Genetics of Apoptosis

Bax to mitochondria during apoptosis is accompanied by a significant lag period
between Bax translocation and cytochrome c release (e.g., 1–5 h) (Saikumar et al.,
1998; McGinnis et al., 1999; Putcha et al., 1999; De Giorgi et al., 2002).
Several distinct processes affecting mitochondrial membrane integrity have been
reported to take place once Bax is in place. Increased permeability of the outer
mitochondrial membrane has been demonstrated by the release of multiple proteins
normally retained within the intermembrane space and by accessibility of the inner
membrane electron transport complexes to exogenous cytochrome c (Vander Heiden
et al., 1997; Kluck et al., 1999). There may be an upper limit to Bax-mediated outer
membrane permeability, as cytochrome c fused to green fluorescent protein (GFP),
with a molecular mass of 45 kDa, remains localized to mitochondria following Bax
expression in yeast, while the 14-kDa cytochrome c is released (Roucou et al., 2000).

2.5

Proapoptotic functions: permeability transition pores

There is an extensive body of evidence to support an interplay between the
mitochondrial effects of Bax-type proteins and the mitochondrial permeability
transition pore (PTP) (Brenner et al., 2000; Shimizu et al., 2001). The PTP is a
calcium-activated high conductance channel in the inner mitochondrial membrane
that is implicated in mitochondrial membrane depolarization and osmotic swelling
of mitochondria. Bax-induced release of mitochondrial cytochrome c is inhibited,
according to some, but not all, reports, by cyclosporin A (CsA), an inhibitor of the
PTP (Jurgensmeier et al., 1998; Marzo et al., 1998; Narita et al., 1998; Pastorino et
al., 1998). Bax-induced lethality and cytochrome c release in yeast are also
compromised in strains with deletions of porin or the adenine nucleotide translocator
(ANT), outer and inner membrane components of the PTP, respectively (Marzo et
al., 1998; Shimizu et al., 1999). Bax was reported by different groups to bind VDAC,
the mammalian porin, and ANT (Marzo et al., 1998; Narita et al., 1998). Finally,
reconstitution of VDAC and the proapoptotic proteins Bax or Bak in synthetic
liposomes resulted in formation of pores large enough to pass cytochrome c (Shimizu
et al., 1999).
Ca2+-induced PTP activation results in osmotic swelling of the matrix space with
secondary rupture of the less-expandable outer mitochondrial membrane and release
of cytochrome c and other proteins in the intermembrane space. Although
mitochondrial swelling is described in apoptosis, it is not universally observed and,
even when present, may follow rather than coincide with cytochrome c release. This
mechanism can also be discounted in cell-free systems, in which mitochondrial
swelling can be prevented by suspending mitochondria in hypertonic solutions
(Scarlett and Murphy, 1997). Observations of inhibition of Bax-induced cytochrome
c release by CsA in the absence of mitochondrial swelling are possibly explained by
heterogeneity in the mitochondrial population (with swelling of a small fraction
contributing cytochrome c) or a transient PTP activation insufficient for osmotic
swelling (Pastorino et al., 1999; Gogvadze et al., 2001). However, the possibility that

54 GENETICS OF APOPTOSIS