Ure2, a prion precursor with homology to glutathione S-transferase, protects Saccharomyces cerevisiae cells from heavy metal ion and oxidant toxicity

Therefore, we have investigated the possibility of additional Ure2 functions. To circumvent repeatedly reported problems associated with in vitro enzyme assays, we adopted a more genetic approach, i.e., asking whether ure2 ∆ mutants exhibit greater sensitivity than isogenic wild type strains to a range of glutathione S-transferase substrates and compounds generating oxidative stress in S. cerevisiae . We show that Ure2 is indeed required for detoxification of glutathione S-transferase substrates and cellular oxidants. Ure2 shares not only structural homology with various glutathione S-transferases, but ure2 mutations exhibit phenotypes similar to those of mutations in known S. cerevisiae and pombe glutathione S-transferase genes. results were observed whether glutamate or ammonia was used as nitrogen source. Growth in the presence of nickel sulfate had little demonstrable effect on NCR-sensitive gene expression using GAP1 as the NCR-sensitive reporter gene. These data suggest that regulation of URE2 expression is unlikely to be induced at moderate levels of perturbant, and that Ure2’s protection of cells from toxic compounds does not demonstrably diminish its ability to regulate NCR-sensitive gene expression. This work identifies a new function for Ure2 in Saccharomyces cerevisiae , i.e., participation in heavy metal ion and oxidant detoxification. Deletion of URE2 results in hypersensitivity to cadmium ions, hydrogen peroxide and, to a lesser extent, nickel ions. ure2 mutants are also slightly hypersensitive to diamide, a glutathione cycling reagent, but only when ammonia is used as sole nitrogen source. There is no convincing hypersensitivity to diamide when glutamate is used in place of ammonia, suggesting that hypersensitivity, if it exists, is seen only when NADPH pools are diminished. Our data also offer a possible explanation of negative results encountered in attempts to demonstrate Ure2-mediated glutathione S-transferase activity using CDNB as substrate. We found CDNB to be only slightly more toxic to ure2 ∆ cells than wild type, suggesting that Ure2 may play only a peripheral role at best in its detoxification.


Introduction
Several neurodegenerative conditions derive from the same pathogenetic mechanism, i.e., a change in protein conformation, polymerization, and plaque formation.
These conditions have been called conformational diseases such as Alzheimers and the prionoses. Recent studies have demonstrated that a protein associated with such disease, amyloid β-protein, protects neurons from metal-induced oxidative damage (1). Neither the molecular basis for this activity nor how it is affected by environment and neuronal metabolism is yet known. The use of eukaryotic model systems, such as the yeast Saccharomyces cerevisiae, has greatly facilitated our acquisition of information about mammalian proteins, their functions, interactions and how their synthesis and activities are regulated and integrated. In particular, the genetic study of prions has been facilitated by Wickner's discovery that S. cerevisiae Ure3 possesses prion-like characteristics (2).
Ure3, the prion form of the nitrogen regulatory protein Ure2, has been well studied in an attempt to gain further insight into the changes that accompany polymerization and the cellular proteins that impact on that process (3-7a). Significant emphasis has been placed on determining whether metabolic activity influences Ure2 à Ure3 conversion (8)(9)(10)(11)(12)(13).
Ure2 à Ure3 conversion has been reported to decrease in an mks1∆, in a strain expressing the constitutively active dominant Ras2 Val19 allele (8), and in strains where the intracellular pool of glutamate is enlarged (11).
Ure2 was originally identified as a mutated genetic locus that permits cells, growing with ammonia as nitrogen source, to transport the pyrimidine precursor ureidosuccinate; wild type cells are unable to do this (14,15). The ure2 mutation was subsequently found -4-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from to possess a pleiotropic phenotype, in which transcriptional repression of many genes encoding proteins needed to transport and degrade poor nitrogen sources become resistant to nitrogen catabolite repression, i.e., repression no longer occurs in the presence of a good nitrogen source.
