Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis

Nature Cell Biology volume 19, pages 318–328 (2017)Cite this article

Subjects

Abstract

Mitochondria drive apoptosis by releasing pro-apoptotic proteins that promote caspase activation in the cytosol. The rhomboid protease PARL, an intramembrane cleaving peptidase in the inner membrane, regulates mitophagy and plays an ill-defined role in apoptosis. Here, we employed PARL-based proteomics to define its substrate spectrum. Our data identified the mitochondrial pro-apoptotic protein Smac (also known as DIABLO) as a PARL substrate. In apoptotic cells, Smac is released into the cytosol and promotes caspase activity by inhibiting inhibitors of apoptosis (IAPs). Intramembrane cleavage of Smac by PARL generates an amino-terminal IAP-binding motif, which is required for its apoptotic activity. Loss of PARL impairs proteolytic maturation of Smac, which fails to bind XIAP. Smac peptidomimetics, downregulation of XIAP or cytosolic expression of cleaved Smac restores apoptosis in PARL-deficient cells. Our results reveal a pro-apoptotic function of PARL and identify PARL-mediated Smac processing and cytochrome c release facilitated by OPA1-dependent cristae remodelling as two independent pro-apoptotic pathways in mitochondria.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Proteomic identification of PARL substrates.
Figure 2: Two-step processing of Smac in mitochondria.
Figure 3: Loss of PARL confers apoptotic resistance.
Figure 4: Cytochrome c but not Smac is released from PARL-deficient mitochondria to the cytosol during apoptosis.
Figure 5: PARL cleavage allows the release of Smac and PGAM5 from mitochondria following apoptotic induction.
Figure 6: Loss of PARL prevents interaction between Smac and XIAP in the cytosol.
Figure 7: PARL-driven apoptosis depends on XIAP inhibition.
Figure 8: Cytosolic Smac promotes apoptosis in PARL−/− cells.

Similar content being viewed by others

References

  1. Chan, D. C. Fusion and fission: interlinked processes critical for mitochondrial health. Annu. Rev. Genet. 46, 265–287 (2012).

    CAS  PubMed  Google Scholar 

  2. Schrepfer, E. & Scorrano, L. Mitofusins, from mitochondria to metabolism. Mol. Cell 61, 683–694 (2016).

    CAS  PubMed  Google Scholar 

  3. Wai, T. & Langer, T. Mitochondrial dynamics and metabolic regulation. Trends Endocrinol. Metab. 27, 105–117 (2016).

    CAS  PubMed  Google Scholar 

  4. Youle, R. J. & van der Bliek, A. M. Mitochondrial fission, fusion, and stress. Science 337, 1062–1065 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Quiros, P. M., Langer, T. & Lopez-Otin, C. New roles for mitochondrial proteases in health, ageing and disease. Nat. Rev. Mol. Cell Biol. 16, 345–359 (2015).

    CAS  PubMed  Google Scholar 

  6. Liesa, M. & Shirihai, O. S. Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure. Cell Metab. 17, 491–506 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Ehses, S. et al. Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1. J. Cell Biol. 187, 1023–1036 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Griparic, L., Kanazawa, T. & van der Bliek, A. M. Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage. J. Cell Biol. 178, 757–764 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Head, B., Griparic, L., Amiri, M., Gandre-Babbe, S. & van der Bliek, A. M. Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells. J. Cell Biol. 187, 959–966 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Song, Z., Chen, H., Fiket, M., Alexander, C. & Chan, D. C. OPA1 processing controls mitochondrial fusion and is regulated by mRNA splicing, membrane potential, and Yme1L. J. Cell Biol. 178, 749–755 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Anand, R. et al. The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission. J. Cell Biol. 204, 919–929 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. MacVicar, T. & Langer, T. OPA1 processing in cell death and disease—the long and short of it. J. Cell Sci. 129, 2297–2306 (2016).

    CAS  PubMed  Google Scholar 

  13. Cipolat, S. et al. Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126, 163–175 (2006).

    CAS  PubMed  Google Scholar 

  14. Frezza, C. et al. OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126, 177–189 (2006).

    CAS  PubMed  Google Scholar 

  15. Korwitz, A. et al. Loss of OMA1 delays neurodegeneration by preventing stress-induced OPA1 processing in mitochondria. J. Cell Biol. 212, 157–166 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Jiang, X., Jiang, H., Shen, Z. & Wang, X. Activation of mitochondrial protease OMA1 by Bax and Bak promotes cytochrome c release during apoptosis. Proc. Natl Acad. Sci. USA 111, 14782–14787 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Pernas, L. & Scorrano, L. Mito-morphosis: mitochondrial fusion, fission, and cristae remodeling as key mediators of cellular function. Annu. Rev. Physiol. 78, 505–531 (2016).

