Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation - PubMed (original) (raw)

Osamu Yamaguchi 1, Ayako Hashimoto 2, Shungo Hikoso 1, Toshihiro Takeda 1, Takafumi Oka 1, Hiroki Yasui 1, Hiromichi Ueda 1, Yasuhiro Akazawa 1, Hiroyuki Nakayama 3, Manabu Taneike 4, Tomofumi Misaka 4, Shigemiki Omiya 4, Ajay M Shah 4, Akitsugu Yamamoto 5, Kazuhiko Nishida 4, Yoshinori Ohsumi 6, Koji Okamoto 2, Yasushi Sakata 1, Kinya Otsu 4

Affiliations

Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation

Tomokazu Murakawa et al. Nat Commun. 2015.

Abstract

Damaged mitochondria are removed by mitophagy. Although Atg32 is essential for mitophagy in yeast, no Atg32 homologue has been identified in mammalian cells. Here, we show that Bcl-2-like protein 13 (Bcl2-L-13) induces mitochondrial fragmentation and mitophagy in mammalian cells. First, we hypothesized that unidentified mammalian mitophagy receptors would share molecular features of Atg32. By screening the public protein database for Atg32 homologues, we identify Bcl2-L-13. Bcl2-L-13 binds to LC3 through the WXXI motif and induces mitochondrial fragmentation and mitophagy in HEK293 cells. In Bcl2-L-13, the BH domains are important for the fragmentation, while the WXXI motif facilitates mitophagy. Bcl2-L-13 induces mitochondrial fragmentation in the absence of Drp1, while it induces mitophagy in Parkin-deficient cells. Knockdown of Bcl2-L-13 attenuates mitochondrial damage-induced fragmentation and mitophagy. Bcl2-L-13 induces mitophagy in Atg32-deficient yeast cells. Induction and/or phosphorylation of Bcl2-L-13 may regulate its activity. Our findings offer insights into mitochondrial quality control in mammalian cells.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Molecular characteristics of Bcl2-L-13.

(a) Schematic representation of mouse Bcl2-L-13 domain structure. The predicted BH1-4 domains, WXXL/I motifs and transmembrane domain (TMD) are indicated. (b) Bcl2-L-13 failed to induce apoptosis in HEK293A cells. Western blot analysis of cleaved caspase 3 in whole-cell lysates from cells expressing the indicated HA-tagged Bcl2-L-13 constructs. Cell lysates with 100 μM etoposide treatment for 24 h were shown as a positive control. (c) Sequence alignment of the WXXL/I motif in Atg32, human and mouse Bcl2-L-13, and human FUNDC1 and NBR1. (d) Yeast two-hybrid assay to examine the interaction between LC3B and wild-type or the indicated mutant HA-Bcl2-L-13. (e) HEK293A cells were transfected with wild-type or mutant HA-Bcl2-L-13. Forty-eight h after transfection, cells were lysed and incubated with GST-LC3B. The co-precipitated proteins with GST-LC3B were detected by immunoblotting for HA. Coomassie brilliant blue staining was performed to visualize GST or GST-LC3B. Right panel represents densitometric analysis of the band for HA. The value for cells transfected with Bcl2-L-13 in each experiment was set equal to 1 (_n_=3). Results are shown as the mean±s.e.m. *P<0.05 versus all other groups. (f) HEK293A cells were treated with 5 μM CCCP and 20 nM bafilomycin A1 for 1 h. Then, cells were lysed and immunoprecipitated with anti-Bcl2-L-13 antibody. Co-precipitated LC3 were detected by immunoblotting. Right panel represents densitometric analysis of the band for LC3. The value for cells treated with CCCP and immunoprecipitated with anti-Bcl2-L-13 antibody was set equal to 1 (_n_=3). Results are shown as the mean±s.e.m. *P<0.05 versus all other groups. (g) Mitochondrial fraction from HEK293A cells in the mitochondrial buffer (Buffer) alone or in buffer containing Na2CO3 or Triton X-100. The pellets (P) and supernatant fractions (S) were immunoblotted with the indicated antibodies. (h) Mitochondrial fraction was digested with proteinase K in the absence or presence of Triton X-100 and immunoblotted with the indicated antibodies. A one-way ANOVA followed by Tukey–Kramer's post hoc test was used for statistical analysis.

