Translocation of a Bak C-Terminus Mutant from Cytosol to Mitochondria to Mediate Cytochrome c Release: Implications for Bak and Bax Apoptotic Function

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.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.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

A molecular mechanics analysis of [Co(medien)(subscript n)(dien)₂₋(subscript n)]³⁺ {n=0-2} (dien = diethylenetriamine or 1,5-diamino-3-azapentane; medien = 3-methyl-1,5-diamino-3 azapentane), and X-ray structural studies of the mer- and s-fac-[Co(medien)(dien)]³⁺ cations

Crystals of mer-[Co(medien)(dien)]Br₂(ClO₄)·H₂O (1 crystallize in the monoclinic space group P21/n with unit cell dimensions a=12.206(2), b=12.744(1), c=12.712(2) Å, β=94.99(1)°, V=1969.9 ų, Z=4; crystals of s-fac- [Co(medien)(dien)] (S₂O₆)₁.₅·0.5H₂O (2) are monoclinic, space group P2₁ with a=9.772(3), b=10.512(2), c=19.263(4) Å, β=92.24(2)°, V=2014.2 ų, Z=4. The structures were refined by a full-matrix least-squares procedures in each case. At convergence, final R0.065, Rw0.067 for 2578 reflections with I⩾2.5σ (I for (1) and R0.061, Rw0.061 for 2845 reflections for (2). A molecular mechanics study of the isomers of the bis (tridentate)cobalt(III) complexes [Co(medien)(subscript n)(dien)₂₋(subscript n)]³⁺,n=0−2, (dien = diethylenetriamine or 1,5-diamino-3-azapentane; medien = 3-methyl-1,5-diamino-3-azapentane) is reported. For the [Co(dien)₂]³⁺ isomers, and the mer and s-fac isomers of [Co(medien)(dien)]³⁺, the structural details are satisfactorily modelled by MM2. For both the [Co(dien)₂]³⁺ and [Co(medien)(dien)]³⁺ systems, the MM2 procedure sbustantially overestimates the stability of the mer geometric isomers relative to the fac forms, although the relative stabilities of the s-fac/ u-fac forms correspond well with the observed isomer ratios. The calculated isomers ratios for the [Co(medien)₂]³⁺ system agree with those found experimentally. The apparent variance in the capability of the MM2 procedure to predict the relative stabilities of the isomers in the three analogous systems is discussed

Bak regulation is independent of initial subcellular localization.

<p>(<b>A</b>) Schematic of Bak-mediated apoptosis initiated by Noxa-Mcl-1 signalling in MEFs. The four prosurvival proteins expressed in MEF (Mcl-1, Bcl-x_L, Bcl-w and Bcl-2) are depicted, together with their preferential binding of BH3-only proteins (Noxa, Bim and Bad) and of Bak and Bax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Willis1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Chen1" target="_blank">[46]</a>. The Noxa-Mcl-1-Bak pathway to apoptosis is indicated (<i>bold</i>). (<b>B</b>) Expression of Bim_S^NOXA preferentially mediates apoptosis via either Bak or Bak/BaxCS. <i>bak^−/−bax^−/−</i>MEFs expressing the indicated Bak and Bax variants were retrovirally infected with Bim_S or with Bim_S containing the Bad or Noxa BH3 domains (Bim_S^BAD and Bim_S^NOXA). Percentage cell death at 36 h (normalized to the efficiency of infection) is expressed as the mean ± SEM of three independent experiments. Statistical significance for treatment when compared to Bak; **p<0.01.</p