Genetics of Apoptosis

mildly weakened by the potentially redundant expression of other pro- and
antiapoptotic family members capable of partnering with Bcl-2 or Bax. However,
attempts to identify substitute binding partners for Bcl-2 in Bax-/- thymocytes were
unsuccessful (Knudson and Korsmeyer, 1997).

2.3

Proapoptotic functions: yeast models

Demonstration of direct cytotoxic activities for the proapoptotic Bax-type BH
proteins has proved to be substantially easier than assaying direct survival effects of
Bcl-2. One of the more compelling arguments has been the conservation of Bax-
induced cytotoxicity with features similar to mammalian cell death in Saccharomyces
cerevisiae, which lacks Bcl-2 homologs and, arguably, a comparable endogenous death
pathway (Ligr et al., 1998; Del Carratore et al., 2002). Reed and coworkers originally
reported Bax-induced lethality in budding yeast, which could be rescued by
coexpression of Bcl-2 (Hanada et al., 1995). Respiratory-deficient petite strains are
resistant to Bax killing, although a growth-arrest phenotype is still present (Greenhalf
et al., 1996). Bax is recruited to yeast mitochondria following induction in galactose-
containing media (Zha, H. et al., 1996). The mitochondrial consequences of Bax
expression in yeast include cytochrome c egress (Manon et al., 1997), initial
mitochondrial membrane hyperpolarization (Minn et al., 1999; Gross et al., 2000),
ROS production (Gross et al., 2000), and outer membrane permeabilization (Priault
et al., 1999), recapitulating Bax-induced events in mammalian cells.

2.4

Proapoptotic functions: membrane permeability

Based on the recognition that the x-ray Bcl-xL fold is related to structures of the known
pore-forming proteins, diphtheria toxin T domain and bacterial colicins, Minn et al.
reported pore-forming activities for Bcl-xL (Minn et al., 1997). Similar properties
have been identified with Bcl-2, Bax, Bcl-xs, and Bid (Schendel et al., 1997;
Schlesinger et al., 1997; Schendel et al., 1999). Escape of mitochondrial proteins
enclosed in the intermembrane space, including cytochrome c and AIF, through large
pores in the outer mitochondrial membrane, is one of several competing theories for
the relocalization of these proteins during apoptosis.
Introduction of Bax by transient transfection or inducible expression assays triggers
cell death, usually accompanied by mitochondrial cytochrome c release. Under these
conditions, Bax delivery to mitochondria occurs almost simultaneously with
cytochrome c release (Pastorino et al., 1998; Rosse et al., 1998). Similar kinetics are
observed for galactose or tetracycline-regulated expression in yeast (Manon et al.,
1997). Addition of recombinant Bax (0.2–4 μM) to mitochondria in cell-free systems
liberates cytochrome c within minutes (Jurgensmeier et al., 1998; Eskes et al., 1998;
2000). In many cell types, Bax is predominantly a soluble protein in the cytoplasm
that traffics to mitochondria following apoptotic signals. Recruitment of endogenous

MAKING SENSE OF THE BCL-2 FAMILY OF APOPTOSIS REGULATORS 53