exposed hydrophobic TM domain targets Bak for degradation. In contrast, Bax lacking the C-segment was stable (data not shown), perhaps due to a strong TM:groove interaction. TM-driven degradation is not likely to contribute to turnover of wild-type Bak, as the TM domain is normally membrane-inserted. In relation to Bak conformation, truncating the whole C-terminus (BakDCT) encouraged unfolding of the N-terminus, as a portion of the protein failed to form a C14:C166 crosslink (Figure 1E) [8], and exposure of N-terminal epitopes could be detected (data not shown). Others have reported N-terminal epitope exposure in Bax after truncation of the C-terminal 21 residues [30], although this effect must be indirect as the N- and C-termini are not in contact in the Bax structure [38]. Bak regulation is independent of mitochondrial localization Conversion of Bak to a semi-cytosolic protein (i.e. Bak/BaxCS) did not alter its regulation by other Bcl-2 proteins. We were able to test this in cells where Noxa-Mcl-1 signalling preferentially initiates Bak-mediated rather than Bax-mediated apoptosis. In that system, Bak/BaxCS responded like Bak rather than like Bax. In addition, a mitochondrial Bax protein (S184L) responded like Bax rather than like Bak. Thus, differences in the regulation of Bak and Bax [24,25] are best explained by specific binding of Bak and Bax with other Bcl-2 proteins such as Mcl-1, with this binding being largely unaffected by initial subcellular localization. Conclusions The C-termini are important for Bak and Bax stability in the cytosol and mitochondrial OM, for their translocation and insertion into the OM, and for their conformation change and oligomeriza- tion to finally form the apoptotic pore. Central to these functions is conversion from a TM:groove to a TM:membrane conformation either before apoptosis (Bak), or during apoptosis (Bax, Bak/ BaxCS). The conversion is dependent not only by residues within the TM domain and the groove, but by residues within the C- segment. Further studies are needed to confirm whether, during apoptosis, converting from a TM:groove to a TM:membrane conformation is triggered by TM dislodgement from the groove by BH3-only proteins, post-translational modification, or changes to the intracellular environment [7,27,38,47,51]. Materials and Methods Ethics Statement We used approximately 20 week old wild type or bak2/2 C57BL-6 mice for this study. All animal care and experimental procedures were conducted according to the guidelines of the Australian Code of Practive for the Care and Use of Animals for Scientific Purposes, 7th Edition 2004, and the WEHI Animal Ethics Committee, and were approved by the Royal Melbourne Hospital Animal Ethics Committee (Approval #2009.012). Cell culture, retroviral infection, induction of cell death, apoptosis assays Mouse embryonic fibroblasts (MEFs) derived from bak2/2bax2/2 mice were generated and transformed with SV40 large T as described [23]. MEFs were maintained in Dulbecco’s Modified Eagles medium supplemented with 10% fetal calf serum, 250 mM L-asparagine and 55 mM 2-mercaptoethanol. Variants of human Bak and Bax were generated by site-directed mutagenesis (primer sequences available on request). Variants were then stably expressed in bak2/2bax2/2 MEFs by retroviral Mutagenesis of the Bak Membrane Anchor infection of pMX-IG (IRES-GFP) retroviral constructs that were first introduced into Phoenix cells by FuGENE 6-mediated transfection according to the manufacturer’s protocol (Roche). Viral supernatants were then used to infect MEFs as previously described [46]. Polyclonal populations of Bak- and Bax-expressing cells (GFP-positive) were selected by FACSorting. For stable expression of FLAG-Bak, FLAG-Bak/BaxCS or FLAG-Bcl-xL, constructs were cloned into a pMIH (IRES-Hygro) retroviral vector and polyclonal populations of infected cells selected by serial passage in hygromycin. To induce apoptosis, cells were treated with apoptotic stimuli (100 J/m2 UV; 1 mM actinomycin D (ActD); 10 mM etoposide; 50 mg/ml cycloheximide) and incubated at 37uC for 24 h, unless indicated otherwise. Where indicated, cells were also co-incubated in the presence of the broad range caspase inhibitor Q-VD.oph (50 mM, Enzyme Systems, CA). To measure cell death, floating cells were combined with trypsin-detached cells, resuspended in phosphate-buffered saline containing 5 mg/ml propidium iodide, and incubated on ice for 5 min before measuring the percentage non-viable cells (propidium iodide positive) by flow cytometry. Where indicated, the cell death data was analyzed by two-tailed Student’s t-test. Bak stability after protein synthesis inhibition To estimate protein half-life, bak2/2bax2/2 cells expressing Bak and Bax mutants were treated with cycloheximide (50 mg/ml). At the indicated time points cells were harvested, and cell lysate immunoblotted for Bak (Cat #B5929, Sigma), Bax (N20, Santa Cruz Biotechnologies), or for b-actin (Sigma) as a loading control. Bak subcellular localization and membrane insertion To assess the subcellular localization of each Bak variant, MEFs were harvested, washed in ice-cold PBS, and the cell membrane permeabilized by resuspending cells at 16107 cells ml21 in permeabilization buffer (20 mM HEPES/KOH pH 7.5, 100 mM sucrose, 2.5 mM MgCl2, 100 mM KCl, 1 mM DTT, 0.025% digitonin, Complete protease inhibitors (Roche), and in most cases 4 mg/ml pepstatin A (Sigma)). After incubation on ice for 10 min, cell membrane permeabilization was verified by uptake of trypan blue, the cells centrifuged at 13,000 g for 5 min and equivalent amounts of cytosol (Cyt) and membrane (Memb) fractions immunoblotted for Bak, HSP70 as a cytosolic marker (HSP70 antibody is a gift from Dr W. Welch and Dr R. Anderson). To assess membrane insertion of Bak, membrane fractions were resuspended in 0.1 M Na2CO3 (pH 11.5) and incubated on ice for 20 min. pH was neutralized with 0.1 M HCl and incubated for 5 min before addition of 106 nuclease buffer (400 mM Tris HCl, 100 mM MgSO4, 10 mM CaCl2) and 1 unit of DNAase I (Promega), and incubation at 37uC for 10 min. Samples were centrifuged at 13,000 g for 10 min and supernatant and pellet fractions immunoblotted for Bak. Detection of Bak conformation by disulphide bond formation MEFs were permeabilized by 0.025% digitonin in cross-linking buffer (20 mM HEPES/KOH pH 7.5, 100 mM sucrose, 2.5 mM MgCl2, 50 mM KCl), and membranes resuspended in the same buffer without digitonin. For disulphide bond formation, cytosol and membrane fractions were incubated with the redox catalyst copper(II)(1,10-phenanthroline)3 (CuPhe) on ice for 30 min. The CuPhe stock was 30 mM CuSO4 and 100 mM 1,10-phenanthro- line in 4:1 water/ethanol and diluted 100-fold into the sample. Oxidizing conditions were quenched by 20 mM EDTA to chelate PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e31510