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.

AIF translocates from mitochondria to the nucleus under apoptotic conditions, degrades DNA, and induces apoptosis in yeast. (A) Fluorescence microscopy of cells expressing Aif1p^yEGFP during exponential growth. Mitochondria were visualized with mitochondrial marker DsRed Su1-69. (B) Fluorescence microscopy of exponentially growing cells expressing Aif1p^yEGFP 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 Aif1p^yEGFP 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 Δaif1 and wild type after treatment with 0.4 mM H₂O₂ for 4 h during early exponential growth. Data represent mean ± SEM. (G) Survival of Aif1p^FLAG 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 Aif1p^FLAG 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 Aif1p^FLAG 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.

Putative pathways of Aif1p death functions. (A) Survival of Aif1p^FLAG 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) Aif1p^FLAG 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 Aif1p^FLAG and vector control in wild type and Δcpr1 background after a 20-h induction with or without 0.4 mM H₂O₂. Data represent mean ± SEM. (D) Survival of Aif1p^FLAG 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 Δaif1 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.