    CAS  PubMed  Google Scholar 

  18. Herlan, M., Vogel, F., Bornhovd, C., Neupert, W. & Reichert, A. S. Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA. J. Biol. Chem. 278, 27781–27788 (2003).

    CAS  PubMed  Google Scholar 

  19. McQuibban, G. A., Saurya, S. & Freeman, M. Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537–541 (2003).

    CAS  PubMed  Google Scholar 

  20. Sesaki, H., Southard, S. M., Hobbs, A. E. & Jensen, R. E. Cells lacking Pcp1p/Ugo2p, a rhomboid-like protease required for Mgm1p processing, lose mtDNA and mitochondrial structure in a Dnm1p-dependent manner, but remain competent for mitochondrial fusion. Biochem. Biophys. Res. Commun. 308, 276–283 (2003).

    CAS  PubMed  Google Scholar 

  21. Duvezin-Caubet, S. et al. OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria. Mol. Biol. Cell 18, 3582–3590 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ishihara, N., Fujita, Y., Oka, T. & Mihara, K. Regulation of mitochondrial morphology through proteolytic cleavage of OPA1. EMBO J. 25, 2966–2977 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Freeman, M. Rhomboids, signalling and cell biology. Biochem. Soc. Trans. 44, 945–950 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Jin, S. M. et al. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J. Cell Biol. 191, 933–942 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Sekine, S. et al. Rhomboid protease PARL mediates the mitochondrial membrane potential loss-induced cleavage of PGAM5. J. Biol. Chem. 287, 34635–34645 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 8, e1000298 (2010).

    PubMed  PubMed Central  Google Scholar 

  27. Morais, V. A. et al. PINK1 loss-of-function mutations affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling. Science 344, 203–207 (2014).

    CAS  PubMed  Google Scholar 

  28. Chen, G. et al. A regulatory signaling loop comprising the PGAM5 phosphatase and CK2 controls receptor-mediated mitophagy. Mol. Cell 54, 362–377 (2014).

    CAS  PubMed  Google Scholar 

  29. Wang, Z., Jiang, H., Chen, S., Du, F. & Wang, X. The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 148, 228–243 (2012).

    CAS  PubMed  Google Scholar 

  30. Shi, G. et al. Functional alteration of PARL contributes to mitochondrial dysregulation in Parkinson’s disease. Hum. Mol. Genet. 20, 1966–1974 (2011).

    CAS  PubMed  Google Scholar 

  31. Heinitz, S., Klein, C. & Djarmati, A. The p.S77N presenilin-associated rhomboid-like protein mutation is not a frequent cause of early-onset Parkinson’s disease. Mov. Disord. 26, 2441–2442 (2011).

    PubMed  Google Scholar 

  32. Civitarese, A. E. et al. Regulation of skeletal muscle oxidative capacity and insulin signaling by the mitochondrial rhomboid protease PARL. Cell Metab. 11, 412–426 (2010).

    CAS  PubMed  Google Scholar 

  33. Venne, A. S., Vogtle, F. N., Meisinger, C., Sickmann, A. & Zahedi, R. P. Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55. J. Proteome Res. 12, 3823–3830 (2013).

    CAS  PubMed  Google Scholar 

  34. Ghezzi, D. et al. Mutations in TTC19 cause mitochondrial complex III deficiency and neurological impairment in humans and flies. Nat. Genet. 43, 259–263 (2011).

    CAS  PubMed  Google Scholar 

  35. Mokry, D. Z., Abrahao, J. & Ramos, C. H. Disaggregases, molecular chaperones that resolubilize protein aggregates. An. Acad. Bras. Cienc. 87, 1273–1292 (2015).

    CAS  PubMed  Google Scholar 

  36. Horibata, Y. & Sugimoto, H. StarD7 mediates the intracellular trafficking of phosphatidylcholine to mitochondria. J. Biol. Chem. 285, 7358–7365 (2010).

