(Received for publication, December 18, 1995)
From the
Yeast lacking copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (SOD), catalase T, or metallothionein were studied using long term stationary phase (10-45 days) as a simple model system to study the roles of antioxidant enzymes in aging. In well aerated cultures, the lack of either SOD resulted in dramatic loss of viability over the first few weeks of culture, with the CuZnSOD mutant showing the more severe defect. The double SOD mutant died within a few days. The severity reversed in low aeration; the CuZnSOD mutant remained viable longer than the manganese SOD mutant. To test whether reactive oxygen species generated during respiration play an important role in the observed cellular death, growth in nonfermentable carbon sources was measured. All strains grew under low aeration, indicating respiratory competence. High aeration caused much reduced growth in single SOD mutants, and the double mutant failed to grow. However, removal of respiration via another mutation dramatically increased short term survival and reversed the known air-dependent methionine and lysine auxotrophies. Our results suggest strongly that mitochondrial respiration is a major source of reactive oxygen species in vivo, as has been shown in vitro, and that these species are produced even under low aeration.
Many theories of aging are based on the hypothesis that aging is caused by oxidative damage as well as other macromolecular modifications that lead to the accumulation of random intracellular molecular defects(1, 2, 3, 4) . Recent studies have supported this idea by demonstrating a correlation between increased superoxide dismutase activity, increased life-span, and decreased oxidative damage in fruit flies and nematodes(5, 6, 7) . We sought to investigate this hypothesis further but in a simpler eucaryotic system. We report here the development of a new in vivo model system for the role of oxidative stress in aging based on the stationary phase of the simple eucaryote Saccharomyces cerevisiae.
A large number of in vitro studies strongly implicate the respiratory chain (8, 9, 10) as a significant source of superoxide and hydrogen peroxide in eucaryotic systems(8, 11, 12) . However, model systems to investigate this issue in vivo have been lacking. The relative importance of the various known reactive oxygen species under various conditions is also often uncertain; hydroxyl radical produced from metal-catalyzed reactions of superoxide and hydrogen peroxide is frequently invoked. Recently, however, it has become abundantly clear that superoxide in some instances can have an important toxicity of its own (13, 14, 15, 16) and that levels occurring naturally in vivo are high enough to cause damage. Hydrogen peroxide has long been known to be toxic, but the extent to which it is a naturally occurring toxin under normal conditions has also been difficult to evaluate.
S. cerevisiae, like most
other eucaryotes, contains CuZnSOD ()(product of the SOD1 gene) in the cytosol and MnSOD (product of the SOD2 gene) in the mitochondria (reviewed in (14) ). These
enzymes catalyze the disproportionation of
O
, producing O
and
H
O
. Together with small molecule antioxidants,
such as glutathione and ascorbate; other antioxidant enzymes, such as
catalases and peroxidases; and metal chelating proteins, such as
metallothionein, they allow aerobes to survive under O
,
presumably by minimizing oxidative damage(14) .
The
importance of cytoplasmic SOD is demonstrated by the high sensitivity
to dioxygen shown by S. cerevisiae and Escherichia coli devoid of SOD. In both organisms, the loss of SOD activity is
associated with slow growth in aerobic conditions, with higher mutation
rates(17, 18) , and with specific biosynthetic
defects. (sod1 yeast require lysine and
methionine for aerobic growth(19) , whereas sod
E. coli require branched chain
amino acids(20, 21) ). In some cases these effects are
known to be due to the inhibitory effect of superoxide on iron sulfur
cluster proteins (16, 22) . sod2
mutants of S. cerevisiae are little affected when grown
in air with glucose as the carbon source. However, they are highly
sensitive to hyperoxia and grow poorly in normoxia in carbon sources
that require respiration for their metabolism.
The full growth cycle of a yeast culture begins with log phase and progresses through the diauxic shift to true stationary phase. In log phase, the cells use glucose to make energy via glycolysis. The diauxic shift occurs when nutrients become limited and energy metabolism shifts to respiration, at which point mitochondrial proteins are synthesized, growth rate slows, and cells utilize ethanol and other two and three carbon compounds for energy. During this phase, glycogen and trehalose are synthesized and stored, and the cells become much more resistant to stress. In the total absence of any nutrients, yeast cells enter true stationary phase. No cell division occurs; the metabolic rate slows; and cells can survive for weeks to months. Many markers, such as increased heat shock resistance and glycogen accumulation, have been utilized to define stationary phase in yeast, although it appears that the best definition of stationary phase is the ability to survive for prolonged periods without added nutrients(23) .
Stationary
phase yeast resemble most of the cells of multicellular organisms in
two important aspects: 1) most energy comes from mitochondrial
respiration and 2) the cells have exited from the cell cycle, i.e., have entered the G phase. In addition,
damage accumulates over time in stationary phase. This damage cannot be
diluted, because cell division and new synthesis are not occurring, and
thus must be prevented or repaired.
Almost all published studies of yeast cells have been performed on actively growing cultures with ample nutrients and a fermentable carbon source (glucose), in spite of the fact that these conditions are relatively rare in nature and, when they do occur, do not persist indefinitely. In such log phase fermentative growth, the cells depend more heavily on glycolysis than respiration for energy and growth, and each individual cell is exposed to oxidative stress for a short period of time before it divides, allowing dilution of any accumulated damage.
