Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria

Abstract

Bax is a Bcl-2-family protein with pro-apoptotic activity that can form channels in lipid membranes. The protein has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. Recombinant human Bax isolated in the presence of detergent was found to be present as an oligomer with an apparent molecular mass of approx. 160000 Da on gel filtration. When Bax was isolated in the absence of detergent the purified protein was monomeric with an apparent molecular mass of 22000 Da. Bax oligomers formed channels in liposomes and triggered cytochrome c release from isolated mitochondria, whereas monomeric Bax was inactive in both respects. Incubation of the monomeric Bax with 2% octyl glucoside induced formation of oligomers that displayed channel-forming activity in liposomes and triggered cytochrome c release from mitochondria. Triton X-100, Nonidet P-40 and n-dedecyl maltoside also activated monomeric Bax, whereas CHAPS had no activating effect. In cytosolic extracts from mouse liver, Bax migrated at a molecular mass of 24000 Da on gel filtration, whereas after incubation of the cytosol with 2% octyl glucoside Bax migrated at approximately 140000 Da. These results show that oligomeric Bax possesses channel-forming activity whereas monomeric Bax has no such activity.

Bax and other pro-apoptotic Bcl-2 family "killer-proteins" and their victim the mitochondrion

“…In normal cells or tissues Bax is predominantly localized in the cytosol as a monomer (Hsu and Youle 1998;Antonsson et al 2000). Bax translocation from the cytosol to the mitochondria after apoptotic stimulation has been demonstrated in several systems.…”

Staurosporine-induced differentiation of the RGC-5 cell line leads to apoptosis and cell death at the lowest differentiating concentration

“…Furthermore, caspase 3/ 7 activity was induced by staurosporine in a dose-dependent manner, whereas the increase in Bax to Bcl-2 ratio was the most pronounced at 200 nM, and dropped to baseline at 300 nM of staurosporine. The expression of the proapoptotic protein Bax is elevated during apoptosis and leads to cytochrome c release from the mitochondrion [17][18][19], whereas the anti-apoptotic protein Bcl-2 is an antagonist of cytochrome c release [20]. Therefore, an increased Bax-toBcl-2 ratio indicates a pro-apoptotic expression profile.…”

Mitochondrial control of neuron death and its role in neurodegenerative disorders

“…Due to the kinetic and thermodynamic properties of the transport systems and to the [Ca 2+]c values during resting conditions (0,1-0,2 mM) or stimulated (0,5-3 mM) conditions, the prediction is that, under physiological conditions, Ca 2+ accumulation in mitochondria turns out to be negligible. However, experiments in which [Ca 2+]m was determined using specifically targeted recombinant aequorin (60) (1) and Ca 2+ release is mediated by Ca 2+/Na+ (2) or Ca 2+/H+ (3) antiporter systems. Electron transport chain, ETC (4) and Na+/H+ antiporter system (5) …”

“…It seems that this release induced by these factors concerns only the outer membrane, and does not imply changes in the potential of the mitochondrial membrane or mitochondrial volume prior to the release of Cyt c. The Bax pathway seems to be related to its capacity to form channels in lipid membranes. Its structure displays similarity with the pore-forming domains of diphtheria toxin and the bacterial colicins (2). Bax can be present in monomeric or oligomeric forms, and only the last one is able to form channels in lipid membranes (2).…”

“…Its structure displays similarity with the pore-forming domains of diphtheria toxin and the bacterial colicins (2). Bax can be present in monomeric or oligomeric forms, and only the last one is able to form channels in lipid membranes (2). Bid, similar to Bax, displays a cytoplasmic localization, but to activate apoptosis, it requires partially hydrolysation by proteases, such as caspase-S or granzyme B (29), and to undergo subsequent NH2-terminal myristilation in order to translocate to mitochondria.…”