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

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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 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.

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Abbreviations

Smac/DIABLO:

second mitochondria-derived activator of caspase/direct inhibitor of apoptosis (IAP)-binding protein with low pI

XIAP:

X-chromosome linked inhibitor of apoptosis

Cyt-c:

cytochrome c

GFP:

Green Fluorescence Protein

YFP:

Yellow Fluorescence Protein

z-VAD-fmk:

N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone

VDAC:

voltage dependent anion channel

Apaf-1:

apoptotic protease-activating factor-1

UV:

ultra-violet light

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Authors and Affiliations

  1. Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    L. L. Zhou, L. Y. Zhou, K. Q. Luo & D. C. Chang (Prof.)

  2. Bioengineering Graduate Program, Department of Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

    K. Q. Luo

Authors
  1. L. L. Zhou
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  2. L. Y. Zhou
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  3. K. Q. Luo
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  4. D. C. Chang
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Correspondence to D. C. Chang.

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Zhou, L.L., Zhou, L.Y., Luo, K.Q. et al. Smac/DIABLO and cytochrome c are released from mitochondria through a similar mechanism during UV-induced apoptosis. Apoptosis 10, 289–299 (2005). https://doi.org/10.1007/s10495-005-0803-9

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  • DOI: https://doi.org/10.1007/s10495-005-0803-9

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