nix and related BNIP3 protein from the indicated species were aligned using the ClustalW2 program (EMBL-EBI, Cambridge, UK). The published LIR sequences were aligned manually alongside Nix/BNIP3 for comparison. The shaded regions indicate highly conserved residues in the two putative LIRs. Consensus symbols ^* (identical residues), : (conserved substitution) and . (semi-conserved substitution) are valid for the Nix/BNIP3 alignment only. (B) A schematic of Nix showing its domain organization and the point mutations used in this study to identify LIRs. (C) Lysates of COS7 cells transfected with Flag-wt-Nix or the indicated mutants were incubated with immobilized GST-LC3A. The coprecipitated proteins were detected by western blotting (WB) with Flag antibodies. BNIP3, Bcl2/E1B 19kDa-interacting protein 3-like protein; Dr, Danio reio; GST, glutathione-S-transferase; Hs, Homo sapiens; LIR, LC3-interacting region; Mm, Mus musculus; wt, wild type; Xl, Xenopus laevis.

Figure 3

Nix recruits EGFP-GABARAP-L1 to stressed mitochondria in reconstituted Nix^−/− MEFs. Nix^−/− MEFs cotransfected with EGFP-GABARAP-L1 and wt-Nix (A), or Nix-W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP-GABARAP-L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag-Nix-Cy3 and GABARAP-L1 using the ‘colocalization' highlighter plug-in for ImageJ software (NIH, Bethesda, MA, USA). (C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP-treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann–Whitney t-test. CCCP, carbonyl cyanide m-chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.

Figure 4

Mitochondrial clearance is impaired in Nix^−/− reticulocytes transduced with Nix-W35A. (A) Flow cytometry of reticulocyte-enriched blood, cultured in vitro for 3 days, stained with MTR. The blood was taken from mice transplanted with Nix^−/− bone marrow and reconstituted with wt-Nix and Nix-W35A viruses. Reticulocytes were induced by phenylhydrazine treatment. The viruses also express GFP. (B) Mitochondrial clearance in the GFP-positive fraction of the reticulocyte-enriched blood, as described in panel (A). Mice were reconstituted with wt-Nix, LIR-W35A or empty vector viruses. Data from Nix^−/− and Nix^+/+ mice are shown for comparison. (C) Anti-Flag, anti-GFP and anti-β-actin western blots of fetal liver cells transduced with Flag-Nix and Flag-W35A-Nix viruses, normalized to total protein. GFP, green fluorescent protein; LIR, LC3-interacting region; MTR, MitoTracker Red; wt, wild type.