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.

Abbreviations used: ANT, adenine nucleotide translocase; GFP, green fluorescent protein; RFP, red fluorescent protein; t-Bid, truncated Bid; VDAC, voltage-dependent anion channel.

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