When URE2 was cloned and sequenced, it was found to possess homology to known glutathione S-transferases (16). Homology between Ure2 and glutathione S-transferases now extends to the level of its crystal structure (17)(18)(19). Two additional structures were determined using crystals in which glutathione or two of its analogues were bound to Ure2 (19). In spite of these structural characteristics, multiple attempts to demonstrate that Ure2 catalyzes a glutathione S-transferase reaction have been unsuccessful (7,16,20). There are also characteristics of the Ure2 three-dimensional structure that prompt the question of whether it would even be expected to possess S-transferase activity. Most specifically, a residue that participates in catalysis by known glutathione S-transferases (i.e., cysteine or histidine in β-class; tyrosine or serine residues in eukaryotic classes) is not present in Ure2 (17). This residue is critical because it destabilizes the cysteine S-H bond, thereby facilitating formation of the active thiolate anion (GS -). The region of Ure2 that binds glutathione does contain an asparagine, Asn124, which some, but not all investigators, suggest may be situated at a location and distance that are consistent with permitting it to function in destabilization of this critical S-H bond (19).
Whether or not Ure2 is a glutathione S-transferase is a question that has remained open and tantalizing for two decades and is increasingly important to future studies of Ure3 prion formation and the impact of environmental and metabolic influences on it.
-5-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from Therefore, we have investigated the possibility of additional Ure2 functions. To circumvent repeatedly reported problems associated with in vitro enzyme assays, we adopted a more genetic approach, i.e., asking whether ure2∆ mutants exhibit greater sensitivity than isogenic wild type strains to a range of glutathione S-transferase substrates and compounds generating oxidative stress in S. cerevisiae. We show that Ure2 is indeed required for detoxification of glutathione S-transferase substrates and cellular oxidants. Ure2 shares not only structural homology with various glutathione Stransferases, but ure2 mutations exhibit phenotypes similar to those of mutations in known S. cerevisiae and pombe glutathione S-transferase genes.  representative of the effects we report. During this work, we noted wild type strain-tostrain differences in overall sensitivity to various perturbants. However, the patterns of sensitivity in wild type vs. mutant cells were always the same. For this reason a wild type control was included on all petri plates so that wild type vs. mutant comparisons could be -7- Northern blot analyses were performed as described (24) using the PCR-generated probes that were radiolabeled with the Invitrogen RAD Prime DNA Labeling System.

Results
Ure2 is required for protection against heavy metals. Ure2 possesses clear structural homology with θor βclasses of glutathione S-transferases. Unfortunately, attempts in multiple laboratories to demonstrate glutathione S-transferase enzyme activity for Ure2 have been unsuccessful (7,16,20). Although negative results are rarely reported in detail, the lack of success might derive from inherent instability in the enzyme, an observed characteristic of glutathione S-transferases, or performing the assay with substrates that are not the preferred ones for the putative transferase (25)(26)(27)(28). The difficulties experienced in attempts to assay an enzyme activity prompted us to take a step backwards from in vitro assays and ask more simply whether Ure2 is required for protection of cells against the growth inhibitory effects of compounds reported to be glutathione Stransferase substrates in other organisms.
Assays unable to demonstrate Ure2-dependent glutathione S-transferase were performed with the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB) (7, markedly inhibited growth of the ure2∆ mutant relative to wild type, though at the concentrations used, cadmium ions were far more toxic (note differences in the times of incubation) (Fig. 1). In addition, the ure2 mutant phenotype was much tighter with cadmium than nickel ions. Equally important, growth was inhibited to roughly the same degree regardless of whether ammonia or glutamate was provided as sole nitrogen source. This is a positive indication that observed sensitivity didn't derive indirectly from the influence of the ure2 mutation on NADPH levels in the cell.
Although growth differences of wild type and ure2∆ strains in Fig. 1 are marked, we were concerned that they might derive trivially from the fact that ure2 mutants in some strain backgrounds grow a bit slower than wild type. Therefore, we asked, did slow growth of the ure2∆ in the presence of nickel or cadmium ions derive from loss of ability to detoxify the metal ions, or was it just a manifestation of the fact that ure2 mutants grow slower? To assess this possibility, we streaked the wild type and ure2∆ strains on the same media used for the metal ions toxicity test, but in the absence of the metal ions.
As shown in the bottom panels of Fig. 1, the ure2∆ strain does grow a bit slower than wild type. Therefore, ure2∆ hypersensitivity to nickel ions is not as great as depicted in the top two panels, but is still present, and can be increased if the nickel ion concentration is raised to 0.9 mM (data not shown). For cadmium, the difference in growth is dramatic.
In fact, there was no growth of the ure2∆ mutant even after four days incubation on YEPD-cadmium plates (Fig. 2).
