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      Movement of Bax from the Cytosol to Mitochondria during Apoptosis

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          Abstract

          Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH 2 termini of Bax, Bcl-2, and Bcl-X L. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP–Bcl-2 and GFP–Bcl-X L had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP–Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP–Bax is a soluble protein, in contrast to organelle-bound GFP–Bcl-2. The diffuse localization of GFP–Bax did not change with coexpression of high levels of Bcl-2 or Bcl-X L. However, upon induction of apoptosis, GFP–Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP–Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP– Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.

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          Most cited references 41

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          Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.

          A cell-free system based on cytosols of normally growing cells is established that reproduces aspects of the apoptotic program in vitro. The apoptotic program is initiated by addition of dATP. Fractionation of cytosol yielded a 15 kDa protein that is required for in vitro apoptosis. The absorption spectrum and protein sequence revealed that this protein is cytochrome c. Elimination of cytochrome c from cytosol by immunodepletion, or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Adding back cytochrome c to the cytochrome c-depleted extracts restored their apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.
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            Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death.

            Bcl-2 protein is able to repress a number of apoptotic death programs. To investigate the mechanism of Bcl-2's effect, we examined whether Bcl-2 interacted with other proteins. We identified an associated 21 kd protein partner, Bax, that has extensive amino acid homology with Bcl-2, focused within highly conserved domains I and II. Bax is encoded by six exons and demonstrates a complex pattern of alternative RNA splicing that predicts a 21 kd membrane (alpha) and two forms of cytosolic protein (beta and gamma). Bax homodimerizes and forms heterodimers with Bcl-2 in vivo. Overexpressed Bax accelerates apoptotic death induced by cytokine deprivation in an IL-3-dependent cell line. Overexpressed Bax also counters the death repressor activity of Bcl-2. These data suggest a model in which the ratio of Bcl-2 to Bax determines survival or death following an apoptotic stimulus.
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              Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death.

              The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.
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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                1 December 1997
                : 139
                : 5
                : 1281-1292
                Affiliations
                [* ]Biochemistry Section, Surgical Neurology Branch, and []Light Microscopy Facility, Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
                Article
                2140220
                9382873
                Categories
                Article

                Cell biology

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