    CAS  PubMed  Google Scholar 

  37. Du, C., Fang, M., Li, Y., Li, L. & Wang, X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33–42 (2000).

    CAS  PubMed  Google Scholar 

  38. Chai, J. et al. Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 406, 855–862 (2000).

    CAS  PubMed  Google Scholar 

  39. Verhagen, A. M. et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43–53 (2000).

    CAS  PubMed  Google Scholar 

  40. Chao, J. R. et al. Hax1-mediated processing of HtrA2 by Parl allows survival of lymphocytes and neurons. Nature 452, 98–102 (2008).

    CAS  PubMed  Google Scholar 

  41. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Mossmann, D., Meisinger, C. & Vogtle, F. N. Processing of mitochondrial presequences. Biochim. Biophys. Acta 1819, 1098–1106 (2012).

    CAS  PubMed  Google Scholar 

  43. Burri, L. et al. Mature DIABLO/Smac is produced by the IMP protease complex on the mitochondrial inner membrane. Mol. Biol. Cell 16, 2926–2933 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Salvesen, G. S. & Duckett, C. S. IAP proteins: blocking the road to death’s door. Nat. Rev. Mol. Cell Biol. 3, 401–410 (2002).

    CAS  PubMed  Google Scholar 

  45. Holcik, M. & Korneluk, R. G. XIAP, the guardian angel. Nat. Rev. Mol. Cell Biol. 2, 550–556 (2001).

    CAS  PubMed  Google Scholar 

  46. Mannhold, R., Fulda, S. & Carosati, E. IAP antagonists: promising candidates for cancer therapy. Drug Discov. Today 15, 210–219 (2010).

    CAS  PubMed  Google Scholar 

  47. Rettinger, E. et al. SMAC mimetic BV6 enables sensitization of resistant tumor cells but also affects Cytokine-Induced Killer (CIK) cells: a potential challenge for combination therapy. Front. Pediatr. 2, 75 (2014).

    PubMed  PubMed Central  Google Scholar 

  48. Li, W. et al. BV6, an IAP antagonist, activates apoptosis and enhances radiosensitization of non-small cell lung carcinoma in vitro. J. Thorac. Oncol. 6, 1801–1809 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Eckelman, B. P., Salvesen, G. S. & Scott, F. L. Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family. EMBO Rep. 7, 988–994 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Seeger, J. M. et al. Elevated XIAP expression alone does not confer chemoresistance. Br. J. Cancer 102, 1717–1723 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Okada, H. et al. Generation and characterization of Smac/DIABLO-deficient mice. Mol. Cell Biol. 22, 3509–3517 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Schug, Z. T. & Gottlieb, E. Cardiolipin acts as a mitochondrial signalling platform to launch apoptosis. Biochim. Biophys. Acta 1788, 2022–2031 (2009).

    CAS  PubMed  Google Scholar 

  53. Jost, P. J. et al. XIAP discriminates between type I and type II FAS-induced apoptosis. Nature 460, 1035–1039 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Saunders, C. et al. CLPB variants associated with autosomal-recessive mitochondrial disorder with cataract, neutropenia, epilepsy, and methylglutaconic aciduria. Am. J. Hum. Genet. 96, 258–265 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Wortmann, S. B. et al. CLPB mutations cause 3-methylglutaconic aciduria, progressive brain atrophy, intellectual disability, congenital neutropenia, cataracts, movement disorder. Am. J. Hum. Genet. 96, 245–257 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Kanabus, M. et al. Bi-allelic CLPB mutations cause cataract, renal cysts, nephrocalcinosis and 3-methylglutaconic aciduria, a novel disorder of mitochondrial protein disaggregation. J. Inherit. Metab. Dis. 38, 211–219 (2015).

    CAS  PubMed  Google Scholar 

  57. Capo-Chichi, J. M. et al. Disruption of CLPB is associated with congenital microcephaly, severe encephalopathy and 3-methylglutaconic aciduria. J. Med. Genet. 52, 303–311 (2015).

    CAS  PubMed  Google Scholar 

  58. Yang, L. et al. Haploinsufficiency for Stard7 is associated with enhanced allergic responses in lung and skin. J. Immunol. 194, 5635–5643 (2015).

    CAS  PubMed  Google Scholar 

  59. Kashkar, H. et al. XIAP targeting sensitizes Hodgkin lymphoma cells for cytolytic T-cell attack. Blood 108, 3434–3440 (2006).

