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Comparative Study
. 2005 Apr 6;24(7):1375-86.
doi: 10.1038/sj.emboj.7600614. Epub 2005 Mar 17.

Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space

Hidenori Otera  1 , Shigenori Ohsakaya, Zen-Ichiro Nagaura, Naotada Ishihara, Katsuyoshi Mihara
Affiliations
Comparative Study

Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space

Hidenori Otera et al. EMBO J. .

Abstract

Apoptosis-inducing factor (AIF) is a mitochondrial intermembrane flavoprotein that is translocated to the nucleus in response to proapoptotic stimuli, where it induces nuclear apoptosis. Here we show that AIF is synthesized as an approximately 67-kDa preprotein with an N-terminal extension and imported into mitochondria, where it is processed to the approximately 62-kDa mature form. Topology analysis revealed that mature AIF is a type-I inner membrane protein with the N-terminus exposed to the matrix and the C-terminal portion to the intermembrane space. Upon induction of apoptosis, processing of mature AIF to an approximately 57-kDa form occurred caspase-independently in the intermembrane space, releasing the processed form into the cytoplasm. Bcl-2 or Bcl-XL inhibited both these events. These findings indicate that AIF release from mitochondria occurs by a two-step process: detachment from the inner membrane by apoptosis-induced processing in the intermembrane space and translocation into the cytoplasm. The results also suggest the presence of a unique protease that is regulated by proapoptotic stimuli in caspase-independent cell death.

