The absence of Sod1p leads to some unexpected metabolic consequences in S. cerevisiae, and this work was undertaken to increase our understanding of the reasons for these changes. Our original observation, made many years ago, was that sod1Δ strains of yeast grow more slowly and to a lower final cell density in liquid cultures, particularly in defined medium. In the current study, by measuring glucose and ethanol levels in the medium over time, we find that, compared to WT, the sod1Δ cells consume similar (or slightly higher) amounts of glucose and accumulate similar (or slightly higher) amounts of ethanol per cell in the medium at early stages. However, once glucose levels fall, the sod1Δ cultures fail to switch to utilization of ethanol and abruptly stop growing, or, in other words, the diauxic shift does not take place.
A normal diauxic shift is accompanied by accumulation and then utilization of the storage carbohydrate glycogen. In the sod1
Δ strain, there was no accumulation of glycogen (). Our results do not fully address the underlying reasons for the failure of glycogen to accumulation. One possibility is that glucose-6-phosphate, the allosteric activator of glycogen synthase as well as the starting material for glycogen synthesis, is chronically low in the sod1
Δ yeast due to competition from an overactive pentose phosphate pathway. (The pentose phosphate pathway is upregulated in sod1
Δ strains, presumably to cope with the excess demand for NADPH [21
]. If this pathway takes priority and uses most of the G6P synthesized, it could result in a lack of substrate for glycogen synthesis.) Alternatively, some regulatory process(es) involved in the diauxic shift or in glycogen synthesis itself may be affected by the presence of excess superoxide.
Because the diauxic shift is, in effect, a response to a change in the carbon source (from glucose to ethanol), we tested the effect of changing the timing of the carbon source shift. Interestingly, we found this timing to be critical—sod1Δ or paraquat-treated WT yeast moved from glucose medium to fresh medium with ethanol as the carbon source during log phase (after 8 hours of growth) can adapt to the respiratory carbon source and grow. However, if they have reached the diauxic shift or stationary phases (24 or 48 hr in culture), they do not grow after transfer (). The carbon source present in the original culture is also important, as raffinose-grown sod1Δ cells grew when transferred to ethanol, while glucose-grown cells did not (). It is important to note this defect is not due to a global inability to respire (petite phenotype) since (1) sod1Δ cells grow on the respiratory carbon source, lactate (data not shown) and (2) raffinose-grown sod1Δ cells and glucose-grown sod1Δ cells transferred early continue growing when moved to ethanol (). This behavior points to a difficulty in executing the diauxic shift under certain conditions rather than an absolute deficiency.
We conclude that sod1
Δ has a defect in its ability to respond to changing carbon sources at later stages of glucose-fueled fermentative growth. One explanation for this behavior could be that insufficient energy is available to fuel the execution of the switch in the sod1
Δ mutant strains. Lack of glycogen accumulation could be responsible, as it is normally used during the shift. Since this strain diverts more glucose to the pentose phosphate pathway to provide extra NADPH reducing power [21
], it may be that it uses up glucose prematurely, leaving no material to fuel the diauxic shift. The fact that the switch is more easily made from raffinose to ethanol than from glucose to ethanol supports this idea: Since glucose represses many respiratory genes and raffinose does not, the changes in cellular machinery required to go from glucose- to ethanol-fueled growth are much larger than those required to go from raffinose to ethanol. It is possible that the sod1
Δ strain is unable to muster the resources to make the more demanding switch from glucose.
An alternative explanation involves signaling—excess O2
- and/or H2
may interfere with some signaling mechanism or deliver a false signal. In mammalian cells, H2
is used as a second messenger to regulate growth [22
], and something similar may happen in yeast, although it has not been definitively shown [23
]. By this analogy, it is possible that excess superoxide-derived H2
might continue to drive the growth program inappropriately even as glucose levels drop.
sod1Δ yeast growing on glucose consumed approximately twice as much oxygen as WT at early and late stages of culture (). Reasoning that this might be due to increased mitochondrial content on the sod1Δ strain, we used several different assays, including flow cytometry using a dye sensitive to mitochondrial membrane potential, mitochondrial DNA copy number relative to that of nuclear DNA (), and levels of common mitochondrial and cytosolic protein markers (), to compare the relative mitochondrial mass in mutant and WT strains. In each of these determinations of mitochondrial mass, the sod1Δ yeast show an elevated signal—approximately twice that of the WT—which is in agreement with the twofold increase in oxygen consumption we observe in the mutant strain. It should be noted that the increase is seen in the flow cytometry experiment, in which the uptake of dye is dependent on membrane potential, that is, on mitochondrial function. If the mitochondrial mass was the same in the two strains, it would have meant that the sod1Δ strain had a higher membrane potential than the WT, which seems unlikely. If respiration was less efficient in the sod1Δ strain, then the dye uptake should have been lower and the approximately two fold difference would not have been observed. Therefore, taken together, these data convinced us that the increased oxygen consumption can be attributed to increased respiration due to an increase in functional mitochondria, i.e., that the effect is not due to inefficient use of oxygen or uncoupling, which would not have engendered extra mitochondrial volume or altered gene expression.
