Phb2 is required for cell proliferation and apoptotic resistance. (A) Incorporation of ³H-thymidine in MEFs after Cre-transduction. Data represent mean ± standard deviations of three independent experiments. (***) P < 0.001. (B) Growth curves of PHB2-deficient and control MEFs. Cells (5 × 10⁴) were plated on 60-mm dishes and Cre-transduced when indicated. Triplicates of cell samples were counted per time point. (**) P < 0.01; (***) P < 0.001. (C) TUNEL staining of Phb2^fl/fl MEFs treated with Cre-recombinase when indicated. DNase I-treated MEFs were analyzed in parallel for control. Bar, 10 μm. (D) Cre-induced expression of Phb2 in Phb2-deficient MEFs. Phb2^fl/fl MEFs were stably transfected with the CAGs–NeoR–STOP–IRES–EGFP constructs harboring the Phb2 cDNA (Phb2^fl/fl∷Phb2). Cre-mediated recombination results in the inactivation of the endogenous, floxed Phb2 alleles and, simultaneously, in the removal of the floxed transcriptional STOP cassette, allowing expression of the Phb2 transgene under control of the CAGs promoter. IRES-EGFP expression was used as a reporter. (E) Caspase activation in Phb2^fl/fl and Phb2^fl/fl∷Phb2 cells in the presence of etoposide. Cell lines were transduced with Cre-recombinase when indicated and treated for 5 h with increasing concentrations of etoposide (0, 0.5, 1, 5, 10, 15 μM). Cell lysates were analyzed by SDS-PAGE and immunoblotting using antibodies directed against caspase-3, PARP, and PHB2. β-Actin was used as a loading control. (F) Caspase activation in Phb2^fl/fl and Phb2^fl/fl∷Phb2 cells by various intrinsic and extrinsic apoptotic stimuli. After Cre-transduction, cells were treated with staurosporine (STS, 1 μM), actinomycin D (ActD, 1 μg/mL), or TNF-α (10 ng/mL) for 5 h. Cell lysates were analyzed as in E. (G) Cytochrome c release from prohibitin-deficient mitochondria in the presence of actinomycin D. Cells transduced with Cre-recombinase when indicated were cultivated in the presence or absence of actinomycin D (1 μg/mL) for 5 h and analyzed by immunofluorescence. Nuclear DNA was stained with DAPI. Bar, 10 μm. (H) Quantification of cytochrome c release from prohibitin-deficient mitochondria. Data represent mean ± standard deviation of three independent experiments. Approximately 300 cells of each type were analyzed. (***) P < 0.001.

Defective cristae morphogenesis in Phb2^−/− cells. (A) Fragmentation of mitochondria in prohibitin-deficient MEFs. Cell lines were transfected with mito-DsRed, treated with Cre-recombinase when indicated, and analyzed after 72 h by fluorescence microscopy. Bar, 10 μm. (B) Quantification of mitochondrial morphology in control (Phb2^fl/fl∷Phb2) and prohibitin-deficient MEFs. Cells containing tubular (white bars) or fragmented (black bars) mitochondria were classified. More than 200 cells were scored per experiment. (C) Defective mitochondrial ultrastructure in Phb2^−/− cells. Representative transmission electron micrographs of mitochondria in the following cell lines are shown: Phb2^fl/fl (panel a), Phb2^fl/+ +Cre (panel b), Phb2^fl/fl∷Phb2 +Cre (panel c), and Phb2^fl/fl +Cre (panels d–g). Bar, 500 nm. (D) Quantification of cristae morphology in Phb2^fl/fl, Phb2^fl/fl∷Phb2, and Phb2^fl/fl MEFs transduced with Cre-recombinase when indicated. Approximately 50% of cells with disorganized cristae morphology contain vesicular cristae structures. Approximately 100 sections of individual cells were scored per experiment. (E) Three-dimensional reconstructions from serial transmission electron microscopy sections of Phb2^fl/fl cells (panel a) transduced with Cre-recombinase (panel b). Bar, 500 nm.