    CAS  PubMed  Google Scholar 

  60. Luciano, P., Geoffroy, S., Brandt, A., Hernandez, J. F. & Geli, V. Functional cooperation of the mitochondrial processing peptidase subunits. J. Mol. Biol. 272, 213–225 (1997).

    CAS  PubMed  Google Scholar 

  61. Venne, A. S. et al. An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana. Proteomics 15, 2458–2469 (2015).

    CAS  PubMed  Google Scholar 

  62. Burkhart, J. M., Schumbrutzki, C., Wortelkamp, S., Sickmann, A. & Zahedi, R. P. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics. J. Proteomics 75, 1454–1462 (2012).

    CAS  PubMed  Google Scholar 

  63. Thingholm, T. E., Palmisano, G., Kjeldsen, F. & Larsen, M. R. Undesirable charge-enhancement of isobaric tagged phosphopeptides leads to reduced identification efficiency. J. Proteome Res. 9, 4045–4052 (2010).

    CAS  PubMed  Google Scholar 

  64. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    CAS  PubMed  Google Scholar 

  65. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  PubMed  Google Scholar 

  66. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    CAS  PubMed  Google Scholar 

  67. Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Google Scholar 

Download references

Acknowledgements

We thank E. Barth for technical support in electron microscopy and other laboratory members for their support. This work was supported a Japan Society for the Promotion of Science (JSPS) Fellowship for Research Abroad to S.S. and a Reinhart Koselleck grant of the Deutsche Forschungsgemeinschaft to T.L. R.P.Z. and A.S.V. thank the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin and the Bundesministerium für Bildung und Forschung for financial support. We thank B. de Strooper (VIB, Leuven) for the Parl−/− MEFs and E. Shoubridge (McGill University, Montreal) for the pBABE-puro vector.

Author information

Authors and Affiliations

  1. Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany

    Shotaro Saita, Hendrik Nolte, Kai Uwe Fiedler, Hamid Kashkar, Marcus Krüger & Thomas Langer

  2. Center for Molecular Medicine (CMMC), University of Cologne, Cologne 50931, Germany

    Hamid Kashkar, Marcus Krüger & Thomas Langer

  3. Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), University of Cologne, Cologne 50931, Germany

    Hamid Kashkar

  4. Leibniz Institute for Analytical Sciences (ISAS), Dortmund 44227, Germany

    A. Saskia Venne & René P. Zahedi

Authors
  1. Shotaro Saita
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hendrik Nolte
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kai Uwe Fiedler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hamid Kashkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. Saskia Venne
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. René P. Zahedi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marcus Krüger
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thomas Langer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.S. and T.L. designed the research. S.S. and K.U.F. performed the experiments. H.N. and M.K. performed the mass spectrometric analysis for differential proteomics. A.S.V. and R.P.Z. performed the ChaFRADIC analysis. H.K. provided essential tools and reagents. S.S. and T.L. wrote the manuscript with the comments of the other authors.

Corresponding author

Correspondence to Thomas Langer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PARL-based proteomics.

(a) Proteomic strategies for the identification of PARL substrate proteins in mitochondria. Left panel; determination of N-terminal peptides of mitochondrial proteins quantitative affinity purification proteomics. (b) iTRAQ based ratios were log2 transformed and biological replicates were plotted against each other. Reproducibility was determined by Pearson correlation coefficient: r > 0.4 (after filtering proteins with higher standard deviations than 99% of the complete population). Proteins that were found with extreme ratios (>0.99 quantile) are highlighted by their gene name. (c) Isolation of mitochondria from wild type (WT) and PARL−/− HEK293 cells by Percoll gradient centrifugation. Fraction 3 was collected and analysed by ChaFRADIC (Supplementary Table 1). (d) SDS-PAGE and immunoblot analysis of cellular fractions of wild type (WT) and PARL−/− HEK293 cells. p, precursor; i, intermediate; m, mature form. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 2 Characterization of Smac/DIABLO in PARL deficient cells.

