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Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity.

Wilson-Annan J, O'Reilly LA, Crawford SA, Hausmann G, Beaumont JG, Parma LP, Chen L, Lackmann M, Lithgow T, Hinds MG, Day CL, Adams JM, Huang DC - J. Cell Biol. (2003)

Bottom Line: We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis.To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w.These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia.

ABSTRACT
Prosurvival Bcl-2-like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

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Bim BH3 fusion drives Bcl-w into the membrane fraction. (A) Expression of FLAG-tagged Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w fusion proteins in FDC-P1 cells. Representative clones (unfilled histograms) were stained with the anti–FLAG M2 followed by FITC-conjugated goat anti–mouse antibody and analyzed by flow cytometry. Filled histograms indicate control staining of the parental FDC-P1 cells. (B and C) BH3/Bcl-w fusions are localized at the mitochondria. Immunogold EM revealing wtBH3/Bcl-w (B) or 4EBH3/Bcl-w (C) in FDC-P1 cells (×37K). Arrows indicate some gold particles located on mitochondria. (D) wtBH3/Bcl-w, but not 4EBH3/Bcl-w, associates with the membrane fraction of healthy cells. The cells were lysed in buffer containing 0.025% digitonin before fractionation. (E) The COOH-terminal domain of Bcl-w is essential for targeting Bcl-w into the membrane fraction. Fractionation of cells transiently expressing wtBH3/Bcl-w (left) or wtBH3/Bcl-w ΔC29 (right). Equivalent fractions were resolved by SDS-PAGE and then blotted.
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fig5: Bim BH3 fusion drives Bcl-w into the membrane fraction. (A) Expression of FLAG-tagged Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w fusion proteins in FDC-P1 cells. Representative clones (unfilled histograms) were stained with the anti–FLAG M2 followed by FITC-conjugated goat anti–mouse antibody and analyzed by flow cytometry. Filled histograms indicate control staining of the parental FDC-P1 cells. (B and C) BH3/Bcl-w fusions are localized at the mitochondria. Immunogold EM revealing wtBH3/Bcl-w (B) or 4EBH3/Bcl-w (C) in FDC-P1 cells (×37K). Arrows indicate some gold particles located on mitochondria. (D) wtBH3/Bcl-w, but not 4EBH3/Bcl-w, associates with the membrane fraction of healthy cells. The cells were lysed in buffer containing 0.025% digitonin before fractionation. (E) The COOH-terminal domain of Bcl-w is essential for targeting Bcl-w into the membrane fraction. Fractionation of cells transiently expressing wtBH3/Bcl-w (left) or wtBH3/Bcl-w ΔC29 (right). Equivalent fractions were resolved by SDS-PAGE and then blotted.

Mentions: As the fusion constructs behaved as expected, to allow tests of their function, we stably expressed them in FDC-P1 cells and derived at least three independent clones of each construct expressing comparable levels of the chimeras (Fig. 5 A). First, we confirmed that the tethered BH3 domain did not affect the predominant mitochondrial localization of Bcl-w (Fig. 5, B and C). Next, we examined the membrane binding properties of the chimeras (Fig. 5 D and Table I). The wtBH3/Bcl-w construct was predicted to bind membranes more tightly, because its COOH terminus should be displaced, mimicking the conformation of wild-type Bcl-w with a BH3 protein bound. Indeed, fractionation of healthy cells revealed that a substantial proportion of the wtBH3/Bcl-w appeared in the pellet fraction, as observed with Bcl-w only after cell death induction (Fig. 1). In contrast, the 4EBH3/Bcl-w chimera, as expected, appeared almost entirely in the soluble fraction, like wild-type Bcl-w (Fig. 5 D). Significantly, the translocation of wtBH3/Bcl-w requires its COOH-terminal domain, because the chimera with that domain excised no longer firmly attached to the membranes (Fig. 5 E). Hence, it is the displaced “tail” that mediates tight membrane binding.


Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity.

