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Bax directly induces release of cytochrome c from isolated mitochondria - PubMed

  • ️Thu Jan 01 1998

Bax directly induces release of cytochrome c from isolated mitochondria

J M Jürgensmeier et al. Proc Natl Acad Sci U S A. 1998.

Abstract

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.

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Figures

Figure 1
Figure 1

Bax induces Cyt c release from isolated mitochondria. In A, mitochondria were incubated for 1 hr at 30°C with 1 μM Bax, 150 μM Ca2+, both, or neither of these reagents. Mitochondria were then pelleted by centrifugation, and the resulting supernatants were subjected to SDS/PAGE immunoblot analysis by using an anti-Cyt c antibody. Complete pelleting of mitochondria and input of equivalent amount of mitochondria was verified in every experiment by immunoblot analysis of the pellets and supernatant fractions by using an antibody to the integral inner membrane protein F1-β-ATPase (lanes 7 and 8 and data not shown). As a control, mitochondria were also treated with 0.2% (vol/vol) Triton X-100, which released all Cyt c from these organelles. Data are representative of over 12 experiments performed with 3 independent preparations of Bax protein. In B, 293T cells were transfected with 5 μg of pcDNA3 or pcDNA3-Bax. Cells were harvested after 1 day, processed to obtain cytosolic and organellar (pellet) subfractions, and aliquots (25 μg total protein) were analyzed by SDS/PAGE by using antibodies specific for Cyt c, β-actin (cytosolic protein) and F1-β-ATPase (mitochondrial matrix protein). No F1-β-ATPase was detected in the cytosolic fraction (data not shown). In C, mitochondria were similarly incubated with various concentrations of recombinant Bax protein and Cyt c release was measured by immunoblotting (representative of two experiments). In D, mitochondria were incubated with 1 μM Bax protein, 50 μM BH3 peptide, both, or neither of these reagents. Cyt c release was measured 1 hr later as above (n = 2).

Figure 2
Figure 2

Bax induces release of mitochondrial factors that trigger processing and activation of cytosolic caspases. In A, supernatants from mitochondria that had been incubated for 1 hr with either 1 μM Bax or diluent control (C) were added to purified cytosol. Alternatively, 1 μM Bax or diluent control were added directly to cytosol. After incubation at 30°C for 0.5 hr, caspase activity was measured by hydrolysis of DEVD-AFC. Data represent micromolar amount of AFC released per time, where samples are normalized for total cytosolic protein content. In B, mitochondria were added to cytosolic extracts with or without 1 μM Bax. After incubation at 30°C for 1 hr, the mitochondria were pelleted by centrifugation and caspase activity was measured in the resulting supernatant. In C, an aliquot of cytosolic fractions was subjected to SDS/PAGE immunoblot assay by using anti-Cyt c antibody. As an additional control (Left), cytosol was treated with 10 μM Cyt c, as described (25). Closed and open arrows indicate the unprocessed zymogen of caspase-3 and the processed form associated with active enzyme, respectively.

Figure 3
Figure 3

Bax does not induce mitochondrial permeability transition. Mitochondria were placed into a cuvette and absorbance was measured by using a double-beam spectrophotometer at 520 nm. In some cases, samples were pretreated with 10 μM CsA. Either 1 μM Bax or 150 μM Ca2+ was added (arrow) and optical density was monitored for the ensuing 75 min (data representative of five experiments).

Figure 4
Figure 4

Effects of Bcl-XL, CsA, and zVAD-fmk on Bax-induced release of Cyt c from mitochondria. Mitochondria were treated (sometimes in duplicate) with 1 μM Bax or diluent control (C), with or without other reagents, and Cyt c release into supernatants was measured 1 hr later by immunoblotting. In A, 12 μM Bcl-XL was added to some samples simultaneously with Bax, as indicated. In B, 10 μM CsA or 10 μM zVAD-fmk was added 5 min prior to Bax. In C, the mitochondrial pellets were analyzed in addition to the supernatants (25 μg). Bax binding to mitochondria was determined by SDS/PAGE immunoblot analysis by using an anti-Bax antibody.

Figure 5
Figure 5

Bcl-XL inhibits processing of caspase-3 induced by Bax plus mitochondria. Mitochondria were added to cytosolic extracts followed by addition of 1 μM Bax or diluent control (C), with or without various concentrations of Bcl-XL protein or zVAD-fmk. After removing mitochondria by centrifugation, aliquots of the cytosol were analyzed by SDS/PAGE immunoblotting with antibodies specific for either caspase-3 (Upper) or Cyt c (Lower). The positions of the unprocessed pro-form of caspase-3 (filled arrow) and processed ≈17-kDa large subunit of caspase-3 (open arrow) are shown. The slightly slower migrating band represents a partially processed form of the large subunit of caspase-3 (25, 51).

Figure 6
Figure 6

Effects of Bcl-XL and CsA on caspase activation induced by Bax plus mitochondria. Cytosol was treated at 30°C for 1 hr with mitochondria, 1 μM Bax, diluent control (C), or various combinations of these in the presence or absence of (A) 3–12 μM Bcl-XL or (B) 10 μM CsA. Caspase activity was measured by DEVD-AFC hydrolysis.

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