We report here the results of an investigation of the roles of the antioxidant enzymes in stationary phase survival in yeast. We have found that both CuZnSOD and MnSOD play a major role in cell survival, whereas catalase T and metallothionein have little effect. In addition, studies of short term survival of respiration-deficient strains indicate that both SODs play an important role in protection against the toxic products formed during mitochondrial respiration.
Figure 1:
Aerobic
growth deficiencies of SOD null mutants in glucose and nonfermentable
carbon sources. Cells were grown in SC medium with the indicated carbon
source. A, 2% glucose, OD was measured at the
indicated times. Cells were grown shaking in flasks to achieve normal
aeration (normoxia) as described under ``Materials and
Methods.'' B, cells were grown in 2% lactate or 2%
ethanol as indicated. For normal aeration flasks were inoculated in
flasks at an initial OD
of 0.01. For low aeration, tubes
were inoculated at an initial OD
of 0.1. The data shown
were taken at 90 h for the normal aeration experiment and at 82 h for
low aeration. The data shown are representative; the experiment was
repeated three times. W.T., wild
type.
The same strains were grown in SC with either ethanol or lactate as
the carbon source under conditions of either high or low aeration (Fig. 1B). With low aeration, all three mutant strains
grew nearly as well as wild type in lactate and were able to grow, but
less well, in ethanol. These results indicate that the strains are able
to respire and therefore that they must contain functional
mitochondria; thus they are potentially competent to enter stationary
phase. Under high aeration in ethanol, the mutant strains showed much
reduced growth, and the double mutant was unable to grow at all. In
lactate, under high aeration, the sod2 mutant also did poorly, and the double mutant did not grow at
all; but, somewhat surprisingly, the sod1
strain grew nearly as well as wild type. When growth in air on
rich medium plates (YPD or YPE) was tested, results similar to the high
aeration liquid culture data were obtained (data not shown). The
absence of cytoplasmic catalase or of metallothionein (CUP1)
did not affect growth under any of these conditions (data not shown).
Figure 2:
Long
term survival of sod and ctt1
mutants in normal aeration (normoxia).
Cells were allowed to grow in flasks for 48 h and then washed three
times in water and reincubated in water. Viability was monitored at the
indicated days by plating onto YPD plates and counting the colonies
formed. Experiments were repeated five times with similar results. A
representative experiment is shown.
, EG103 (wt);
, EG223 (ctt1
);
, EG110 (sod2
);
, EG118 (sod1
);
, EG133 (sod1
, sod2
).
Comparison between the
survival rates of the sod1 and sod2
mutants (Fig. 2) indicates that
there is some cross-talk between the two compartments despite the fact
that the two SODs have different subcellular locations. The fact that
the EG110 (sod2
) strain survives as well as
wild type in the early stages of this experiment indicates that the
cytoplasmic CuZnSOD can at least temporarily compensate for the loss of
the mitochondrial MnSOD. Nevertheless, the fact that MnSOD does itself
have an important role to play is apparent from the very poor survival
of EG133 (sod1
, sod2
) after a few days relative to EG118 (sod1
).
Cells were incubated in medium
containing excess amounts of lysine and methionine in order to
eliminate the possibility that these known auxotrophies might be
responsible for the early loss of viability seen in the sod1 null mutants. A 3-fold excess of these
amino acids did not reverse the viability loss (data not shown).
Figure 3:
Transformation with wild type yeast or
human CuZnSOD gene restored wild type survival to sod1 and sod1
2
yeast
strains. Incubations were performed as indicated in the legend to Fig. 2. Strains were transformed with either the control plasmid
(YEP351) or the same plasmid carrying either the yeast or the human
CuZnSOD gene.
, EG103 (wt) with YEP351;
, EG118 (sod1
) with YEP351;
, EG118 (sod1
) with human wild type SOD1 gene;
EG118 (sod1
) with yeast
wild type SOD1 gene;
, EG133 (sod1
, sod2
) with
YEP351;
, EG133 with yeast wild type SOD1 gene.
Figure 4:
Long term survival of sod mutants in low aeration (hypoxia). Cells
were allowed to grow in SC medium with glucose in tubes for 5 days and
were then washed three times in water and reincubated in water.
Experiments were repeated five times with similar results; a
representative experiment is shown.
, EG103 (wt);
, EG110 (sod2
);
, EG118 (sod1
);
, EG133 (sod1
, sod2
).
Figure 5:
Oxygen uptake by sod strains with and without coq3
deletions. Oxygen consumption was measured on aliquots of cells
taken from cultures grown in high aeration conditions at 12 h of
incubation (late log phase). Similar results were obtained at 24 h of
incubation and in early log phase (data not shown). Cells from the same
flasks were plated to monitor viability (see Fig. 6).
