Genetics of Apoptosis

involving the mitochondrial pathway, cytochrome c is released into the cytosol. The
central role of cytochrome c in apoptosis has been demonstrated in cell-free systems
in vitro, through injection of cytochrome c into cells, and in apoptotic cells (Liu, X.
et al., 1996; Bossy-Wetzel et al., 1998; Zhivotovsky et al., 1998). Using green
fluorescent protein (GFP)-tagged cytochrome c, Goldstein et al. (2000) showed that
cytochrome c release is a fast process. Once the release has been initiated, all
mitochondria in the cell lose their cytochrome c within approximately 5 min.
However, despite an apparently complete depletion of cytochrome c, the
mitochondria retain or regain their membrane potential. In the cytosol, cytochrome
c induces complex formation with Apaf-1, dATP, and procaspase-9. The procaspase
is autoactivated, and the active enzyme subsequently cleaves and activates downstream
caspases, such as caspase-3. The executioner caspases cleave various cellular substrates,
ultimately leading to cell death (Li et al., 1997).
Endonuclease G and AIF translocate to the nucleus after release from the
mitochondria. The endonuclease induces DNA fragmentation in a caspase-
independent manner (van Loo, 2001). Recent studies have identified a gene in
C.elegans, cps-6, with strong similarities to the mammalian endonuclease G (Parrish
et al., 2001). AIF is an oxidoreductase and possesses no detectable DNAse activity;
however, it has also been implicated in caspase-independent DNA fragmentation
(Susin et al., 1999; Miramar et al., 2001). These results suggest the existence of a
parallel apoptosis-signaling pathway independent of caspase activity. The
endonuclease G pathway appears to be evolutionary conserved from the worm up to
mammals.
Smac/DIABLO is a 25-kDa protein that, when released into the cytosol, binds to
inhibitor of apoptosis proteins (IAPs). IAPs are a group of proteins that bind to
activated caspases and in that way inhibit their activity. Smac/DIABLO competes for
binding to the IAPs and thereby prevents the IAPs from inhibiting activated caspases
(Srinivasula et al., 2001). The first four N-terminal amino acids (Ala-Val-Pro-Ile-)
of the mature Smac/DIABLO protein have been shown to interact with BIR domains
in the IAP proteins (Chai et al., 2000; Srinivasula et al., 2001). The presence of IAPs
in the cytosol ensures that the apoptotic signaling cascade is not accidentally induced,

138 GENETICS OF APOPTOSIS

attached to the outer side of the inner mitochondrial membrane. During apoptosis

as by damaged mitochondria in a cell. However, when there is a massive release of

cytochrome c and Smac/DIABLO from the mitochondria, Smac/DIABLO inhibition

of the IAPs ensures a fast propagation of the caspase cascade and execution of the

apoptosis death program.

10.

Bax and mitochondrial respiration

Besides inducing permeabilization of the outer mitochondrial membrane, Bax is

thought to have effects on components of the respiratory chain in the inner