al., 1996), caspases (Kang et al., 1999), and Apaf-1/CED-4 (James et al., 1997), has
been found to be lethal for yeast.
Further research indicated that the resulting cell death is indeed of apoptotic nature.
Ligr et al. (1998) have shown that bax (a Bcl-2 family member)-mediated cell death
in S. cerevisiae is accompanied by typical features of apoptosis such as externalization
of phosphatidylserine at the surface of the cytoplasmic membrane, membrane
blebbing, chromatin condensation and margination, and DNA cleavage.
Simultaneous expression of bcl-xL prevents these effects and cell death.
In addition, human bak (another proapoptotic gene of the bax/bcl-2 family)
induces cell death in S.pombe accompanied with an apoptotic phenotype:
condensation and fragmentation of chromatin, vacuolization of cytoplasm, DNA
cleavage, and dissolution of the nuclear envelope (Ink et al., 1997). These alterations
can also be suppressed by the expression of bcl-xL.
Furthermore, mutant forms of Bcl-xL, an antiapoptotic Bcl-2 family member
lacking Bax-binding activity, can prevent bax-induced death in yeast (Tao et al., 1997;
Minn et al., 1999) This indicates that Bcl-xL acts downstream of Bax, perhaps by
competing for binding to a common target, which may be part of the conserved
apoptotic machinery.
Recently, Pavlov et al. (2001) found a novel high-conductance channel of
mitochondria. The activity of this channel correlates with the presence of Bax in the
mitochondrial outer membrane and is absent in mitochondria from yeast cells
overexpressing antiapoptotic bcl-2. The pore diameter of approximately 4 nm,
inferred from the largest conductance state of this channel, is sufficient to allow
diffusion of cytochrome c and even larger proteins. This channel, named the
‘mitochondrial apoptosis-induced channel,’ is a candidate for the outer-membrane
pore through which cytochrome c and possibly other factors leave mitochondria
during apoptosis (see Chapter 7).
Reed and coworkers used the cytotoxicity of Bax to S.cerevisiae to select
antiapoptotic effectors from a human gene library and to identify yeast mutations
preventing cell death. Both strategies were successful, and the results could be
extended to mammalian apoptosis: bax expression was not lethal for a mutant in the
mitochondrial F0F1-ATPase. Oligomycin, an inhibitor of this enzyme, prevented
cell death in both S.cerevisiae and mammalian cells (Matsuyama et al., 1998).
Expression of a human gene, BI-1, rescued bax-expressing yeast. Its gene product is
located predominately at the endoplasmic reticulum, with a small portion at the
mitochondrial membrane. It interacts with Bcl-2, but not Bax or Bak. When
overexpressed in mammalian cells, it suppresses apoptosis induced by Bax, growth
factor withdrawal, or various drugs, but not by Fas (Xu and Reed, 1998). A BI- 1
homolog has been described in plants. Its overexpression reduces cell death caused
by heterologous expression of mammalian bax in Arabidopsis (KawaiYamada et al.,
2001).
Heterologous expression of a regulator caspase gene coding for human procaspase-8
or -10 in yeast results in production of the efficiently processed, active caspase (Kang
et al., 1999). Caspase-10 activity has little effect on yeast-cell viability, whereas
142 GENETICS OF APOPTOSIS