To test whether it was the ure2 mutation in TCY-5 that was specifically responsible for increased metal ion sensitivity, we complemented the mutation by transforming -10-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from strains RR114 (ure2∆) with pRR529 (URE2). TCY5 (wild type) transformed with vector YEp24 was the positive control. URE2 pRR529 restored the growth of strain RR114 in the presence of nickel or cadmium ions to wild type levels (Fig. 3A). If Ure2 is itself responsible for wild type detoxification of metal ions and is the limiting entity in metal ion detoxification, one might expect to see increased ability to cope with high levels of environmental metal ions when URE2 is over-expressed. As shown in Fig. 3B, overexpression of URE2 did not generate increased resistance to 0.6 mM nickel sulfate relative to wild type. However, when the nickel ion concentration was increased to 0.9 mM, over-expression of URE2 did result in detectably increased protection relative to the wild type strain transformed with vector YEp24.
In studies of this nature, one of the most significant problems is to distinguish whether observed effects derive from direct or indirect participation of the gene product being tested. This is an especially serious consideration when that product is known, as Ure2 is, to negatively regulate NCR-sensitive, Gln3 and Gat1-mediated transcription in the presence of a good nitrogen source (31)(32)(33). By this reasoning, loss of metal ion detoxification in a ure2∆ mutant might derive indirectly from increased Gln3/Gat1mediated transcription. To test this possibility, we transformed wild type strain STCY32 with ADH1-GAT1 pRA27 or vector control pEG202, and tested the metal ion sensitivity of the transformants. Over-expression of GAT1 in this way, has been previously shown to result in both increased Gat1 and Gln3-mediated transcription and to render that transcription largely resistant to NCR (23,34). In addition, we tested metal ion sensitivity with glutamate as sole nitrogen source rather than ammonia since glutamate generates -11-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from much less NCR than ammonia in these strains which in turn would further increase Gln3/Gat1-mediated gene expression. Rather than increasing metal ion sensitivity, as occurs when URE2 is deleted, over-expression of GAT1 resulted in greater nickel ion resistance than observed when the wild type strain was transformed with control pEG202 (Fig. 4). The effect of GAT1 over-expression, though visible with cadmium ions, was less striking. When this experiment was repeated in strain BY4741, parent of the consortium-generated set of yeast gene deletion strains, pEG202 and pRA27 containing transformants grew indistinguishably in the presence of cadmium chloride or nickel sulfate (data not shown). These data and those in Fig. 4 argue against the possibility that metal ion hypersensitivity in a ure2∆ results from its only demonstrated function, negatively regulating GATA-factor-mediated transcription.
Since Ure2 regulation of Gln3/Gat1-mediated transcription influences expression of GDH1 and GDH2, another conceivable explanation of the Ure2 requirement for metal ion detoxification might be that it alters the level of reducing equivalents (NADPH) required to maintain glutathione in its active, reduced form. Although this explanation was circumstantially addressed in Fig. 1 by comparing metal ion sensitivity with glutamate vs. ammonia as nitrogen source, it can be addressed more specifically by comparing metal ion sensitivity of wild type and gdh1∆ mutant cells. In gdh1∆ mutants, ammonia is assimilated without the consumption of NADPH because it occurs through the combined action of the GLN1 (glutamine synthetase) and GLT1 (glutamate synthase, GOGAT) gene products (35). As shown in Fig. 5, wild type and gdh1∆ strains are equally able to detoxify nickel ions. If anything, the gdh1∆ mutant may grow slightly better than wild -12-  (7,20), we compared wild type and ure2∆ strain sensitivity to CDNB. As shown in Fig.   6, wild type colonies are larger than a ure2∆ mutant after 54 hours of incubation in the presence of CDNB. At 68 hours incubation, the difference in growth between the two strains, though still apparent, is much less drastic. Similar results were observed with minimal glutamate medium ( Fig. 6 and data not shown). However, the growth difference seen at 68 hours is not convincingly different from that of wild type and ure2∆ strains growing in the absence of perturbant (Fig. 1, bottom two panels). Therefore, if loss of Ure2 generates CDNB hypersensitivity it is modest at best. This correlates well with the inabilities of multiple investigators, including ourselves, to detect in vitro glutathionation of CDNB.
