Bax directly induces release of cytochrome c from isolated mitochondria.
Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induced caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induced caspase processing and activation. Recombinant Bcl-XL protein abrogated Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP had no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria.
Jürgensmeier
J M
JM
Program on Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Xie
Z
Z
Deveraux
Q
Q
Ellerby
L
L
Bredesen
D
D
Reed
J C
JC
D000818:Animals; D017209:Apoptosis; D002474:Cell-Free System; D016572:Cyclosporine / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008930:Mitochondria, Liver / Q000187:drug effects*; D008933:Mitochondrial Swelling / Q000187:drug effects; D010539:Permeability; D011518:Proto-Oncogene Proteins / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; C494827:Bax protein, mouse; C494828:Bax protein, rat; C494812:Bcl2l1 protein, mouse; C494813:Bcl2l1 protein, rat; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D016572:Cyclosporine; D003546:Cysteine Endopeptidases