Genetics of Apoptosis

8.1

Release mechanisms

The controversial question is how Bax and other multidomain proteins induce the
release of cytochrome c and other proteins from the mitochondrial intermembrane
space.
Two main mechanisms for permeabilization of the outer mitochondrial membrane
have been proposed: formation of specific channels or pores in the outer membrane,
or opening of the permeability transition pore (PTP) that results in mitochondria
matrix swelling and rupture of the outer membrane. The following models have been
proposed (Figure 4): (i) Bax forms channels itself. (ii) Bax destabilizes the
mitochondrial membrane, inducing ‘lipic holes’ (Basanez et al., 1999). (iii, iv) Bax
forms chimeric channels with VDAC or ANT. (v) Bax triggers opening of the
mitochondrial permeability transition pore (PTP). In the large number of
publications addressing these questions during the last few years, arguments for and
against each of these models can be found. Most studies are based on experiments
performed on artificial membranes, isolated mitochondrial membranes, and extracted
mitochondrial proteins or recombinant proteins. In intact mitochondria, and even
more so in the cell, it is difficult to establish the molecular mode of action and
distinguish between the various models.

8.2

Bax channels

As described above, recombinant Bax is able to form channels in artificial membranes
without any additional proteins (Antonsson et al., 1997). These ‘Bax-alone’ channels
are able to permeabilize lipid membranes to cytochrome c (Saito et al., 2000).
However, when we closely examined the quaternary structure of artificially
oligomerized recombinant Bax and Bax oligomers extracted from mitochondria of
apoptotic HeLa or HEK cells, the oligomers differed in size (Antonsson et al., 2001).
The oligomers of recombinant Bax showed molecular masses of 80 and 160 kDa,
respectively, whereas Bax oligomers extracted from mitochondria of apoptotic cells
were found to be 96 and 250 kDa. This would suggest that either the number of
monomeric Bax subunits is different or that Bax oligomers isolated from
mitochondria contain additional proteins. Furthermore, cross-linking experiments
suggested that the oligomers contain Bax reactive bands that do not correspond to
multimers of Bax molecules, indicating the presence of other, still unidentified
proteins. However, the Bax oligomers did not comigrate with either VDAC or ANT,
showing that these proteins are not part of the stable Bax oligomers. In a study by
Mikhailov et al. (2002), rat kidney cells were deprived of ATP to induce apoptosis
and the cells subsequently treated with cross-linking reagents. In these apoptotic cells,
Bax oligomers up to hexamers were identified on Western blots. All Bax-reactive
bands corresponded to multimers of Bax molecules, suggesting that no other proteins
had been cross-linked to the Bax oligomers under the conditions used in these

132 GENETICS OF APOPTOSIS