Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy

Abstract

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. Bnip3 is an atypical BH3-only protein that is known to cause mitochondrial dysfunction and cell death. Interestingly, Bnip3 can also protect against cell death by inducing mitochondrial autophagy. The mechanism for this process, however, remains poorly understood. Bnip3 contains a C-terminal transmembrane domain that is essential for homodimerization and proapoptotic function. In this study, we show that homodimerization of Bnip3 is also a requirement for induction of autophagy. Several Bnip3 mutants that do not interfere with its mitochondrial localization but disrupt homodimerization failed to induce autophagy in cells. In addition, we discovered that endogenous Bnip3 is localized to both mitochondria and the endoplasmic reticulum (ER). To investigate the effects of Bnip3 at mitochondria or the ER on autophagy, Bnip3 was targeted specifically to each organelle by substituting the Bnip3 transmembrane domain with that of Acta or cytochrome b(5). We found that Bnip3 enhanced autophagy in cells from both sites. We also discovered that Bnip3 induced removal of both ER (ERphagy) and mitochondria (mitophagy) via autophagy. The clearance of these organelles was mediated in part via binding of Bnip3 to LC3 on the autophagosome. Although ablation of the Bnip3-LC3 interaction by mutating the LC3 binding site did not impair the prodeath activity of Bnip3, it significantly reduced both mitophagy and ERphagy. Our data indicate that Bnip3 regulates the apoptotic balance as an autophagy receptor that induces removal of both mitochondria and ER.

FIGURE 1.

Homodimerization of Bnip3 is essential for induction of autophagy. A, HeLa cells overexpressing Bnip3 or Bnip3 mutants were scored for the presence of GFP-LC3-positive autophagosomes. Bnip3, but not Bnip3ΔTM, significantly increased the number of autophagosomes/cell compared with β-gal-infected cells. *, p < 0.05. n = 3. B, Bnip3, but not Bnip3ΔTM, significantly increased the number of cells positive for autophagy compared with β-gal-infected cells. *, p < 0.05. n = 3. Shown is a representative Western blot analysis for endogenous LC3I/II. C, Bnip3, but not the homodimerization-deficient mutants, significantly increased the number of GFP-LC3 positive autophagosomes/cell compared with Myc-transfected control cells. *, p < 0.05. n = 3. D, Bnip3, but not the homodimerization-deficient mutants, induced significant autophagy compared with Myc-transfected control cells. *, p < 0.05. n = 4. Representative Western blot analysis for endogenous LC3I/II. E, the presence of GABARAP-GFP-positive autophagosomes in HeLa cells overexpressing Bnip3. Bnip3, but not the mutants, significantly increased the number of GABARAP-GFP-positive autophagosomes/cell compared with Myc-transfected control cells. *, p < 0.05. n = 3. Representative images are shown. F, Bnip3, but not the mutants, induced autophagy in a significant number of cell compared with Myc-transfected control cells. *, p < 0.05. n = 4.

FIGURE 2.

Bnip3 interacts with LC3 but not GABARAP. A, the presence of a conserved LIR in Bnip3 and Nix. B, Bnip3, but not Bnip3ΔTM, coimmunoprecipitates (IP) with GFP-LC3 in HeLa cells. C, Bnip3G180F and Bnip3H173A have reduced interaction with LC3 compared with Bnip3. D, Bnip3 does not interact with GABARAP-GFP.

FIGURE 3.

Disrupting the interaction between Bnip3 and LC3 reduces mitophagy. A, a point mutation in the conserved LIR disrupts the interaction between Bnip3 and LC3. Bnip3W18A failed to coimmunoprecipitate (IP) with LC3 in HeLa cells. Equal expression levels of Bnip3 and Bnip3W18A were confirmed by Western blot analysis. Actin was used as a loading control. B, representative images of HeLa cells infected with Bnip3 or Bnip3W18A plus GFP-LC3. Mitophagy was determined by assessing colocalization (white arrow) between GFP-LC3-positive autophagosomes and Tom20-labeled mitochondria using fluorescence microscopy. C, quantitation of the number of cells positive for mitophagy. Bnip3W18A induced significantly less mitophagy compared with Bnip3. *, p < 0.05. n = 4. D, mitophagy was also confirmed by Western blotting for cytochrome c oxidase subunit IV (COX IV), a mitochondrial inner membrane protein, in HeLa cells infected with β-gal, Bnip3, or Bnip3W18A. GAPDH was used as a loading control. E, Western blot analysis for endogenous LC3I/II. F, quantitation of the number of GFP-LC3-positive autophagosomes per cell. n = 4. n.s., not significant. G, HeLa cells overexpressing β-gal, Bnip3, or Bnip3W18A were scored for induction of autophagy (n = 3). H, HeLa cells overexpressing β-gal, Bnip3, or Bnip3W18A stained with tetramethylrhodamine methyl ester (TMRM) to measure mitochondrial membrane potential (n = 4).

