Limits...
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.

Show MeSH

Related in: MedlinePlus

Apoptotic signals trigger membrane integration of Bcl-w and its oligomerization. (A) FLAG–Bcl-w is absent from the mitochondria after subcellular fractionation. FLAG-Bcl-w– expressing FDC-P1 cells were separated into heavy membrane, cytosolic (C), and mitochondrial (mit) fractions. Equal cell numbers of the total cell lysate (Lys), homogenate (H), or the fractions were resolved by SDS-PAGE. (B) Tighter association of FLAG–Bcl-w with membranes after γ irradiation (10 Gy). Healthy or irradiated FD/FLAG–Bcl-w cells were fractionated into the soluble (s) and pellet (p) fractions using lysis buffer containing 0.025% digitonin. (C) Endogenous Bcl-w is soluble in healthy FDC-P1 cells but damage signals induce membrane association resistant to alkali treatment. Lysates prepared from healthy cells (control) or cells exposed to 100 nM staurosporine (STS) or γ irradiation (10 Gy) or deprived of IL-3 were fractionated into the soluble (s, top) or pellet (p, bottom) fractions. (D) Damage signals trigger membrane association of endogenous Bcl-w in HeLa cells. Soluble (s) and pellet (p) fractions of healthy HeLa cells (control) or 24 h after treatment with 50 Jm−2 UV irradiation (UV), 1.0 μg/ml etoposide (VP16), or 10 nM staurosporine (STS). (E and F) Bcl-w is a soluble monomeric protein in healthy cells, but damage signals cause its oligomerization. Lysates from FD/FLAG–Bcl-w cells were fractionated by gel filtration chromatography after lysis without detergent (E) or with 1% digitonin (F). Equivalent portions of the indicated fractions were loaded. By comparison with the elution of standard size markers as indicated, FLAG–Bcl-w appears to be a soluble, monomeric protein in healthy cells (E and top panel of F) but forms larger complexes after damage signals (10 Gy γ irradiation; F, bottom). The blots were probed for FLAG–Bcl-w with the anti–FLAG 9H1 or for the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172834&req=5

fig1: Apoptotic signals trigger membrane integration of Bcl-w and its oligomerization. (A) FLAG–Bcl-w is absent from the mitochondria after subcellular fractionation. FLAG-Bcl-w– expressing FDC-P1 cells were separated into heavy membrane, cytosolic (C), and mitochondrial (mit) fractions. Equal cell numbers of the total cell lysate (Lys), homogenate (H), or the fractions were resolved by SDS-PAGE. (B) Tighter association of FLAG–Bcl-w with membranes after γ irradiation (10 Gy). Healthy or irradiated FD/FLAG–Bcl-w cells were fractionated into the soluble (s) and pellet (p) fractions using lysis buffer containing 0.025% digitonin. (C) Endogenous Bcl-w is soluble in healthy FDC-P1 cells but damage signals induce membrane association resistant to alkali treatment. Lysates prepared from healthy cells (control) or cells exposed to 100 nM staurosporine (STS) or γ irradiation (10 Gy) or deprived of IL-3 were fractionated into the soluble (s, top) or pellet (p, bottom) fractions. (D) Damage signals trigger membrane association of endogenous Bcl-w in HeLa cells. Soluble (s) and pellet (p) fractions of healthy HeLa cells (control) or 24 h after treatment with 50 Jm−2 UV irradiation (UV), 1.0 μg/ml etoposide (VP16), or 10 nM staurosporine (STS). (E and F) Bcl-w is a soluble monomeric protein in healthy cells, but damage signals cause its oligomerization. Lysates from FD/FLAG–Bcl-w cells were fractionated by gel filtration chromatography after lysis without detergent (E) or with 1% digitonin (F). Equivalent portions of the indicated fractions were loaded. By comparison with the elution of standard size markers as indicated, FLAG–Bcl-w appears to be a soluble, monomeric protein in healthy cells (E and top panel of F) but forms larger complexes after damage signals (10 Gy γ irradiation; F, bottom). The blots were probed for FLAG–Bcl-w with the anti–FLAG 9H1 or for the indicated proteins.

Mentions: Confocal microscopy of HeLa cells has indicated that Bcl-w is located on the mitochondria (O'Reilly et al., 2001), and we have confirmed that observation with both HeLa cells and FDC-P1 myeloid cells (unpublished data). In view of its clear mitochondrial localization in microscopic studies (see next section in Results), we were surprised to find that Bcl-w did not behave like an organelle-associated protein on sub-cellular fractionation. Irrespective of the method used, only an insignificant amount of Bcl-w was recovered from membrane fractions. For example, none was detectable in purified mitochondria (Fig. 1 A). Moreover, Bcl-w behaved entirely as a cytosolic protein on subcellular fractionation of healthy FDC-P1 or HeLa cells lysed in low digitonin buffer (Fig. 1 B, 0 h; Fig. 1, C and D, control), as did the endogenous protein in several other cell types, NIH3T3, Jurkat, and T cells (unpublished data). We also fractionated lysates made without detergents, either by Dounce homogenization, needle (26 gauge) lysis, freeze/thawing, or nitrogen cavitation. In every case, rather than appearing in the pellet fraction, where proteins of the mitochondrial membrane (voltage-dependent anion channel [VDAC]/Porin) and intermembrane space (cytochrome c) were found, Bcl-w fractionated with soluble proteins such as Bax (Fig. 1 A; unpublished data). In contrast, Bcl-2, which is reported to be an integral membrane protein (Chen-Levy et al., 1989; Janiak et al., 1994), was found exclusively in the pellet fraction (Fig. 1 D, control). Thus, in healthy cells, Bcl-w appears to be only weakly bound to membranes.


