Dimerization of mitophagy receptor BNIP3L/NIX is essential for recruitment of autophagic machinery

Abstract

Mitophagy is a conserved intracellular catabolic process responsible for the selective removal of dysfunctional or superfluous mitochondria to maintain mitochondrial quality and need in cells. Here, we examine the mechanisms of receptor-mediated mitophagy activation, with the focus on BNIP3L/NIX mitophagy receptor, proven to be indispensable for selective removal of mitochondria during the terminal differentiation of reticulocytes. The molecular mechanisms of selecting damaged mitochondria from healthy ones are still very obscure. We investigated BNIP3L dimerization as a potentially novel molecular mechanism underlying BNIP3L-dependent mitophagy. Forming stable homodimers, BNIP3L recruits autophagosomes more robustly than its monomeric form. Amino acid substitutions of key transmembrane residues of BNIP3L, BNIP3L^G204A or BNIP3L^G208V, led to the abolishment of dimer formation, resulting in the lower LC3A-BNIP3L recognition and subsequently lower mitophagy induction. Moreover, we identified the serine 212 as the main amino acid residue at the C-terminal of BNIP3L, which extends to the intermembrane space, responsible for dimerization. In accordance, the phosphomimetic mutation BNIP3L^S212E leads to a complete loss of BNIP3L dimerization. Thus, the interplay between BNIP3L phosphorylation and dimerization indicates that the combined mechanism of LIR phosphorylation and receptor dimerization is needed for proper BNIP3L-dependent mitophagy initiation and progression.Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; Baf A1: bafilomycin A₁; BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CoCl₂: cobalt (II) chloride; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PHB2: prohibitin 2; PI: propidium iodide; PINK1: PTEN induced kinase 1; TM: transmembrane domain; TOMM20: translocase of outer mitochondrial membrane 20.

Keywords: Autophagy; BNIP3L/NIX; dimerization; mitophagy; selective autophagy.

Figure 1.

Glycine 204 and 208 in the BNIP3L transmembrane domain are important for BNIP3L dimerization. (A) A scheme of BNIP3L domain organization: LC3-interacting region (LIR), BCL2 homology domain 3 (BH3), and transmembrane domain (TM). The alignment shows amino acid sequences of BNIP3L and related BNIP3 TM domains from the indicated species. The shaded regions indicate highly conserved glycine residues in the TM domains. Consensus symbols “*” (identical residues), “:” (conserved substitution) and “.” (semi-conserved substitution). (B) Western blot analysis of BNIP3L dimer temperature stability. HEK293 cells overexpressing Flag-BNIP3L WT or Flag-BNIP3L^G204A mutant were lysed in RIPA buffer containing 0.5% SDS and boiled for the indicated period of time (C) Western blot of HEK293 lysates overexpressing different GFP-BNIP3L mutants with or without dimerization ability. (D) Representative immunofluorescence images of BNIP3L dimerization mutants co-localizing to the mitochondria. Nuclei were stained with DAPI (blue), green signals are GFP-BNIP3L proteins, and red represents mitochondrial marker TOMM20. BNIP3L and TOMM20 colocalization is reflected as a yellow color. (E) Western blot of HEK293 subcellular fractionation for detecting the localization of GFP-BNIP3L WT or the indicated mutants. TOMM20 was used as a marker for the outer mitochondrial membrane or mitochondrial fraction (mito.). Abbreviations: BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CAEEL: Caenorhabditis elegans; cyto: cytosolic fraction; DANRE: Danio reio; LIR: LC3-interacting region; BNIP3L ΔLIR: recombinant BNIP3L lacking LC3-interacting region; BNIP3L^G202A: recombinant BNIP3L with substituted glycine 202 with alanine; BNIP3L^G204A: recombinant BNIP3L with substituted glycine 204 with alanine; BNIP3L^G208V: recombinant BNIP3L with substituted glycine 208 with valine; mito: mitochondrial fraction; TM: transmembrane; XENLA: Xenopus laevis; WT: wild type

Figure 2.

