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 mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates.
  
  
  RESULTS
  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
  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.
authors.affiliation: Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.
authors.forename: Hyunmi
authors.initials: H
authors.lastname: Lee
authors.forename: Jimmy A
authors.initials: JA
authors.lastname: Rotolo
authors.forename: Judith
authors.initials: J
authors.lastname: Mesicek
authors.forename: Tuula
authors.initials: T
authors.lastname: Penate-Medina
authors.forename: Andreas
authors.initials: A
authors.lastname: Rimner
authors.forename: Wen-Chieh
authors.initials: WC
authors.lastname: Liao
authors.forename: Xianglei
authors.initials: X
authors.lastname: Yin
authors.forename: Govind
authors.initials: G
authors.lastname: Ragupathi
authors.forename: Desiree
authors.initials: D
authors.lastname: Ehleiter
authors.forename: Erich
authors.initials: E
authors.lastname: Gulbins
authors.forename: Dayong
authors.initials: D
authors.lastname: Zhai
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
authors.forename: Adriana
authors.initials: A
authors.lastname: Haimovitz-Friedman
authors.forename: Zvi
authors.initials: Z
authors.lastname: Fuks
authors.forename: Richard
authors.initials: R
authors.lastname: Kolesnick
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002417:Cattle; D002518:Ceramides / Q000378:metabolism*; D037341:Fumonisins / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D008970:Molecular Weight; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010539:Permeability / Q000187:drug effects / Q000528:radiation effects; D020836:Protein Structure, Quaternary; D011839:Radiation, Ionizing; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002518:Ceramides; D037341:Fumonisins; D051028:bcl-2-Associated X Protein; C056933:fumonisin B1; D010088:Oxidoreductases; C108138:dihydroceramide desaturase