We describe here a screen for antioxidant genes in addition to TSA1
that are important in combating the oxidative stress that occurs in zinc-limited cells. This analysis identified SOD1
, encoding cytosolic Cu/Zn-superoxide dismutase, as critically important for low zinc growth. Sod1 was shown previously to be important for tolerating such varied stresses as oxidant insult, heat shock, hyperosmosis, freeze-thaw stress, stationary phase growth arrest, and ER stress 
. To our knowledge, this is the first demonstration of the essential role that Sod1 plays in supporting zinc-limited growth.
It was previously found that Sod1 is also required for resistance to high zinc 
. Thus, Sod1 is required at both extremes of zinc status. Intriguingly, despite the fact that high zinc induces oxidative stress, the contribution of Sod1 to zinc tolerance is not dependent on its SOD activity and therefore does not involve Sod1's antioxidant role. A zinc binding but catalytically inactive Sod1 allele, Sod1H46C
, conferred wild type zinc tolerance on a sod1Δ
mutant strain while an active, Mn-binding SOD did not 
. These observations led to the hypothesis that Sod1 serves as a “zinc sink” and, by binding zinc in the cell, helps buffer cytosolic zinc and thereby contributes to zinc tolerance. It has been estimated that there are ~500,000 molecules of Sod1 protein per cell under zinc-replete conditions 
so the amount of zinc that is bound by this one protein is substantial. These observations initially suggested to us that Sod1 may be playing a similar role in low zinc by serving as a zinc reservoir and supplying zinc to other proteins under those conditions. However, our results indicate that Sod1 enzymatic activity is required in low zinc suggesting that it is needed to metabolize increased levels of superoxide anion that accumulate in these cells. The source of ROS in low zinc is still unknown and this is an issue we are currently addressing. Our results indicate that decreased Sod1 activity is not the primary source. Other possible sources under investigation include mitochondrial dysfunction and/or ER stress.
Sod1 and the Tsa1 peroxiredoxin are both critical for low zinc growth. Sod1 converts O2−
) while Tsal metabolizes H2
O. Thus, it is conceivable that these two proteins function primarily in a sequential manner with Sod1 generating H2
and Tsa1 further reducing that oxidant. If true, this would suggest that O2−
is the primary form of ROS generated in zinc-limited cells. This hypothesis remains to be tested. However, our observation that the tsa1Δ sod1Δ
double mutant has elevated ROS when compared to either single mutant suggests that these proteins are not functioning solely in a sequential fashion. It is intriguing that substantially higher amounts of zinc in the growth medium are required to suppress the sod1Δ
mutant than the tsa1Δ
mutant; the low zinc growth defect of the tsa1Δ
mutant was suppressed by as little as 10 µM added zinc 
while the sod1Δ
mutant required greater than 10-fold more zinc for suppression. While the mechanism underlying this difference is unknown, it clearly shows that Sod1 is needed for optimal cell growth during even modest zinc limitation while Tsa1 is only required under the most severely zinc-deficient conditions.
We noted that Sod1 activity is decreased by about 50% in zinc-limited cells. Protein levels also decreased to a similar extent despite no change in mRNA levels. These results suggest that if Sod1 fails to acquire its zinc cofactor soon after translation, the protein is degraded. While the mechanism of Sod1 turnover in yeast has not been studied, both proteasomal and autophagic degradation of SOD has been observed in mammalian cells 
. Fifty percent of Sod1 molecules represent ~250,000 atoms of zinc. The minimum number of total zinc atoms per yeast cell required for growth is ~5×106 
. Thus, decreased Sod1 accumulation represents a considerable decrease (~5%) in the total zinc demand of the cell.
Our findings may have important relevance with regard to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder in humans. ALS is characterized by the progressive loss of motor neurons due to cell death. Approximately 10% of ALS cases are inherited and a subset of these are caused by dominant mutations in human Cu/Zn-SOD. While the mechanism of neuronal cell death in both heritable and spontaneous cases of ALS is not known, there is a consensus that it results from the accumulation of toxic SOD protein aggregates 
. Zinc appears to play an important role in influencing this aggregation process 
. For example, it has been shown that zinc inhibits the aggregation of both wild type and ALS mutant SOD in vitro 
. In addition, some ALS-causing mutations reduce the affinity of the SOD protein for its zinc cofactor 
. Zinc-deficient wild type and ALS-causing SOD proteins induce apoptosis when delivered to motor neurons in vitro 
. Finally, high dietary zinc was found to improve survival in a mouse model of heritable ALS 
. Formation of the toxic SOD aggregates is also thought to be favored by increased oxidative stress due to the formation of intermolecular disulfide bonds 
. Thus, nutritional zinc deficiency may play a role in the etiology of ALS for two reasons. First, decreased zinc availability will reduce the amount of the metal ion available for binding to the zinc site. Several studies demonstrate decreased SOD activity in zinc-deficient mammalian cells 
. Second, the increased oxidative stress associated with zinc deficiency could potentially increase the likelihood of aggregate formation.
Finally, aside from TSA1
, we did not identify any other antioxidant genes that are as important for low zinc growth. Genes playing little if any apparent role include those encoding the four other peroxiredoxins, TSA2
, and DOT5
. In addition, we found no major role for CTT1
, which encodes the cytosolic isozyme of catalase. This result was unexpected given our recent observations indicating that CTT1
expression is up-regulated in zinc-deficient cells and may be a direct target of Zap1 regulation 
. No major roles were found for the glutathione peroxidases nor for transcription factors YAP1
, and MSN4
that are normally involved in oxidative stress responses. This result is consistent with our observation that the targets of these transcription factors are not induced in zinc-limited cells despite the increased oxidative stress 
. The level of oxidative stress in zinc deficiency may be insufficient to cause a response by these factors or, alternatively, their activity may be impaired under these conditions. One important caveat to these observations is that our analysis would not have detected small effects on growth rates that might have been caused by these mutations and, therefore, small but important contributions could have been missed. In addition, because we only analyzed strains carrying single mutations, we would not have identified genes that contribute to low zinc growth but are redundant with other genes. Thus, the full repertoire of genes required for tolerance of the oxidative stress of zinc deficiency remains to be defined.