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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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Bcl-w is predominantly a mitochondrial protein in healthy and dying cells. (A and B) Overexpressed FLAG–Bcl-w in FDC-P1 (A; ×52K) or NIH3T3 (B, ×37K) cells is mainly mitochondrial. Representative immunogold electron microscopic images of cells stained with the anti–Bcl-w 13F9 antibody were detected with 18-nm gold–conjugated goat anti–rat antibody. (C) Scanty staining for endogenous Bcl-w around the mitochondria in parental FDC-P1 cells. The four insets show Bcl-w staining around mitochondria (×27.5K). (D) Bcl-2 is present on the nuclear/ER membranes as well as the outer mitochondrial membranes of FDC-P1 cells. Overexpressed Bcl-2 detected with the mouse monoclonal anti–Bcl-2-100 antibody and revealed using 20-nm gold–conjugated goat anti–mouse antibody. (E and F) Bcl-w remains closely associated with the mitochondria after damage signals. Staining of FD/FLAG–Bcl-w cells 24 h after (E) 10 Gy γ irradiation or (F) IL-3 deprivation (×37K). Arrowhead, ER; arrow, mitochondria; arrow with ball on end, nuclear membrane. N, nucleus.
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fig2: Bcl-w is predominantly a mitochondrial protein in healthy and dying cells. (A and B) Overexpressed FLAG–Bcl-w in FDC-P1 (A; ×52K) or NIH3T3 (B, ×37K) cells is mainly mitochondrial. Representative immunogold electron microscopic images of cells stained with the anti–Bcl-w 13F9 antibody were detected with 18-nm gold–conjugated goat anti–rat antibody. (C) Scanty staining for endogenous Bcl-w around the mitochondria in parental FDC-P1 cells. The four insets show Bcl-w staining around mitochondria (×27.5K). (D) Bcl-2 is present on the nuclear/ER membranes as well as the outer mitochondrial membranes of FDC-P1 cells. Overexpressed Bcl-2 detected with the mouse monoclonal anti–Bcl-2-100 antibody and revealed using 20-nm gold–conjugated goat anti–mouse antibody. (E and F) Bcl-w remains closely associated with the mitochondria after damage signals. Staining of FD/FLAG–Bcl-w cells 24 h after (E) 10 Gy γ irradiation or (F) IL-3 deprivation (×37K). Arrowhead, ER; arrow, mitochondria; arrow with ball on end, nuclear membrane. N, nucleus.

Mentions: To resolve the apparent discrepancy between the predominance of Bcl-w in the soluble fraction after subcellular fractionation (Fig. 1) and its clear mitochondrial localization in confocal microscopic studies, we reevaluated its localization by immunogold EM. In accord with confocal studies, a well-characterized rat monoclonal antibody raised against Bcl-w (13F9) (O'Reilly et al., 2001) revealed most (66%) of the FLAG–Bcl-w molecules around the mitochondria of both FDC-P1 (Fig. 2 A) and NIH3T3 (Fig. 2 B) cells, and an anti-FLAG monoclonal antibody gave an equivalent staining pattern (unpublished data). Little FLAG–Bcl-w was found on the nuclear and ER membranes, whereas FLAG–Bcl-2 was prevalent on those membranes (Lithgow et al., 1994) (Fig. 2 D). As expected, the anti–Bcl-w 13F9 antibody revealed far fewer endogenous Bcl-w molecules in parental FDC-P1, NIH3T3, or HeLa cells (Fig. 2 C; unpublished data), but examination of a number of fields confirmed that the endogenous protein was mainly mitochondrial in all three lines.


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)

Bcl-w is predominantly a mitochondrial protein in healthy and dying cells. (A and B) Overexpressed FLAG–Bcl-w in FDC-P1 (A; ×52K) or NIH3T3 (B, ×37K) cells is mainly mitochondrial. Representative immunogold electron microscopic images of cells stained with the anti–Bcl-w 13F9 antibody were detected with 18-nm gold–conjugated goat anti–rat antibody. (C) Scanty staining for endogenous Bcl-w around the mitochondria in parental FDC-P1 cells. The four insets show Bcl-w staining around mitochondria (×27.5K). (D) Bcl-2 is present on the nuclear/ER membranes as well as the outer mitochondrial membranes of FDC-P1 cells. Overexpressed Bcl-2 detected with the mouse monoclonal anti–Bcl-2-100 antibody and revealed using 20-nm gold–conjugated goat anti–mouse antibody. (E and F) Bcl-w remains closely associated with the mitochondria after damage signals. Staining of FD/FLAG–Bcl-w cells 24 h after (E) 10 Gy γ irradiation or (F) IL-3 deprivation (×37K). Arrowhead, ER; arrow, mitochondria; arrow with ball on end, nuclear membrane. N, nucleus.
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Related In: Results  -  Collection

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fig2: Bcl-w is predominantly a mitochondrial protein in healthy and dying cells. (A and B) Overexpressed FLAG–Bcl-w in FDC-P1 (A; ×52K) or NIH3T3 (B, ×37K) cells is mainly mitochondrial. Representative immunogold electron microscopic images of cells stained with the anti–Bcl-w 13F9 antibody were detected with 18-nm gold–conjugated goat anti–rat antibody. (C) Scanty staining for endogenous Bcl-w around the mitochondria in parental FDC-P1 cells. The four insets show Bcl-w staining around mitochondria (×27.5K). (D) Bcl-2 is present on the nuclear/ER membranes as well as the outer mitochondrial membranes of FDC-P1 cells. Overexpressed Bcl-2 detected with the mouse monoclonal anti–Bcl-2-100 antibody and revealed using 20-nm gold–conjugated goat anti–mouse antibody. (E and F) Bcl-w remains closely associated with the mitochondria after damage signals. Staining of FD/FLAG–Bcl-w cells 24 h after (E) 10 Gy γ irradiation or (F) IL-3 deprivation (×37K). Arrowhead, ER; arrow, mitochondria; arrow with ball on end, nuclear membrane. N, nucleus.
Mentions: To resolve the apparent discrepancy between the predominance of Bcl-w in the soluble fraction after subcellular fractionation (Fig. 1) and its clear mitochondrial localization in confocal microscopic studies, we reevaluated its localization by immunogold EM. In accord with confocal studies, a well-characterized rat monoclonal antibody raised against Bcl-w (13F9) (O'Reilly et al., 2001) revealed most (66%) of the FLAG–Bcl-w molecules around the mitochondria of both FDC-P1 (Fig. 2 A) and NIH3T3 (Fig. 2 B) cells, and an anti-FLAG monoclonal antibody gave an equivalent staining pattern (unpublished data). Little FLAG–Bcl-w was found on the nuclear and ER membranes, whereas FLAG–Bcl-2 was prevalent on those membranes (Lithgow et al., 1994) (Fig. 2 D). As expected, the anti–Bcl-w 13F9 antibody revealed far fewer endogenous Bcl-w molecules in parental FDC-P1, NIH3T3, or HeLa cells (Fig. 2 C; unpublished data), but examination of a number of fields confirmed that the endogenous protein was mainly mitochondrial in all three lines.

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