Expression of L-OPA1^Δ restores mitochondrial morphology in Phb2^−/− cells. (A) Flow chart for complementation experiments using OPA1 variants. MEFs were transiently transfected twice within 4 h with plasmids indicated and subjected to Cre-transduction. After incubation for a further 72 h, cells were analyzed by immunoblotting and fluorescence microscopy. (B) Immunoblot analysis of Phb2^fl/fl MEFs transfected with plasmids and transduced with Cre-recombinase as indicated. Endogenous OPA1 isoforms and transfected Flag-tagged OPA1 variants were detected with OPA1- and Flag-specific antibodies, respectively. The arrow indicates the presence of the L-OPA1^Δ in PHB2-depleted MEFs after transfection. β-Actin was used as a loading control. (C) Restoration of tubular mitochondria in prohibitin-deficient MEFs upon expression of L-OPA1^Δ. Phb2^fl/fl cells complemented with PHB2 when indicated were transfected with mito-DsRed and the indicated plasmids, treated with Cre-recombinase, and analyzed after 72 h by fluorescence microscopy. Bar, 10 μm. (D) Quantification of mitochondrial morphology in prohibitin-deficient MEFs transfected with mito-DsRed and OPA1 variants. Cells containing tubular (black bars) or fragmented (white bars) mitochondria were classified. More than 200 cells were scored in three independent experiments. (**) P < 0.01. Error bars indicate ±standard deviations. (E) Restoration of cristae morphology in Phb2^−/− cells upon expression of L-OPA1^Δ. MEFs were transfected with the indicated plasmids, and mitochondrial morphology was assessed by transmission electron microscopy. Bar, 500 nm. (F) Quantification of deficiencies in cristae morphology in Phb2^−/− cells upon expression of S-OPA1 and L-OPA1^Δ. Approximately 100 sections of individual cells were scored per experiment. (***) P < 0.001. Error bars indicate standard deviations.

Conditional inactivation of murine Phb2 in vivo and in vitro. (A) Schematic representation of the wild-type Phb2 locus (WT), the targeting vector (TV), the targeted Phb2^fl(neo)/+ locus after homologous recombination (FN), the conditional Phb2^fl/fl locus after Flpe-mediated recombination (F), and the knockout locus upon Cre-mediated recombination (Δ). Positive and negative selection markers (Neo^R and TK), exons (black bars), FRT and loxP sites (black ovals and black triangles, respectively), external probes (black boxes, A and B), locations of PCR primers (black arrows, P1–P3), and relevant HindIII restriction sites (H) are indicated. (B) Southern blot analysis of germline transmitted offspring harboring the targeted Phb2^fl(neo)/+ locus. Genomic DNA isolated from ES cells and tail biopsies was digested with HindIII, hybridized, and analyzed by autoradiography. (C) PCR analysis of DNA isolated from MEFs. Amplified DNA fragments for the wild-type (WT), floxed (flox), and knockout (Δ) locus are shown. (D) Immunoblot analysis of total protein lysates. MEFs, transduced with Cre-recombinase when indicated, were lysed and analyzed by immunoblotting using PHB1- and PHB2-specific antibodies. A cross-reacting band was used as a loading control. (E) RT–PCR analysis of Phb1 and Phb2 transcripts in Phb2-deficient and control MEFs. Transcripts of β-actin were used as control.

Cell proliferation depends on mitochondrial targeting of PHB2. (A) N-terminal amino acids of murine PHB2. Residues highly conserved among prohibitins are shown in black, and homologous amino acids are shown in gray. Arginine residues replaced by alanine at positions 11, 17, 48, and 54 of PHB2 are shown. The N terminus of PHB2ΔN44 is indicated by an arrowhead. (B) Membrane potential-dependent import of PHB2 into mitochondria. PHB2 was synthesized in a cell-free system in the presence of ³⁵S-methionine and imported for 30 min at 25°C into isolated murine liver mitochondria. The membrane potential across the inner membrane was dissipated by adding valinomycin prior to (−ΔΨ) or after completion (+ΔΨ) of import. Nonimported preproteins were degraded by trypsin treatment (50 μg/mL). The samples were analyzed by SDS-PAGE and autoradiography. Input corresponds to 10%. (C) Import of PHB2 mutant variants into isolated mitochondria. Import experiments using ³⁵S-labeled PHB2 and mutant variants were performed as described in B. Import reactions were quantified by PhosphorImaging analysis. (D) Mitochondrial targeting of PHB2 variants in vivo. MEFs were transfected with EGFP-tagged murine PHB2 and mito-DsRed and analyzed by fluorescence microscopy. PHB2 hybrid proteins carrying mutations at the positions indicated are schematically shown. Bar, 10 μm. (E) Proliferation of stable cell lines expressing PHB2 variants monitored by ³H-thymidine incorporation. Stable cell lines expressing PHB2 mutants (as in D) and IRES-EGFP were established as described in Figure 2D. Data represent mean values ± standard deviation of three independent experiments. (F) Immunoblot analysis of MEF cell lines expressing PHB2 or mutant variants thereof. Cell extracts were analyzed by SDS- PAGE and immunoblotting using PHB1- and PHB2-specific antibodies. The α-subunit of the F₁ particle of complex V (Suα) was used as a loading control. (G) RT–PCR analysis of Phb1 and Phb2 transcripts in various MEF cell lines. Phb2 transcripts derived from the genomic locus (Phb2) and the transgene (Phb2*) were amplified using allele-specific primer pairs. Transcripts of Gapdh were used as control.

