Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of 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 Mg(2+), 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 (A(520)) 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.
Doran
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Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
Halestrap
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D000263:Adenylate Kinase / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D003911:Dextrans / Q000494:pharmacology; D019281:Dimerization; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D010455:Peptides / Q000494:pharmacology; D010539:Permeability; D018272:Porins / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017208:Rats, Wistar; D012402:Rotenone / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology; D050994:Voltage-Dependent Anion Channels; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D003574:Cytochrome c Group; D003911:Dextrans; D010455:Peptides; D018272:Porins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050994:Voltage-Dependent Anion Channels; D051028:bcl-2-Associated X Protein; D012402:Rotenone; C032456:antimycin; C017645:polyaspartate; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D013395:Sucrose; D000968:Antimycin A; D011189:Potassium Chloride; D016572:Cyclosporine; D000263:Adenylate Kinase