Mutagenesis of the Bak Membrane Anchor Figure 4. Bak/BaxCS translocates to mitochondria and following tBid releases cytochrome c. (A) Cytosolic and mitochondrial Bak/BaxCS both contribute to permeabilization of MEF mitochondria. Cytosol and membrane fractions from bak2/2bax2/2 MEFs or from bak2/2bax2/2 MEFs expressing either Bak or Bak/BaxCS were combined as shown, and incubated at 30uC in the presence of 100 nM tBid for 0, 30 or 60 mins, or without tBid for 60 mins (60-). Supernatant (Cyt) and membrane (Memb) fractions were immunoblotted for cytochrome c and for Bak. (B) Cytosolic Bak/BaxCS is sufficient to permeabilize mouse liver mitochondria. MEF cytosol fractions derived as in (A) were combined with mitochondria isolated from wild- type (bak+/+) or bak2/2 mouse liver, and incubated at 37uC with or without 100 nM tBid for 60 min. Supernatant (Cyt) and membrane (Memb) fractions were immunoblotted for cytochrome c and for Bak. Note that Bak/BaxCS levels appear high compared to the endogenous mouse Bak, possibly due to different recognition by the anti-Bak antibody. (C) Mcl-1 is higher in MEF membranes than in mouse liver mitochondria. MEF membranes and mouse liver mitochondria at the concentrations used in (A) and (B) were examined for levels of Mcl-1, Bcl-xL and VDAC1 by immunoblotting. Results are representative of two or more independent experiments. doi:10.1371/journal.pone.0031510.g004 (Figure 5A) [42,43]. Based on these findings, we used disulphide linkage to examine whether the cytosolic populations of Bax and Bak/BaxCS adopt a TM:groove interaction. We first substituted the two endogenous cysteines in Bax (C62 and C126) and in Bak/ BaxCS (C14 and C166) for serine. Then cysteine residues were placed in the TM and groove at positions predicted to interact in Bax (D98C/S184C), and at equivalent positions in Bak/BaxCS (S117C/Q202C). As controls, single cysteine substitutions in Bax (D98C and S184C) and Bak/BaxCS (S117C and Q202C) were also examined. Each Bax and Bak/BaxCS cysteine variant retained proapoptotic function when stably expressed in bak2/2bax2/2MEFs (Figure S6 and unpublished data). We first noted that subcellular localization was altered by the cysteine substitutions, as cysteine in the TM of both Bax (S184C) and Bak/BaxCS (Q202C) increased localization to the membrane fraction (Figure 5B, lanes 4). As cysteine is more hydrophobic than serine or glutamine, it may encourage membrane insertion, as previously reported for Bax in which S184 was substituted with a more hydrophobic residue (BaxS184V, S184A and S184L) [30,44]. To then test whether cysteines in the TM and groove were in close proximity in cytosolic Bax and Bak/BaxCS, we tested whether the two cysteines could form an intramolecular disulphide bond after addition of the oxidant, copper (I,II) phenanthroline (CuPhe). As predicted by the Bax structure, intramolecular PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e31510