Figure 2

Figure 2. The function of Bcl2-L-13 in mitochondrial dynamics.

(a,b) Expression level of Bcl2-L-13 mutants. Immunoblot of whole-cell lysates from HEK293A cells expressing various HA-tagged Bcl2-L-13 constructs 48 h after transfection (a). HEK293A cells were transfected with Bcl2-L-13-specific siRNA (siBcl2-L-13) or with a non-targeting control siRNA (siCTRL), and analysed by immunoblotting with anti-Bcl2-L-13 antibody (b). (c) Mitochondrial morphology in HEK293A cells transfected with empty vector or HA-Bcl2-L-13 was analysed using anti-ATP synthase and anti-HA antibodies. Boxed areas for ATP synthase staining are shown at higher magnification (high) in the right panels. Scale bar, 10 μm. (d) Electron micrographs of mitochondria in HEK293A cells transfected with empty vector or HA-Bcl2-L-13. Images at higher magnification are shown in the right panels. Scale bar, 1 μm in the left panels and 200 nm in the right panels. (e) Representative z-stack confocal images of mitochondrial morphologies in HEK293A cells transfected with siCTRL or siBcl2-L-13 using anti-ATP synthase antibody. Scale bar, 10 μm. (f) Electron micrographs of mitochondria in HEK293A cells transfected with siCTRL or siBcl2-L-13. Images at higher magnification are shown in the right panels. Scale bar, 5 μm in the left panels and 2 μm in the right panels. (g) HEK293A cells transfected with GFP-Bcl2-L-13 were strained with TMRE. Scale bar, 10 μm.

Figure 3

Figure 3. The role of BH domains and LIR motifs in Bcl2-L-13-mediated mitochondrial fragmentation.

(a) Schematic diagrams of Bcl2-L-13 mutants in the BH domains and LIR. (b–e) HEK293A cells transfected with wild-type or mutant HA-Bcl2-L-13 constructs were immunostained for ATP synthase and HA. Boxed areas for ATP synthase staining are shown at higher magnification in the right panels. (b) LIR mutants, (c) TMD deletion mutant (ΔTM), (d) BH domain deletion mutants and (e) BH domain amino acid mutants. Scale bar, 10 μm.

Figure 4

Figure 4. Drp1 is not essential for Bcl2-L-13-induced mitochondrial fragmentation.

(a) HEK293A cells were transfected with Drp1-specific siRNA (siDrp1) or with a non-targeting control siRNA (siCTRL), and after 48 h analysed by immunoblotting for Drp1. (b) Drp1 knockdown HEK293A cells were transfected with empty vector or HA-Bcl2-L-13 for 48 h and then immunostained with the indicated antibodies along with MitoTracker Deep Red. Scale bar, 10 μm. Boxed areas in the MitoTracker Deep Red images are shown at higher magnification (high) in right panels. (c) Transfected cells in (b) were scored according to their mitochondrial morphology as fragmented, tubular or elongated patterns, and the percentages of distribution are shown in the bar graphs. More than 80 cells were counted for each group (_n_=3). Results are shown as the mean±s.e.m. *P<0.05 versus corresponding control group (Student's _t_-test). (d) Lysates from HEK293A cells transfected with empty vector, HA-Bcl2-L-13 or siBcl2-L-13 were subjected to immunoblotting with the indicated antibodies. α-tubulin was used as a loading control. (e) HEK293A cell lysates expressing empty vector or HA-Bcl2-L-13 were subjected to SDS–gel electrophoresis in the absence of 2-mercaptoethanol followed by immunoblotting with anti-Drp1 antibody. (f) HEK293A cell lysates expressing empty vector or HA-Bcl2-L-13 were subjected to immunoblotting with the indicated antibodies targeted to proteins involved in mitochondrial fusion. (g) Bak knockdown HEK293A cells were transfected with empty vector or HA-Bcl2-L-13 for 48 h and then immunostained with the indicated antibodies. Scale bar, 10 μm. Boxed areas in the ATP synthase staining are shown at higher magnification (high) in the right panels. (h) Bax knockdown HEK293A cells were transfected with empty vector or HA-Bcl2-L-13 for 48 h and then immunostained with the indicated antibodies. Scale bar, 10 μm. Boxed areas in the ATP synthase staining are shown at higher magnification (high) in the right panels.

