Yeast Ynl305cp contains a bona fide BH3 domain. (A) Sequence comparison of BH3 domains of indicated BCL-2 family members and Ynl305cp. Following sequences were used: BAX (Homo sapiens, Q07812), BAX (Danio rerio, AF231015), HRK (H. sapiens, NP_003797), BAD (H. sapiens, CAG46757), BIM (H. sapiens, O43521), NOXA (also known as PMAIP1, Mus musculus, BAA95781), PUMA (also known as BBC3, H. sapiens, Q9BXH1), BID (H. sapiens, P55957) BID (M. musculus, P70444), EGL-1 (Caenorhabditis elegans, O61667) and Ynl305cp (S. cerevisiae, AAT92698). (B, C) Survival determined by clonogenicity (B) and quantification of ROS accumulation (DHE → Eth) (C) of Ybh3p- or Ybh3p^ΔBH3-overexpressing cells or corresponding vector control treated or not with 120 mM acetic acid or 0.6 mM H₂O₂ (mean±s.e.m., n=6). (D) Quantification of DNA fragmentation using TUNEL staining of Ybh3p- or Ybh3p^ΔBH3-overexpressing cells or corresponding vector control after treatment with or without 120 mM acetic acid or 0.6 mM H₂O₂, respectively. In each experiment, 30 000 cells were analysed using flow cytometry (mean±s.e.m., n=4). (E) Quantification of phosphatidylserine externalization and loss of membrane integrity using AnnexinV/PI-co-staining of Ybh3p- or Ybh3p^ΔBH3-overexpressing cells or corresponding vector control after treatment with or without 120 mM acetic acid or 0.6 mM H₂O₂, respectively. In each experiment, 30 000 cells were analysed using flow cytometry (mean±s.e.m., n=4). (F) Representative micrographs of AnnexinV (green)/PI (red)-co-staining of cells overexpressing Ybh3p or harbouring the empty vector after treatment with or without 120 mM acetic acid or 0.6 mM H₂O₂, respectively. ^*P<0.05, ^**P<0.01 and ^***P<0.001.

The yeast BH3 domain induces apoptotic changes in isolated mitochondria as well as in mammalian and yeast cells. (A) Amino-acid sequence alignment of the native yeast BH3 domain and the BH3^D295K and BH3^L290K point mutants. (B) Immunoblot analysis of WT yeast cells for FLAG-tagged Ybh3p, Ybh3p^ΔBH3, Ybh3p^D295K or Ybh3p^L290K. (C, D) Death determined by clonogenicity (C) and quantification of ROS accumulation using DHE → Eth conversion (D) of yeast cells overexpressing Ybh3p, Ybh3p^ΔBH3, Ybh3p^D295K or Ybh3p^L290K after treatment with 120 mM acetic acid for 1 or 4 h. Death rates were calculated by setting the survival of vector control cells to 100% and thus 0% death (mean±s.e.m., n=8). (E–H) Swelling of mouse liver mitochondria as indicated by loss of absorbance (ABS) at 490 nm. Mitochondria were pre-incubated or not with 5 μM CsA, followed by the administration of 2.5 μM (E) or 5 μM (F) of native (BH3) or mutated (BH3^L290K) peptide or indicated concentrations of Ca²⁺(G). To permit direct comparison, swelling after 14 min under all conditions analysed was depicted in (H). Representative experiments are shown, with data representing mean±s.e.m. of three replicates. (I, J) Immunoblot analysis of supernatants and mitochondrial pellets (I) from mouse liver mitochondria incubated with the indicated concentration of native (BH3) or mutated (BH3^L290K) peptide using an antibody specific for cyt c. For supernatants, the signal intensities of cyt c were quantified and normalized to VDAC intensities of deployed mitochondria (J) (mean±s.e.m., n=3). (K) Non-small cell lung cancer (A549) cells were incubated with 30 μM of the native (BH3) or mutated (BH3^L290K) peptide, followed by the flow cytometric assessment of Δψ_m dissipation (DiOC₆(3)^low) and loss of plasma membrane integrity (PI+) (mean±s.e.m., n=5). (L) A549 cells transfected with a control siRNA (siUNR) or with siRNA targeting BAX, BAK or with a combination of these were treated with 30 μM of the native (BH3) or mutated (BH3^L290K) peptide followed by the flow cytometric assessment of Δψ_m dissipation (DiOC₆(3)^low) and loss of plasma membrane integrity (PI+). Data represent mean±s.e.m. and one representative experiment is shown with n=3. ^*P<0.05, ^**P<0.01 and ^***P<0.001.

