Mutagenesis of the Bak Membrane Anchor Figure 2. Basic residues in the C-segment are necessary for Bak stability and function. (A) C-terminal sequence of Bak variants indicating which basic residues in the C-segment that were substituted with serine. (B) Substitution of basic residues in the C-segment reduces Bak proapoptotic function. bak2/2bax2/2 MEFs expressing Bak, BakRRS, BakRSS or BakSSS were left untreated, or treated with UV or etoposide for 24 h. Percentage cell death is expressed as the mean 6 SEM from three independent experiments. Statistical significance for treatment when compared to Bak; *p,0.05, **p,0.01. Upper panel is a western blot of cell lysates immunoblotted for Bak, and for b-actin as a loading control. (C) Substitution of basic residues in the C-segment destabilizes Bak. Cells from (B) were incubated with cycloheximide for up to 24 h and cell lysates immunoblotted for Bak, and for b-actin as a loading control. Note that due to low expression of BakSSS, 4-fold total protein was loaded onto the gels. (D) Substitution of basic residues does not prevent targeting to membranes. Cells from (B) were left untreated or treated with UV, separated into cytosolic and membrane fractions, and immunoblotted for Bak and for the cytosolic marker HSP70. Results are representative of two or more independent experiments. doi:10.1371/journal.pone.0031510.g002 membranes from bak2/2bax2/2 MEF (Figure 4A, lanes 6,7, 10 and 11), indicating that both the cytosolic and mitochondrial portions of Bak/BaxCS are required to reach the threshold of Bak necessary for cytochrome c release in this assay. Blotting for Bak indicated that this threshold (lanes 2 and 3) includes resident mitochondrial Bak/BaxCS (e.g. lane 5) as well as cytosolic Bak/ BaxCS recruited to mitochondria in the absence of tBid (e.g. lane 9) and in the presence of tBid (e.g. lane 10). In a second cell-free assay (Figure 4B), MEF cytosol fractions were incubated with mouse liver mitochondria which contain very low levels of Bcl-xL and Mcl-1 [41]. Cytosolic Bak/BaxCS was now able to mediate complete cytochrome c release by tBid (Figure 4B, lane 4). Again, a portion of Bak/BaxCS was recruited to mitochondria in the absence of tBid, with further protein recruited during tBid incubation (lanes 3 and 4). While the recruited Bak/BaxCS (lanes 3 and 4) appears greater than the resident mouse Bak in bak+/+ liver mitochondria (lanes 5 and 6), the anti-Bak antibody was raised against a peptide derived from the human 23–38 sequence that differs from that of mouse Bak. Notably, in this assay the cytosolic Bak/BaxCS protein was solely responsible for cytochrome c release as liver mitochondria from bak2/2 mice are essentially devoid of Bak and Bax [41]. A direct comparison of Mcl-1 levels in the two sources of mitochondria (Figure 4C) suggests that a low Mcl-1 level in mouse liver mitochondria confers a low threshold of Bak required for cytochrome c release. In summary, in these cell-free assays, cytosolic Bak/BaxCS could translocate to the mitochondrial fraction, and contribute to cytochrome c release initiated by tBid (Figure 4). These findings, together with evidence of translocation following apoptotic signalling in cells (Figure 3 and Figures S3 and S5), indicate that in cells undergoing apoptosis (Figure 3), both the cytosolic and mitochondrial fractions of Bak/BaxCS are functional. A TM: Groove interaction contributes to the cytosolic localization of Bax and Bak/BaxCS As discussed above, structure and mutagenesis studies found that cytosolic Bax is stabilized by a TM:groove interaction possibly involving hydrogen bonding between S184 (TM) and D98 (groove) PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e31510