Growth, Differentiation and Sexuality

Besides ROS, mitochondrial lipid oxidation
may also play a role in the Bax-induced PCD.
Priault et al. (2002) have demonstrated that there
is a substantial decrease in the amount of fatty
acids and in the unsaturation index (unsaturated/saturated
ratio) in mitochondria isolated
from Bax-expressing yeast cells. This change
is linked to mitochondrial respiration-induced
oxidation or peroxidation of different classes
of mitochondrial phospholipid. By a kinetic
study, there is a strong link between Bax-induced
cell death and Bax-induced mitochondrial lipid
oxidation in the wild-type (WtB1) yeast. This
link is further supported by the assay that known
inhibitors of lipid oxidation (α-tocopherol and
resveratrol) decrease Bax-induced cell death in
yeast (Priault et al. 2002).
More recently, a direct link between ROS
production and cell death has been uncovered
by Pozniakovsky et al. (2005). These authors
have established that elevated ROS level is essential
in pheromone- and amiodarone-induced
cell-death cascade in yeast, because quenching
of ROS by antioxidant NAC (N-acetyl cysteine)
prevented fragmentation of mitochondrial filaments
and cell death. Amiodarone (2-butyl-3benzofuranyl-4-[diethylamino]-ethoxyl-3,5-diiodophenylketone
hydrochloride) is a fungicide, a Ca ++
channel blocker, and it causes an increase of
cytosolic Ca ++ concentration that triggers mitochondrial
membrane potential increase, followed
by elevated ROS production (see references in Pozniakovsky
et al. 2005). Moreover, glutathione is an
endogenous means to overcome oxidative stress.
A yeast strain (YPH98gsh1) lacking glutathione,
caused by a deletion of the GSH1 gene coding for
G-glutamyl-cysteine synthetase, exhibits symptoms
of apoptosis, and this strain is supersensitive
to treatments with H2O2 (Madeo et al. 1999).
The gcs1/gcs1 null mutant of C. albicans lacking
glutathione also results in increased ROS production
and apoptosis (Baek et al. 2004). Besides, the
apoptotic phenotype can be suppressed in yeast by
oxygen radical scavengers such as free radical spin
traps (e.g., 5 mM PBN or 0.5 mM TMPO), and by
anaerobic growth conditions (Madeo et al. 1999).
C. Mitochondrial Respiration in Apoptosis
There is another controversy whether mitochondrial
respiration is essential for PCD. Ludovico
et al. (2002) have shown that PCD induced by
Programmed Cell Death 175
acetic acid in S. cerevisiae is mitochondriadependent:
the Rho − , the null ATP10 mutant
lacking mitochondrial ATPase, and the null
CYC3 mutant all fail to undergo PCD. The petite
mutant of S. cerevisiae is also resistant to
Bax-induced PCD (Greenhalf et al. 1996; Gross
et al. 2000; Harris et al. 2000). Other studies
have shown yeast respiration-deficient mutants
to suffer a reduced and a delayed (by 12 h), but
not abrogated, Bax-induced cell killing (Matsuyama
et al. 1998). On the other hand, oxidative
phosphorylation appears not to be a factor,
because the wild-type yeast and mitochondrial
mutants follow the same kinetics in Bax-induced
growth arrest and cell killing (Kiˇsˇsová et al. 2000).
Surprisingly, in the yeast Kluyveromyces lactis,
which is petite-negative and strictly aerobic,
mitochondrial respiration is not required for
Bax-induced cell killing. In fact, Rho − mutant is
more sensitive to Bax, with plating efficiency at
0%, compared to the wild type at 60% and the
mgi1-1 (ATPase) mutant at 20% (Poliakova et al.
2002).
Part of the controversy may rest with the way
Bax is expressed in yeast. Most of the experiments
reported above were done with BAX driven
by Gal10 promoter. This could be problematic
when dealing with fermentation conditions, since
Gal10 promoter is strongly repressed by glucose,
and yeast respiration-deficient mutants grow
poorly in a galactose medium. A system was
devised whereby the Bax expression is driven
by a tetracycline-off (tetO) promoterthatallows
Bax and Bcl-xL expression under fermentative
as well as respiratory conditions (Priault et al.
1999a). There is no difference in Bax-induced
growth arrest under any growth conditions using
glucose or mannose as a carbon source, where
glucose induces a strong catabolic repression
of respiratory enzymes but mannose does not.
The most revealing results are the kinetics of
cell killing in different mutants under different
growth conditions (Fig. 9.1). Under fermentative
conditions using glucose as the carbon source,
the wild-type (WtB1) yeast cells are resistant to
Bax-induced cell killing, whereas under respiratory
conditions using lactate as the carbon source,
they are rapidly killed. It is interesting to note
that, under respiro-fermentative conditions (cf.
non-repressive sugars such as mannose), the Bax
effect is intermediate. Similar result is seen in
the adenine nucleotide carrier (AtB1) defective
mutant (Priault et al. 1999a).