Summary In response to many apoptotic stimuli, oligomerization of Bax is essential for mitochondrial outer membrane permeabilization and the ensuing release of cytochrome c. These events are accompanied by mitochondrial fission that appears to require Drp1, a large GTPase of the dynamin superfamily. Loss of Drp1 leads to decreased cytochrome c release by a mechanism that is poorly understood. Here we show that Drp1 stimulates tBid-induced Bax oligomerization and cytochrome c release by promoting tethering and hemifusion of membranes in vitro. This function of Drp1 is independent of its GTPase activity and relies on arginine 247 and the presence of cardiolipin in membranes. In cells, overexpression of Drp1 R247A/E delays Bax oligomerization and cell death. Our findings reveal a novel function of Drp1 and provide a new insight into the mechanism of Bax oligomerization.

BCL-2 homology 3 (BH3)-only proteins of the BCL-2 family such as tBID and BIM EL assist BAX-type proteins to breach the permeability barrier of the outer mitochondrial membrane, thereby allowing cytoplasmic release of cytochrome c and other active inducers of cell death normally confined to the mitochondrial intermembrane space. However, the exact mechanism by which tBID and BIM EL aid BAX and its close homologues in this mitochondrial protein release remains enigmatic. Here, using pure lipid vesicles, we provide evidence that tBID acts in concert with BAX to 1) form large membrane openings through both BH3-dependent and BH3-independent mechanisms, 2) cause lipid transbilayer movement concomitant with membrane permeabilization, and 3) disrupt the lipid bilayer structure of the membrane by promoting positive monolayer curvature stress. None of these effects were observed with BAX when BIM EL was substituted for tBID. Based on these data, we propose a novel model in which tBID assists BAX not only via protein-protein but also via protein-lipid interactions to form lipidic pore-type nonbilayer structures in the outer mitochondrial membrane through which intermembrane prodeath molecules exit mitochondria during apoptosis.Mitochondria usually play a crucial role in the cellular commitment to apoptosis through the release of a variety of prodeath molecules from the intermembrane space into the cytosol (1). This process is tightly controlled by BCL-2 family proteins, which exert their function primarily, although not exclusively, at the level of the OMM 1 (2-4). Members of the BCL-2 family possess up to four conserved regions called BCL-2 homology (BH) domains and can be either proapoptotic or antiapoptotic. Based on these criteria, BCL-2 family members can be divided into three subgroups. Members of the first subgroup, exemplified by BCL-2, contain four BH domains and act predominantly as death inhibitors. Members of the second subgroup, exemplified by BAX, contain BH1-BH3 domains and promote apoptosis in most cellular contexts. Finally members of the third subgroup share only the BH3 domain (BH3-only proteins) and appear to function invariably as death agonists. Two of the most highly studied and important BH3-only proteins are BID and BIM.BID and BIM must cooperate with multidomain proapoptotic members to kill cells (5-7). However, it is unclear exactly how BID and BIM function in concert with BAX-type proteins to induce the release of mitochondrial intermembrane apoptogenic factors. One popular model holds that BID and BIM share a common mode of action via BH3-mediated binding to BAX-type proteins at the OMM (8). This physical interaction is believed to trigger a conformational change of multidomain proapoptotic members, resulting in their intramembraneous oligomerization and OMM permeabilization. Other not necessarily mutually exclusive mechanisms of action proposed for BID and BIM include (i) binding to and neutralization or reversal of prosurvival BCL-2-type family member function (6, 7, 9, 10), (ii) modulation ...

Cholesterol metabolism is deregulated in carcinogenesis, and cancer cells exhibit enhanced mitochondrial cholesterol content whose role in cell death susceptibility and cancer therapy has not been investigated. Here, we describe that mitochondria from rat or human hepatocellular carcinoma (HC) cells (HCC) or primary tumors from patients with HC exhibit increased mitochondrial cholesterol levels. HCC sensitivity to chemotherapy acting via mitochondria is enhanced upon cholesterol depletion by inhibition of hydroxymethylglutaryl-CoA reductase or squalene synthase (SS), which catalyzes the first committed step in cholesterol biosynthesis. HCC transfection with siRNA targeting the steroidogenic acute regulatory protein StAR, a mitochondrial cholesterol-transporting polypeptide which is overexpressed in HCC compared with rat and human liver, sensitized HCC to chemotherapy. Isolated mitochondria from HCC with increased cholesterol levels were resistant to mitochondrial membrane permeabilization and release of cytochrome c or Smac/DIABLO in response to various stimuli including active Bax. Similar behavior was observed in cholesterol-enriched mitochondria or liposomes and reversed by restoring mitochondrial membrane order or cholesterol extraction. Moreover, atorvastatin or the SS inhibitor YM-53601 potentiated doxorubicin-mediated HCC growth arrest and cell death in vivo. Thus, mitochondrial cholesterol contributes to chemotherapy resistance by increasing membrane order, emerging as a novel therapeutic niche in cancer therapy. [Cancer Res 2008;68(13):5246-56]