(a) Fractionation of wild type (WT) and PARL−/− HEK293 cells into post nuclear (PNS), mitochondrial (mito), microsomal and cytosolic fractions. Samples were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature. (b) siRNA-mediated downregulation of Smac/DIABLO in wild type (WT) and PARL−/− HEK293 cells for 72 h (or with control siRNA). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature. (c) Transient expression of Smac/DIABLOHA in wild type (WT) and PARL−/− (KO) HEK293 cells for 24 h (reagent 1, GeneJuice; reagent 2, Lipofectamine 2000). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature. (d) Quantification of different Smac/DIABLO forms in wild type (WT) and PARL−/− HEK293 cells expressing PARLFLAG or PARLS277A−FLAG when indicated. Left panel; cell lysates were analysed by SDS-PAGE and immunoblotting. Right panel; quantification of fluorescence intensities using the infrared Odyssey System (n = 3 independent experiments; mean values ± SD). p, precursor; i, intermediate; m, mature. (e–g) SDS-PAGE and immunoblot analysis of cell extracts of wild type (WT) and PARL-deficient (KO) MEFs (e), HeLa cells (f), HCT116 (g) cells. Different clones of PARL−/− HeLa and HCT116 cells were analysed. a–e, OPA1 forms, p, precursor; i, intermediate; m, mature form. ∗unspecific cross-reaction. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 3 Processing of Smac/DIABLO.

(a) Hydropathy plot of the N-terminal 90 amino acids of Smac/DIABLO. The red bar indicates a predicted transmembrane domain (W42–A60). (b) Accumulation of the intermediate form of Smac/DIABLO upon downregulation of MPP in wild type (WT) and PARL−/− HEK293 cells. siRNAs directed against α- and β-Mpp (or control siRNA) were transfected for 72 h. Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. ∗unspecific cross-reaction. (c) siRNA-mediated downregulation of MPP in wild type (WT) and PARL−/− HEK293 cells expressing Smac/DIABLOHA (for 24 h) when indicated. Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. ∗unspecific cross-reaction. (d) Recombinant Smac/DIABLOHA (synthesized in rabbit reticulocyte lysate) was incubated for 1 h at 30 °C with mitochondrial extracts of wild type (WT) and PARL−/− HEK293 cells in the absence or presence of CCCP, o-phenanthroline, DCI, PMSF or cOmplete inhibitor mix. Samples were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. (e,f) Downregulation of (e) IMMPL1/2L or (f) HTRA2/Omi in PARL wild type (WT) and PARL−/− HEK293 cells for 72 h (or with control siRNA). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. ∗unspecific cross-reaction. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 4 Increased apoptotic resistance of PARL-deficient MEFs and HeLa cells.

(a) Total RNA was extracted from wild type (WT) and PARL−/− HeLa or HCT116 cells complemented with PARLFLAG or PARLS277A−FLAG and subjected to RT and real-time PCR analysis of human PARL mRNA. (n = 3 independent experiments; mean values ± SD). (b) Wild type (WT) and Parl−/− MEFs were treated for 8 h with staurosporine (STS, 1 μM), actinomycin D (Act D, 1 μM), or a combination of TNFα (10 ng ml−1) and CHX (10 μg ml−1). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. ∗unspecific cross-reaction. (c) Quantification of PARP cleavage of experiments in b (n = 3 independent experiments; mean values ± s.d.). P values were calculated by one-way ANOVA and indicated in figure. A P value of <0.05 was considered statistically significant. (d) Viability of wild type and Parl−/− MEFs (complemented with PARLFLAG when indicated) after incubation for 8 h with staurosporine (STS, 0.1 μM) (n = 3 independent experiments; mean values ± s.d.). Cell viability was assessed monitoring cellular ATP levels. P values were calculated by one-way ANOVA and indicated in figure. A P value of <0.05 was considered statistically significant. (e) Relative confluency of wild type (WT) and PARL−/− HeLa cells in the presence of actinomycin D (Act D, 1 μM). Images of the cells after 16 h Act D treatment are shown in the lower panel. Scale bars, 100 μm. Unprocessed original scans of blots are shown in Supplementary Fig. 9. Statistical source data can be found in Supplementary Table 3.

Supplementary Figure 5 The loss of PARL does not affect mitochondrial morphology.

(a) Immunofluorescence analysis of wild type (WT) and PARL−/− HEK293 cells using antibodies directed against ATP5β (green) and TOMM20 (red), and examined by confocal microscopy. Higher-magnification views of the boxed areas were shown. Scale bars, 10 μm or 2 μm (magnified image). (b,c) Immunofluorescence analysis of wild type (WT) and PARL−/− HCT116 cells (b), and HeLa cells (c) using antibodies directed against TOMM20 (green) and ATP5β (red), and examined by confocal microscopy. Higher magnifications of the boxed areas are shown. Scale bars, (b), 10 μm or 5 μm (magnified image); (c) 20 μm or 5 μm (magnified image). (d) Transmission electron microscopic analysis of wild type (WT) and PARL−/− HeLa cells (complemented with PARLFLAG when indicated). Scale bars, 1 μm. (e) BAX oligomerization in PARL-deficient HCT116 cells. Wild type (WT) and PARL−/− HCT116 cells were incubated with TNFα (20 ng ml−1) and CHX (10 μg ml−1) for 6 h. Mitochondria were isolated and analysed by BN-PAGE or SDS-PAGE, followed by immunoblotting with the indicated antibodies. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 6 Quantification of XIAP mRNA and protein in human and mouse cell lines.

