Smac/DIABLO and cytochrome c are released from mitochondria through a similar mechanism during UV-induced apoptosis.
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 YFP-labeled 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.
Zhou
L L
LL
Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Zhou
L Y
LY
Luo
K Q
KQ
Chang
D C
DC
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D045304:Cytochromes c / Q000378:metabolism*; D004789:Enzyme Activation; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D050356:Lipid Metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010539:Permeability; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays
D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; D001426:Bacterial Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D045304:Cytochromes c