Apoptosis-inducing factor is a target for ubiquitination through interaction with XIAP

Abstract

X-linked inhibitor of apoptosis (XIAP) is an inhibitor of apoptotic cell death that protects cells by caspase-dependent and independent mechanisms. In a screen for molecules that participate with XIAP in regulating cellular activities, we identified apoptosis-inducing factor (AIF) as an XIAP binding protein. Baculoviral IAP repeat 2 of XIAP is sufficient for the XIAP/AIF interaction, which is disrupted by Smac/DIABLO. In healthy cells, mature human AIF lacks only the first 54 amino acids, differing significantly from the apoptotic form, which lacks the first 102 amino-terminal residues. Fluorescence complementation and immunoprecipitation experiments revealed that XIAP interacts with both AIF forms. AIF was found to be a target of XIAP-mediated ubiquitination under both normal and apoptotic conditions, and an E3 ubiquitin ligase-deficient XIAP variant displayed a more robust interaction with AIF. Expression of either XIAP or AIF attenuated both basal and antimycin A-stimulated levels of reactive oxygen species (ROS), and when XIAP and AIF were expressed in combination, a cumulative decrease in ROS was observed. These results identify AIF as a new XIAP binding partner and indicate a role for XIAP in regulating cellular ROS.

FIG. 1.

AIF is an XIAP-associated factor. (A) D148A/W310A XIAP-TAP was expressed in HEK 293 cells. A cell lysate was prepared and subjected to TAP. Total protein in input and final eluted fractions was visualized by SDS-PAGE and Coomassie blue staining (left and middle panels), showing XIAP bait protein and associated factors. Eluted fractions were pooled, precipitated with trichloroacetic acid, and digested with trypsin, and peptides were identified by mass spectrometry. Eluted fractions were also immunoblotted for the presence of AIF and Smac (right panel). (B) Untransfected HEK 293 cell lysate was precipitated with either beads alone or beads bound to an AIF-specific antibody, and the presence of XIAP in precipitated complexes was assessed by immunoblot analysis. Numbers at left of the panels are molecular masses in kilodaltons.

FIG. 2.

BIR2 of XIAP binds AIF; mature AIF begins at residue 55. (A) HEK 293 cells were transfected with plasmids encoding various XIAP domain truncations in fusion with GST along with a plasmid encoding full-length human AIF. Cell lysates were prepared, and XIAP was precipitated with glutathione (GSH) beads. The presence of AIF in precipitated complexes was determined by immunoblotting. (B) Full-length human AIF-TAP was expressed in HEK 293 cells. AIF was precipitated from cell lysates using IgG-Sepharose beads; eluted proteins were then separated by SDS-PAGE and transferred to PVDF membranes. Following Coomassie blue staining, the AIF band was excised and subjected to Edman degradation in order to determine the mature amino terminus. Numbers at left of panels A and B are molecular masses in kilodaltons. (C) Primary sequence of full-length human AIF. The underlined sequence is one of four AIF peptides recovered from analysis of XIAP-TAP. The gray arrow is the first residue of mature AIF as reported by reference ; the black arrow is the first residue of mature human AIF as determined by this study.

FIG. 3.

XIAP binds AIF variants and mediates AIF ubiquitination. (A) HEK 293 cells were transfected with a plasmid encoding WT-XIAP along with control, full-length AIF, Ub-Δ54AIF, Ub-Δ102AIF, met-Δ54AIF, or met-Δ102AIF expression plasmids. Cell lysates were then prepared and precipitated with anti-XIAP. The presence of AIF in precipitated complexes was determined by immunoblot analysis for the FLAG tag present at the carboxy terminus of each AIF protein (top panel). Equivalent expression of XIAP and AIF variants was confirmed by immunoblotting input lysates with anti-FLAG (middle panel) or anti-XIAP (bottom panel). The black line present in the bottom panel indicates removal of a single empty lane solely for the purpose of clarity. (B) HEK 293 cells were transiently transfected with His-tagged ubiquitin and plasmids expressing full-length AIF-FLAG, Ub-Δ54AIF-FLAG, and Ub-Δ102AIF-FLAG in the absence and presence of an XIAP expression plasmid. Ubiquitinated material was then precipitated using Ni-NTA beads, and the presence of FLAG-tagged proteins (AIF) in precipitated complexes was detected by immunoblot analysis. Numbers at left of panel A and at right of panel B are molecular masses in kilodaltons.

