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. 2006 Aug 1;103(31):11573-8.
doi: 10.1073/pnas.0603007103. Epub 2006 Jul 24.

Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration

Cristina Muñoz-Pinedo  1 , Ana Guío-Carrión, Joshua C Goldstein, Patrick Fitzgerald, Donald D Newmeyer, Douglas R Green
Affiliations

Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration

Cristina Muñoz-Pinedo et al. Proc Natl Acad Sci U S A. .

Abstract

The release of mitochondrial intermembrane space proteins to the cytosol is a key event during apoptosis. We used in situ fluorescent labeling of proteins tagged with a short tetracysteine-containing sequence to follow the release of Smac, Omi, adenylate kinase-2, cytochrome c, and apoptosis-inducing factor (AIF) during apoptosis and compared the release with that of cytochrome c tagged with GFP in individual cells observed over time. We observed a caspase-independent, simultaneous release of cytochrome c, Smac, Omi, and adenylate kinase-2. Although AIF release also was caspase-independent and commenced with that of the other proteins, it proceeded much more slowly and incompletely from mitochondria, perhaps because of a requirement for a secondary event. These results suggest that these proteins are released through the same mitochondrial pore and that apoptosis may not be regulated through a selective release of individual mitochondrial proteins. The timing and extent of AIF release makes it unlikely that it is involved in the induction of apoptosis, either upstream or downstream of mitochondrial outer membrane permeabilization.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
TC-tagged proteins display mitochondrial localization. HeLa-cyt.c-GFP cells were stably (Smac, Omi, and AK2) or transiently (AIF) transfected with the vector encoding the corresponding TC-tagged protein and stained with the red dye ReAsH. (Scale bars: 20 μm.) Arrow indicates a cell transfected with AIF-TC; other cells in the field did not express the protein. Full-resolution images are shown in Fig. 5.
Fig. 2.
Fig. 2.
Smac, AK2, and Omi are released with cytochrome c. (A) HeLa-cyt.c-GFP cells stably expressing Smac-TC were stained with the red dye ReAsH or the green dye FlAsH and treated with the indicated inducers. (A Upper) Scaled average and SD of the punctate/diffuse index are shown of 10 cells from the same experiment, aligned to the time of first detected Smac-FlAsH release (t = 0). Cells also were stained with the mitochondrial dye tetramethylrhodamine ethyl ester (TMRE); TMRE brightness index was calculated by averaging the values of the average intensity of the pixels within each cell. (A Lower) Scaled average of the punctate/diffuse index for Smac-ReAsH and cytochrome c aligned to the time of cytochrome c release (n = 7). Images were taken every 3 min with a ×40 objective. (B) HeLa-cyt.c-GFP cells stably transduced with AK2-TC were stained with ReAsH and treated with the indicated inducers. Graphs show the scaled average of the punctate/diffuse index from 7 (Upper) or 17 (Lower) cells. (C) HeLa-cyt.c-GFP cells were transiently transfected with a construct encoding Omi-TC. Cells were stained with ReAsH, typically 24 to 40 h after transfection, and treated with the indicated inducers. (C Upper) Graph from a representative experiment showing the scaled punctate/diffuse index of cytochrome c-GFP and Omi-TC (average and SD of five cells). (C Middle) Graph from an experiment in which cells were treated with staurosporine in the presence of 100 μM zVAD-fmk. Pictures were taken every 3 min. Images from the same experiment are shown in C Lower. Additional cells are shown in Movie 2. (Scale bar: 10 μm.)
Fig. 3.
Fig. 3.
AIF-TC is released slowly and in a caspase-independent manner. HeLa-cyt.c-GFP cells were transiently transfected with a construct encoding AIF-TC and stained with the red dye ReAsH typically 24 to 40 h after transfection. Images were taken every 4 min after appropriate incubation times with the inducer, as described in Methods. (A) Time shown is time of total incubation with ActD. (Scale bars: 10 μm.) (B) Average and SD of four cells from an experiment in which cells were treated with staurosporine in the presence of quinoline-Val-Asp-CH2-difluorophenoxy (qVD-OPH).
Fig. 4.
Fig. 4.
AIF-GFP is released slowly and in a caspase-independent manner. (A–C) HeLa cells stably expressing cytochrome c-TC were transiently transfected with a construct encoding murine AIF-GFP. Pictures in A show the colocalization of AIF-GFP with the mitochondrial marker Mitotracker Red (MTR). (Scale bars: 10 μm.) (B and C) Cells were stained with the red dye ReAsH to label cytochrome c-TC (abbreviated cyt.c-TC) and treated with the indicated inducers. Images were taken every 3 min. Propidium iodide was added to the medium to monitor cell death. (D and E) HeLa cells stably expressing cyt.c-TC were transiently transfected with a construct encoding human AIF-GFP and stained with ReAsH. Graph in D shows the average and SD of punctate/diffuse index for AIF-GFP and cyt.c-TC of six cells aligned to the time of cytochrome c release. (E) Images were taken every 3 min. Numbers show time (in minutes) after drug addition. (Scale bar: 10 μm.)
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