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Bcl-X(L) specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum.

Klee M, Pimentel-Muiños FX - J. Cell Biol. (2005)

Bottom Line: Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant.These results reveal a previously unidentified role of Bak in regulating reticular conformation.Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, 37007 Spain.

ABSTRACT
Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-X(L) provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

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Inhibition of Bak-induced apoptosis is not sufficient to reveal cytoplasmic vacuolization. (A) Quantitation of vacuolated cells after transfection with Bak in the presence of different apoptotic inhibitors. 293T cells were transfected with a mix of the indicated plasmids and a GFP-expressing plasmid. C9.DN and p35 denote a dominant-negative version of caspase-9 and the viral caspase inhibitor p35, respectively. When indicated, the pan-caspase inhibitor z-VAD.fmk (100 μM) was added 1 h after transfection. Cytoplasmic vacuolization was scored as in Fig. 1 B. (B) Death inhibitors unable to reveal vacuolization block Bak apoptosis to the same extent as Bcl-XL. 293T cells were transfected with a mix of the indicated expression plasmids. Z-VAD.fmk (100 μM) was added 1 h after transfection. Cells were lysed 24 h after transfection. Shown are blots probed with anti-PARP (top) and anti-AU (bottom) antibodies. The latter confirms equal transfection efficiency. (C) Quantitation of cells showing cytoplasmic vacuolization after transfection with Bak and Bcl-XL in the presence of different inhibitors of apoptosis. 293T cells were transfected and treated as in A, and scored as in Fig. 1 B.
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fig2: Inhibition of Bak-induced apoptosis is not sufficient to reveal cytoplasmic vacuolization. (A) Quantitation of vacuolated cells after transfection with Bak in the presence of different apoptotic inhibitors. 293T cells were transfected with a mix of the indicated plasmids and a GFP-expressing plasmid. C9.DN and p35 denote a dominant-negative version of caspase-9 and the viral caspase inhibitor p35, respectively. When indicated, the pan-caspase inhibitor z-VAD.fmk (100 μM) was added 1 h after transfection. Cytoplasmic vacuolization was scored as in Fig. 1 B. (B) Death inhibitors unable to reveal vacuolization block Bak apoptosis to the same extent as Bcl-XL. 293T cells were transfected with a mix of the indicated expression plasmids. Z-VAD.fmk (100 μM) was added 1 h after transfection. Cells were lysed 24 h after transfection. Shown are blots probed with anti-PARP (top) and anti-AU (bottom) antibodies. The latter confirms equal transfection efficiency. (C) Quantitation of cells showing cytoplasmic vacuolization after transfection with Bak and Bcl-XL in the presence of different inhibitors of apoptosis. 293T cells were transfected and treated as in A, and scored as in Fig. 1 B.

Mentions: Because Bak overexpression triggers efficient death (Fig. 1 E; Chittenden et al., 1995b), it is possible that the generation of cytoplasmic vacuolae is a simultaneous phenomenon normally obscured by the dominant apoptotic activity. In this scenario, cotransfected Bcl-XL would reveal the phenotype by simply inhibiting cell death. A prediction of this model is that Bak expression should induce vacuolization in the presence of alternative blockers of apoptosis. However, treatment with different caspase inhibitors was not sufficient to reveal the phenotype in Bak-transfected cells (Fig. 2 A), although all tested reagents suppressed PARP processing to the same extent as Bcl-XL did (Fig. 2 B). While this result could also indicate that caspases are necessary for vacuolization, caspase inhibition during coexpression of Bak and Bcl-XL did not reduce the proportion of vacuolated cells (Fig. 2 C), ruling out a role for these proteases in the observed phenomenon. Together, these data show that Bak normally lacks the capability of inducing cytoplasmic vacuolization, and suggest the need for an activation step provided by Bcl-XL. Alternatively, Bcl-XL, and not Bak, could be the effector molecule in this context.


Bcl-X(L) specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum.

