scholarly journals Biphasic translocation of Bax to mitochondria

2002 ◽  
Vol 367 (1) ◽  
pp. 169-178 ◽  
Author(s):  
Michela CAPANO ◽  
Martin CROMPTON
Keyword(s):  
Green Fluorescent Protein ◽  
Cytochrome C ◽  
Fluorescent Protein ◽  
Adenine Nucleotide ◽  
Phase 1 ◽  
Mitochondrial Outer Membrane ◽  
Second Phase ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release ◽  
Green Fluorescent

Using green fluorescent protein-tagged Bax, we demonstrate that Bax is sequestered from the cytosol of cardiomyocytes in two distinct phases following the induction of apoptosis with staurosporine. In the first phase, lasting several hours, Bax removal from the cytosol was relatively small. In the second phase, Bax was very largely removed from the cytosol and sequestered into large aggregates associated with the mitochondria. To test which of the phases involved cytochrome c release, cells were transfected with a red fluorescent protein—cytochrome c fusion. The cytochrome c fusion protein was accumulated by mitochondria of healthy cells and was released by staurosporine in phase 1. When green fluorescent protein—Bax was immunoprecipitated from extracts of cells in phase 1 and phase 2, the voltage-dependent anion channel (mitochondrial outer membrane) and the adenine nucleotide translocase (mitochondrial inner membrane) were also precipitated. These data support a two-phase model of Bax translocation in which Bax targets the mitochondrial intermembrane contact sites and releases cytochrome c in the first phase, and is then packaged into large aggregates on mitochondria in the second.

scholarly journals The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in ΔpH formation across peroxisomal membranes

2004 ◽  
Vol 381 (3) ◽  
pp. 581-585 ◽  
Author(s):  
Francesco M. LASORSA ◽  
Pasquale SCARCIA ◽  
Ralf ERDMANN ◽  
Ferdinando PALMIERI ◽  
Hanspeter ROTTENSTEINER ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Imaging ◽  
Fluorescent Protein ◽  
Adenine Nucleotide ◽  
Ph Sensitive ◽  
Peroxisomal Membrane ◽  
Transport Modes ◽  
Proton Movement ◽  
Green Fluorescent

The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a ΔpH across the peroxisomal membrane, acidic inside.

scholarly journals Death-signal-induced relocalization of cyclin-dependent kinase 11 to mitochondria

2005 ◽  
Vol 392 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Yongmei Feng ◽  
Maria E. Ariza ◽  
Anne-Christine Goulet ◽  
Jiaqi Shi ◽  
Mark A. Nelson
Keyword(s):  
Cytochrome C ◽  
Fas Ligand ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Cyclin Dependent Kinase ◽  
Cytochrome C Release ◽  
Ligand Interaction ◽  
Protein Levels ◽  
Induced Apoptosis ◽  
Green Fluorescent

Fas receptor–Fas ligand interaction appears to be important in carcinogenesis, tumour outgrowth and metastasis. Emerging evidence suggests that CDK11 (cyclin-dependent kinase 11) plays a role in apoptosis and melanoma development. Here, we show that CDK11p110 protein kinase was cleaved after induction of apoptosis by Fas. The N-terminal portion of CDK11p110, CDK11p60, was translocated from the nucleus to the mitochondria. The targeting of CDK11p60 to mitochondria occurred as early as 12 h after treatment. Overexpression of EGFP (enhanced green fluorescent protein)-tagged CDK11p60 could partially break down the mitochondrial membrane potential, induce cytochrome c release and promote apoptosis. Reduction of endogenous CDK11p110 protein levels with siRNA (small interfering RNA) resulted in the suppression of both cytochrome c release and apoptosis. In addition, subcellular fractionation studies of Fas-mediated apoptosis demonstrated that CDK11p60 was associated with the mitochondrial import motor, mitochondrial heat shock protein 70. Taken together, our data suggest that CDK11p60 can contribute to apoptosis by direct signalling at the mitochondria, thereby amplifying Fas-induced apoptosis in melanoma cells.

scholarly journals Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites

2000 ◽  
Vol 348 (2) ◽  
pp. 343-350 ◽  
Author(s):  
Elena DORAN ◽  
Andrew P. HALESTRAP
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Adenine Nucleotide ◽  
Permeability Transition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release ◽  
Membrane Rupture ◽  
Contact Sites

