An AIF orthologue regulates apoptosis in yeast - PubMed (original) (raw)

. 2004 Sep 27;166(7):969-74.

doi: 10.1083/jcb.200404138. Epub 2004 Sep 20.

Paula Ludovico, Eva Herker, Sabrina Büttner, Silvia M Engelhardt, Thorsten Decker, Alexander Link, Astrid Proksch, Fernando Rodrigues, Manuela Corte-Real, Kai-Uwe Fröhlich, Joachim Manns, Céline Candé, Stephan J Sigrist, Guido Kroemer, Frank Madeo

Affiliations

An AIF orthologue regulates apoptosis in yeast

Silke Wissing et al. J Cell Biol. 2004.

Abstract

Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1).

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Figures

Figure 1.

Figure 1.

Ynr074cp (Aif1p) is the yeast homologue of AIF. Alignment of Homo sapiens (Hs_AIF), Mus musculus (Mm_AIF), Dictyostelium discoideum (Dd_AIF), Caenorhabditis elegans (Ce_AIF) AIF, and Homo sapiens AMID (Hs_AMID) amino-acid sequences with the protein encoded by ORF YNR074C. Black boxes indicate amino acid identity and gray boxes indicate amino acid similarity.

Figure 2.

Figure 2.

AIF translocates from mitochondria to the nucleus under apoptotic conditions, degrades DNA, and induces apoptosis in yeast. (A) Fluorescence microscopy of cells expressing Aif1pyEGFP during exponential growth. Mitochondria were visualized with mitochondrial marker DsRed Su1-69. (B) Fluorescence microscopy of exponentially growing cells expressing Aif1pyEGFP and DsRed-NLS (nuclear staining) after an apoptotic stimulus with 0.6 mM H2O2 for 5 h on SCD. (C) Fluorescence microscopy of chronological aged yeast cells expressing Aif1pyEGFP and DsRed-NLS (nuclear staining) after 5 d growing on SCD. (D) Immunoblot of cellular fractions of untreated cells during exponential growth expressing endogenous yEGFP-tagged AIF1 expressing (lanes 2, 4, 6) and controls (lanes 1, 3, 5). Blot probed with antibodies against GFP, or Cox2p, or with mAB 414, a mAb immunoreacting with both p110, a nuclear pore protein and with a 55-kD cytosolic protein (Aris and Blobel, 1989). (E) Import of in vitro–synthesized 35S-labeled Aif1p precursor into isolated mitochondria. Wild-type and Δtom5 mitochondria were incubated with Aif1p precursor protein. Wild-type mitochondria (M) and mitoplasts (MP) were treated with proteinase K (PK). (F) Survival of Δ_aif1_ and wild type after treatment with 0.4 mM H2O2 for 4 h during early exponential growth. Data represent mean ± SEM. (G) Survival of Aif1pFLAG overexpressor and vector control after a 20-h induction on galactose with and without H2O2. Data represent mean ± SEM. (H) TUNEL and DAPI staining of Aif1pFLAG overexpressor and vector control after a 20-h induction on galactose with H2O2. (I) Degradation of 1 μg purified plasmid DNA by 2.5 μg cell extracts from Aif1pFLAG overexpressor. (J) Purification of Aif1p after recombinant expression in E. coli under denaturing conditions, and after refolding via dialysis. IC, induced control; L, lysate; M, marker; FT, flow through; W, wash; E, eluate; and refolded Aif1p. (K) Time course of the degradation of isolated yeast nuclei by purified refolded Aif1p. (L) Degradation of 1 μg plasmid DNA with different concentrations of purified refolded Aif1p. Bars, 5 μm.

Figure 3.

Figure 3.

Putative pathways of Aif1p death functions. (A) Survival of Aif1pFLAG overexpressor and vector control in wild type and yca1 disruptant background after a 20-h induction on galactose with and without 0.4 mM H2O2. Data represent mean ± SEM. (B) Aif1pFLAG overexpressor and vector control grown for 20 h on galactose with or without 0.4 mM H2O2 were analyzed in vivo for caspase activity by FITC-VAD-fmk staining using flow cytometry for quantification. (C) Survival of yeast cells upon moderate overexpression of Aif1pFLAG and vector control in wild type and Δ_cpr1_ background after a 20-h induction with or without 0.4 mM H2O2. Data represent mean ± SEM. (D) Survival of Aif1pFLAG overexpressor and vector control in wild type after a 20-h induction on galactose with or without 0.4 mM H2O2 and with 50 μg/ml Cyclosporin A (CsA) as indicated. Data represent mean ± SEM. (E) Chronological aging of wild type and Δ_aif1_ cells. Asterisks indicate a significant difference in an independent t test for days 3 and 5 at 0.02 and for day 7 at 0.075 level.

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References

    1. Aris, J.P., and G. Blobel. 1989. Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins. J. Cell Biol. 108:2059–2067. - PMC - PubMed
    1. Arnoult, D., B. Gaume, M. Karbowski, J.C. Sharpe, F. Cecconi, and R.J. Youle. 2003. Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. EMBO J. 22:4385–4399. - PMC - PubMed
    1. Blanchard, F., M.E. Rusiniak, K. Sharma, X. Sun, I. Todorov, M.M. Castellano, C. Gutierrez, H. Baumann, and W.C. Burhans. 2002. Targeted destruction of DNA replication protein Cdc6 by cell death pathways in mammals and yeast. Mol. Biol. Cell. 13:1536–1549. - PMC - PubMed
    1. Candé, C., N. Vahsen, I. Kouranti, E. Schmitt, E. Daugas, C. Spahr, J. Luban, R.T. Kroemer, F. Giordanetto, C. Garrido, et al. 2004. AIF and cyclophilin A cooperate in apoptosis-associated chromatinolysis. Oncogene. 23:1514–1521. - PubMed
    1. Cregan, S.P., A. Fortin, J.G. MacLaurin, S.M. Callaghan, F. Cecconi, S.W. Yu, T.M. Dawson, V.L. Dawson, D.S. Park, G. Kroemer, and R.S. Slack. 2002. Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death. J. Cell Biol. 158:507–517. - PMC - PubMed

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