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Drosophila IAP antagonists form multimeric complexes to promote cell death.

Sandu C, Ryoo HD, Steller H - J. Cell Biol. (2010)

Bottom Line: In addition, we show that Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo.Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis.Our results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, Strang Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, New York, NY 10065, USA.

ABSTRACT
Apoptosis is a specific form of cell death that is important for normal development and tissue homeostasis. Caspases are critical executioners of apoptosis, and living cells prevent their inappropriate activation through inhibitor of apoptosis proteins (IAPs). In Drosophila, caspase activation depends on the IAP antagonists, Reaper (Rpr), Head involution defective (Hid), and Grim. These proteins share a common motif to bind Drosophila IAP1 (DIAP1) and have partially redundant functions. We now show that IAP antagonists physically interact with each other. Rpr is able to self-associate and also binds to Hid and Grim. We have defined the domain involved in self-association and demonstrate that it is critical for cell-killing activity in vivo. In addition, we show that Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo. Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis. Our results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis.

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Rpr translocates to the mitochondria through physical interaction with Hid. (A) BT549 cells expressing Rpr-HA and GFP-XIAP. Rpr was stained with an anti-HA antibody. “Overlay” represents a composite image of Rpr (red), GFP-XIAP (green), and nuclei (DAPI, blue) staining. Bar, 20 µm. (B) BT549 cells transiently transfected with GFP-XIAP and mitochondrial RFP (mtRFP) plasmids. “Overlay” indicates GFP-XIAP (green), mitochondria (red), and nuclei (blue). Bar, 20 µm. (C) BT549 cells cotransfected with Hid-HA and GFP-XIAP plasmids. “Overlay” indicates Hid (red), GFP-XIAP (green), and nuclei (blue). Bar, 20 µm. (D) BT549 cells cotransfected with GFP-Rpr and Hid-HA plasmids. “Overlay” shows Rpr (green), Hid (red), and nuclei (blue) staining. Bar, 20 µm. (E) S2R+ Drosophila cells transiently transfected with a mCherryDIAP1 plasmid (left image) or with mCherryDIAP1 and Hid-Myc plasmids (right image). Each image shows the overlay of DIAP1 (red) and nuclei (blue) staining. Bar, 5 µm. (F) S2R+ Drosophila cell, transiently transfected with a Rpr-HA plasmid, followed by immunostaining with anti-HA and anti-Cyt C antibodies. “Overlay” indicates Rpr (red), Cyt C (green), and nuclei (blue) staining. Bar, 5 µm. (G) S2R+ Drosophila cell cotransfected with Rpr-HA and Hid-Myc plasmids. Cells were immunostained with an anti-HA antibody and an anti-Myc antibody. “Overlay” represents Rpr (green), Hid (red), and nuclei (blue) staining. Bar, 5 µm.
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fig4: Rpr translocates to the mitochondria through physical interaction with Hid. (A) BT549 cells expressing Rpr-HA and GFP-XIAP. Rpr was stained with an anti-HA antibody. “Overlay” represents a composite image of Rpr (red), GFP-XIAP (green), and nuclei (DAPI, blue) staining. Bar, 20 µm. (B) BT549 cells transiently transfected with GFP-XIAP and mitochondrial RFP (mtRFP) plasmids. “Overlay” indicates GFP-XIAP (green), mitochondria (red), and nuclei (blue). Bar, 20 µm. (C) BT549 cells cotransfected with Hid-HA and GFP-XIAP plasmids. “Overlay” indicates Hid (red), GFP-XIAP (green), and nuclei (blue). Bar, 20 µm. (D) BT549 cells cotransfected with GFP-Rpr and Hid-HA plasmids. “Overlay” shows Rpr (green), Hid (red), and nuclei (blue) staining. Bar, 20 µm. (E) S2R+ Drosophila cells transiently transfected with a mCherryDIAP1 plasmid (left image) or with mCherryDIAP1 and Hid-Myc plasmids (right image). Each image shows the overlay of DIAP1 (red) and nuclei (blue) staining. Bar, 5 µm. (F) S2R+ Drosophila cell, transiently transfected with a Rpr-HA plasmid, followed by immunostaining with anti-HA and anti-Cyt C antibodies. “Overlay” indicates Rpr (red), Cyt C (green), and nuclei (blue) staining. Bar, 5 µm. (G) S2R+ Drosophila cell cotransfected with Rpr-HA and Hid-Myc plasmids. Cells were immunostained with an anti-HA antibody and an anti-Myc antibody. “Overlay” represents Rpr (green), Hid (red), and nuclei (blue) staining. Bar, 5 µm.

Mentions: Although Rpr is known to localize to mitochondria (Olson et al., 2003a; Abdelwahid et al., 2007), prediction tools used to assess protein localization failed to identify any motifs for specific subcellular localization. This raises the possibility that Rpr localizes to the mitochondria through a novel mechanism. Thus, we decided to investigate Rpr localization by ectopic coexpression experiments in human BT549 cells, as well as in Drosophila S2R+ cells. We specifically followed the distribution of Rpr-HA, as well as XIAP, a human IAP member that is known to bind Rpr (Holley et al., 2002). Rpr-HA (Fig. 4 A) as well as GFP-fused XIAP (Fig. 4 B) was found to be spread diffusely throughout the cytoplasm. Similar experiments using Rpr-Myc and GFP-Rpr confirmed the broad distribution of Rpr in BT549 cells (unpublished data). In contrast, Hid, which has a mitochondrial targeting sequence, localizes exclusively to mitochondria and triggers GFP-XIAP translocation to this organelle (Fig. 4 C). This experiment confirms the ability of the IAP antagonist to interact with other proteins and recruit them to mitochondria. We also coexpressed GFP-Rpr and Hid in the BT549 cells and monitored any changes in the intracellular distribution of the proteins. Consistent with the ability of Rpr to bind Hid in vitro, the presence of Hid prompted GFP-Rpr distribution to change into a mitochondrial pattern (Fig. 4 D). The experiments above suggest that Rpr is not a mitochondrial protein, per se, but it is recruited to mitochondria by interaction with a mitochondrial-anchored protein. To further validate these results, we performed a similar set of experiments in Drosophila S2R+ cells. As in BT549 cells, we observed that mCherry-DIAP1 is distributed evenly throughout the cytoplasm of S2R+, but after cotransfection with Hid, mCherry-DIAP1 is translocated to mitochondria in a Hid-like pattern (Fig. 4 E). When Rpr was expressed transiently in S2R+ cells, it shows an occasional punctate staining that is only coincidental with cytochrome c (Cyt C) staining (Fig. 4 F). However, after cotransfection with Hid, Rpr’s colocalization with Hid becomes obvious (Fig. 4 G). In sum, our experiments suggest that Rpr is a soluble protein that displays a diffuse distribution throughout the cell, and coexpression with Hid leads to Rpr relocation to the mitochondria. Additionally, these experiments underline Hid’s ability to recruit DIAP1 and its human homologue XIAP to the mitochondrial membrane. To test whether the recruitment of Rpr, XIAP, and DIAP1 is indeed dependent on Hid’s MTS, we have performed coexpression experiments with HidΔMTS and Rpr, XIAP, or DIAP1. HidΔMTS localizes to the nucleus in BT549 and S2R+ cells and triggers nuclear localization of Rpr, XIAP, and DIAP1 (Fig. S1), suggesting that indeed the observed mitochondrial localization of Rpr, XIAP, and DIAP1 is dependent on mitochondrial localization of Hid. Because Rpr and Hid overexpression induce cell death, we tested whether this might induce a change in the intracellular localization of these proteins. However, addition of the caspase inhibitor zVAD-FMK did not affect localization of Rpr or Hid (Fig. S2).