PHB2 controls cleavage of OPA1 but does not affect respiratory activities. (A) Immunoblot analysis of Phb2^fl/fl and Phb2^fl/fl∷Phb2 cells transduced with Cre-recombinase. Cell lysates were analyzed by SDS-PAGE and immunoblotting using OPA1-specific, PHB1-specific, PHB2-specific, and, for control, β-actin-specific antibodies. (B) Maintenance of mitochondrial membrane potential in prohibitin-deficient MEFs. The cell lines indicated were stained with the fluorescent dye JC-1 and analyzed by flow cytometry at 590 nm. Dissipation of the membrane potential with CCCP and unstained cells were used as controls. (C) Oxygen consumption in permeabilized control (n = 5; white bars) and Phb2^−/− MEFs (n = 3; black bars) after Cre-mediated inactivation of Phb2 under conditions of substrate-driven respiration. Data represent mean value ± standard deviations. (D) Relative activities of respiratory chain and TCA cycle enzymes in control (n = 5, white bars) and Phb2^−/− MEFs (n = 4, black bars) after Cre-mediated inactivation of Phb2. (CII) Succinate quinone dichlorophenol indophenol reductase; (CII + CIII) succinate cytochrome c reductase; (CIII) decylubiquinol cytochrome c reductase; (CIV) cytochrome c oxidase; (CS) citrate synthase; (IDH) isocitrate dehydrogenase. Data represent mean value ± standard deviation.

The loss of L-OPA1 renders Phb2^−/− cells susceptible toward apoptosis. (A) Flow chart for complementation experiments using OPA1 variants. Phb2^−/− cells were transiently transfected twice within 4 h with plasmids indicated and subjected to Cre-transduction. After a further incubation for 72 h, cells were cultivated in the presence of actinomycin D (1 μg/mL), and induction of apoptosis was assessed monitoring cytochrome c release and caspase activation. (B) Restoration of apoptotic resistance of Phb2^−/− cells upon expression of L-OPA1^Δ. After Cre-transduction of Phb2^fl/fl cells and transfection of plasmids encoding S-OPA1 or L-OPA1^Δ as indicated, cytochrome c release was monitored by immunofluorescence using cytochrome c-specific antibodies. Nuclear DNA was stained with DAPI. Representative images are shown. Bar, 10 μm. (C) Quantification of cytochrome c release from prohibitin-deficient mitochondria supplemented with S-OPA1 or L-OPA1^Δ and stimulated with actinomycin D. Approximately 300 cells were counted for each genotype in three independent experiments. (**) P < 0.01. (D) Quantitative assessment of mitochondrial morphology in prohibitin-deficient cells supplemented with S-OPA1 or L-OPA1^Δ and stimulated with actinomycin D. Tubular mitochondria in cells transfected with vector (white bars), S-OPA1 (gray bars), or L-OPA1^Δ (black bars) were classified. Approximately 300 cells were scored for each genotype in three independent experiments. (**) P < 0.01. (E) Expression of L-OPA1^Δ promotes proliferation of Phb2^−/− cells. Growth of prohibitin-deficient MEFs was monitored after transfection of S-OPA1 and L-OPA1^Δ by determining the incorporation of ³H-thymidine into DNA. Data represent the mean ± standard deviation of four independent experiments. (*) P < 0.05; (**) P < 0.01.