cysteine linkage was apparent for the double-cysteine Bax variant (D98C/S184C) in the cytosolic fraction, as under non-reducing conditions (CuPhe) the protein migrated further than the single- cysteine variants (Figure 5C, compare lane 5 to lanes 1–4). Cysteine linkage was also apparent for the double-cysteine variant of Bak/BaxCS (S117C/Q202C) compared to the single-cysteine variants (Figure 5C, compare lane 5 with lanes 1–4). Disulphide linkage was responsible for the fast migration as the reducing agent 2-mercaptoethanol (2ME) prevented faster migration for both double-cysteine variants (Figure 5C, lower gels, lanes 5). Cysteine linkage was not evident in the membrane fractions of the double- cysteine variants, likely due to membrane insertion of the TM domain (Figure 5C, lanes 6). In summary, a TM:groove interaction was evident both in cytosolic Bax and in cytosolic Bak/BaxCS. We next examined whether Bak/BaxCS and Bax convert from a TM:groove conformation to a TM:membrane conformation during apoptosis, as depicted for Bax in Figure 5A. Cells expressing the double-cysteine Bak/BaxCS variant (S117C/ Q202C) were treated with etoposide to induce apoptosis, and the cell fractions treated with CuPhe and examined for TM:groove linkage (X-link; Figure 5D). Here, TM:groove linkage was evident not only in the cytosolic fraction but in part of the membrane- associated protein prior to etoposide, presumably due to some Bak/BaxCS being peripherally attached to mitochondria, as indicated by carbonate extraction (Figure 3E) and by partial dissociation from membranes during a 30 min incubation in bak2/ 2bax2/2 MEF cytosol (Figure 4A, lanes 5–8). Notably however, TM:groove linkage in the membrane fraction decreased after etoposide (Figure 5D, compare lanes 2 and 4). (Unfortunately, several experiments with the double-cysteine Bax variant could not clearly distinguish a decrease in TM:groove linkage.) In a second approach, cell extracts from these cells were incubated with tBid (as in Figure 4A), and cytosol and membrane fractions subjected to oxidation and examined for TM:groove linkage (X-link; Figure 5E). tBid caused the double-cysteine Bak/BaxCS variant to translocate to the membrane fraction, and decreased the proportion of protein that could undergo TM:groove linkage (Figure 5E). The observed decrease in the TM:groove conforma- tion of Bak/BaxCS after either etoposide or tBid is consistent with insertion of the TM domain (that contains the Cys202 residue) into the mitochondrial OM. To more directly monitor membrane insertion of the Bak/ BaxCS TM domain, a cysteine labelling approach was used (Figure 5F). Cysteine can be labelled irreversibly by 4-acetamido- 49-((iodoacetyl) amino) stilbene-2,29-disulfonic acid (IASD), except if the cysteine is positioned in hydrophobic environments such as lipid bilayers [45]. Cysteine was thus introduced at position V194 in the TM domain of Bak/BaxCS, and the protein found to retain proapoptotic function (data not shown). Cell lysates were then incubated with or without tBid and subjected to IASD-labelling. Bak/BakCS in membrane fractions showed an increase in molecular weight conferred by the IASD molecule (624 Da). While most of the membrane-associated V194C could be labelled before tBid, labelling decreased after tBid (Figure 5F). Thus, cysteine linkage and cysteine labelling both indicate that Bak/ BaxCS translocation to mitochondria during apoptosis involves conversion from a TM:groove to a TM:membrane conformation, as depicted for Bax in Figure 5A. Regulation of Bak and Bax is independent of initial subcellular localization How Bak and Bax are regulated by prosurvival Bcl-2 proteins is a major enigma in the field. Having shown that Bak/BaxCS is Mutagenesis of the Bak Membrane Anchor semi-cytosolic and that the cytosolic portion of Bak/BaxCS was important for cell death in these cells, we examined whether subcellular localization contributes to the differential regulation of Bak and Bax. For example, do apoptotic signals that preferentially initiate Bak-dependent apoptosis, also initiate Bak/BaxCS-depen- dent apoptosis? To test this we used a retroviral approach to express three BimS variants in MEFs that already stably express different Bak and Bax variants. The three BimS variants were wild type BimS, or BimS in which the BH3 domain was swapped with the BH3 domain of Bad (BimSBAD) or of Noxa (BimSNOXA). The predicted effects of each BimS variant is described in the schematic in Figure 6A, which depicts Mcl-1 as the principal guardian of Bak Figure 6. Bak regulation is independent of initial subcellular localization. (A) Schematic of Bak-mediated apoptosis initiated by Noxa-Mcl-1 signalling in MEFs. The four prosurvival proteins expressed in MEF (Mcl-1, Bcl-xL, Bcl-w and Bcl-2) are depicted, together with their preferential binding of BH3-only proteins (Noxa, Bim and Bad) and of Bak and Bax [20,46]. The Noxa-Mcl-1-Bak pathway to apoptosis is indicated (bold). (B) Expression of BimSNOXA preferentially mediates apoptosis via either Bak or Bak/BaxCS. bak2/2bax2/2MEFs expressing the indicated Bak and Bax variants were retrovirally infected with BimS or with BimS containing the Bad or Noxa BH3 domains (BimSBAD and BimSNOXA). Percentage cell death at 36 h (normalized to the efficiency of infection) is expressed as the mean 6 SEM of three independent experiments. Statistical significance for treatment when compared to Bak; **p,0.01. doi:10.1371/journal.pone.0031510.g006 PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e31510