Abstract
Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, BimEL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.
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Abbreviations
- BafA1:
-
bafilomycin A1
- CCCP:
-
carbonyl cyanide m-chlorophenyl hydrazine
- FRAP:
-
fluorescence recovery after photobleaching
- GFP:
-
green fluorescent protein
- IAP:
-
inhibitor of apoptosis
- IF:
-
immunofluorescence
- LIR:
-
LC3-interacting region
- MIU:
-
motif interacting with ubiquitin
- MDVs:
-
mitochondrial-derived vesicles
- MOMP:
-
mitochondrial outer membrane permeabilization
- OMM:
-
outer mitochondrial membrane
- RING:
-
really interesting new gene
- RFP:
-
red fluorescent protein
- ROI:
-
region of interest
- Smac:
-
second mitochondria-derived activator of caspases
- XIAP:
-
X-chromosome-linked IAP
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Acknowledgements
For constructs, we would like to acknowledge Drs. B van Deurs (Copenhagen University), R Youle (Addgene; plasmid 23956), B Fang (Addgene; plasmid 10952), N Dantuma (Addgene; plasmid 11928) and R Cohen (Addgene; 35527). We thank Dr. C Ackermann of the Nikon Imaging Center Heidelberg for assistance with N-SIM. This work was supported through SBCancer within the Helmholtz Alliance on Systems Biology funded by the Initiative and Networking Fund of the Helmholtz Association (NRB); and the e:Bio Grant No. 0316191 (LysoSys) of the Federal Ministry of Education and Research (BMBF), Germany (AH-B). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Author Contributions
AH-B and NRB conceived and designed the study, performed experiments, analyzed the data and prepared figures. JKL performed electron microscopy experiments. SCC contributed intellectually and edited the manuscript. AH-B and NRB wrote the manuscript.
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Hamacher-Brady, A., Choe, S., Krijnse-Locker, J. et al. Intramitochondrial recruitment of endolysosomes mediates Smac degradation and constitutes a novel intrinsic apoptosis antagonizing function of XIAP E3 ligase. Cell Death Differ 21, 1862–1876 (2014). https://doi.org/10.1038/cdd.2014.101
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DOI: https://doi.org/10.1038/cdd.2014.101
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