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. 2000 Nov 20;192(10):1391-402.
doi: 10.1084/jem.192.10.1391.

Granzyme B-mediated cytochrome c release is regulated by the Bcl-2 family members bid and Bax

J A Heibein  1 , I S Goping, M Barry, M J Pinkoski, G C Shore, D R Green, R C Bleackley
Affiliations

Granzyme B-mediated cytochrome c release is regulated by the Bcl-2 family members bid and Bax

J A Heibein et al. J Exp Med. .

Abstract

Cytotoxic T lymphocytes (CTLs) destroy target cells through a mechanism involving the exocytosis of cytolytic granule components including granzyme B (grB) and perforin, which have been shown to induce apoptosis through caspase activation. However, grB has also been linked with caspase-independent disruption of mitochondrial function. We show here that cytochrome c release requires the direct proteolytic cleavage of Bid by grB to generate a 14-kD grB-truncated product (gtBid) that translocates to mitochondria. In turn, gtBid recruits Bax to mitochondria through a caspase-independent mechanism where it becomes integrated into the membrane and induces cytochrome c release. Our results provide evidence for a new pathway by which CTLs inflict damage and explain the caspase-independent mechanism of mitochondrial dysfunction.

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Figures

Figure 1
Figure 1
GrB-mediated cytochrome c release from isolated mitochondria requires Bid. Purified Jurkat mitochondria were treated with grB and/or Jurkat cytosol for 30 min at room temperature in the presence or absence of 100 μM zVAD-fmk. The mitochondria/cytosol mixture was then centrifuged and the pellets (mitochondria) and supernatants (cytosol) were resolved on 15% polyacrylamide gels followed by transfer to nitrocellulose and immunoblotting for cytochrome c. M, mitochondria; C, cytosol; C (mock), mock immunodepleted cytosol; C (−Bid), cytosol immunodepleted for Bid. These data are representative of three independent experiments.
Figure 1
Figure 1
GrB-mediated cytochrome c release from isolated mitochondria requires Bid. Purified Jurkat mitochondria were treated with grB and/or Jurkat cytosol for 30 min at room temperature in the presence or absence of 100 μM zVAD-fmk. The mitochondria/cytosol mixture was then centrifuged and the pellets (mitochondria) and supernatants (cytosol) were resolved on 15% polyacrylamide gels followed by transfer to nitrocellulose and immunoblotting for cytochrome c. M, mitochondria; C, cytosol; C (mock), mock immunodepleted cytosol; C (−Bid), cytosol immunodepleted for Bid. These data are representative of three independent experiments.
Figure 3
Figure 3
Bax is integrated into the mitochondrial membrane in response to grB and Ad. Jurkat targets were treated either with nothing (control [ctrl]), grB alone, Ad alone, or grB and Ad (grB/Ad) in the presence or absence of 100 μM zVAD-fmk for 3 h at 37°C. After incubation, the plasma membranes of cells were rendered permeable with digitonin. The permeable cells were then centrifuged to separate the pellets (containing mitochondria) from the supernatants (cytosol). Pellets were subjected to an alkaline extraction with 0.1 M Na2CO3 for 30 min on ice followed by centrifugation. The pellets and the prealkaline extraction supernatants were resolved on 15% polyacrylamide gels. Proteins were transferred to nitrocellulose and the blots were probed with polyclonal anti-Bax antibodies. These data are representative of three independent experiments.
Figure 2
Figure 2
Cytosolic Bid is cleaved and translocates to mitochondria in response to grB and Ad. (A) Jurkat targets were treated with either anti-Fas antibodies (lanes 1–12) or grB and Ad (lanes 13–20) in the presence or absence of 100 μM zVAD-fmk. After incubation at 37°C for the times indicated, the plasma membranes of cells were rendered permeable with digitonin. The membrane fraction (containing mitochondria) was then separated from the cytosol by centrifugation. The pellets (mitochondrial fraction) were resolved on 15% polyacrylamide gels followed by transfer to nitrocellulose and immunoblotting for Bid using a rabbit polyclonal anti-Bid antiserum. (B) The supernatant blots from A were stripped and reprobed for cytochrome c using a monoclonal anti–cytochrome c antibody. These data are representative of three independent experiments.
Figure 4
Figure 4
GrB-mediated Bax integration into mitochondria requires Bid. Purified Jurkat mitochondria (M) were treated with grB and cytosol (C) for 30 min at room temperature in the presence or absence of 100 μM zVAD-fmk. Mitochondria were also treated with grB in the presence of mock-immunodepleted cytosol (C [mock]) and cytosol immunodepleted for Bid (C [−Bid]) as well as rBid. After a 30-min incubation at room temperature, the mitochondria were subjected to an alkaline extraction with 0.1 M Na2CO3 for 30 min on ice followed by centrifugation. The pellets were resuspended in buffer A and resolved on 15% polyacrylamide gels, followed by transfer to nitrocellulose and immunoblotting for Bax. These data are representative of three independent experiments.
Figure 5
Figure 5
Bcl-2 blocks grB-mediated Bax insertion and cytochrome c release but not Bid processing and translocation. (A) Vector-only transfected controls (neo) and Bcl-2–overexpressing Jurkats were treated with grB and Ad for the times indicated. After incubation, cells were fractionated and the proteins were resolved by SDS-PAGE on 15% gels and transferred to nitrocellulose. Cytosols were probed with a monoclonal anti–cytochrome c antibody. (B) The mitochondrial fraction from A was probed for Bid using a polyclonal anti-Bid antiserum. (C) Jurkat and Bcl-2–overexpressing Jurkat cells were treated with grB and Ad as indicated followed by subfractionation as described in Materials and Methods. The mitochondria were subjected to alkaline extraction followed by SDS-PAGE on 15% gels and transferred to nitrocellulose. Blots were probed with polyclonal anti-Bax antibodies. (D) Mitochondrial fractions from C were overexposed, scanned, and then analyzed with Image Gauge software to determine relative band intensities.
Figure 5
Figure 5
Bcl-2 blocks grB-mediated Bax insertion and cytochrome c release but not Bid processing and translocation. (A) Vector-only transfected controls (neo) and Bcl-2–overexpressing Jurkats were treated with grB and Ad for the times indicated. After incubation, cells were fractionated and the proteins were resolved by SDS-PAGE on 15% gels and transferred to nitrocellulose. Cytosols were probed with a monoclonal anti–cytochrome c antibody. (B) The mitochondrial fraction from A was probed for Bid using a polyclonal anti-Bid antiserum. (C) Jurkat and Bcl-2–overexpressing Jurkat cells were treated with grB and Ad as indicated followed by subfractionation as described in Materials and Methods. The mitochondria were subjected to alkaline extraction followed by SDS-PAGE on 15% gels and transferred to nitrocellulose. Blots were probed with polyclonal anti-Bax antibodies. (D) Mitochondrial fractions from C were overexposed, scanned, and then analyzed with Image Gauge software to determine relative band intensities.
Figure 6
Figure 6
Model for regulation by Bcl-2 family members of grB-mediated cytochrome c release. GrB may activate caspase-3 directly, or indirectly through the proteolytic activation of Bid. Bid is cleaved to generate gtBid that translocates to mitochondria where it recruits Bax through a mechanism antagonized by Bcl-2. Bax becomes integrated into the mitochondrial membrane and induces cytochrome c release. Cytochrome c acts as a cofactor with Apaf-1, caspase-9, and ATP/dATP to foster the proteolytic activation of caspase-3. ΔΨm, inner mitochondrial membrane potential.
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