Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apopt osis, possibly by neutralizing one or more members of the IAP family of apo ptosis inhibitory proteins. Smac has been shown to exit mitochondria and en ter the cytosol during apoptosis triggered by UV- or gamma -irradiation. He re, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpr essing cells. Thus, in addition to modulating apoptosis-associated mitochon drial cytochrome c release, Bcl-2 also regulates Smac release, suggesting t hat both molecules may escape via the same route. However, whereas cell str ess-associated mitochondrial cytochrome c release was largely caspase indep endent, release of Smac/ DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associ ated cytochrome c and Smac/ DIABLO release from mitochondria do not occur v ia the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a ca spase-catalysed event that occurs downstream of cytochrome c release.