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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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A version of Bak lacking the BH3 domain induces cytoplasmic vacuolization in the absence of cotransfected Bcl-XL. (A) Confocal analysis of cytoplasmic vacuolization induced by expression of Bak-ΔBH3. 293T cells were transfected with a mix of plasmids expressing Bak-ΔBH3 and GFP. Cells were fixed, mounted, and analyzed by confocal microscopy 24 or 36 h (as indicated) after transfection. (B) Quantitation of vacuolization in cells transfected with Bak-ΔBH3 or Bak-ΔBH3-ΔBH1. 293T cells were transfected with the indicated plasmids, along with a GFP-expressing plasmid. 36 h later the proportion of GFP-expressing cells showing vacuolization was determined as in Fig. 1 B. (C) AU-Bak, AU-Bak-ΔBH3, and AU-Bak-ΔBH3-ΔBH1 are expressed to comparable levels. 293T cells were transfected, lysed, and processed as in Fig. 1 F. Shown is a Western blot probed with an anti-AU antibody.
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fig3: A version of Bak lacking the BH3 domain induces cytoplasmic vacuolization in the absence of cotransfected Bcl-XL. (A) Confocal analysis of cytoplasmic vacuolization induced by expression of Bak-ΔBH3. 293T cells were transfected with a mix of plasmids expressing Bak-ΔBH3 and GFP. Cells were fixed, mounted, and analyzed by confocal microscopy 24 or 36 h (as indicated) after transfection. (B) Quantitation of vacuolization in cells transfected with Bak-ΔBH3 or Bak-ΔBH3-ΔBH1. 293T cells were transfected with the indicated plasmids, along with a GFP-expressing plasmid. 36 h later the proportion of GFP-expressing cells showing vacuolization was determined as in Fig. 1 B. (C) AU-Bak, AU-Bak-ΔBH3, and AU-Bak-ΔBH3-ΔBH1 are expressed to comparable levels. 293T cells were transfected, lysed, and processed as in Fig. 1 F. Shown is a Western blot probed with an anti-AU antibody.

Mentions: Bcl-XL has been shown to bind and inhibit Bak and Bax BH3 domains, thus antagonizing their apoptotic activities (Chittenden et al., 1995a; Simonen et al., 1997). Therefore, the capacity of Bcl-XL to reveal the vacuolating potential of Bak might as well involve BH3 domain inhibition. To test this possibility, we created a deleted version of Bak lacking the BH3 domain (Bak-ΔBH3) and evaluated its ability to induce the observed phenomenon in the absence of Bcl-XL. Transfection experiments indicated that this Bak mutant has an autonomous ability to induce cytoplasmic vacuolization (Fig. 3 A). Quantitation studies showed only a slight decrease in the percentage of vacuolated cells compared with cultures transfected with Bak and Bcl-XL (Fig. 3 B). Both Bak versions were expressed to similar levels (Fig. 3 C). These data confirm that Bak, and not Bcl-XL, is the active molecule in this context, and suggest that Bcl-XL reveals this function by binding and inactivating the BH3 domain of Bak.


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

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

A version of Bak lacking the BH3 domain induces cytoplasmic vacuolization in the absence of cotransfected Bcl-XL. (A) Confocal analysis of cytoplasmic vacuolization induced by expression of Bak-ΔBH3. 293T cells were transfected with a mix of plasmids expressing Bak-ΔBH3 and GFP. Cells were fixed, mounted, and analyzed by confocal microscopy 24 or 36 h (as indicated) after transfection. (B) Quantitation of vacuolization in cells transfected with Bak-ΔBH3 or Bak-ΔBH3-ΔBH1. 293T cells were transfected with the indicated plasmids, along with a GFP-expressing plasmid. 36 h later the proportion of GFP-expressing cells showing vacuolization was determined as in Fig. 1 B. (C) AU-Bak, AU-Bak-ΔBH3, and AU-Bak-ΔBH3-ΔBH1 are expressed to comparable levels. 293T cells were transfected, lysed, and processed as in Fig. 1 F. Shown is a Western blot probed with an anti-AU antibody.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171806&req=5

fig3: A version of Bak lacking the BH3 domain induces cytoplasmic vacuolization in the absence of cotransfected Bcl-XL. (A) Confocal analysis of cytoplasmic vacuolization induced by expression of Bak-ΔBH3. 293T cells were transfected with a mix of plasmids expressing Bak-ΔBH3 and GFP. Cells were fixed, mounted, and analyzed by confocal microscopy 24 or 36 h (as indicated) after transfection. (B) Quantitation of vacuolization in cells transfected with Bak-ΔBH3 or Bak-ΔBH3-ΔBH1. 293T cells were transfected with the indicated plasmids, along with a GFP-expressing plasmid. 36 h later the proportion of GFP-expressing cells showing vacuolization was determined as in Fig. 1 B. (C) AU-Bak, AU-Bak-ΔBH3, and AU-Bak-ΔBH3-ΔBH1 are expressed to comparable levels. 293T cells were transfected, lysed, and processed as in Fig. 1 F. Shown is a Western blot probed with an anti-AU antibody.
Mentions: Bcl-XL has been shown to bind and inhibit Bak and Bax BH3 domains, thus antagonizing their apoptotic activities (Chittenden et al., 1995a; Simonen et al., 1997). Therefore, the capacity of Bcl-XL to reveal the vacuolating potential of Bak might as well involve BH3 domain inhibition. To test this possibility, we created a deleted version of Bak lacking the BH3 domain (Bak-ΔBH3) and evaluated its ability to induce the observed phenomenon in the absence of Bcl-XL. Transfection experiments indicated that this Bak mutant has an autonomous ability to induce cytoplasmic vacuolization (Fig. 3 A). Quantitation studies showed only a slight decrease in the percentage of vacuolated cells compared with cultures transfected with Bak and Bcl-XL (Fig. 3 B). Both Bak versions were expressed to similar levels (Fig. 3 C). These data confirm that Bak, and not Bcl-XL, is the active molecule in this context, and suggest that Bcl-XL reveals this function by binding and inactivating the BH3 domain of Bak.

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