(a) Immunoblot analysis of cell lysates of wild type (WT) or PARL−/− cells using the indicated antibodies. ∗unspecific cross-reaction. (b) Quantification of experiments in c (n = 3 independent experiments; mean values ± s.d.). P values were calculated by one-way ANOVA and indicated in figure. A P value of <0.05 was considered statistically significant. Statistical source data can be found in Supplementary Table 3. Unprocessed original scans of blots are shown in Supplementary Fig. 9.

Supplementary Figure 7 Depletion of XIAP restores apoptosis in PARL-deficient cells.

(a) After transfection with Xiap (or with control) siRNA for 48 h, wild type (WT) and PARL−/− (KO) HCT116 cells were incubated with TNFα (20 ng ml−1) and CHX (10 μg ml−1). Cell lysates were analysed by SDS-PAGE and immunoblotting. p, precursor; i, intermediate; m, mature form. ∗unspecific cross-reaction. (b) Quantification of PARP cleavage in experiments described in a (n = 3 independent experiments); mean values ± s.d.). P values were calculated by one-way ANOVA and indicated in figure. A P value of <0.05 was considered statistically significant. Unprocessed original scans of blots are shown in Supplementary Fig. 9. Statistical source data can be found in Supplementary Table 3.

Supplementary Figure 8 Cytosolic, mature Smac/DIABLO is sufficient to drive apoptosis in PARL−/− cells.

(a, left) Wild type (WT) and PARL−/− HCT116 cells were incubated with TNFα (20 ng ml−1) and CHX (10 μg ml−1) for 6 h in the absence or presence of the Smac/DIABLO mimetic BV6 (1 μM). (a, right) Wild type (WT) and PARL−/− HeLa cells were incubated with staurosporine (STS, 0.1 μM) for 6 h in the absence or presence of the Smac/DIABLO mimetic BV6 (2.5 μM). p, precursor; i, intermediate; m, mature form. ∗unspecific cross-reaction. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (b) Smac/DIABLO-deficient HeLa cells were generated by CRISPR/Cas9-mediated genome editing. Variants of Smac/DIABLO predicted to be expressed are shown. The red asterisk indicates the CRISPR/Cas9 targeted site. (c) Domain organization of Ubiquitin-Smac/DIABLO (Δ55)-DsRed and Ubiquitin-Smac/DIABLO (Δ59)-DsRed. (d) Two pathways for the release of pro-apoptotic proteins from mitochondria. BAX/BAK-induced apoptosis requires PARL-mediated processing and cytosolic translocation of Smac/DIABLO and the release of cytochrome c, which is facilitated by OPA1-dependent cristae remodeling and cardiolipin oxidation.

Supplementary information

Supplementary Information

Supplementary Information (PDF 19513 kb)

Supplementary Table 1

Supplementary Information (XLSX 1209 kb)

Supplementary Table 2

Supplementary Information (XLSX 2051 kb)

Supplementary Table 3

Supplementary Information (XLSX 106 kb)

Supplementary Table 4

Supplementary Information (XLSX 14 kb)

Supplementary Table 5

Supplementary Information (XLSX 31 kb)

Act D treated HeLa cells (related to Supplementary Fig. 4e).

Relative confluency of wild type (WT) HeLa cells in the presence of actinomycin D (Act D, 1 μM). Images were taken every 5 min for 16 h. (MOV 7923 kb)

Act D treated PARL−/− HeLa cells (related to Supplementary Fig. 4e).

Relative confluency of PARL−/− (KO) HeLa cells in the presence of actinomycin D (Act D, 1 μM). Images were taken every 5 min for 16 h. (MOV 10865 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Saita, S., Nolte, H., Fiedler, K. et al. PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis. Nat Cell Biol 19, 318–328 (2017). https://doi.org/10.1038/ncb3488

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3488

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing