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Role for X-linked Inhibitor of apoptosis protein upstream of mitochondrial permeabilization.

Owens TW, Foster FM, Valentijn A, Gilmore AP, Streuli CH - J. Biol. Chem. (2009)

Bottom Line: Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak.We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified.Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

ABSTRACT
Apoptosis is controlled by a signaling equilibrium between prosurvival and proapoptotic pathways, such that unwanted apoptosis is avoided, but when required it occurs rapidly and efficiently. Many apoptosis regulators display dual roles, depending upon whether a cell has received an apoptotic stimulus or not. Here, we identify a novel and unexpected function for X-linked inhibitor of apoptosis (XIAP) that occurs when apoptosis is triggered under physiological conditions. We show that in response to loss of survival signals provided by cell adhesion, endogenous XIAP translocates from the cytosol into a mitochondrial 400-kDa complex and that this occurs very early in the apoptosis process. Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak. Thus, although XIAP suppresses apoptosis in healthy cells, our data indicate that XIAP may contribute to it in response to a proapoptotic signal such as loss of extracellular matrix-dependent survival signaling. We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified. Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

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Membrane association of XIAP corresponds with ability to induce MOMP. A, MECs transiently expressing RFP-XIAP were stained with mitochondrial HSP70 (green). Note close association of RFP-XIAP with mtHSP70 (arrows and enlarged view on right). B, to confirm mitochondrial association of RFP-XIAP, cells were co-transfected with RFP-XIAP and a V5-epitope tagged form of Bid that constitutively localizes to mitochondria (p11BidV5). C and D, MEF cells transiently expressing Myc-XIAP were either left adherent (Adh) or detached for 60 min (D60) prior to fractionation. Samples were separated by SDS-PAGE only (C) or by BN-PAGE followed by SDS-PAGE (D) and immunoblotted for XIAP. Arrow and arrowhead indicate Myc-XIAP and endogenous XIAP, respectively. Note that the cytosolic Myc-XIAP was in a complex of the same size as the endogenous protein (compare lanes 6–12 D, upper panel, and Fig. 1C, top panel). E and F, MECs transiently expressing RFP, RFP-XIAP (XIAP), RFP-BIR3-RING, or RFP-BIR3 were scored for cytochrome c release. G, cytosolic and membrane fractions of MEF cells, untransfected or transiently expressing Myc-XIAP, Myc-BIR3-RING, or Myc-BIR3 were analyzed by immunoblotting for Myc and reprobing for XIAP. Arrowhead indicates endogenous XIAP. Arrows indicate transfected, membrane-localized XIAP fragments. Note that BIR3 is only present in the cytosolic fraction and that Myc-BIR3 is only detected by the Myc antibody because it does not contain the epitope for the XIAP antibody.
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Figure 4: Membrane association of XIAP corresponds with ability to induce MOMP. A, MECs transiently expressing RFP-XIAP were stained with mitochondrial HSP70 (green). Note close association of RFP-XIAP with mtHSP70 (arrows and enlarged view on right). B, to confirm mitochondrial association of RFP-XIAP, cells were co-transfected with RFP-XIAP and a V5-epitope tagged form of Bid that constitutively localizes to mitochondria (p11BidV5). C and D, MEF cells transiently expressing Myc-XIAP were either left adherent (Adh) or detached for 60 min (D60) prior to fractionation. Samples were separated by SDS-PAGE only (C) or by BN-PAGE followed by SDS-PAGE (D) and immunoblotted for XIAP. Arrow and arrowhead indicate Myc-XIAP and endogenous XIAP, respectively. Note that the cytosolic Myc-XIAP was in a complex of the same size as the endogenous protein (compare lanes 6–12 D, upper panel, and Fig. 1C, top panel). E and F, MECs transiently expressing RFP, RFP-XIAP (XIAP), RFP-BIR3-RING, or RFP-BIR3 were scored for cytochrome c release. G, cytosolic and membrane fractions of MEF cells, untransfected or transiently expressing Myc-XIAP, Myc-BIR3-RING, or Myc-BIR3 were analyzed by immunoblotting for Myc and reprobing for XIAP. Arrowhead indicates endogenous XIAP. Arrows indicate transfected, membrane-localized XIAP fragments. Note that BIR3 is only present in the cytosolic fraction and that Myc-BIR3 is only detected by the Myc antibody because it does not contain the epitope for the XIAP antibody.

Mentions: To determine whether XIAP influences MOMP through a direct association with mitochondria, we examined the subcellular localization of tagged XIAP using fractionation (Myc-XIAP) and immunofluorescence (RFP-XIAP). The transfected XIAP was present in both the cytosolic and membrane fractions (Fig. 4D), and in the latter it was located adjacent to mitochondria but was not associated with other organelle membranes (Fig. 4, A and B, and supplemental Fig. S8). Importantly, the exogenous XIAP was present in membranes even in attached cells, and detaching the cells from ECM caused no further enrichment in the membrane fraction (Fig. 4C). This membrane-associated Myc-XIAP was constitutively in the high molecular weight complex that is only formed by endogenous XIAP following ECM withdrawal (Fig. 4D, lower panel, lane 3). These results suggest that exogenous XIAP activates the intrinsic pathway by mimicking a function of endogenous XIAP, which is normally only revealed once the cell has received an apoptotic insult.


Role for X-linked Inhibitor of apoptosis protein upstream of mitochondrial permeabilization.