Deletion of YBH3 protects against H₂O₂, acetic acid, BAX expression and ageing. (A, B) Survival determined by clonogenicity (A) and quantification of ROS accumulation (DHE → Eth) (B) of wild-type (WT) and Δybh3 cells treated or not with acetic acid or H₂O₂ (mean±s.e.m., n=8). (C) Flow cytometric quantification of phosphatidylserine externalization and loss of membrane integrity using AnnexinV/PI co-staining of cells described in (A) (mean±s.e.m., n=4). (D) Survival determined by clonogenicity upon heterologous expression of murine BAX in WT and Δybh3 cells. Viability was determined after 20 h of BAX expression (mean±s.e.m., n=8). (E) Immunoblot analysis of WT and Δybh3 cells expressing BAX. (F) Replicative lifespan of WT and Δybh3 cells. A representative experiment is shown with n⩾40. Indicated P-value (calculated using Tarone-Ware) refers to median lifespan of Δybh3 compared with WT. (G, H) Survival determined by clonogenicity (G) and quantification of ROS accumulation using DHE → Eth conversion (H) of WT and Δybh3 cells during chronological ageing on glycerol-containing media. Colony forming units of WT cells at day 1 were set to 100%. One representative ageing experiment is shown, with data representing mean±s.e.m. of six independent cultures. (I, J) Flow cytometric quantification of TUNEL staining (I) and AnnexinV/PI-co-staining (J) of chronologically aged WT and Δybh3 cells at indicated days during ageing (mean±s.e.m., n=4). See also Supplementary Figure S5. ^**P<0.01 and ^***P<0.001.

Knockdown of the human orthologues of Cor1p (QCR1) and Mir1p (PHC) reduces BAX aggregation and attenuates apoptosis in U2OS cells. (A, B) U2OS cells stably expressing GFP-BAX were transfected with siRNAs against human COR1 (siQCR1) or MIR1 (siPHC) or with an siRNA against an unrelated sequence (siUNR) for 48 h and subsequently treated with MTX or left untreated for 12 h. Nuclei were counterstained with Hoechst 33342 and subjected to automated image acquisition and analysis. Representative images (A) and quantitative analysis of GFP-BAX aggregation (B) are shown (mean±s.e.m., n=8). (C, D) U2OS transfected with siRNA and treated with 1 μM MTX or left untreated were immunostained with anti-cleaved caspase3 antibody followed by anti-rabbit Alexa488 conjugated antibody. Upon counterstaining with Hoechst 33342, automated image acquisition was performed. Representative images (C) and quantitative analysis (D) of cleaved-caspase3/Alexa488 signals are shown (mean±s.e.m., n=4). (E, F) U2OS cells treated as described in (C) were analysed for Δψ_m using the membrane potential-sensitive dye CMTMRos and counterstained with Hoechst 33342. Representative images (E) and quantitative analyses (F) are shown (mean±s.e.m., n=8). (G) U2OS cells treated as described in (C) were stained with PI and the membrane potential-sensitive dye DiOC₆(3) and subjected to flow cytometric quantification. The percentage of cells displaying loss of mitochondrial membrane potential alone (DiOC₆(3)^low/PI⁻) or in combination with plasma membrane rupture (DiOC₆(3)^low/PI⁺) is plotted (mean±s.e.m., n=3). (H) Validation of the siRNA-mediated knockdown using specific antibodies for PHC, QCR1 and actin. ^***P<0.001.

BCL-X_L interacts with Ybh3p and inhibits Ybh3p-mediated death. (A, B) Survival determined by clonogenicity (A) and quantification of ROS accumulation using DHE → Eth conversion (B) of cells overexpressing Ybh3p alone or co-expressing Ybh3p and BCL-X_L and the corresponding vector controls upon treatment with or without 120 mM acetic acid. Survival was normalized to untreated control cultures (mean±s.e.m., n=5). (C) Protein extracts of cells expressing BCL-X_L either alone or in combination with FLAG-tagged Ybh3p as well as of cells harbouring both empty vectors were used to immunoprecipitate FLAG-tagged Ybh3p using agarose beads coupled with a monoclonal anti-FLAG antibody. Immunoprecipitates (eluates) as well as whole protein extracts were analysed via immunoblot for BCL-X_L and Ybh3p. (D, E) Survival determined by clonogenicity (D) and quantification of ROS accumulation using DHE → Eth conversion (E) of WT and Δuth1 cells overexpressing Ybh3p or harbouring the empty vector upon treatment with or without 120 mM acetic acid or 0.6 mM H₂O₂. Survival was normalized to untreated isogenic vector control (mean±s.e.m., n=6). (F) Immunoblot analysis of WT and Δuth1 cells for FLAG-tagged Ybh3p. ^**P<0.01 and ^***P<0.001.

