The Mitochondrial F0F1-ATPase Proton Pump Is Required for Function of the Proapoptotic Protein Bax in Yeast and Mammalian Cells

Abstract

The proapoptotic mammalian protein Bax associates with mitochondrial membranes and confers a lethal phenotype when expressed in yeast. By generating Bax-resistant mutant yeast and using classical complementation cloning methods, subunits of the mitochondrial F0F1-ATPase proton pump were determined to be critical for Bax-mediated killing in S. cerevisiae. A pharmacological inhibitor of the proton pump, oligomycin, also partially abrogated the cytotoxic actions of Bax in yeast. In mammalian cells, oligomycin also inhibited Bax-induced apoptosis and activation of cell death proteases. The findings imply that an intact F0F1-ATPase in the inner membrane of mitochondria is necessary for optimal function of Bax in both yeast and mammalian cells.

A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed

“…Immunoblots of mitochondrial and cytosolic fractions revealed that the small molecules that afforded protection in Figure 2G were able to delay Raptinal-induced cytochrome c release and subsequent caspase-9 activation (Figure 2H). These results are consistent with previous suggestions that proton transport may be required for Bax-mediated mitochondrial outer membrane permeabilization (Matsuyama et al, 1998). Utilizing a variety of small molecule modulators in combination with rapid induction of the intrinsic pathway by Raptinal has helped to further validate the importance of voltage dependent anion channel, coupled mitochondrial respiration, electron transport chain function and ATP synthase activity in apoptosis induction.…”

A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed

“…Immunoblots of mitochondrial and cytosolic fractions revealed that the small molecules that afforded protection in Figure 2G were able to delay Raptinal-induced cytochrome c release and subsequent caspase-9 activation (Figure 2H). These results are consistent with previous suggestions that proton transport may be required for Bax-mediated mitochondrial outer membrane permeabilization (Matsuyama et al, 1998). Utilizing a variety of small molecule modulators in combination with rapid induction of the intrinsic pathway by Raptinal has helped to further validate the importance of voltage dependent anion channel, coupled mitochondrial respiration, electron transport chain function and ATP synthase activity in apoptosis induction.…”

Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H + -ATP synthase

“…The activity of the H + -ATP synthase is required for efficient execution of cell death in C9 liver cells. Consistent with previous findings (19,20,35) we observed that 3h pre-incubation of C9 cells with oligomycin (OL) significantly delayed the cell death response to staurosporine (STS) as assessed both by the analysis of cell nuclei morphology (Fig. 1A) and intracellular DNA content (Fig.…”

The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant

“…Consistent with published results 32 , oligomycin added at the same time as treatment with ultraviolet light reduced the number of cells that released cytochrome-c-GFP at 8 h by about half (data not shown). The duration of cytochrome-c-GFP release in cells treated with oligomycin, zVAD-fmk and ultraviolet light was not significantly different from that in controls treated with ultraviolet light Cyto.…”

A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed

“…Immunoblots of mitochondrial and cytosolic fractions revealed that the small molecules that afforded protection in Figure 2G were able to delay Raptinal-induced cytochrome c release and subsequent caspase-9 activation (Figure 2H). These results are consistent with previous suggestions that proton transport may be required for Bax-mediated mitochondrial outer membrane permeabilization (Matsuyama et al, 1998). Utilizing a variety of small molecule modulators in combination with rapid induction of the intrinsic pathway by Raptinal has helped to further validate the importance of voltage dependent anion channel, coupled mitochondrial respiration, electron transport chain function and ATP synthase activity in apoptosis induction.…”

Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H + -ATP synthase

“…The activity of the H + -ATP synthase is required for efficient execution of cell death in C9 liver cells. Consistent with previous findings (19,20,35) we observed that 3h pre-incubation of C9 cells with oligomycin (OL) significantly delayed the cell death response to staurosporine (STS) as assessed both by the analysis of cell nuclei morphology (Fig. 1A) and intracellular DNA content (Fig.…”

The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant

“…Consistent with published results 32 , oligomycin added at the same time as treatment with ultraviolet light reduced the number of cells that released cytochrome-c-GFP at 8 h by about half (data not shown). The duration of cytochrome-c-GFP release in cells treated with oligomycin, zVAD-fmk and ultraviolet light was not significantly different from that in controls treated with ultraviolet light Cyto.…”

A Small Molecule that Induces Intrinsic Pathway Apoptosis with Unparalleled Speed

“…Immunoblots of mitochondrial and cytosolic fractions revealed that the small molecules that afforded protection in Figure 2G were able to delay Raptinal-induced cytochrome c release and subsequent caspase-9 activation (Figure 2H). These results are consistent with previous suggestions that proton transport may be required for Bax-mediated mitochondrial outer membrane permeabilization (Matsuyama et al, 1998). Utilizing a variety of small molecule modulators in combination with rapid induction of the intrinsic pathway by Raptinal has helped to further validate the importance of voltage dependent anion channel, coupled mitochondrial respiration, electron transport chain function and ATP synthase activity in apoptosis induction.…”

Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H + -ATP synthase

“…The activity of the H + -ATP synthase is required for efficient execution of cell death in C9 liver cells. Consistent with previous findings (19,20,35) we observed that 3h pre-incubation of C9 cells with oligomycin (OL) significantly delayed the cell death response to staurosporine (STS) as assessed both by the analysis of cell nuclei morphology (Fig. 1A) and intracellular DNA content (Fig.…”

The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant

“…Consistent with published results 32 , oligomycin added at the same time as treatment with ultraviolet light reduced the number of cells that released cytochrome-c-GFP at 8 h by about half (data not shown). The duration of cytochrome-c-GFP release in cells treated with oligomycin, zVAD-fmk and ultraviolet light was not significantly different from that in controls treated with ultraviolet light Cyto.…”