Figure 6:
Lack of respiration prevents the rapid
viability loss of CuZnSOD null mutants. Cells were grown and kept in SC
medium. The results are representative of at least two experiments in
each of which the viability of two independent samples of each strain
was monitored. , EG103 (wt);
, CC103 (coq3
);
, EG118 (sod1
);
, CC118 (sod1
, coq3
);
, EG133 (sod1
, sod2
);
, CC133 (sod1
, sod2
, coq3
).
Figure 7:
The coq3 mutation reverses the
air-dependent methionine and lysine auxotrophies of sod1 strains. sod1
or sod1
, coq3
strains were inoculated at an OD
of 0.01 into
either SC complete medium or SC medium lacking methionine or lysine and
grown under high aeration conditions. OD600 was read at 48 h of
incubation. The results shown are representative; the experiment was
repeated three times.
We have
obtained similar results with strains constructed in another genetic
background, the strain EG225, an sod1 derivative of W303B (data not shown). In this strain, in addition
to petites generated by the coq3 deletion, we had available
strains that were respiration-incompetent due to the deletion of the ATP2 gene, which codes for the beta subunit of the
mitochondrial ATPase(28) . Either kind of petite mutation
improved the survival and allowed growth without lysine or methionine,
compared with the respiration-competent parent (data not shown). These
experiments confirmed that our results were not peculiar to one strain.
Our results show that superoxide dismutase is a major
antioxidant in yeast stationary phase, playing a very important role in
its survival under nongrowing conditions and implying that the
superoxide ion is a major damaging species. Additionally, we show that
mitochondrial respiration is a primary source of superoxide and other
reactive oxygen species in vivo. Previously this conclusion
was based on studies done in vitro. Studies in isolated
mitochondria showed that the majority of the superoxide in eucaryotic
cells was produced in the mitochondria by electron leakage at the
QH2:cytochrome c segment (complex III)(8) ; it has
been estimated that over 80% of the superoxide in S. cerevisiae is produced in this manner(9) . We were interested to note
that the sod1 strains grew much better on
lactate than on other nonfermentable carbon sources. In yeast, through
the action of lactate:cytochrome c oxidoreductase, lactate can
pass electrons directly to cytochrome c, thus bypassing
coQ(38) . This observation supports the idea that much of the
cytoplasmic superoxide generated by respiration in vivo comes
from complex III or before (as has been previously noted in
vitro). Alternatively, it may indicate that steps after CoQ are
less sensitive to oxidative damage; the ability to bypass complex I and
III and send electrons to complex IV by another route would then lead
to better growth.
A recent report (39) concluded that the electron transport chain is extremely tight under normoxic conditions and that superoxide is not produced in toxic amounts when MnSOD is absent. Our results differ from those in indicating a major role for CuZnSOD, as well as for MnSOD, in protecting against mitochondrially derived superoxide in normoxic conditions. In our system, even when MnSOD is present, reactive oxygen species are produced during electron transport in large enough amounts that some are able to evade the mitochondrial antioxidant enzymes and reach the cytoplasm. In the absence of cytoplasmic CuZnSOD, this leakage is apparently enough to cause considerable havoc.
Another study by the same
group(40) , reported that strains of S. cerevisiae deficient in both CuZnSOD and electron transport showed a
substantial growth defect in 21% oxygen compared with the
respiration-competent sod1 parent and
concluded that respiration reduced cytosolic oxidant stress in
vivo. Again, results in our system were different. In two
different sod1
strains, removal of
respiration improved survival and prototrophic growth relative to
similar respiration-competent strains, leading us to conclude that
respiration increased oxidant stress. Differences in strains used or in
the end point measured may account for these discrepancies.
The
differences observed between the single and double sod mutants reinforce the hypothesis that
despite the strong compartmentalization of these two enzymes, there is
significant overlap between their functions. Apparently the two
superoxide dismutase enzymes cooperate but cannot fully replace each
other. Even though the most dramatic defects have been observed for the
cells lacking the cytoplasmic CuZnSOD, the MnSOD is nonetheless
essential in its compartment. The fast loss of viability of CuZnSOD
null mutants under normal but not low aeration may indicate that the
mitochondrial enzymes are able to prevent migration of reactive oxygen
species to the cytoplasm under low but not normal environmental oxygen
levels. Interestingly, the survival rate for the sod2
mutant was not changed by alteration of
the state of aeration nearly as much as was the survival rate for the sod1
mutant, such that MnSOD was more
important than CuZnSOD for survival in low oxygen. This result has
implications for higher organisms, because the dissolved oxygen
concentration that most mammalian cells are exposed to is close to our
low aeration conditions, making it likely that MnSOD plays a more
important role than has been previously realized, in spite of its lower
abundance.
Our results confirm roles of considerable importance for both SODs in stationary phase survival and agree with recently reported results in prokaryotes in E. coli lacking intracellular SOD(41) . In that work, the stationary phase loss of viability was dependent on the presence of oxygen and could be prevented by a synthetic SOD mimic.
In summary, we have developed a simple in vivo model system using long term survival of S. cerevisiae maintained in stationary phase in which to investigate the interplay between damaging and protective mechanisms in nongrowing cells. We believe this is a good model for mammalian cells and will lead to better understanding of the oxidative processes involved in cancer, neurological diseases, and aging.