Biphasic translocation of Bax to mitochondria.
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.
Capano
Michela
M
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
Crompton
Martin
M
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006801:Humans; D007473:Ion Channels; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology; D011233:Precipitin Tests; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D051028:bcl-2-Associated X Protein; D000097573:Red Fluorescent Protein
D003574:Cytochrome c Group; D049452:Green Fluorescent Proteins; D007473:Ion Channels; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D019311:Staurosporine; D051028:bcl-2-Associated X Protein; D000097573:Red Fluorescent Protein; C494826:BAX protein, human; C494828:Bax protein, rat