scholarly journals A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast

2001 ◽  
Vol 155 (5) ◽  
pp. 725-732 ◽  
Author(s):  
Evgeny V. Pavlov ◽  
Muriel Priault ◽  
Dawn Pietkiewicz ◽  
Emily H.-Y. Cheng ◽  
Bruno Antonsson ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Outer Membrane ◽  
Mammalian Cells ◽  
Pore Diameter ◽  
Mitochondrial Outer Membrane ◽  
Channel Activity ◽  
Patch Clamping ◽  
Membrane Pore ◽  
Outer Membranes ◽  
High Conductance

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is ∼4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis–induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

Effects of cytochrome c on the mitochondrial apoptosis-induced channel MAC

2004 ◽  
Vol 286 (5) ◽  
pp. C1109-C1117 ◽  
Author(s):  
Liang Guo ◽  
Dawn Pietkiewicz ◽  
Evgeny V. Pavlov ◽  
Sergey M. Grigoriev ◽  
John J. Kasianowicz ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Outer Membrane ◽  
Cell Types ◽  
Rnase A ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
Mitochondrial Apoptosis ◽  
Noise Type ◽  
Voltage Dependent

Recent studies indicate that cytochrome c is released early in apoptosis without loss of integrity of the mitochondrial outer membrane in some cell types. The high-conductance mitochondrial apoptosis-induced channel (MAC) forms in the outer membrane early in apoptosis of FL5.12 cells. Physiological (micromolar) levels of cytochrome c alter MAC activity, and these effects are referred to as types 1 and 2. Type 1 effects are consistent with a partitioning of cytochrome c into the pore of MAC and include a modest decrease in conductance that is dose and voltage dependent, reversible, and has an increase in noise. Type 2 effects may correspond to “plugging” of the pore or destabilization of the open state. Type 2 effects are a dose-dependent, voltage-independent, and irreversible decrease in conductance. MAC is a heterogeneous channel with variable conductance. Cytochrome c affects MAC in a pore size-dependent manner, with maximal effects of cytochrome c on MAC with conductance of 1.9–5.4 nS. The effects of cytochrome c, RNase A, and high salt on MAC indicate that size, rather than charge, is crucial. The effects of dextran molecules of various sizes indicate that the pore diameter of MAC is slightly larger than that of 17-kDa dextran, which should be sufficient to allow the passage of 12-kDa cytochrome c. These findings are consistent with the notion that MAC is the pore through which cytochrome c is released from mitochondria during apoptosis.

scholarly journals Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

2009 ◽  
Vol 20 (8) ◽  
pp. 2276-2285 ◽  
Author(s):  
Blanca Schafer ◽  
Joel Quispe ◽  
Vineet Choudhary ◽  
Jerry E. Chipuk ◽  
Teddy G. Ajero ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Pore Formation ◽  
Outer Membrane Proteins ◽  
Mitochondrial Outer Membrane ◽  
Outer Membranes ◽  
Mitochondrial Outer Membrane Permeabilization ◽  
In Vitro Systems ◽  
Large Round ◽  
Key Features

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the “pores” and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin.

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 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.

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.

Structure of crystalline VDAC, the voltage-gated channel in the mitochondrial outer membrane

1992 ◽  
Vol 50 (1) ◽  
pp. 440-441
Author(s):  
X.W. Guo ◽  
P.R. Smith ◽  
M. Radermacher ◽  
C.A. Mannella
Keyword(s):  
Neurospora Crassa ◽  
Outer Membrane ◽  
Repeat Unit ◽  
Mitochondrial Outer Membrane ◽  
Lattice Contraction ◽  
Outer Membranes ◽  
Voltage Gated ◽  
Lateral Contraction ◽  
Gated Channel ◽  
Projection Images

The voltage-gated, mitochondrial channel, VDAC, is formed by a 31-kDa outer-membrane polypeptide. Crystalline arrays of this channel, produced by phospholipase A2 treatment of Neurospora crassa mitochondrial outer membranes, consist of groups of 6 channels repeated on a parallelogram (“oblique”) lattice (a=13.3nm, b=11.5nm, γ = 109°). These membrane crystals are polymorphic, i.e. lateral contraction is triggered by a polyanion which also decreases VDAC’s gating potential. Projection images of unstained, frozen-hydrated VDAC arrays indicate that lattice contraction is accompanied by changes in the distribution of protein away from the hexameric repeat unit.

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