can block membrane insertion and proapoptotic function [27,28,29,30,31]. Two or more basic residues in the extreme C- terminus (i.e. within the C-segment) may assist insertion of the TM domain across the OM, as observed for other mitochondrial tail- anchored proteins [30,32,33]. Whether the TM domain inserts spontaneously across the mitochondrial membrane remains con- troversial [27,28], however peptides equivalent to the C-termini of Bak (24 residues) and of Bax (24 residues) can integrate into model membranes in the absence of chaperones or receptors [34,35]. Bak is integrated in the OM in healthy cells, whereas Bax is largely cytosolic until its translocation to mitochondria after apoptotic signalling [36]. A portion of Bax that is peripherally attached to mitochondria in healthy cells can retrotranslocate upon binding Bcl-xL [37]. Cytosolic Bax is proposed to sequester its TM domain in a hydrophobic surface groove through an interaction involving hydrogen bonding between S184 in the TM domain and D98 in the groove [30,38]. While others have examined how the C- termini of Bax, Bcl-xL, and Bcl-2 control mitochondrial targeting [30,31,39,40], this has not been examined for Bak. To understand how Bak is targeted to mitochondria, and to address whether differences in Bak and Bax localization might contribute to their differential regulation, we mutated the C- terminus of Bak. Removing the C-segment (C-terminal six residues), or the basic residues within, decreased mitochondrial targeting and protein stability, thereby decreasing proapoptotic function. Notably, replacing the C-segment of Bak with that from Bax converted Bak to a relatively stable, semi-cytosolic protein (named Bak/BaxCS) that could translocate to mitochondria. Translocation of both Bak/BaxCS and Bax following apoptotic signalling suggests similar activation mechanisms for Bak and Bax. Furthermore, the semi-cytosolic localization of Bak/BaxCS did not alter its regulation by Mcl-1. Results The Bak C-segment is required for stability, mitochondrial targeting and proapoptotic function To identify elements in the Bak C-terminus that are required for OM targeting and integration, the whole C-terminus or just the C- segment was truncated (BakDCT, BakDCS; Figure 1A). Following stable expression in bak2/2bax2/2 MEFs, both truncated proteins failed to mediate cytochrome c release and apoptosis after exposure to UV or etoposide (Figure 1B and Figure S1). BakDCT was relatively stable, as protein expression levels were very high, and half-life as assessed by incubation in cycloheximide was similar to that of Bak (Figures 1B and C). In contrast, BakDCS was unstable as indicated by low expression level, low half-life, and degradation following UV treatment (Figures 1B, C, D), perhaps due to an exposed TM domain targeting the protein for degradation. The truncated proteins were inefficient at targeting membranes both before and after apoptotic signalling (Figures 1D and E). In Bax, truncation of the C-terminus (23 residues) or C- segment (188WKKMG192) also blocked mitochondrial transloca- tion and function (data not shown), as reported previously [27,30,31]. Thus, the C-termini, including the C-segments, are required for targeting stably expressed Bak and Bax to mitochondria. Notably, the Bak C-terminus was also necessary for apoptotic conformation change and oligomerization, as BakDCT essentially remained in the non-activated conformation, and failed to dimerize following UV treatment (Figure 1E). Moreover, BakDCT failed to change conformation following addition of the proapop- totic BH3-only protein tBid to cell extracts [8]. Mutagenesis of the Bak Membrane Anchor Basic residues in the Bak C-segment govern stability and function Basic residues in the C-segment of tail-anchored proteins can assist in membrane-integration and in targeting specific mem- branes [30,32,33]. To examine the role of the three basic residues in the Bak C-segment, one, two, or all three, were replaced with serine (Figure 2A). Substitution of all three basic residues (BakSSS) had a similar effect to C-segment truncation, as the protein was unstable as shown by low expression level and low half-life, with cytochrome c release and apoptosis greatly decreased (Figures 2B and C and Figure S1). Substitution of the last two basic residues (BakRSS) slightly blocked apoptotic function (Figure 2B and Figure S1) despite high protein levels (Figure 2B) and efficient membrane targeting (Figure 2D). This diminished function of BakRSS may be due to targeting to non-mitochondrial mem- branes, as reported for Bcl-xL when either basic residue in the C- segment was replaced [39], although attempts to examine this by both cell fractionation and confocal microscopy were not conclusive (data not shown). Finally, substitution of a single basic residue at two positions had little effect on localization or apoptotic function (BakRRS, BakSRK; Figures 2B and D and data not shown). While we have not tested whether the second or third basic residue alone (e.g. BakSRS or BakSSR) may efficiently target Bak to mitochondria, our data suggest that two basic residues in the Bak C-segment are necessary for efficient proapoptotic function, as shown for Bcl-xL [39]. However, a third basic residue may contribute to Bak stability, as BakRRS had a slightly lower half-life compared to Bak, and many vertebrate Bak proteins contain three basic residues in the C-segment. Substitution of the Bak C-segment with that of Bax preserves Bak stability and function, but confers cytosolic localization To explore the role of the C-termini in the differential localization of Bak and Bax we generated three chimeras in which the whole Bak C-terminus, or just the TM domain or C-segment was replaced with that of Bax (Figure 3A). Bak containing the Bax C-terminus (Bak/ BaxCT) exhibited the characteristics of wild-type Bak in terms of expression level, half-life, mitochondrial localization, oligomeriza- tion and proapoptotic function (Figures 3B, C, D, E, F, G and Figure S1). Thus, the Bax C-terminus could act as a fully functional membrane anchor for Bak. The protein was fully membrane-bound prior to apoptosis, presumably due to inability of the Bax TM domain to be sequestered in the Bak groove. Bak containing the Bax TM domain (Bak/BaxTM) was relatively unstable. The protein displayed reduced proapoptotic function, short half-life, and rapid degradation following UV (Figures 3B, C, D, E and Figure S1). This chimera also exhibited impaired targeting to membranes (Figures 3D and E), suggesting that the secondary structure of the ‘‘mixed’’ C-terminus may not be optimal for membrane insertion. As the membrane-integrated population was also unstable (Figures 3D and E), the Bak C- terminus may not only be important for targeting and insertion, but also for stability of the protein once membrane anchored. Notably, Bak containing the Bax C-segment (Bak/BaxCS) was fully functional in mediating apoptosis, as indicated by cytochrome c release and by loss of cell viability in a caspase-dependent manner (Figure 3B and Figures S1 and S2). Accordingly, the protein exhibited normal half-life (Figure 3C). However, instead of being located solely in the mitochondrial fraction, a significant portion of the protein was now cytosolic in healthy cells (Figures 3D and E). The portion of Bak/BaxCS that did locate to mitochondria was partially inserted as indicated by carbonate PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e31510