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Figures

Figure 1
Figure 1
Intracellular and intramitochondrial localization of AIF variants. (A) Schematic representation of AIF variants and their intracellular or intramitochondrial localizations. The N-terminal segments of AIF from the first to the indicated residues were ligated to the N-terminus of GFP-FLAG, or the N-terminal 17 residues or TMS of AIF-GFP-FLAG was deleted (ΔN17 and AIFΔH, respectively). (B) HeLa cells expressing the indicated constructs were permeabilized and the expressed proteins were detected by GFP fluorescence and anti-FLAG antibody under confocal microscopy. Magnification, × 630; bar=20 μm.
Figure 2
Figure 2
Determination of the processing site of AIF precursor during mitochondrial import. (A) Schematic representation of AIF and its truncation mutants. Numbers represent the positions in the AIF precursor amino-acid sequence. Predicted TMS is shown in black boxes and apoptosis-dependent processing site (101/102) is indicated on the top of the figure. The MPP-processed site deduced by N-terminal sequencing is shown. (B) N-terminal segment of ∼50 amino-acid residues is cleaved by MPP. Mitochondria from rat liver and HeLa cells, cell-free synthesized AIFΔN52, AIFΔN101, and full-length AIF, and cell-free synthesized full-length AIF precursor that had been incubated with purified MPP were subjected to SDS–PAGE and subsequent immunoblot analysis using anti-AIF antibodies. (C) In vitro-synthesized 35S-labeled AIF precursor and pSu9-DHFR (lane 1) were incubated with or without purified MPP (lanes 2 and 3, respectively) or with isolated rat liver mitochondria (lane 4). The reaction mixtures were analyzed by SDS–PAGE and autoradiography. Solid and open arrowheads represent precursor proteins (p) and mature proteins (m), respectively. (D) The MPP-processing site of AIF was probed using the alanine-substituted mutant. N120-GFP-FLAG (N120) and the mutant carrying R51A-M53-A-S55/56A mutations (N120mut) were expressed in HeLa cells and the lysates were analyzed by SDS–PAGE and immunoblotting using anti-FLAG antibody.
Figure 3
Figure 3
Determination of transmembrane topology of AIF. (A) The isolated mitochondria harboring N120-GFP or N120ΔH-GFP were separated into five aliquots. Four of them were incubated with or without proteinase K under the indicated conditions. The remaining aliquot was sonicated in the presence of 1 M NaCl, followed by ultracentrifugation to separate into the supernatant (S100) and membrane (P100) fractions. All these fractions were subjected to SDS–PAGE and subsequent immunoblot analysis using antibodies against the indicated proteins. (B) HeLa cells transfected with N120-GFP-FLAG carrying an internal T7-tag insertion in the residue position of 59/60 (N120-60T7) were fixed and treated with 0.4 mg/ml digitonin (a–f) or 2.0 mg/ml digitonin (g–l). The expressed proteins were detected by GFP fluorescence (green) and either anti-FLAG (shown in red; b, h) or anti-T7 antibodies (shown in red; e, k) under confocal microscopy. Merged images are also shown (c, f, i, l). Magnification, × 630; bar=20 μm.
Figure 4
Figure 4
Apoptosis-induced processing and mitochondrial release of AIF. (A) Schematic representation of the AIF constructs and their intracellular location. (B) Subcellular distribution of various AIF constructs in response to actinomycin D treatment. HeLa cells expressing the indicated constructs (48 h after transfection) were treated with or without actinomycin D for 12 h in the presence of zVAD-fmk. The cells were examined by fluorescence confocal microscopy for GFP. The cells with a cytoplasmic GFP pattern were counted. Each histogram indicates mean±s.d. in three fields of at least 100 cells within a representative experiment. Magnification, × 630; bar=20 μm. (C) HeLa cells expressing the indicated constructs were treated with or without actinomycin D in the absence or presence of zVAD-fmk. Total cell lysates were subjected to SDS–PAGE followed by immunoblot analysis using anti-FLAG antibody. The lysate from N120ΔN101-GFP-FLAG-expressing cells was used as a size reference (lane 3). (D) HeLa cells expressing the indicated constructs were treated with or without actinomycin D in the presence of zVAD-fmk. The cells were fractionated into membrane and cytosolic fractions, and each fraction was analyzed by immunoblotting using antibodies against FLAG (for AIF constructs) or against the indicated proteins. Open and closed arrowheads indicate the GFP fusion proteins corresponding to unprocessed and processed forms, respectively. Asterisks represent uncharacterized minor bands detected in the absence of zVAD-fmk. Note that processed forms of N120 and N110 were detectable in mock-treated cells (closed arrowhead in lanes 2 and 6). Apoptosis might be brought about in the cells to a slight extent by mock treatment with Lipofectamine to induce this processing.
Figure 5
Figure 5
Processing of AIF is essential for the mitochondrial release by proapoptotic stimuli. (A) Subcellular distribution of full-length AIF/3xFLAG and ΔN101AIF/3xFLAG lacking the IMS localization signal. HeLa cells expressing the indicated constructs were treated with or without 20 μM actinomycin D in the presence of zVAD-fmk. The cells were stained with anti-FLAG antibodies (red) and DAPI (blue), and examined by fluorescence confocal microscopy. The number of cells exhibiting cytoplasmic patterns for each construct was counted and is shown in the right panel. Other conditions are same as in (B). (B) HeLa cells expressing AIF/3xFLAG were treated as described in (A), and then fractionated into membrane and cytosolic fractions, and each fraction was analyzed by immunoblotting using antibodies against the indicated proteins. (C) Schematic representation of AIF/3xFLAG and the truncation mutant. TMS is shown in yellow boxes and apoptosis-dependent processing site (101/102) is indicated on the top of the figure. (D) Subcellular distribution of full-length AIF/3xFLAG and AIFΔ96–110/3xFLAG. (E) HeLa cells expressing AIFΔ96–110/3xFLAG were treated with actinomycin D in the presence or absence of zVAD-fmk. They were fractionated into membrane and cytosolic fractions, and each fraction was analyzed by immunoblotting using antibodies against the indicated proteins.
Figure 6
Figure 6
Membrane orientation of apoptosis-dependent processing site of AIF. (A) Schematic representation of the T7-tag-scanned constructs. MPP processing site and apoptosis-dependent processing site (101/102) are indicated. T7-tag sequence was inserted at sites 59/60, 89/90, 99/100, and 109/110 of N120-GFP to create N120-60T7, N120-90T7, N120-100T7, and N120-110T7, respectively. H, predicted TMS. (B) HeLa cells expressing the indicated constructs were fixed and treated with 0.4 mg/ml digitonin (left panel) or 2.0 mg/ml digitonin (right panel). The expressed proteins were detected by GFP fluorescence (shown in green) and anti-T7 antibody (shown in red) under confocal microscopy. Merged images are also shown. Magnification, × 630; bar=20 μm.
Figure 7
Figure 7
Time course of actinomycin D-induced release of AIF-GFP, cytochrome c, and smac/DIABLO as revealed by fluorescence microscopy. HeLa cells expressing AIF-GFP were treated with actinomycin D in the presence of zVAD-fmk. At the indicated time points, the cells were examined by confocal microscopy for GFP (AIF-GFP) together with immunofluorescence microscopy using antibodies against cytochrome c (left panel) or Smac/DIABLO (right panel). Note that cytochrome c and smac/DIABLO, but not AIF-GFP, were efficiently released to the cytoplasm after 4 h incubation.
Figure 8
Figure 8
Bcl-2 and Bcl-XL inhibit both proteolytic processing and mitochondrial release of AIF. (A) HeLa cells expressing AIF/3xFLAG were cotransfected with an empty vector, mycBcl-2 vector, or 6xHis-Bcl-XL vector. Cells were treated with actinomycin D in the presence of zVAD-fmk and stained with anti-FLAG antibody. The cells exhibiting the cytoplasmic pattern were counted and are indicated by the histograms. Other conditions are same as in Figure 5. (B) HeLa cells expressing AIF/3xFLAG as described above were analyzed by SDS–PAGE followed by immunoblotting using anti-FLAG antibody. (C) HeLa cells and HeLa/XL cells transiently expressing AIF/3xFLAG were treated with staurosporine (left panel) or etoposide/VP16 (right panel) in the presence or absence of zVAD-fmk. They were then fractionated into membrane and cytosolic fractions, which were analyzed by SDS–PAGE and subsequent immunoblotting using antibodies against the indicated proteins. (D) HeLa and HeLa/XL cells were treated with actinomycin D in the presence or absence of zVAD-fmk. Other conditions are as described in (C). Processing and export of the endogenous AIF were examined using anti-AIF antibodies. The asterisk represents nonspecific bands detected with anti-AIF antibodies.
Figure 9
Figure 9
A model for proteolytic processing-dependent release of AIF from mitochondrial IMS upon induction of apoptosis. Apoptotic information from the cytoplasm is transmitted by proapoptotic Bcl-2 family proteins to alter mitochondrial environments (Step 1), thus triggering a switch that activates the responsible protease(s) or renders AIF accessible to constitutively active protease(s) (Step 2). The IMS portion of AIF is proteolytically cleaved from the inner membrane (Step 3). The mature AIF fragment is released from the mitochondrial IMS to the cytoplasm, possibly through a specific channel (Step 4). Bcl-2 blocks not only the initiation of the apoptotic process on the mitochondrial surface by proapoptotic Bcl-2 family proteins, but inhibits AIF processing in the IMS.
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