FIG. 4.

Fluorescence complementation analysis of the XIAP/AIF interaction. (A and B) Full-length AIF (A) and Ub-Δ102AIF (B) in fusion with YFP at the carboxy terminus (here shown in green) were expressed in HEK 293 cells. Cells were stained with Mitotracker Red (red staining) and Hoechst stain (blue staining). The cellular localization of AIF proteins was then determined by confocal microscopy. (C) BiFC was used to examine the XIAP/AIF interaction. YN-XIAP was coexpressed with Ub-Δ102AIF-YC. The green fluorescence observed is indicative of a cytoplasmic interaction between XIAP and Δ102 AIF. Cells were additionally stained with both Mitotracker Red (red) and Hoechst stain (blue).

FIG. 5.

H467A-XIAP immunoprecipitates all AIF variants. (A) HEK 293 cells were transfected with a plasmid encoding H467A-XIAP along with control, full-length AIF, Ub-Δ54AIF, Ub-Δ102AIF, met-Δ54AIF, or met-Δ102AIF expression plasmids. XIAP was then immunoprecipitated from cell lysates, and the presence of AIF was determined by immunoblot analysis using anti-FLAG (top panel). Equivalent expression of XIAP (bottom panel) and AIF variants (middle panel) was confirmed by immunoblotting input lysates. The black line present in the bottom panel indicates removal of a single empty lane solely for the purpose of clarity. Note that this experiment was carried out in parallel to that shown for WT-XIAP in Fig. 3A so that efficiency of AIF coprecipitation could be compared between the two XIAP variants. Numbers at left are molecular masses in kilodaltons.

FIG. 6.

XIAP ubiquitinates AIF following induction of apoptosis. HEK 293 cells were transfected with His-tagged ubiquitin and either control plasmids (lanes 1 and 2), full-length AIF-FLAG (lanes 3 and 4), or full-length AIF-FLAG and XIAP expression plasmids (lanes 5 and 6), in the absence (lanes 1, 3, and 5) and presence (lanes 2, 4, and 6) of a plasmid encoding Bax. Ubiquitinated material was then precipitated using Ni-NTA beads, and the presence of FLAG-tagged proteins (AIF) in precipitated complexes was detected by immunoblot analysis. Numbers at right are molecular masses in kilodaltons.

FIG. 7.

Smac/DIABLO disrupts the XIAP/AIF interaction. HEK 293 cells were transfected with a plasmid encoding WT-XIAP along with either control, DIABLO-HA, Δ1A-DIABLO-HA, or A1G-DIABLO-HA expression plasmid. Cell lysates were then precipitated with anti-XIAP, and the presence of AIF (anti-AIF, top panel) or Smac/DIABLO (anti-HA, second panel from top) in immune complexes was determined by immunoblot analysis. Equivalent expression of AIF (third panel from top) and Smac/DIABLO (bottom panel) was assessed by immunoblot analysis of input lysates. Numbers at left are molecular masses in kilodaltons.

FIG. 8.

AIF does not prevent XIAP-mediated caspase inhibition. HEK 293 cells were transfected with the indicated plasmids, cell lysates were prepared, and the activation of caspase 3 was determined by incubation with the fluorogenic caspase 3 substrate DEVD-7-amino-4-trifluoromethyl coumarin.

FIG. 9.

Effects of XIAP/AIF expression of ROS formation. HEK 293 cells were transfected with control or XIAP expression plasmids in the absence and presence of either full-length AIF, Ub-Δ54 AIF, or Ub-Δ102 AIF. Cells were then left untreated or treated with antimycin A and stained with the ROS-indicating dye CM-H₂-DCFDA, and cellular ROS levels were determined by flow cytometry. Representative histograms of untreated (gray) and antimycin A-treated (black) cells are shown, and values shown are the average ±1 standard deviation of three replicate measurements.