Drosophila IAP antagonists form multimeric complexes to promote cell death.

Sandu C, Ryoo HD, Steller H - J. Cell Biol. (2010)

Rpr translocates to the mitochondria through physical interaction with Hid. (A) BT549 cells expressing Rpr-HA and GFP-XIAP. Rpr was stained with an anti-HA antibody. “Overlay” represents a composite image of Rpr (red), GFP-XIAP (green), and nuclei (DAPI, blue) staining. Bar, 20 µm. (B) BT549 cells transiently transfected with GFP-XIAP and mitochondrial RFP (mtRFP) plasmids. “Overlay” indicates GFP-XIAP (green), mitochondria (red), and nuclei (blue). Bar, 20 µm. (C) BT549 cells cotransfected with Hid-HA and GFP-XIAP plasmids. “Overlay” indicates Hid (red), GFP-XIAP (green), and nuclei (blue). Bar, 20 µm. (D) BT549 cells cotransfected with GFP-Rpr and Hid-HA plasmids. “Overlay” shows Rpr (green), Hid (red), and nuclei (blue) staining. Bar, 20 µm. (E) S2R+ Drosophila cells transiently transfected with a mCherryDIAP1 plasmid (left image) or with mCherryDIAP1 and Hid-Myc plasmids (right image). Each image shows the overlay of DIAP1 (red) and nuclei (blue) staining. Bar, 5 µm. (F) S2R+ Drosophila cell, transiently transfected with a Rpr-HA plasmid, followed by immunostaining with anti-HA and anti-Cyt C antibodies. “Overlay” indicates Rpr (red), Cyt C (green), and nuclei (blue) staining. Bar, 5 µm. (G) S2R+ Drosophila cell cotransfected with Rpr-HA and Hid-Myc plasmids. Cells were immunostained with an anti-HA antibody and an anti-Myc antibody. “Overlay” represents Rpr (green), Hid (red), and nuclei (blue) staining. Bar, 5 µm.
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fig4: Rpr translocates to the mitochondria through physical interaction with Hid. (A) BT549 cells expressing Rpr-HA and GFP-XIAP. Rpr was stained with an anti-HA antibody. “Overlay” represents a composite image of Rpr (red), GFP-XIAP (green), and nuclei (DAPI, blue) staining. Bar, 20 µm. (B) BT549 cells transiently transfected with GFP-XIAP and mitochondrial RFP (mtRFP) plasmids. “Overlay” indicates GFP-XIAP (green), mitochondria (red), and nuclei (blue). Bar, 20 µm. (C) BT549 cells cotransfected with Hid-HA and GFP-XIAP plasmids. “Overlay” indicates Hid (red), GFP-XIAP (green), and nuclei (blue). Bar, 20 µm. (D) BT549 cells cotransfected with GFP-Rpr and Hid-HA plasmids. “Overlay” shows Rpr (green), Hid (red), and nuclei (blue) staining. Bar, 20 µm. (E) S2R+ Drosophila cells transiently transfected with a mCherryDIAP1 plasmid (left image) or with mCherryDIAP1 and Hid-Myc plasmids (right image). Each image shows the overlay of DIAP1 (red) and nuclei (blue) staining. Bar, 5 µm. (F) S2R+ Drosophila cell, transiently transfected with a Rpr-HA plasmid, followed by immunostaining with anti-HA and anti-Cyt C antibodies. “Overlay” indicates Rpr (red), Cyt C (green), and nuclei (blue) staining. Bar, 5 µm. (G) S2R+ Drosophila cell cotransfected with Rpr-HA and Hid-Myc plasmids. Cells were immunostained with an anti-HA antibody and an anti-Myc antibody. “Overlay” represents Rpr (green), Hid (red), and nuclei (blue) staining. Bar, 5 µm.
Mentions: Although Rpr is known to localize to mitochondria (Olson et al., 2003a; Abdelwahid et al., 2007), prediction tools used to assess protein localization failed to identify any motifs for specific subcellular localization. This raises the possibility that Rpr localizes to the mitochondria through a novel mechanism. Thus, we decided to investigate Rpr localization by ectopic coexpression experiments in human BT549 cells, as well as in Drosophila S2R+ cells. We specifically followed the distribution of Rpr-HA, as well as XIAP, a human IAP member that is known to bind Rpr (Holley et al., 2002). Rpr-HA (Fig. 4 A) as well as GFP-fused XIAP (Fig. 4 B) was found to be spread diffusely