Percoll-purified rat liver mitochondria were shown to contain BAX dimer and rapidly (< 2 min) release 5-10% of their cytochrome c when incubated in a standard KCl incubation medium under energized conditions. This release was not accompanied by release of adenylate kinase (AK), another intermembrane protein, and was not inhibited by Mg2+, dATP, inhibitors of the permeability transition or ligands of the peripheral benzodiazepine receptor. However, release was greatly reduced by the presence of 5% (w/v) dextran (40 kDa), which caused a decrease in the light scattering (A520) of mitochondrial suspensions. Dextran also inhibited both mitochondrial oxidation of exogenous ferrocytochrome c in the presence of rotenone and antimycin, and respiratory-chain-driven reduction of exogenous ferricytochrome c. Hypo-osmotic medium or digitonin treatment of mitochondria caused a large additional release of both cytochrome c and AK that was not blocked by dextran. Polyaspartate, which stabilizes the low conductance state of the voltage-dependent anion channel (VDAC), increased cytochrome c release. VDAC and BAX are both found at the contact sites between the inner and outer membranes and dextran is known to stabilize these contact sites in isolated mitochondria. Thus our data suggest that regulation of a specific permeability pathway for cytochrome c may be mediated by changes in protein-protein interactions within contact sites. The adenine nucleotide translocase is known to bind to VDAC and thus provides an additional link between the specific cytochrome c release pathway and the permeability transition.

scholarly journals VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release

2001 ◽  
Vol 155 (6) ◽  
pp. 1003-1016 ◽  
Author(s):  
Muniswamy Madesh ◽  
György Hajnóczky
Keyword(s):  
Cytochrome C ◽  
Hepg2 Cells ◽  
Mitochondrial Membrane ◽  
Mitochondrial Permeability Transition ◽  
Anion Channel ◽  
Permeability Transition ◽  
Mitochondrial Outer Membrane ◽  
Inner Mitochondrial Membrane ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release

Enhanced formation of reactive oxygen species (ROS), superoxide (O2·−), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2·− elicited rapid and massive cytochrome c release (CCR), whereas H2O2 failed to induce any release. Both O2·− and H2O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2·−-induced CCR. Furthermore, O2·− alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2·−-induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2·− exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2·−-induced CCR did not depend on Bax translocation to mitochondria. O2·−-induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2·− triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.

scholarly journals Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein

2002 ◽  
Vol 368 (3) ◽  
pp. 915-921 ◽  
Author(s):  
Xavier ROUCOU ◽  
Sylvie MONTESSUIT ◽  
Bruno ANTONSSON ◽  
Jean-Claude MARTINOU
Keyword(s):  
Cytochrome C ◽  
Plasma Membranes ◽  
Mitochondrial Protein ◽  
Mitochondrial Outer Membrane ◽  
Caspase 8 ◽  
Mitochondrial Membranes ◽  
Voltage Dependent Anion Channel ◽  
Cytochrome C Release ◽  
Isolated Mitochondria

In response to various apoptotic stimuli, Bax, a pro-apoptotic member of the Bcl-2 family, is oligomerized and permeabilizes the mitochondrial outer membrane to apoptogenic factors, including cytochrome c. Bax oligomerization can also be induced by incubating isolated mitochondria containing endogenous Bax with recombinant tBid (caspase-8-cleaved Bid) in vitro. The mechanism by which Bax oligomerizes under these conditions is still unknown. To address this question, recombinant human full-length Bax was purified as a monomeric protein. Bax failed to oligomerize spontaneously in isolated mitochondria or in liposomes composed of either cardiolipin or lipids extracted from mitochondria. However, in the presence of tBid, the protein formed large complexes in mitochondrial membranes and induced the release of cytochrome c. tBid also induced Bax oligomerization in isolated mitochondrial outer membranes, but not in other membranes, such as plasma membranes or microsomes. Moreover, tBid-induced Bax oligomerization was inhibited when mitochondria were pretreated with protease K. The presence of the voltage-dependent anion channel was not required either for Bax oligomerization or for Bax-induced cytochrome c release. Finally, Bax oligomerization was reconstituted in proteoliposomes made from mitochondrial membrane proteins. These findings imply that tBid is necessary but not sufficient for Bax oligomerization; a mitochondrial protein is also required.

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