Figure 5

Figure 5. The role of LIR in Bcl2-L-13-induced mitophagy.

(a) Immunoblots for LC3. HEK293A cells transfected with the indicated vectors were treated with or without 20 nM of bafilomycin A1 for 1 h. The two blots from the top were originated from the same transfer membrane, but exposure time for the second blot (short) was shorter than that for the top blot. Right upper and lower panels represent densitometric analysis of the ratio of LC3-II to GAPDH in non-treated groups on the top blot and that in bafilomycin A1-treated groups on the second blot, respectively. The value for cells transfected with empty vector in each experiment was set equal to 1 (_n_=3). (b) mKeima expressing HEK293A cells were excited using 559 nm laser. Scale bar, 10 μm. (c) Colocalization of autophagosomes and mitochondria in mKeima expressing cells transfected with GFP-LC3 and wild-type or LIR mutant HA-Bcl2-L-13. Forty-eight h after transfection, cells were treated with E64d and pepstatin A for 4 h prior to analysis. Images in the box at higher magnification are shown in the right panels. Scale bar, 10 μm. (d) Colocalization of lysosomes and mitochondria in mKeima-expressing HEK293A cells transfected with the indicated vectors for 48 h. Cells were stained with LysoTracker before microscopic analysis. Scale bar, 10 μm. Images in the box at higher magnification are shown in the right panels. (e) Quantification of the number of LysoTracker-positive dots colocalized with mKeima in cells from (d). More than 60 cells were counted for each group (_n_=3). (f) Electron micrographs of autophagosomes engulfing mitochondria in an HEK293A cell transfected with HA-Bcl2-L-13. E64d and pepstatin A were added 4 h before fixation. Scale bar, 500 nm. Right graph shows the number of mitophagic structures, which were counted in cell areas of more than 0.165 mm2 for each group (_n_=3). (g) Mitochondrial DNA (mtDNA) normalized to nuclear DNA (nDNA) (_n_=7). All quantitative results are shown as the mean±s.e.m. *P<0.05 versus all other groups. A one-way ANOVA followed by Tukey–Kramer's post hoc test was used for statistical analysis.

Figure 6

Figure 6. Bcl2-L-13-induced mitophagy is Parkin-independent.

(a) Parkin knockdown in HEK293A cells stably expressing mKeima was achieved by transfection with control siRNA (siCTRL) or Parkin-specific siRNA (siParkin). Then, the cells were transfected with empty vector or HA-Bcl2-L-13 and stained with LysoTracker. Scale bar, 10 μm. Images at higher magnification of areas in the box are shown in right panels. Right graph, quantification of the number of LysoTracker dots colocalized with mKeima. More than 30 cells were counted for each group (_n_=3). Values represent the mean±s.e.m. *P<0.05 versus siCTRL- or siParkin- and vector-transfected cells (A one-way ANOVA and Tukey–Kramer's post hoc test). (b) HeLa cells transfected with pMT-mKeima-Red and the indicated plasmids for 48 h were stained with LysoTracker. Scale bar, 10 μm. (c) Selective mitochondria elimination by Parkin or Bcl2-L-13. HeLa cells expressing FLAG-Parkin or HA-Bcl2-L-13 were incubated with 10 μM CCCP for 48 h. Cells were stained with DAPI and antibodies for Tom20 and tag such as FLAG (Parkin) or HA (Bcl2-L-13). Outlines demarcate the edges of transfected cells. Scale bar, 10 μm.

Figure 7

Figure 7. Bcl2-L-13 can substitute for Atg32 to induce mitophagy.