FIGURE 4.

Endogenous Bnip3 localizes to mitochondria and ER in HeLa cells and in heart tissue. A, after 24 h of normoxia or hypoxia, HeLa cells were subjected to subcellular fractionation into cytosol, mitochondria (MITO), and ER-rich fractions. The fractions were separated by SDS-PAGE and immunoblotted with Bnip3, Tom20, or calnexin antibodies. B, hearts from adult rats were fractionated into cytosol (CYTO), mitochondria, and ER/SR-enriched fractions. The fractions were separated by SDS-PAGE and immunoblotted with Bnip3, Tom20, calnexin, or GAPDH antibodies.

FIGURE 5.

Bnip3 induces autophagy from both the mitochondria and the ER. A, representative images of HeLa cells transfected with vector, Bnip3, Bnip3Acta, or Bnip3cb5 plus GFP-LC3. B, quantitation of the number of GFP-LC3-positive autophagosomes per cell. n = 5. *, **, and ***, p < 0.05 versus Myc. C, quantitation of cells with activated autophagy. Bnip3, Bnip3Acta, and Bnip3cb5 significantly activated autophagy in HeLa cells compared with Myc cells. Data are mean ± S.E. n = 3. *, **, and ***, p < 0.05 versus Myc. D, Bnip3Acta and Bnip3cb5 coimmunoprecipitate (IP) with GFP-LC3 in HeLa cells. E, isolation of autophagosomes from HeLa cells using anti-GFP-linked to magnetic beads. Western blot analysis of isolated autophagosomes showed an increase in the number of autophagosomes and the presence of mitochondria and ER proteins in the autophagosomes in cells overexpressing Bnip3. Results are representative of three independent experiments.

FIGURE 6.

Bnip3 promotes autophagy of ER. HeLa cells overexpressing vector, Bnip3, or Bnip3cb5 plus GFP-LC3 were fixed and stained with anti-calnexin. ER autophagy was determined by assessing colocalization between GFP-LC3-positive autophagosomes and calnexin-labeled ER using fluorescence microscopy. A, representative images of HeLa cells. The yellow line indicates the segment used for line scan analysis. B, colocalization of GFP-LC3 and calnexin was quantified with line scan analysis using ImageJ software. The line scan shows colocalization between autophagosomes (GFP-LC3, green line) and the ER (calnexin, red line) in Bnip3 and Bnip3cb5 but not Myc-transfected cells. C, surface volume rendering of autophagosomes revealed ER inside autophagosomes in Bnip3cb5-overexpressing cells. D, quantitation of ERphagy. Bnip3 and Bnip3cb5 induced significant autophagy of ER compared with Myc-transfected control cells. * and **, p < 0.05. n = 3.

FIGURE 7.

Disrupting the interaction between Bnip3cb5 and LC3 reduces ERphagy. A, a point mutation in the conserved LIR disrupts the interaction between Bnip3cb5 and LC3. Bnip3cb5W18A failed to coimmunoprecipitate (IP) with LC3 in HeLa cells. Equal expression levels of Bnip3cb5 and Bnip3cb5W18A were confirmed by Western blot analysis. B, ERphagy was determined by assessing colocalization between GFP-LC3-positive autophagosomes and calnexin-labeled ER using fluorescence microscopy. Bnip3cb5W18A induced significantly less ERphagy compared with Bnip3cb5. *, p < 0.05. n = 3. C, quantitation of the number of autophagosomes/cell in cells infected with β-gal, Bnip3, or Bnip3cb5W18A. * and **, p < 0.05 versus β-gal. n = 3. D, quantitation of cells positive for activation of autophagy after infection with β-gal, Bnip3, or Bnip3cb5W18A. Bnip3 and Bnip3cb5 induced autophagy in a significant number of cells compared with β-gal. * and **, p < 0.05 versus β-gal. n = 3. E, Western blot analysis for endogenous LC3I/II.

FIGURE 8.

A schematic illustrating the mechanism by which Bnip3 targets mitochondria for removal by autophagosomes. Inactive Bnip3 exists as a monomer in the mitochondria. Upon activation, Bnip3 undergoes homodimerization via the transmembrane domain. The conformational change in the homodimer facilitates the interaction between the LIR in Bnip3 and LC3.