Klee M, Pimentel-Muiños FX - J. Cell Biol. (2005)

Inhibition of Bak-induced apoptosis is not sufficient to reveal cytoplasmic vacuolization. (A) Quantitation of vacuolated cells after transfection with Bak in the presence of different apoptotic inhibitors. 293T cells were transfected with a mix of the indicated plasmids and a GFP-expressing plasmid. C9.DN and p35 denote a dominant-negative version of caspase-9 and the viral caspase inhibitor p35, respectively. When indicated, the pan-caspase inhibitor z-VAD.fmk (100 μM) was added 1 h after transfection. Cytoplasmic vacuolization was scored as in Fig. 1 B. (B) Death inhibitors unable to reveal vacuolization block Bak apoptosis to the same extent as Bcl-XL. 293T cells were transfected with a mix of the indicated expression plasmids. Z-VAD.fmk (100 μM) was added 1 h after transfection. Cells were lysed 24 h after transfection. Shown are blots probed with anti-PARP (top) and anti-AU (bottom) antibodies. The latter confirms equal transfection efficiency. (C) Quantitation of cells showing cytoplasmic vacuolization after transfection with Bak and Bcl-XL in the presence of different inhibitors of apoptosis. 293T cells were transfected and treated as in A, and scored as in Fig. 1 B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171806&req=5

fig2: Inhibition of Bak-induced apoptosis is not sufficient to reveal cytoplasmic vacuolization. (A) Quantitation of vacuolated cells after transfection with Bak in the presence of different apoptotic inhibitors. 293T cells were transfected with a mix of the indicated plasmids and a GFP-expressing plasmid. C9.DN and p35 denote a dominant-negative version of caspase-9 and the viral caspase inhibitor p35, respectively. When indicated, the pan-caspase inhibitor z-VAD.fmk (100 μM) was added 1 h after transfection. Cytoplasmic vacuolization was scored as in Fig. 1 B. (B) Death inhibitors unable to reveal vacuolization block Bak apoptosis to the same extent as Bcl-XL. 293T cells were transfected with a mix of the indicated expression plasmids. Z-VAD.fmk (100 μM) was added 1 h after transfection. Cells were lysed 24 h after transfection. Shown are blots probed with anti-PARP (top) and anti-AU (bottom) antibodies. The latter confirms equal transfection efficiency. (C) Quantitation of cells showing cytoplasmic vacuolization after transfection with Bak and Bcl-XL in the presence of different inhibitors of apoptosis. 293T cells were transfected and treated as in A, and scored as in Fig. 1 B.
Mentions: Because Bak overexpression triggers efficient death (Fig. 1 E; Chittenden et al., 1995b), it is possible that the generation of cytoplasmic vacuolae is a simultaneous phenomenon normally obscured by the dominant apoptotic activity. In this scenario, cotransfected Bcl-XL would reveal the phenotype by simply inhibiting cell death. A prediction of this model is that Bak expression should induce vacuolization in the presence of alternative blockers of apoptosis. However, treatment with different caspase inhibitors was not sufficient to reveal the phenotype in Bak-transfected cells (Fig. 2 A), although all tested reagents suppressed PARP processing to the same extent as Bcl-XL did (Fig. 2 B). While this result could also indicate that caspases are necessary for vacuolization, caspase inhibition during coexpression of Bak and Bcl-XL did not reduce the proportion of vacuolated cells (Fig. 2 C), ruling out a role for these proteases in the observed phenomenon. Together, these data show that Bak normally lacks the capability of inducing cytoplasmic vacuolization, and suggest the need for an activation step provided by Bcl-XL. Alternatively, Bcl-XL, and not Bak, could be the effector molecule in this context.

Bottom Line: Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant.These results reveal a previously unidentified role of Bak in regulating reticular conformation.Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, 37007 Spain.

ABSTRACT
Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-X(L) provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

Show MeSH
Related in: MedlinePlus