in MEFs (see Figure 4C), and describes the binding profiles of Noxa, Bim and Bad to prosurvival proteins, and the binding profiles of prosurvival proteins to Bak and Bax [18,19,20,46]. We used bak2/2bax2/2MEFs that had been stably transfected with Bak, Bak/BaxCS, Bax or a mitochondria-targeted BaxS184L variant [44]. In each of these cells lines, expression of BimS initiated apoptosis (Figure 6B), explained by strong binding of BimS to all prosurvival proteins (and perhaps direct activation of both Bak and Bax by BimS). In contrast, expression of BimSBAD failed to induce apoptosis in any cell line, explained by weak binding of Bad to Mcl-1. Notably, BimSNOXA efficiently initiated apoptosis in cells expressing Bak because Noxa binds strongly to Mcl-1 to leave Bak unguarded. In contrast, BimSNOXA did not kill cells expressing Bax because Noxa does not bind to three Bax guardians (Bcl-2, Bcl-w and Bcl-xL). Thus, this approach distinguishes between Bak- and Bax-mediated apoptosis. We then noted that BimSNOXA was still able to initiate cell death via Bak/ BaxCS as efficiently as via wild-type Bak, indicating that the semi- cytosolic locale of Bak/BaxCS did not alter its regulation. We note that in these experiments, Noxa expression alone was sufficient to kill bak2/2bax2/2 MEFs expressing human Bak, presumably because Mcl-1 is the principal guardian of Bak in these cells and because Noxa can directly activate Bak [47]. This contrasts with earlier studies in which Noxa required co-expression of Bad to kill bax2/2MEFs [46]. One possibility is that in the present studies, enforced expression of hBak has altered the profile of prosurvival proteins to make Mcl-1 dominant. Consistent with this, altering the prosurvival profile by overexpressing Bcl-xL made the cells resistant to Noxa-induced killing (Figure S7). Bak and Bax regulation in these cells was also tested by initiating apoptosis with UV, cycloheximide and actinomycin D, three agents that are thought to initiate Bak-dependent apoptosis by degrading Mcl-1 and/or by increasing Noxa expression [20,26,48]. Accordingly, these stimuli preferentially triggered apoptosis via Bak or Bak/BaxCS, whereas etoposide induced apoptosis equally via Bak, Bak/BaxCS or Bax (Figure S8). The major role of Mcl-1 in regulating Bak and Bak/BaxCS in these cells is supported by experiments in which both Bak and Bak/ BaxCS readily co-precipitated with Mcl-1, while Bax and BaxS184L did so only poorly (Figure S9). Thus, Bak-mediated apoptosis initiated by targeting Mcl-1 is independent of the initial intracellular localization of Bak. Discussion The Bak transmembrane domain and C-segment both contribute to mitochondrial targeting and membrane insertion The C-termini of tail-anchored proteins, including Bak and Bax, contain an a-helical transmembrane domain often flanked by basic residues [32,33]. Accordingly, in Bak, truncation of the whole C-terminus (25 residues) rendered Bak cytosolic and non- functional when stably expressed in bak2/2bax2/2 MEFs (Figure 1) [29], as reported for transient transfection of HeLa cells [28]. Likewise, truncation of the Bax C-terminal 23 residues also blocked its function in bak2/2bax2/2MEFs (data not shown), as recently shown for Bax with GFP fused to the C-terminus [31]. Truncation of just the C-segment also rendered Bak largely cytosolic and non-functional, as reported for truncation of the C- segment of Bax [30]. Substitution of the three basic residues in the Bak C-segment also significantly decreased half-life and function. Thus, our results based on stable expression of Bak and Bax in Bak/Bax-deficient cells verify that the C-terminus of each protein is necessary for targeting and insertion into the mitochondrial Mutagenesis of the Bak Membrane Anchor outer membrane, and show that the C-segment within these regions plays a vital role. Somewhat curiously, the C-terminus, or C-terminus-dependent mitochondrial localization, was also need- ed for Bak activation and conformation change, as truncation of the C-terminus essentially prevented Bak conformation change in response to tBid or UV (Figure 1E) [8]. Bak/BaxCS, a semi-cytosolic functional form of Bak A semi-cytosolic form of Bak, Bak/BaxCS, was generated by replacing the Bak C-segment (RRFFKS) with that of Bax (KKMG). This chimera retained proapoptotic function despite its significant distribution in the cytosol, suggesting that it may translocate like Bax to mitochondria and permeabilize the OM. Indeed, translocation of the cytosolic portion to mitochondria was observed in cell-free assays and in cells. Notably, recruitment of cytosolic Bak/Bax to MEF mitochondria was necessary to reach the threshold of mitochondrial Bak needed for OM permeabilization in cell-free assays, suggesting that Bak/BaxCS translocation is also necessary for OM permeabilization in intact MEF cells. The ability of cytosolic Bak/BaxCS and of cytosolic Bax to translocate to mitochondria, oligomerize and permeabilize the OM during apoptosis suggests a conserved mode of activation for Bak and Bax. In the cytosol, Bak/BaxCS was able to sequester the hydrophobic TM domain in the hydrophobic surface groove, as shown by cysteine linkage. A similar TM:groove interaction in cytosolic Bax was also evident, as proposed by structural and mutagenesis studies [42,43]. A TM:groove interaction was less evident in mitochondrial Bak/BaxCS, especially following apo- ptotic signalling, indicating that the TM domain moves from the hydrophobic groove to insert into the hydrophobic OM. Actual insertion into the OM following tBid was indicated by decreased labelling of cysteine introduced into the TM domain of Bak/ BaxCS. A TM:groove interaction in cytosolic Bak/BaxCS also implies that although the Bak groove appears ‘‘closed’’ in the structure of non-activated Bak [49], it may ‘‘open’’ to accommodate the TM domain while remaining non-activated. Thus, plasticity evident in the hydrophobic grooves of the prosurvival proteins Bcl-xL and A1 [50] may extend to the hydrophobic groove in Bak. Indeed, a recent study showed that the Bak groove could transiently bind activator BH3-only proteins, initiating Bak activation [47]. A much greater change in the groove is presumably involved in Bak activation, where the groove opens to expose the BH3 domain and then bind the BH3 domain of another activated Bak molecule, resulting in symmetric dimers [8,29]. How the Bax C-segment in Bak/BaxCS promotes a TM:groove interaction is not clear. It is possible that the Bax C-segment (KKMG) forms opportunistic interactions with negatively charged residues on the groove surface (e.g. E105 and E109 in Bak a-helix 4), which then stabilize TM binding to the body of the protein. Alternatively, the hybrid of Bak TM domain with Bax C-segment may hinder membrane insertion, and so skew Bak/BaxCS towards a TM:groove interaction in the cytosol. It is unlikely that the Bax C-segment acts by altering Bak/BaxCS binding to proteins in the cytosol or mitochondria, as cells sorted for low or high levels of Bak/BaxCS exhibited a similar distribution of cytosolic vs mitochondrial Bak/BaxCS (data not shown). The role of the C-terminus in Bak stability and conformation Certain changes to the Bak C-termini decreased protein stability or altered conformation, highlighting the importance of this region in Bak function. Bak half-life was decreased by truncating the C- segment (but not the whole C-terminus) presumably because an PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e31510