Abstract
Translocation of proteins from the mitochondrial intermembrane space to the cytoplasm is a critical event during apoptosis. There are several methods for assaying this event cited in the literature. In this chapter, we highlight separation of cytosolic and mitochondrial fractions of cultured cells using digitonin as the method for measuring cytochrome c release that, in our hands has been the simplest and most reproducible.
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References
Vaux, D. L., Cory, S., and Adams, J. M. (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335, 440–442.
Yang, J., Liu, X., Bhalla, K., et al. (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129–1132.
Waterhouse, N. J., Ricci, J. E., and Green, D. R. (2002) And all of a sudden it’s over: mitochondrial outer-membrane permeabilization in apoptosis. Biochimie 84, 113–121.
Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer, D. D. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136.
Zou, H., Li, Y., Liu, X., and Wang, X. (1999) An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274, 11,549–11,556.
Salvesen, G. S. and Dixit, V. M. (1999) Caspase activation: the induced-proximity model. Proc. Natl. Acad. Sci. USA 96, 10,964–10,967.
Verhagen, A. M., Silke, J., Ekert, P. G., et al. (2002) HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J. Biol. Chem. 277, 445–454.
Verhagen, A. M., Ekert, P. G., Pakusch, M., et al. (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102, 43–53.
van Loo, G., van Gurp, M., Depuydt, B., et al. (2002) The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity. Cell Death Differ. 9, 20–26.
Suzuki, Y., Imai, Y., Nakayama, H., et al. (2001) A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol. Cell 8, 613–621.
Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33–42.
Waterhouse, N. J., Goldstein, J. C., von Ahsen, O., et al. (2001) Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J. Cell Biol. 153, 319–328.
Goldstein, J. C., Waterhouse, N. J., Juin, P., et al. (2000) The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat. Cell Biol. 2, 156–162.
Waterhouse, N. J., Goldstein, J. C., Kluck, R. M., et al. (2001) The (Holey) study of mitochondria in apoptosis. Methods Cell Biol. 66, 365–391.
Finucane, D. M., Bossy-Wetzel, E., Waterhouse, N. J., et al. (1999) Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J. Biol. Chem. 274, 2225–2233.
Sutton, V. R., Davis, J. E., Cancilla, M., et al. (2000) Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation. J. Exp. Med. 192, 1403–1414.
Pinkoski, M. J., Waterhouse, N. J., Heibein, J. A., et al. (2001) Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway. J. Biol. Chem. 276, 12,060–12,067.
Buzzard, K. A., Giaccia, A. J., Killender, M., and Anderson, R. L. (1998) Heat shock protein 72 modulates pathways of stress-induced apoptosis. J. Biol. Chem. 273, 17,147–17,153.
Waterhouse, N., Kumar, S., Song, Q., et al. (1996) Heteronuclear ribonucleoproteins C1 and C2, components of the spliceosome, are specific targets of interleukin 1beta-converting enzyme-like proteases in apoptosis. J. Biol. Chem. 271, 29,335–29,341.
Murphy, B. M., O’Neill, A. J., Adrain, C., et al. (2003) The apoptosome pathway to caspase activation in primary human neutrophils exhibits dramatically reduced requirements for cytochrome c. J. Exp. Med. 197, 625–632.
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© 2004 Humana Press Inc., Totowa, NJ
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Waterhouse, N.J., Steel, R., Kluck, R., Trapani, J.A. (2004). Assaying Cytochrome c Translocation During Apoptosis. In: Dickson, R.C., Mendenhall, M.D. (eds) Signal Transduction Protocols. Methods in Molecular Biology, vol 284. Humana Press. https://doi.org/10.1385/1-59259-816-1:307
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DOI: https://doi.org/10.1385/1-59259-816-1:307
Publisher Name: Humana Press
Print ISBN: 978-1-58829-245-2
Online ISBN: 978-1-59259-816-8
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