Mutants with defects in glutathione S-transferase genes are often found to exhibit increased sensitivity not only to preferred S-transferase substrates, but also to compounds that are not detoxified by direct conjugation to glutathione. Two compounds in this category are hydrogen peroxide and diamide. Hydrogen peroxide is detoxified in two ways: by peroxidation, an activity found in some mammalian theta-class GSTs and in S. pombe GST3 or by GSH conjugation of cellular products that are oxidized by -13-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from hydrogen or other peroxides (25,26,28). Diamide, also oxidizes cellular proteins and other constituents, but in addition, depletes the reduced-glutathione pool because it is detoxified via glutathione-dependent reduction (36). ure2∆ colonies, grown in the presence of diamide, were smaller than wild type after 51 hours of incubation in minimal-glutamate or -ammonia medium (Fig. 7, top two panels). Differences in growth, however, were less marked after 72 hours of incubation (Fig. 7, middle two panels). Sensitivity of the ure2∆ to diamide was greater than observed for CDNB, but less than for nickel ions when ammonia was used as nitrogen source. In contrast, when glutamate was used as nitrogen source, differences in growth were not different than that seen in the bottom two panels of Fig. 1. It is important to note that diamide was the only perturbant in which different growth patterns were observed on glutamate vs. ammonia medium. This difference correlates with the facts that (i) ammonia assimilation places a greater drain on the NADPH pool than does glutamate, and (ii) the only cellular constituent used in detoxifying diamide is NADPH. Therefore, it is conceivable that apparent ure2∆ hypersensitivity to diamide derives indirectly from effects of the mutation on NADPH metabolism, or alternatively that hypersensitivity can only be visualized at lower NADPH concentrations.
Mutants lacking glutathione S-transferases have been reported to also become hypersensitive to hydrogen peroxide (25). Therefore, we compared hydrogen peroxide toxicity in wild type and ure2∆ strains. Hydrogen peroxide was more toxic to ure2∆ mutants than any of the other non-metal ion perturbants (Fig. 7, bottom two panels). In contrast to what occurred with diamide, hypersensitivity to hydrogen peroxide exhibited -14-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from by the ure2∆ strain was equivalent whether ammonia or glutamate was provided as nitrogen source. Hypersensitivity was also observed in hydrogen peroxide containing YEPD medium (Fig. 2, bottom panel). In sum, ure2 mutations, though most sensitive to heavy metal ions, exhibit the same pleiotropic hypersensitivity to oxidants, hydrogen peroxide being most toxic, whose detoxification does not involve direct conjugation to glutathione as seen in gst1,gst2,gst3 mutants of Schizosaccharomyces pombe (25).
Ure3 is capable of protecting cells from heavy metals. URE3 and ure2 mutants were originally isolated from the same selection (14,15). Therefore, it is not surprising that URE3 exhibits the same phenotype as ure2 mutations, except for being a bit more leaky.
Prior to this work, the only known ure2 phenotype has been resistance to NCR (14,15,(31)(32)(33). Identification of a new ure2 phenotype prompts the question of whether URE3 strains possess a similar one, as is the case for negative regulation of GATA-factormediated transcription. The question is pertinent because there is a strong correlation between Ure2-Gln3 and Ure2-Gat1 complex formation and Ure2's ability to inhibit NCR-sensitive transcription in the presence of a good nitrogen source, i.e., the Ure2 regulatory activity appears to be associated with a stoichiometric reaction between Gln3 and itself. Since Ure2, in its Ure3 prion form, is a polymer, it is not too surprising that it cannot simultaneously interact with itself and Gln3. Indeed, portions of the Ure2 molecule that interact with Gln3 also participate in prion formation (37). If we assume that Ure2 does possess glutathione S-transferase activity, it may not be as adversely affected by Ure3 prion formation because this activity is catalytic rather than stoichiometric. Consistent with this possibility, glutathione has been reported to bind to To answer the above question, we used three strains generously provided by Edskes and Wickner: YHE711 (wild type, ure-o), TIFY3 (ure2:G418), and YHE731 (strain YHE711 into which URE3 was cytoduced). Since Ure3 can be lost from some cells during storage in glycerol (H. Edskes, personal communication), we streaked out all three strains on YEPD and then scored the phenotypes of multiple isolates on glucose ammonia + USA medium. Wild type ure-o cells (all strains are ura2, a prerequisite of the plate assay) will not grow in this medium, whereas ure2 and ure3 mutants will. We indeed found that a few of the "ure3" isolates had become identical to the wild type, i.e. no longer able to grow on ammonia + USA medium. We assayed the metal ion sensitivity of seven randomly chosen URE3 clones, using wild type and ure2 mutants as controls. As shown in Fig. 8, URE3 and ure2 clones grew similarly in glucose ammonia + USA medium, but exhibited opposite phenotypes in the presence of metal ions. The URE3 clones were just as resistant as the wild type to both environmental insults. Note that there was some cross-feeding of the "wild type" (ura2) strain. This did not occur when the streaked cells were more distantly separated.

