Abstract
BackgroundEvidence 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 mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates.Methodology/Principal FindingsMCRMs, 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/SignificanceOur 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.
Highlights
We recently reported that the ceramide is an essential element in the mitochondrial phase of apoptosis in the C. elegans germline [1]
In agreement with previous studies [44], Bax insertion occurred without apparent translocation from cytosol, but rather from a Bax pool loosely attached to unirradiated HeLa mitochondria but not inserted into MOM [37,45]
This pool, which is readily removed by 100 mM Na2CO3 washing and does not spontaneously induce mitochondrial outer membrane permeabilization (MOMP) [45,46], represents the large majority of endogenous Bax co-isolated with HeLa mitochondria
Summary
We recently reported that the ceramide is an essential element in the mitochondrial phase of apoptosis in the C. elegans germline [1]. A complementary pathway involving mitochondrial outer membrane permeabilization (MOMP) initiates the commitment phase of the apoptotic response. MOMP is regulated either by opening of the inner mitochondrial membrane permeability transition pore or by insertion of pro-apoptotic Bcl-2 family members into the MOM. Bax contains three Bcl-2 homology domains (BH1-3) and a C-terminal transmembrane (TM) domain, arranged in 9 a-helices [3,4] This spatial configuration is reminiscent of the structure of a-helical pore-forming toxins, including diphtheria toxin, colicins and dendotoxin [4]. Evidence indicates that Bax functions as a ‘‘lipidic’’ pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. We show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previouslyunrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates
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