Dimerization increases BNIP3L activity as a mitophagy receptor. (A) GST affinity isolation showing BNIP3L ability for LC3A binding. Affinity isolation with purified GST-LC3A was performed against GFP-BNIP3L WT or indicated TM mutants: BNIP3L^G202A, BNIP3L^G204A, and BNIP3L^G208V (lower blot). Upper blot shows a western blot of 10% of TCL input used for the affinity-isolation reaction. GAPDH was used as a loading control. The graph represents a quantitative analysis of GST affinity-isolation binding efficiency between BNIP3L dimer and monomer to GST-fused LC3A. Binding efficiency was shown as a percentage of GST affinity isolation from a total of 10% TCL input in an affinity-isolation assay. Densitometric scans of immunoblots were obtained from three independent experiments and analyzed in Image Lab. T-test statistical analysis was used to compare differences between GFP-WT BNIP3L dimer and monomer LC3A binding, as well as between GFP-BNIP3L^G202A dimer and monomer. (B) Recruitment of autophagosomes on mitochondria overexpressing different GFP-BNIP3L mutants in HeLa cells upon mitophagy induction. HeLa cells were transfected with different GFP-BNIP3L proteins and treated with CCCP for 2 h to induce mitophagy. GFP-BNIP3L proteins are represented as green and autophagosomes labeled by LC3A as red. The quantification was based on the analysis of the total number of LC3A puncta in 100 cells per each WT BNIP3L or indicated mutants. The data are represented as fold-change relative to the WT BNIP3L (mean ± SD) from three independent experiments (significance assessed by one-way ANOVA with Tukey’s multiple comparisons test). (C) Mitochondrial removal upon CCCP treatment in cells overexpressing GFP-BNIP3L WT, BNIP3L∆LIR, BNIP3L^G204A, or BNIP3L^G208V proteins monitored by flow cytometry. Transfected cells were treated for 24 h with CCCP or in combination with Baf A1 to induce mitophagy. Two-way ANOVA with Tukey’s multiple comparisons test analysis was used to compare differences between mitochondrial removal in GFP-BNIP3L (WT BNIP3L, BNIP3L∆LIR, BNIP3L^G204A, and BNIP3L^G208V) transfected cells. All data were analyzed in GraphPad Prism 8. Statistical significance: *P = < 0.05, **P = < 0.01; ***P = < 0.001; ns, not significant; error bars indicate standard deviation, n = 3

Figure 3.

Phosphorylation at the C-terminal BNIP3L disrupts its dimerization. (A) Domain organization of the BNIP3L protein and amino acid alignment of the C-terminal of BNIP3L and BNIP3 proteins from the indicated species. The shaded region indicates the Ser212 residue that is important for dimerization. (B) Western blot analysis of HEK293 overexpressing GFP-BNIP3L WT, GFP-BNIP3L^S212A, GFP-BNIP3L^212Stop, GFP-BNIP3L^S212E, and GFP-BNIP3L∆C-terminal mutants. (C) GST affinity isolation of GST-LC3A against GFP-BNIP3L WT, GFP-BNIP3L^S212A, and GFP-BNIP3L^S212E mutants (right blot). Left blot shows BNIP3L expression in 10% of cell lysates used in the affinity-isolation reaction with GAPDH as a loading control. Quantification of the GST affinity-isolation binding efficiency between BNIP3L dimer and monomer to GST-fused LC3A was showed as a percentage of GST binding from a total of 10% TCL input in the affinity-isolation assay. Densitometric scans of immunoblots were obtained from three independent experiments and analyzed in Image Lab. T-test statistical analysis was used to compare differences in LC3A binding between GFP-BNIP3L WT or BNIP3L^S212A dimer and monomer. (D) Immunofluorescence microscopy of the BNIP3L C-terminal mutant and its localization to the mitochondria. Nuclei were stained with DAPI (blue), green signals are GFP-BNIP3L proteins, and red represents mitochondrial marker TOMM20. BNIP3L and TOMM20 colocalization is reflected by a yellow color. (E) Western blot analysis of HEK293 subcellular fractionation for detecting the localization of GFP-BNIP3L WT or GFP-BNIP3L^S212A/E mutants. TOMM20 was used as a marker for the outer mitochondrial membrane. (F) Recruitment of autophagosomes on damaged mitochondria overexpressing GFP-BNIP3L WT, BNIP3L^G204A, BNIP3L^S212A, or BNIP3L^S212E. HeLa cells were transfected with indicated BNIP3L C-terminal dimerization mutants and treated with CCCP for 2 h to induce mitophagy. Quantification of the LC3A puncta was performed by analyzing the number of LC3A dots for 100 cells per each BNIP3L plasmid in three independent experiments (the data are represented as mean ± SD of fold-change against GFP-WT BNIP3L). One-way ANOVA with Tukey’s multiple comparisons test was used to compare the difference in autophagosomal recruitment between BNIP3L mutants. (G) Quantification of mitochondrial removal using GFP-BNIP3L WT, BNIP3L^S212A, or BNIP3L^S212E by flow cytometry. Transfected HEK293 cells were treated with CCCP or CoCl₂ and in combination with Baf A1 for 24 h. Two-way ANOVA with Tukey’s multiple comparisons test was used to compare differences between mitochondrial removal in GFP-BNIP3L WT, BNIP3L^S212A, and BNIP3L^S212E. Data were analyzed in GraphPad Prism 8. Statistical significance: *P = < 0.05, **P = < 0.01, ***P = < 0.001; ns, not significant; error bars indicate standard deviation, n = 3 (F), n = 2 (G)