(a,b) Yeast transfected with wild-type or LIR mutant HA-Bcl2-L-13 or empty vector were collected at the indicated time points after induction of mitophagy and subjected to western blotting for mCherry. The red arrows depict free mCherry generated by mitophagy. Yeast strains are wild-type (WT) or _atg32_Δ in (a) or _atg32_Δ or _atg7_Δ derivatives in (b) expressing a mitochondrial matrix-targeted DHFR-mCherry. Quantitative analysis for free mCherry in (a) is shown in the right graph (_n_=3). The value for wild-type yeast strain was set equal to 1. Results are shown as the mean±s.e.m. *P<0.05 versus all other groups. (c) HEK293 cells were transfected with mCherry-Tom22 and wild-type or the indicated mutant HA-Bcl2-L-13. Seventy-two h after transfection, cells were lysed and subjected to western blotting for mCherry. Processed mCherry-Tom22 was detected as free mCherry. Quantitative analysis for free mCherry is shown in the right graph (_n_=3). The value for vector transfected cells was set equal to 1. Results are shown as the mean±s.e.m. *P<0.05 versus all other groups. A one-way ANOVA followed by Tukey–Kramer's post hoc test was used for statistical analysis.

Figure 8

Figure 8. Physiological role of Bcl2-L-13 in mitochondrial homeostasis.

(a,b) HEK293A cells transfected with siRNA were incubated with 5 μM CCCP for 4 h and stained with anti-ATP synthase antibody. Mitochondrial morphology was estimated in (b). More than 70 cells were counted for each group (_n_=3). *P<0.05 versus all other groups. Scale bar, 10 μm. (c) HEK293A cells expressing mKeima were transfected with siBcl2-L-13, treated with 5 μM CCCP for 4 h and stained with LysoTracker. Quantitative analysis for LysoTracker-positive dots colocalized with mKeima is shown in the right graph. More than 50 cells were counted for each group (_n_=3). *P<0.05 versus control. (d) HEK293A cells were treated with 5 μM CCCP for the indicated time and mitochondrial fraction was subjected to immunoblotting (_n_=3). Data were normalized to the corresponding protein level of Tom20 and the value treated with DMSO at the indicated time was set equal to 1. *P<0.05 versus control. (e) Forty-eight h after transfection, cells lysates were immunoprecipitated with HA antibody. The phosphorylation was detected using anti-Phospho-Serine/Threonine antibody. Lower panel represents densitometric analysis of phospho-Ser/Thr (_n_=3). *P<0.05 versus Bcl2-L-13. (f) Immunoblot for LC3. Lower panel represents densitometric analysis of LC3-II-to-GAPDH ratio (_n_=3). *P<0.05 versus all other groups. (g,h) Colocalization of autophagosomes and mitochondria in HEK293A cells. After 48 h of transfection, cells were treated with E64d and pepstatin A for 4 h and stained with anti-LC3 and anti-ATP synthase antibody. Images in the box at higher magnification are shown in the right panels. The number of LC3 dots colocalized with ATP synthase per cell showing mitochondrial fragmentation in wild-type or mutant Bcl2-L-13 overexpressing cells in (h). At least 30 cells were counted for each group (_n_=3). Scale bar, 10 μm. *P<0.05 versus Bcl2-L-13. Quantitative results are shown as the mean±s.e.m. Student's _t_-test (c,d,e,h) or a one-way ANOVA followed by Tukey–Kramer's post hoc test (b,f) was used for statistical analysis.

Figure 9

Figure 9. Ubiquitination is not involved in Bcl2-L-13-induced mitophagy.

HEK293A cells expressing FLAG-Parkin or HA-Bcl2-L-13 were incubated with 10 μM CCCP for 3 h. Cells were stained antibodies for Tom20 and ubiquitin. Scale bar, 10 μm.

References

    1. Westermann B. Mitochondrial fusion and fission in cell life and death. Nat. Rev. Mol. Cell. Biol. 11, 872–884 (2010). - PubMed
    1. Mizushima N. & Komatsu M. Autophagy: renovation of cells and tissues. Cell 147, 728–741 (2011). - PubMed
    1. Kim I., Rodriguez-Enriquez S. & Lemasters J. J. Selective degradation of mitochondria by mitophagy. Arch. Biochem. Biophys. 462, 245–253 (2007). - PMC - PubMed
    1. Mao K., Wang K., Liu X. & Klionsky D. J. The scaffold protein Atg11 recruits fission machinery to drive selective mitochondria degradation by autophagy. Dev. Cell. 26, 9–18 (2013). - PMC - PubMed
    1. Twig G. et al. Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J. 27, 433–446 (2008). - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources