BACKGROUND: One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to 
permeabilize the mitochondrial outer membrane during apoptosis. While Bax is 
mostly cytosolic and translocates to mitochondria following an apoptotic 
stimulus, Bak is constitutively integrated within the outer membrane. Membrane 
anchorage occurs via a C-terminal transmembrane domain that has been studied in 
Bax but not in Bak, therefore what governs their distinct subcellular 
distribution is uncertain. In addition, whether the distinct subcellular 
distributions of Bak and Bax contributes to their differential regulation during 
apoptosis remains unclear.
METHODOLOGY/PRINCIPAL FINDINGS: To gain insight into Bak and Bax targeting to 
mitochondria, elements of the Bak C-terminus were mutated, or swapped with those 
of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of 
three basic residues within the C-segment destabilized Bak. Replacing the Bak 
C-segment with that from Bax rescued stability and function, but unexpectedly 
resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, 
both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in 
their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS 
translocated to the mitochondrial outer membrane, inserted its transmembrane 
domain, oligomerized, and released cytochrome c. Despite this Bax-like 
subcellular distribution, Bak/BaxCS retained Bak-like regulation following 
targeting of Mcl-1.
CONCLUSIONS/SIGNIFICANCE: Residues in the C-segment of Bak and of Bax contribute 
to their distinct subcellular localizations. That a semi-cytosolic form of Bak, 
Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates 
that Bak and Bax share a conserved mode of activation. In addition, the 
differential regulation of Bak and Bax by Mcl-1 is predominantly independent of 
the initial subcellular localizations of Bak and Bax.