A logical extension of this reasoning says that inappropriate glucose derepression could be occurring in the sod1Δ yeast. We explored this possibility by measuring lacZ expression driven by the CYC1 promoter. This promoter is turned on by the transcription factor Hap2,3,4,5 when glucose becomes limiting and is thus a good indicator of glucose repression status. We found that this promoter is more active in the sod1Δ strain, indicating some level of glucose derepression.
Why glucose repression is affected is not clear from our work to date. One possible explanation is that some step in the glucose sensing and signaling pathway, from glucose sensing by the Snf3/Rtg2 complex, to the Ras/cAMP cascade, to transcriptional activation of Hap4 and other genes is impaired by excess superoxide. Another possibility is that it could be a natural regulatory response to an elevated energy requirement in the mutant strain relative to WT yeast. sod1
Δ yeast are subject to greater oxidative stress than WT, leading to higher turnover of damaged cellular components and, perhaps more importantly, an increased difficulty in maintaining redox balance. To regenerate cellular reducing equivalents in the mutant strain (i.e.,
NADH and NADPH, which are required to maintain an optimal GSH:GSSG ratio), increased flux through the pentose phosphate pathway is required [21
], diverting glycolytic intermediates that would otherwise be put toward ATP generation. Increased mitochondrial ATP production (via increased mitochondrial mass) seems a reasonable response to the altered energy demands of the mutant cell. A third possibility is that an increase in respiration can function as a physiological response to create a “relief valve” when oxidative stress is high. Bonawitz et al
] proposed this idea to explain the increased life span observed in tor1
Δ yeast strains, which also show glucose derepression. Increased oxygen consumption could certainly lower ROS generation, and a role for TOR signaling in the phenotypes observed in sod1
Δ yeast is possible, but these possibilities will have to be addressed through further research.
Although the phenomena we observe in sod1Δ yeast could arise from some strategic or “rational” response by the cell to its situation of elevated superoxide stress, it is equally possible that all of these defects arise simply because excess superoxide interferes with the ability of the cell to signal the necessity for and/or properly execute its metabolic changes. Particularly for the diauxic shift defect, everything we have seen is consistent with the notion that the sod1Δ yeast cultures are stuck in a state prior to transition into stationary phase, incapable of completing some of the required steps (e.g., glycogen accumulation, optimal ethanol consumption level), while “spinning their wheels” by continuously carrying out the steps they can complete until nutrients run out and catastrophic failure occurs.
Interestingly, the growth of sod1
Δ in raffinose is more WT-like than its growth in glucose, supporting the idea that a derepressed condition (or an inability to repress) is good for these mutants. The difference in final cell density between WT and sod1
Δ that is observed in glucose medium virtually disappears for growth in raffinose, indicating a rescue of the growth phenotype of sod1
Δ cells (). Thus, the sod1
Δ cells seem to do best on a “dual-purpose” carbon source, which both supports glycolysis and allows higher levels of respiration. This compromise might allow more glucose to be diverted to the pentose phosphate pathway while extracting higher energy yield from the remaining glucose via
mitochondrial respiration. Indeed, it has been noted that glucose derepression turns on the expression of both Sod1p and Sod2p prior to the presumed oxidative stress generated by respiration, and thus can be considered a preventative measure against oxidative stress [26
Our studies demonstrating effects on the diauxic shift and glucose repression provide a more complete picture of the metabolic implications of oxidative stress in yeast. These phenomena may be particular to this model organism and closely related species—certainly cells in higher multicellular organisms are exposed to far less variation in oxygen levels and nutritional supply and thus may have lost those response pathways over the course of evolution. On the other hand, cancer cells are known to undergo a shift to aerobic glycolytic metabolism, which may be related at some level. It is also interesting to compare the effects of Sod1p deletion in S. cerevisiae
with those observed in genetically manipulated mice. As described above, the absence of Sod1p in yeast has a significant detrimental effect on the health of the cell and particularly on its rate of growth, but our studies imply that many of these effects can be attributed to defects in metabolic pathways that are specific to yeast. By contrast, sod1−/−
mice appear healthy at birth, suggesting that there are no major defects in the metabolic pathways most critical to growth of mammalian cells. Nevertheless, as they grow, elevated levels of oxidative stress are clearly present in the sod1−/−
mice as evidenced by significantly increased levels of lipid peroxidation, protein carbonyls, oxidative damage to DNA, and DNA mutation rates. Unlike the intact sod1−/−
mice, cultured fibroblasts from these mice do not grow well, possibly because the cultured cells experience elevated levels of oxygen relative to those in the intact animal [27
]. Thus, sod1
Δ yeast may be a better model for individual cultured cells than for whole animals. Overall, the simplicity and flexibility of the budding yeast, Saccharomyces cerevisiae
as a model organism has served scientific researchers well, and will likely continue to do so, provided special care is taken in applying results to higher organisms.