Mutagenesis of the Bak Membrane Anchor Figure 1. The Bak C-segment is essential for stability, membrane targeting and proapoptotic function. (A) C-terminal sequence of Bak variants truncated at the C-terminus. The C-terminus (CT) contains a hydrophobic transmembrane (TM) domain and a hydrophilic C-segment (CS). (B) Truncation of the Bak C-terminus or C-segment blocks proapoptotic function. bak2/2bax2/2MEFs expressing Bak, BakDCT and BakDCS 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,0.001. Upper panel is a western blot of cell lysates immunoblotted for Bak, and for b-actin as a loading control. (C) Truncation of the C-segment reduces half-life. 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 BakDCS, 4-fold total protein was loaded onto the gels. (D) Truncation of the Bak C-terminus prevents membrane targeting. Cells were left untreated or treated with UV, separated into cytosolic and membrane fractions, and immunoblotted for Bak and the cytosolic marker HSP70. (E) Bak lacking the C-terminus fails to undergo conformation change and oligomerization in response to UV. Cells treated as in (D) were exposed to oxidant (CuPhe), separated into cytosolic and membrane fractions, run on non-reducing SDS-PAGE and immunoblotted for Bak. MX, non-activated intramolecular cross-linked monomer; M, non- crosslinked monomer; D, intermolecular crosslinked dimers. Results are representative of two or more independent experiments. doi:10.1371/journal.pone.0031510.g001 resistance (Figures 3E). Following apoptotic signalling, Bak/ BaxCS still underwent conformation change and oligomerization following UV (Figures 3F and G) or actinomycin D (Figure S3), with oligomerization predominantly in the membrane fraction. A variant of this chimera that contains five (WKKMG) rather than four (KKMG) Bax residues displayed similar subcellular localiza- tion and proapoptotic function (Bak/BaxCSb; Figure S4 and data not shown). In summary, Bak/BaxCS remained functional despite significant initial cytosolic localization. To examine in more detail whether the cytosolic fraction of Bak/BaxCS can translocate and permeabilize mitochondria, a series of experiments were performed. Subcellular fractionation showed that after treatment with apoptotic stimlui, Bak/BaxCS is predominantly at mitochondria (Figures 3D, E and G and Figure S3), although partial loss of Bak/BaxCS following UV treatment precluded clear conclusions regarding translocation (e.g. Figures 3D and E). By confocal microscopy, Bak/BaxCS (tagged with FLAG at the N-terminus) also increased at mitochondria in a portion of cells after treatment with etoposide (Figure S5), although translocation was not marked due to the portion of Bak/BaxCS already resident at mitochondria. We then tested translocation and permeabilization in cell-free assays by combin- ing Bak/BaxCS cytosolic extracts with mitochondria from two different sources (Figure 4). When MEF cytosol containing Bak/ BaxCS was ‘‘recombined’’ with MEF membranes containing Bak/ BaxCS, the addition of tBid triggered cytochrome c release (Figure 4A, lanes 2 and 3). However, cytochrome c was not released if either fraction was combined with cytosol or PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e31510