throughout the cytoplasm. Similar experiments using Rpr-Myc and GFP-Rpr confirmed the broad distribution of Rpr in BT549 cells (unpublished data). In contrast, Hid, which has a mitochondrial targeting sequence, localizes exclusively to mitochondria and triggers GFP-XIAP translocation to this organelle (Fig. 4 C). This experiment confirms the ability of the IAP antagonist to interact with other proteins and recruit them to mitochondria. We also coexpressed GFP-Rpr and Hid in the BT549 cells and monitored any changes in the intracellular distribution of the proteins. Consistent with the ability of Rpr to bind Hid in vitro, the presence of Hid prompted GFP-Rpr distribution to change into a mitochondrial pattern (Fig. 4 D). The experiments above suggest that Rpr is not a mitochondrial protein, per se, but it is recruited to mitochondria by interaction with a mitochondrial-anchored protein. To further validate these results, we performed a similar set of experiments in Drosophila S2R+ cells. As in BT549 cells, we observed that mCherry-DIAP1 is distributed evenly throughout the cytoplasm of S2R+, but after cotransfection with Hid, mCherry-DIAP1 is translocated to mitochondria in a Hid-like pattern (Fig. 4 E). When Rpr was expressed transiently in S2R+ cells, it shows an occasional punctate staining that is only coincidental with cytochrome c (Cyt C) staining (Fig. 4 F). However, after cotransfection with Hid, Rpr’s colocalization with Hid becomes obvious (Fig. 4 G). In sum, our experiments suggest that Rpr is a soluble protein that displays a diffuse distribution throughout the cell, and coexpression with Hid leads to Rpr relocation to the mitochondria. Additionally, these experiments underline Hid’s ability to recruit DIAP1 and its human homologue XIAP to the mitochondrial membrane. To test whether the recruitment of Rpr, XIAP, and DIAP1 is indeed dependent on Hid’s MTS, we have performed coexpression experiments with HidΔMTS and Rpr, XIAP, or DIAP1. HidΔMTS localizes to the nucleus in BT549 and S2R+ cells and triggers nuclear localization of Rpr, XIAP, and DIAP1 (Fig. S1), suggesting that indeed the observed mitochondrial localization of Rpr, XIAP, and DIAP1 is dependent on mitochondrial localization of Hid. Because Rpr and Hid overexpression induce cell death, we tested whether this might induce a change in the intracellular localization of these proteins. However, addition of the caspase inhibitor zVAD-FMK did not affect localization of Rpr or Hid (Fig. S2).

Bottom Line: In addition, we show that Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo.Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis.Our results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, Strang Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, New York, NY 10065, USA.

ABSTRACT
Apoptosis is a specific form of cell death that is important for normal development and tissue homeostasis. Caspases are critical executioners of apoptosis, and living cells prevent their inappropriate activation through inhibitor of apoptosis proteins (IAPs). In Drosophila, caspase activation depends on the IAP antagonists, Reaper (Rpr), Head involution defective (Hid), and Grim. These proteins share a common motif to bind Drosophila IAP1 (DIAP1) and have partially redundant functions. We now show that IAP antagonists physically interact with each other. Rpr is able to self-associate and also binds to Hid and Grim. We have defined the domain involved in self-association and demonstrate that it is critical for cell-killing activity in vivo. In addition, we show that Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo. Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis. Our results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis.

Show MeSH