Wilson-Annan J, O'Reilly LA, Crawford SA, Hausmann G, Beaumont JG, Parma LP, Chen L, Lackmann M, Lithgow T, Hinds MG, Day CL, Adams JM, Huang DC - J. Cell Biol. (2003)

Bim BH3 fusion drives Bcl-w into the membrane fraction. (A) Expression of FLAG-tagged Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w fusion proteins in FDC-P1 cells. Representative clones (unfilled histograms) were stained with the anti–FLAG M2 followed by FITC-conjugated goat anti–mouse antibody and analyzed by flow cytometry. Filled histograms indicate control staining of the parental FDC-P1 cells. (B and C) BH3/Bcl-w fusions are localized at the mitochondria. Immunogold EM revealing wtBH3/Bcl-w (B) or 4EBH3/Bcl-w (C) in FDC-P1 cells (×37K). Arrows indicate some gold particles located on mitochondria. (D) wtBH3/Bcl-w, but not 4EBH3/Bcl-w, associates with the membrane fraction of healthy cells. The cells were lysed in buffer containing 0.025% digitonin before fractionation. (E) The COOH-terminal domain of Bcl-w is essential for targeting Bcl-w into the membrane fraction. Fractionation of cells transiently expressing wtBH3/Bcl-w (left) or wtBH3/Bcl-w ΔC29 (right). Equivalent fractions were resolved by SDS-PAGE and then blotted.
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Related In: Results  -  Collection

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fig5: Bim BH3 fusion drives Bcl-w into the membrane fraction. (A) Expression of FLAG-tagged Bcl-w, wtBH3/Bcl-w, or 4EBH3/Bcl-w fusion proteins in FDC-P1 cells. Representative clones (unfilled histograms) were stained with the anti–FLAG M2 followed by FITC-conjugated goat anti–mouse antibody and analyzed by flow cytometry. Filled histograms indicate control staining of the parental FDC-P1 cells. (B and C) BH3/Bcl-w fusions are localized at the mitochondria. Immunogold EM revealing wtBH3/Bcl-w (B) or 4EBH3/Bcl-w (C) in FDC-P1 cells (×37K). Arrows indicate some gold particles located on mitochondria. (D) wtBH3/Bcl-w, but not 4EBH3/Bcl-w, associates with the membrane fraction of healthy cells. The cells were lysed in buffer containing 0.025% digitonin before fractionation. (E) The COOH-terminal domain of Bcl-w is essential for targeting Bcl-w into the membrane fraction. Fractionation of cells transiently expressing wtBH3/Bcl-w (left) or wtBH3/Bcl-w ΔC29 (right). Equivalent fractions were resolved by SDS-PAGE and then blotted.
Mentions: As the fusion constructs behaved as expected, to allow tests of their function, we stably expressed them in FDC-P1 cells and derived at least three independent clones of each construct expressing comparable levels of the chimeras (Fig. 5 A). First, we confirmed that the tethered BH3 domain did not affect the predominant mitochondrial localization of Bcl-w (Fig. 5, B and C). Next, we examined the membrane binding properties of the chimeras (Fig. 5 D and Table I). The wtBH3/Bcl-w construct was predicted to bind membranes more tightly, because its COOH terminus should be displaced, mimicking the conformation of wild-type Bcl-w with a BH3 protein bound. Indeed, fractionation of healthy cells revealed that a substantial proportion of the wtBH3/Bcl-w appeared in the pellet fraction, as observed with Bcl-w only after cell death induction (Fig. 1). In contrast, the 4EBH3/Bcl-w chimera, as expected, appeared almost entirely in the soluble fraction, like wild-type Bcl-w (Fig. 5 D). Significantly, the translocation of wtBH3/Bcl-w requires its COOH-terminal domain, because the chimera with that domain excised no longer firmly attached to the membranes (Fig. 5 E). Hence, it is the displaced “tail” that mediates tight membrane binding.

Bottom Line: We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis.To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w.These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

View Article: PubMed Central - PubMed

Affiliation: Walter and Eliza Hall Institute of Medical Research, Victoria 3050, Australia.

ABSTRACT
Prosurvival Bcl-2-like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

Show MeSH
Related in: MedlinePlus