Owens TW, Foster FM, Valentijn A, Gilmore AP, Streuli CH - J. Biol. Chem. (2009)

Membrane association of XIAP corresponds with ability to induce MOMP. A, MECs transiently expressing RFP-XIAP were stained with mitochondrial HSP70 (green). Note close association of RFP-XIAP with mtHSP70 (arrows and enlarged view on right). B, to confirm mitochondrial association of RFP-XIAP, cells were co-transfected with RFP-XIAP and a V5-epitope tagged form of Bid that constitutively localizes to mitochondria (p11BidV5). C and D, MEF cells transiently expressing Myc-XIAP were either left adherent (Adh) or detached for 60 min (D60) prior to fractionation. Samples were separated by SDS-PAGE only (C) or by BN-PAGE followed by SDS-PAGE (D) and immunoblotted for XIAP. Arrow and arrowhead indicate Myc-XIAP and endogenous XIAP, respectively. Note that the cytosolic Myc-XIAP was in a complex of the same size as the endogenous protein (compare lanes 6–12 D, upper panel, and Fig. 1C, top panel). E and F, MECs transiently expressing RFP, RFP-XIAP (XIAP), RFP-BIR3-RING, or RFP-BIR3 were scored for cytochrome c release. G, cytosolic and membrane fractions of MEF cells, untransfected or transiently expressing Myc-XIAP, Myc-BIR3-RING, or Myc-BIR3 were analyzed by immunoblotting for Myc and reprobing for XIAP. Arrowhead indicates endogenous XIAP. Arrows indicate transfected, membrane-localized XIAP fragments. Note that BIR3 is only present in the cytosolic fraction and that Myc-BIR3 is only detected by the Myc antibody because it does not contain the epitope for the XIAP antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Membrane association of XIAP corresponds with ability to induce MOMP. A, MECs transiently expressing RFP-XIAP were stained with mitochondrial HSP70 (green). Note close association of RFP-XIAP with mtHSP70 (arrows and enlarged view on right). B, to confirm mitochondrial association of RFP-XIAP, cells were co-transfected with RFP-XIAP and a V5-epitope tagged form of Bid that constitutively localizes to mitochondria (p11BidV5). C and D, MEF cells transiently expressing Myc-XIAP were either left adherent (Adh) or detached for 60 min (D60) prior to fractionation. Samples were separated by SDS-PAGE only (C) or by BN-PAGE followed by SDS-PAGE (D) and immunoblotted for XIAP. Arrow and arrowhead indicate Myc-XIAP and endogenous XIAP, respectively. Note that the cytosolic Myc-XIAP was in a complex of the same size as the endogenous protein (compare lanes 6–12 D, upper panel, and Fig. 1C, top panel). E and F, MECs transiently expressing RFP, RFP-XIAP (XIAP), RFP-BIR3-RING, or RFP-BIR3 were scored for cytochrome c release. G, cytosolic and membrane fractions of MEF cells, untransfected or transiently expressing Myc-XIAP, Myc-BIR3-RING, or Myc-BIR3 were analyzed by immunoblotting for Myc and reprobing for XIAP. Arrowhead indicates endogenous XIAP. Arrows indicate transfected, membrane-localized XIAP fragments. Note that BIR3 is only present in the cytosolic fraction and that Myc-BIR3 is only detected by the Myc antibody because it does not contain the epitope for the XIAP antibody.
Mentions: To determine whether XIAP influences MOMP through a direct association with mitochondria, we examined the subcellular localization of tagged XIAP using fractionation (Myc-XIAP) and immunofluorescence (RFP-XIAP). The transfected XIAP was present in both the cytosolic and membrane fractions (Fig. 4D), and in the latter it was located adjacent to mitochondria but was not associated with other organelle membranes (Fig. 4, A and B, and supplemental Fig. S8). Importantly, the exogenous XIAP was present in membranes even in attached cells, and detaching the cells from ECM caused no further enrichment in the membrane fraction (Fig. 4C). This membrane-associated Myc-XIAP was constitutively in the high molecular weight complex that is only formed by endogenous XIAP following ECM withdrawal (Fig. 4D, lower panel, lane 3). These results suggest that exogenous XIAP activates the intrinsic pathway by mimicking a function of endogenous XIAP, which is normally only revealed once the cell has received an apoptotic insult.

Bottom Line: Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak.We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified.Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

ABSTRACT
Apoptosis is controlled by a signaling equilibrium between prosurvival and proapoptotic pathways, such that unwanted apoptosis is avoided, but when required it occurs rapidly and efficiently. Many apoptosis regulators display dual roles, depending upon whether a cell has received an apoptotic stimulus or not. Here, we identify a novel and unexpected function for X-linked inhibitor of apoptosis (XIAP) that occurs when apoptosis is triggered under physiological conditions. We show that in response to loss of survival signals provided by cell adhesion, endogenous XIAP translocates from the cytosol into a mitochondrial 400-kDa complex and that this occurs very early in the apoptosis process. Membrane-associated XIAP induces mitochondrial outer membrane permeabilization leading to cytochrome c and Smac release, which is dependent on Bax and Bak. Thus, although XIAP suppresses apoptosis in healthy cells, our data indicate that XIAP may contribute to it in response to a proapoptotic signal such as loss of extracellular matrix-dependent survival signaling. We suggest that, as with Bcl-2 family proteins, more diverse functions for XIAP exist than previously identified. Moreover, switching the function of proteins from anti- to proapoptotic forms may be a common theme in the efficient execution of cell death.

Show MeSH
Related in: MedlinePlus