Mitochondrial ceramide-rich macrodomains functionalize bax upon irradiation Journal Article
| Authors: | Lee, H.; Rotolo, J. A.; Mesicek, J.; Penate Medina, T.; Rimner, A.; Liao, W. C.; Yin, X.; Ragupathi, G.; Ehleiter, D.; Gulbins, E.; Zhai, D.; Reed, J. C.; Haimovitz-Friedman, A.; Fuks, Z.; Kolesnick, R. |
| Article Title: | Mitochondrial ceramide-rich macrodomains functionalize bax upon irradiation |
| Abstract: | Background: Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized <underline>m</underline>itochondrial <underline>c</underline>eramide-<underline>r</underline>ich <underline>m</underline>acrodomain (MCRM), which we visualize and isolate, into which Bax integrates. Methodology/Principal Findings: MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax. Conclusions/Significance: Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo. © 2011 Lee et al. |
| Keywords: | controlled study; nonhuman; protein function; protein localization; mammalia; confocal microscopy; hela cell; irradiation; cell isolation; protein secretion; caenorhabditis elegans; cellular stress response; cell level; molecular weight; mitochondrion; oligomerization; cell lysate; protein synthesis inhibition; protein isolation; ceramide; sphingosine acyltransferase; lipid composition; protein lipid interaction; cytochrome c; protein bax; mitochondrial permeability; membrane structure; fumonisin b1; liver mitochondrion |
| Journal Title: | PLoS ONE |
| Volume: | 6 |
| Issue: | 6 |
| ISSN: | 1932-6203 |
| Publisher: | Public Library of Science |
| Date Published: | 2011-01-01 |
| Start Page: | e19783 |
| Language: | English |
| DOI: | 10.1371/journal.pone.0019783 |
| PROVIDER: | scopus |
| PMCID: | PMC3113798 |
| PUBMED: | 21695182 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 17 August 2011" - "Art. No.: e19783" - "Source: Scopus" |
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