Effect of heavy metals URE2 and NCR-sensitive transcription. Given Ure2 regulation of
NCR-sensitive expression, we determined whether heavy metal ion treatment affects URE2 expression or the ability of Ure2 to regulate GATA-factor mediated transcription.
We used 0.6 mM nickel sulfate rather than cadmium chloride as the pertubant here because cadmium ions have such a drastic effect on cell growth. URE2 expression increased about two-fold in minimal medium containing nickel sulfate (Fig. 9). Similar -16-

Discussion
This work identifies a new function for Ure2 in Saccharomyces cerevisiae, i.e., participation in heavy metal ion and oxidant detoxification. Deletion of URE2 results in hypersensitivity to cadmium ions, hydrogen peroxide and, to a lesser extent, nickel ions.
ure2 mutants are also slightly hypersensitive to diamide, a glutathione cycling reagent, but only when ammonia is used as sole nitrogen source. There is no convincing hypersensitivity to diamide when glutamate is used in place of ammonia, suggesting that hypersensitivity, if it exists, is seen only when NADPH pools are diminished. Our data also offer a possible explanation of negative results encountered in attempts to demonstrate Ure2-mediated glutathione S-transferase activity using CDNB as substrate.
We found CDNB to be only slightly more toxic to ure2∆ cells than wild type, suggesting that Ure2 may play only a peripheral role at best in its detoxification.
Primary sequence and three-dimensional homology between Ure2 and known glutathione S-transferases, as well as the observed binding of glutathione and glutathione S-transferase substrates to crystallized Ure2 are consistent with the possibility that URE2 encodes a glutathione S-transferase. Our work provides additional support for that argument, i.e., the demonstration that Ure2 is required for metal ion detoxification and increased in vitro activity to catalyze the conjugation of CDNB and glutathione, while overproduction of only Gst3 increased glutathione peroxidase activity using cumene hydroperoxide as substrate (25,38). Two glutathione S-transferase genes have been reported in Saccharomyces cerevisiae, GTT1 and GTT2 (20). Both genes were shown to -19-by guest on March 23, 2020 http://www.jbc.org/ Downloaded from catalyze the glutathione S-transferase reaction using CDNB as substrate. When these investigators similarly assayed Ure2 (20), the results were negative just as they were for others and ourselves (data not shown). Finally, Hynes and his co-workers cloned the gstA gene from Aspergillus nidulans and found its primary protein sequence to be most homologous to Ure2. Among the compounds to which a gstA deletion mutant was hypersensitive, were nickel ions, selenium, diamide, and CDNB (39). However, in contrast to S. cerevisiae Ure2, the A. nidulans GstA protein does not participate in the regulation of GATA-factor-mediated, nitrogen-responsive transcription.
The fact that URE2 expression increased only two-fold in the presence of nickel sulfate suggests that cellular Ure2 levels are normally sufficient for it to function effectively in protecting the cell from heavy metal ions and strong oxidants. Consistent with this suggestion is the fact that over-expression of URE2 (Fig. 3)  Finally, in addition to identifying a new function for Ure2, this work has identified a phenotype that can be used along with growth in ammonia + U.S.A. medium, to distinguish wild type, ure2 and URE3 alleles. URE3 strains are as resistant to nickel and cadmium ions as wild type, while ure2 mutants exhibit markedly increased sensitivity to these ions. Both ure2 and URE3 strains, on the other hand, are resistant to NCR, whereas the wild type is not. If Ure2 is a direct participant in enzyme reactions involving glutathione, the shared and distinguishable phenotypes of ure2 and URE3 mutations may derive from the physical requirements of the two processes assayed, one requires stoichiometric participation of Ure2 while for the other only catalytic participation is involved. This ability to distinguish Ure2 and Ure3 in vivo will prove highly useful in future studies of relationships between the two forms of this fascinating protein. Minimal-ammonia or -glutamate media used in the bottom two panels did not contain any added heavy metal ions.