Growth, Differentiation and Sexuality

174 B.C.K. Lu
(MMP), through ruptures of the outer membrane,
or through molecular channels. Thus, the regulation
of MMP is an important control point for
apoptosis, but how this regulation is achieved
remains controversial (reviewed in Green and
Reed 1998; Mignotte and Vayssiere 1998; Harris
and Thompson 2000).
Mitochondria are the main action sites of Bcl-2
family proteins, which include proapoptotic (Bax
and Bak) and antiapoptotic (Bcl-2 and Bcl-xL)proteins
in mammalian systems. By using a yeast twohybrid
system to test protein–protein interactions,
it has been suggested that homodimerization of
Bax would lead to cell death whereas heterodimerization
between Bax and Bcl-2 would lead to cell
survival (Sato et al. 1994; Zha and Reed 1997). In
yeast, Bax and Bcl-xL are directly targeted to the
outer mitochrondrial membrane (Gross et al. 2000;
Poliaková et al. 2002; Polčic and Forte 2003). The
most informative piece of evidence has been obtained
by Polčic and Forte (2003) by using a system
whereby Bax and Bcl-xL in a yeast strain (CML282)
can be independently and quantitatively regulated.
When BAX is driven by GAL1 promoter or GAL10
promoter (GAL-BAX), the quantity of Bax protein
produced is proportional to the amount of galactose
present in the growth medium. When BCL-
XL is driven by tetO promoter (TET-BCL-XL), in
a CEN plasmid, the quantity of Bcl-xL is proportional
to the amount of doxycycline present in
thegrowthmedium.Theresultsaremostrevealing
– 1% of galactose gives the maximal Bax expression,
but only 0.1% is sufficient to give maximum
killing. Likewise, 0.5 μg/ml of doxycycline
induces the maximum expression of Bcl-xL, but
only 0.1 μg/ml is needed to rescue cells from Baxinduced
cell death at all concentrations of galactose
used. Furthermore, the level of Bcl-xL required for
rescue does not change even if Bax expression is
increased 30- to 40-fold by using multi-copy plasmids
carrying GAL-BAX construct, making it unlikely
for heterodimerization of Bcl-xL with Bax
to account for the rescue. This is consistent with
the finding that a Bcl-xL protein containing a mutation
of amino acid 101 from tyrosine to lysine
(Y101K), which blocks the ability of Bcl-xL to heterodimerize
with Bax, can rescue Bax-induced cell
death in yeast with the same level of expression
(Polčic and Forte 2003). If heterodimerization is
ruled out, then what is the mechanism of cell death
rescue? It would appear that Bax targeted to the mitochondrial
membrane is required for killing, and
indeed, when Bax alone is expressed in yeast, it is
targeted to the mitochondrial membrane in a stable
alkali-resistant manner. When Bax and Bcl-xL are
co-expressed in yeast, a significant amount of Bax
is found in the cytosol (Polčic and Forte 2003). It is
interesting to note that VDAC (voltage-dependent
anion channel) is not required for actions of either
pro- or antiapoptotic members of the Bcl-2 family
(Polčic and Forte 2003).
B. Oxidative Stress in Apoptosis
Evidence has been accumulating that ROS are regulators
of apoptosis. For example, exogenous oxygen
stress by application of hydrogen peroxide induces
an apoptotic cell death in yeast (Madeo et al.
1999; Fröhlich and Madeo 2000; Chen et al. 2003
for review). Cell death induced by acetic acid is accompanied
by a dramatic increase in production
of ROS in yeast (Ludovico et al. 2002). Increased
production of ROS is also found in P. anserina and
Coprinopsis cinerea (C. cinereus) when confronted
with non-self filamentous fungus (Silar 2005). The
yeast ts-mutant cdc48 S565G that exhibits the apoptotic
phenotype also shows an accumulation of
ROS, whereas nonapoptotic mutants of CDC48 or
other cell-cycle mutants (e.g., cdc2, orcdc31) do
not (Madeo et al. 1999). In yeast, oxygen stress is
also induced in aging mother yeast cells (Laun et al.
2001), in chronologically aged cells (Fabrizio et al.
2004; Herker et al. 2004), and in auxotrophic mutant
cells upon starvation of an essential amino acid
(Eisler et al. 2004).
As discussed above, expression of the mammalian
proapoptogenic Bax or Bak in yeast
causes the apoptotic phenotype, and cell death
is accompanied by an accumulation of ROS.
This Bax-induced cell death is suppressed by
co-expression of mammalian Bcl-2 or Bcl-xL (Ligr
et al. 1998; Fröhlich and Madeo 2000; Gross et al.
2000). Furthermore, expression of CED-9 of C.
elegans, as that of Bcl-2 and Bcl-xL of mammals,
can also rescue apoptosis-like cell death induced
by exogenous oxidative and heat stresses in yeast
(Chen et al. 2003). There are conflicting reports
with respect to the associated formation of ROS
and cell death. In one case, Bcl-xL suppresses not
only Bax-induced cell death but also Bax-induced
production of ROS in S. cerevisiae (Gross et al.
2000). In another, Bcl-xL prevents Bax-induced
cell death but not Bax-induced formation of ROS
in K. lactis (Poliakova et al. 2002). The conflicting
results suggest that there are other players in this
complex problem that remain to be uncovered.