Figure 4.

LIR and receptor dimerization jointly enhanced BNIP3L-mediated mitophagy. (A) Western blot analysis of the double ∆LIR and TM/C-terminal BNIP3L mutants. (B) GST affinity isolation of GST-LC3A and GFP-BNIP3L WT, BNIP3L∆LIR, BNIP3L^G204A, BNIP3L∆LIR^G204A, BNIP3L^S212A, BNIP3L^S212E, BNIP3L∆LIR^S212A, or BNIP3L∆LIR^S212E. (C) Recruitment of autophagosomes on damaged mitochondria overexpressing GFP-BNIP3L WT, BNIP3L∆LIR, BNIP3L^G204A, BNIP3L∆LIR^G204A, BNIP3L^S212A, BNIP3L^S212E, BNIP3L∆LIR^S212A, or BNIP3L∆LIR^S212E. Quantification of LC3A puncta was performed by analyzing the number of LC3A dots for 100 cells per each BNIP3L plasmid in three independent experiments (the data are represented as mean ± SD). One-way ANOVA with Tukey’s multiple comparisons test was used to compare the difference in autophagosome recruitment between GFP-BNIP3L WT, BNIP3L∆LIR, BNIP3L^G204A, BNIP3L∆LIR^G204A, BNIP3L^S212A, BNIP3L^S212E, BNIP3L∆LIR^S212A, and BNIP3L∆LIR^S212E. *P = < 0.05, ** = P < 0.01, *** = P < 0.001. (D) Western blot confirmation of BNIP3L silencing in Hela cells transfected with control siRNA or siRNA against BNIP3L for 48 h that were used for the quantification of autophagosome recruitment on CCCP-damaged mitochondria overexpressing the indicated GFP-BNIP3L proteins. (E) Western blot analysis of HEK293 with silenced endogenous BNIP3L and transfected with different GFP-BNIP3L dimerization mutants upon CCCP- or CoCl₂-induced mitophagy (right). Mitochondrial removal in CCCP- or CoCl₂-treated HEK293 cells lacking endogenous BNIP3L overexpressing GFP-BNIP3L dimerization mutants were analyzed by flow cytometry (left). Two-way ANOVA with Tukey’s multiple comparisons test was used to compare the differences in mitochondrial removal in cells with GFP- BNIP3L WT, BNIP3L^S212E, BNIP3L∆LIR, or BNIP3L∆LIR^S212E proteins. *P = < 0.05, **P = < 0.01; ns, not significant; error bars indicate standard deviation, n = 3