Smac/DIABLO and cytochrome c are released from mitochondria through a similar mechanism during UV-induced apoptosis

Abstract

During apoptosis, a key event is the release of Smac/DIABLO (an inhibitor of XIAP) and cytochrome c (Cyt-c, an activator of caspase-9) from mitochondria to cytosol. It was not clear, however, whether the releasing mechanisms of these two proteins are the same. Using a combination of single living-cell analysis and immunostaining techniques, we investigated the dynamic process of Smac and Cyt-c release during UV-induced apoptosis in HeLa cells. We found that YFP-labeled Smac and GFP-labeled Cyt-c were released from mitochondria in the same time window, which coincided with the mitochondrial membrane potential depolarization. Furthermore, using immunostaining, we found that the endogenous Smac and Cyt-c were always released together within an individual cell. Finally, when cells were pre-treated with caspase inhibitor (z-VAD-fmk) to block caspase activation, the process of Smac release, like that of Cyt-c, was not affected. This was true for both YFPlabeled Smac and endogenous Smac. These results suggest that in HeLa cells, both Smac and Cyt-c are released from mitochondria during UV-induced apoptosis through the same permeability transition mechanism, which we believe is triggered by the aggregation of Bax in the outer mitochondrial membrane to form lipid-protein complex.

The mitochondrial gateway to cell death

“…Live cell imaging was used on either staurosporine (STS)-or actinomycin D-treated HeLa cells to show there was coincident and efficient release of cyt c, Smac, Omi, and adenylate kinase II (a nonapoptogenic IMS protein) (25). In accordance with this finding, when HeLa cells were exposed to UV light, simultaneous redistribution of cyt c and Smac was observed (27). However, application of other apoptotic inducers to HeLa cells or other cell types exposed to a variety of treatments reveals that differential release of cyt c and Smac occurs.…”

“…This was demonstrated by real-time confocal photomicrography of live cells expressing fluorescently labeled cyt c and/or Smac while they underwent apoptosis (24)(25)(26)(27). These studies also showed that after the initiation of apoptosis, the onset of release of IMS proteins was not predicable and took place at widely different time intervals after the addition of the apoptotic stimulus.…”

SMAC/Diablo mediates the proapoptotic function of PUMA by regulating PUMA-induced mitochondrial events

“…A recent study showed that SMAC release is suppressed in cytochrome cdeficient cells (Hansen et al, 2006). It has been shown that SMAC and cytochrome c are released together from the mitochondria during UV-induced apoptosis (Zhou et al, 2005). Therefore, questions remain as to how SMAC release affects other mitochondria-related events during apoptosis, and whether its function in regulating mitochondrial events is related to IAP inhibition.…”

Regulation of mitochondrial Smac/DIABLO-selective release by survivin

“…It has been reported both that Smac/DIABLO is released independently of cytochrome c (Deng et al, 2002) and simultaneously with it (Zhou et al, 2005). This discrepancy might be the result of different mechanisms of release after a specific stimulus that eventually converge, as suggested by the requirement of caspase activity for Smac/DIABLO release but not for cytochrome c release (Adrain et al, 2001).…”