Ybh3p translocates to mitochondria and is imported in a TOM-receptor-independent way. (A, B) Fluorescence microscopy of yeast cells expressing GFP-tagged Ybh3p or Ybh3p^ΔBH3 or harbouring the vector control for 16 h before (A) and after (B) treatment with acetic acid. Vacuoles were counterstained using Celltracker Blue. For visualization of mitochondria, the mitochondrial marker DsRed Su1-69 (DsRed-MLS) was co-expressed. (C) Immunoblot analysis of mitochondrial fractions harvested from cells overexpressing FLAG-tagged Ybh3p without or with acetic acid treatment for 2 and 4 h before subcellular fractionation. (D) The cyt c content of mitochondrial fractions harvested from cells overexpressing Ybh3p compared with isogenic vector control after 4 h of acetic acid treatment determined by immunoblot analysis and subsequent quantification of cyt c signal compared with porin signal (Por1p, mitochondrial marker) (mean±s.e.m., n=6). (E) The cyt c content in supernatants after release of cytosolic content via short permeabilization. Cells overexpressing Ybh3p or harbouring the empty vector were pre-treated or not with 120 mM acetic acid for 2 h prior to permeabilization followed by immunoblot analysis. Cyt c signal in supernatants was normalized to cyt c content in whole cell extracts (mean±s.e.m., n=5). (F, G) ³⁵S-labelled Ybh3p was incubated with isolated yeast mitochondria, followed by treatment with proteinase K (F) or carbonate extraction (G) and analysis by NuPAGE and autoradiography. For carbonate extraction, supernatants (SN) and pellets (P) were analysed. Cells were grown in the presence or absence of acetic acid prior to isolation of mitochondria. (H) Yeast mitochondria isolated from cells grown in the presence or absence of 90 mM acetic acid were incubated with ³⁵S-labelled Su9-DHFR for 30 min or with ³⁵S-labelled Ybh3p for the indicated time followed by treatment with proteinase K and analysis by NuPAGE and autoradiography. Isolated mitochondria were incubated with or without trypsin prior to the import reaction. In addition, mitochondria were analysed for Tom22p, Tom70p, Atp2p and Mdh1 using immunoblotting. Where indicated the membrane potential (Δψ) was dissipated prior to the import reaction. See also Supplementary Figure S4. ^*P<0.05 and ^**P<0.01.

Mir1p and Cor1p facilitate mitochondrial import of Ybh3p as well as Ybh3p-mediated breakdown of mitochondrial membrane potential and subsequent cell death. (A) Death and ROS accumulation of wild-type (WT) cells and indicated deletion mutants overexpressing Ybh3p upon treatment with 120 mM acetic acid. Percentage of death as well as fold of DHE → Eth conversion mediated by overexpression of Ybh3p compared with corresponding isogenic vector control was plotted (mean±s.e.m., n=6–8). (B) Immunoblot analysis of Ybh3p^FLAG expression in WT, Δmir1 and Δcor1 cells. (C, D) Representative immunoblot (C) and quantification of mitochondrial full-length Ybh3p^FLAG normalized to porin (Por1p, mitochondrial loading control) (D) in mitochondrial fractions isolated from WT, Δmir1 and Δcor1 cells overexpressing Ybh3p^FLAG treated 120 mM with acetic acid for 2 and 4 h or left untreated (mean±s.e.m., n=12). (E) [³⁵S]-labelled precursor proteins were incubated with yeast mitochondria followed by treatment with proteinase K (+ PK). Plus and minus signs denote whether Δψ_m had been dissipated prior to the import reaction. (F) Yeast mitochondria isolated from WT, Δmir1 and Δcor1 cells were incubated with [³⁵S]-labelled Ybh3p for 1, 5 and 25 min or with [³⁵S]-labelled Tom20p for 1, 4 and 20 min followed by carbonate extraction (pH 10.5) and analysis by NuPAGE and autoradiography. In addition, mitochondrial carbonate resistant pellets from the Ybh3p import reactions were analysed for Por1p and Pam18p as markers for outer and inner membrane using immunoblotting. (G) Measurement of Δψ_m using TMRM in yeast cells overexpressing Ybh3p or harbouring the empty vector. Cells were treated or not with acetic acid or FCCP before flow cytometric analysis. TMRM fluorescence upon Ybh3p overexpression compared with similarly treated vector control was indicated (mean±s.e.m., n=12). (H) Change of Δψ_m using flow cytometric assessment of TMRM fluorescence upon Ybh3p overexpression in WT, Δmir1 and Δcor1 cells treated with acetic acid or FCCP or left untreated. Fold change compared with isogenic vector control was plotted (mean±s.e.m., n=12). See also Supplementary Figure S6 and Supplementary Table S1. ^*P<0.05, ^**P<0.01 and ^***P<0.001.