scholarly journals The Pro-Apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane That Is Enhanced by Cytosol

1999 ◽  
Vol 147 (4) ◽  
pp. 809-822 ◽  
Author(s):  
Ruth M. Kluck ◽  
Mauro Degli Esposti ◽  
Guy Perkins ◽  
Christian Renken ◽  
Tomomi Kuwana ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Cytochrome C Release ◽  
Free System ◽  
Xenopus Egg ◽  
Apoptotic Proteins ◽  
Outer Membrane Permeability

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP

2007 ◽  
Vol 292 (4) ◽  
pp. C1388-C1397 ◽  
Author(s):  
Wenzhi Tan ◽  
Johnathan C. Lai ◽  
Paul Miller ◽  
C. A. Stein ◽  
Marco Colombini
Keyword(s):  
Cytochrome C ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Cytochrome C Release ◽  
Voltage Dependent ◽  
Selective Channel ◽  
Vdac Channels ◽  
Outer Membrane Permeability ◽  
Apoptotic Cascade

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane ( 21 ). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a Ki between 0.2 and 0.5 μM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 μM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 μM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.

scholarly journals The Mitochondrial TOM Complex Is Required for tBid/Bax-induced Cytochrome c Release

2007 ◽  
Vol 282 (38) ◽  
pp. 27633-27639 ◽  
Author(s):  
Martin Ott ◽  
Erik Norberg ◽  
Katharina M. Walter ◽  
Patrick Schreiner ◽  
Christian Kemper ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Outer Membrane ◽  
Recombinant Proteins ◽  
Mitochondrial Membrane ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Outer Mitochondrial Membrane ◽  
Cytochrome C Release ◽  
Unknown Mechanism

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.

scholarly journals Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli

2002 ◽  
Vol 159 (6) ◽  
pp. 923-929 ◽  
Author(s):  
Damien Arnoult ◽  
Philippe Parone ◽  
Jean-Claude Martinou ◽  
Bruno Antonsson ◽  
Jérôme Estaquier ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Outer Membrane ◽  
Actinomycin D ◽  
Membrane Permeabilization ◽  
Mitochondrial Outer Membrane ◽  
Simple Explanation ◽  
Cytochrome C Release ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
Mitochondrial Inner Membrane ◽  
Apoptosis Inducing Factor

Mitochondrial outer membrane permeabilization by proapoptotic Bcl-2 family proteins, such as Bax, plays a crucial role in apoptosis induction. However, whether this only causes the intracytosolic release of inducers of caspase-dependent death, such as cytochrome c, or also of caspase-independent death, such as apoptosis-inducing factor (AIF) remains unknown. Here, we show that on isolated mitochondria, Bax causes the release of cytochrome c, but not of AIF, and the association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization. In cells overexpressing Bax or treated either with the Bax- or Bak-dependent proapoptotic drugs staurosporine or actinomycin D, or with hydrogen peroxide, caspase inhibitors did not affect the intracytosolic translocation of cytochrome c, but prevented that of AIF. These results provide a paradigm for mitochondria-dependent death pathways in which AIF cannot substitute for caspase executioners because its intracytosolic release occurs downstream of that of cytochrome c.

scholarly journals Regulation of Respiration in Permeabilized Muscle Cells: Apparent KM for ADP Shows the Mitochondrial Outer Membrane Permeability

2013 ◽  
Vol 104 (2) ◽  
pp. 447a-448a
Author(s):  
Rafaela Bagur Quetglas ◽  
Minna Karu-Varikmaa ◽  
Kersti Tepp ◽  
Madis Metsis ◽  
Tuuli Kaambre ◽  
...  
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Muscle Cells ◽  
Mitochondrial Outer Membrane ◽  
Regulation Of Respiration ◽  
Outer Membrane Permeability ◽  
Permeabilized Muscle

scholarly journals Resistance to Granzyme B-mediated Cytochrome c Release in Bak-deficient Cells

2001 ◽  
Vol 194 (9) ◽  
pp. 1325-1338 ◽  
Author(s):  
Gui-Qiang Wang ◽  
Eva Wieckowski ◽  
Leslie A. Goldstein ◽  
Brian R. Gastman ◽  
Asaf Rabinovitz ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Outer Membrane ◽  
Knockout Mice ◽  
Granzyme B ◽  
Membrane Permeabilization ◽  
Mitochondrial Outer Membrane ◽  
Target Cells ◽  
Cytochrome C Release ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
S 100

Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

Subcellular Distribution and Redistribution of Bcl-2 Family Proteins in Human Leukemia Cells Undergoing Apoptosis

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2353-2359 ◽  
Author(s):  
Li Jia ◽  
Marion G. Macey ◽  
Yuzhi Yin ◽  
Adrian C. Newland ◽  
Stephen M. Kelsey
Keyword(s):  
Cytochrome C ◽  
Cell Lines ◽  
Western Blotting ◽  
Leukemic Cells ◽  
Mitochondrial Outer Membrane ◽  
Human Leukemia ◽  
Cell Level ◽  
Cytochrome C Release ◽  
Free System ◽  
Whole Cell

It has been suggested that the ratio of Bcl-2 family proapoptotic proteins to antiapoptotic proteins determines the sensitivity of leukemic cells to apoptosis. However, it is believed that Bcl-2 family proteins exert their function on apoptosis only when they target to the mitochondrial outer membrane. The vinblastine-resistant T-lymphoblastic leukemic cell line CEM/VLB100 has increased sensitivity to tumor necrosis factor- (TNF-)–induced cytochrome crelease, mitochondrial respiratory inhibition, and consequently apoptosis, compared with parental CEM cells. However, there was no difference between the two cell lines in the expression of Bcl-2 family proteins Bcl-2, Bcl-XL, Bcl-XS, Bad, and Bax at the whole cell level, as analyzed by Western blotting. Bcl-2 mainly located to mitochondria and light membrane as a membrane-bound protein, whereas Bcl-XL was located in both mitochondria and cytosol. Similar levels of both Bcl-2 and Bcl-XL were present in the resting mitochondria of the two cell lines. Although the proapoptotic proteins Bcl-XS, Bad, and Bax were mainly located in the cytosol, CEM/VLB100 mitochondria expressed higher levels of these proapoptotic proteins. Subcellular redistribution of the Bcl-2 family proteins was detected in a cell-free system by both Western blotting and flow cytometry after exposure to TNF-. The levels of Bcl-2 family proteins were not altered at the whole cell level by TNF-. However, after exposure to TNF-, Bax, Bad, and Bcl-XS translocated from the cytosol to the mitochondria of both cell lines. An increase in Bcl-2 levels was observed in CEM mitochondria, which showed resistance to TNF-–induced cytochrome c release. By contrast, decreased mitochondrial Bcl-2 was observed in CEM/VLB100 cells, which released cytochrome c from the mitochondria and underwent apoptosis as detected by fluorescence microscopy. We conclude that mitochondrial levels of Bcl-2 family proteins may determine the sensitivity of leukemic cells to apoptosis and that, furthermore, these levels may change rapidly after exposure of cells to toxic stimuli.

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