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)

Apoptotic signals trigger membrane integration of Bcl-w and its oligomerization. (A) FLAG–Bcl-w is absent from the mitochondria after subcellular fractionation. FLAG-Bcl-w– expressing FDC-P1 cells were separated into heavy membrane, cytosolic (C), and mitochondrial (mit) fractions. Equal cell numbers of the total cell lysate (Lys), homogenate (H), or the fractions were resolved by SDS-PAGE. (B) Tighter association of FLAG–Bcl-w with membranes after γ irradiation (10 Gy). Healthy or irradiated FD/FLAG–Bcl-w cells were fractionated into the soluble (s) and pellet (p) fractions using lysis buffer containing 0.025% digitonin. (C) Endogenous Bcl-w is soluble in healthy FDC-P1 cells but damage signals induce membrane association resistant to alkali treatment. Lysates prepared from healthy cells (control) or cells exposed to 100 nM staurosporine (STS) or γ irradiation (10 Gy) or deprived of IL-3 were fractionated into the soluble (s, top) or pellet (p, bottom) fractions. (D) Damage signals trigger membrane association of endogenous Bcl-w in HeLa cells. Soluble (s) and pellet (p) fractions of healthy HeLa cells (control) or 24 h after treatment with 50 Jm−2 UV irradiation (UV), 1.0 μg/ml etoposide (VP16), or 10 nM staurosporine (STS). (E and F) Bcl-w is a soluble monomeric protein in healthy cells, but damage signals cause its oligomerization. Lysates from FD/FLAG–Bcl-w cells were fractionated by gel filtration chromatography after lysis without detergent (E) or with 1% digitonin (F). Equivalent portions of the indicated fractions were loaded. By comparison with the elution of standard size markers as indicated, FLAG–Bcl-w appears to be a soluble, monomeric protein in healthy cells (E and top panel of F) but forms larger complexes after damage signals (10 Gy γ irradiation; F, bottom). The blots were probed for FLAG–Bcl-w with the anti–FLAG 9H1 or for the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172834&req=5

fig1: Apoptotic signals trigger membrane integration of Bcl-w and its oligomerization. (A) FLAG–Bcl-w is absent from the mitochondria after subcellular fractionation. FLAG-Bcl-w– expressing FDC-P1 cells were separated into heavy membrane, cytosolic (C), and mitochondrial (mit) fractions. Equal cell numbers of the total cell lysate (Lys), homogenate (H), or the fractions were resolved by SDS-PAGE. (B) Tighter association of FLAG–Bcl-w with membranes after γ irradiation (10 Gy). Healthy or irradiated FD/FLAG–Bcl-w cells were fractionated into the soluble (s) and pellet (p) fractions using lysis buffer containing 0.025% digitonin. (C) Endogenous Bcl-w is soluble in healthy FDC-P1 cells but damage signals induce membrane association resistant to alkali treatment. Lysates prepared from healthy cells (control) or cells exposed to 100 nM staurosporine (STS) or γ irradiation (10 Gy) or deprived of IL-3 were fractionated into the soluble (s, top) or pellet (p, bottom) fractions. (D) Damage signals trigger membrane association of endogenous Bcl-w in HeLa cells. Soluble (s) and pellet (p) fractions of healthy HeLa cells (control) or 24 h after treatment with 50 Jm−2 UV irradiation (UV), 1.0 μg/ml etoposide (VP16), or 10 nM staurosporine (STS). (E and F) Bcl-w is a soluble monomeric protein in healthy cells, but damage signals cause its oligomerization. Lysates from FD/FLAG–Bcl-w cells were fractionated by gel filtration chromatography after lysis without detergent (E) or with 1% digitonin (F). Equivalent portions of the indicated fractions were loaded. By comparison with the elution of standard size markers as indicated, FLAG–Bcl-w appears to be a soluble, monomeric protein in healthy cells (E and top panel of F) but forms larger complexes after damage signals (10 Gy γ irradiation; F, bottom). The blots were probed for FLAG–Bcl-w with the anti–FLAG 9H1 or for the indicated proteins.
Mentions: Confocal microscopy of HeLa cells has indicated that Bcl-w is located on the mitochondria (O'Reilly et al., 2001), and we have confirmed that observation with both HeLa cells and FDC-P1 myeloid cells (unpublished data). In view of its clear mitochondrial localization in microscopic studies (see next section in Results), we were surprised to find that Bcl-w did not behave like an organelle-associated protein on sub-cellular fractionation. Irrespective of the method used, only an insignificant amount of Bcl-w was recovered from membrane fractions. For example, none was detectable in purified mitochondria (Fig. 1 A). Moreover, Bcl-w behaved entirely as a cytosolic protein on subcellular fractionation of healthy FDC-P1 or HeLa cells lysed in low digitonin buffer (Fig. 1 B, 0 h; Fig. 1, C and D, control), as did the endogenous protein in several other cell types, NIH3T3, Jurkat, and T cells (unpublished data). We also fractionated lysates made without detergents, either by Dounce homogenization, needle (26 gauge) lysis, freeze/thawing, or nitrogen cavitation. In every case, rather than appearing in the pellet fraction, where proteins of the mitochondrial membrane (voltage-dependent anion channel [VDAC]/Porin) and intermembrane space (cytochrome c) were found, Bcl-w fractionated with soluble proteins such as Bax (Fig. 1 A; unpublished data). In contrast, Bcl-2, which is reported to be an integral membrane protein (Chen-Levy et al., 1989; Janiak et al., 1994), was found exclusively in the pellet fraction (Fig. 1 D, control). Thus, in healthy cells, Bcl-w appears to be only weakly bound to membranes.

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