INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Many metals are essential for all organisms at trace amounts but can be toxic at higher concentrations. Copper (Cu) is a well-studied important cofactor of a variety of enzymes that are involved in a variety of biochemical processes, such as cytochrome c oxidase, Cu, Zn superoxide dismutase, lysyl oxidase, and dopamine--monooxygenase (21), and plays a critical role in iron (Fe) assimilation (1, 16). However, accumulation of Cu can generate hydroxyl radicals, which cause cellular damage such as oxidation of proteins, cleavage of DNA and RNA, and membrane destruction by lipid peroxidation (11). Therefore, it is necessary for organisms to have elaborate mechanisms to maintain Cu homeostasis by regulation of uptake of the Cu needed to drive particular biochemical processes, by detoxification if Cu is accumulated, and by monitoring of both of these processes. The importance of this Cu homeostasis is revealed by the existence of the two human genetic disorders of Cu homeostasis, Menkes syndrome and Wilson's disease (2, 3, 28, 30).

The yeast Saccharomyces cerevisiae has been extensively used to study Cu homeostasis and for genetic screens that revealed the genes responsible for Cu uptake, subcellular distribution of Cu, and detoxification of Cu at higher levels (4-6, 9, 18, 20, 26, 27, 29, 32). Cu occurs in the environment as the oxidized Cu²⁺ form and is transported as the reduced Cu⁺ form. In summary, this process is mediated by two membrane-associated high-affinity Cu transporters, Ctr1p and Ctr3p, and a cell surface Cu(II) and Fe(III) reductase, Fre1p (6, 13, 18). In the presence of excess Cu, the Ctr1p, Ctr3p, and Fre1p components are down regulated at the transcriptional level through the action of the metalloregulatory transcription factor Mac1p, essentially abolishing high-affinity uptake of Cu (9, 15, 19, 31). Excess levels of Cu are sensed by another transcription factor, Ace1p. Through cooperative binding of Cu to specific cysteine residues of the Ace1p DNA binding domain, Ace1p binds to metal response elements on the promoters of genes, activating such genes as CUP1, CRS5, and SOD1, which are involved in Cu detoxification and protection against oxidative stress (5, 8, 10, 12, 26).

In this study, we demonstrate that the growth inhibition of the yeast S. cerevisiae by Cu and other metal salts is pH dependent. We also establish that His strains containing a lesion in any one of the seven genes that encode a biosynthetic component for the amino acid histidine are more sensitive to Cu, cobalt (Co), or nickel (Ni) salts. Sensitivity to Cu is dependent on the pHs of growth media, such that His strains can grow in the presence of 2.4 mM CuSO₄ at a pH below 6 but do not grow at a pH above 6.5. Also, addition of excess histidine reverses the Cu sensitivity of His strains. Deletion of the high-affinity histidine transporter Hip1p in His strains caused greater sensitivity to Cu and a greater requirement for exogenous histidine to reverse this Cu sensitivity, suggesting an absolute requirement for histidine in Cu resistance. We also report that sensitivity to a large number of metal salts is dependent on the pH of the growth medium and that a functional vacuolar H⁺-ATPase is needed to confer resistance to some metal salts. These findings on the effect of pH and histidine auxotrophy should be considered when metal toxicity in yeast is investigated and when His⁺ plasmids are used to explore the phenotypes of mutations in His strains.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Nomenclature. We used standard genetic nomenclature, including, for example, the phenotypic symbols His⁺ and His for the independence of and requirement for histidine, respectively. HIS3 or HIS3⁺, for example, denotes the wild-type allele, whereas his3 denotes any defective recessive allele, his3- denotes any deleted or disrupted allele, and his3-1, his3-200, etc. denote specific deleted or disrupted alleles.

Yeast strains and media. The strains used in this study are listed in Table 1. Yeast extract (1%)-peptone (2%)-and dextrose (2%) medium (YPD) was used throughout this study, with the metal salts being added after autoclaving at the concentrations listed in Table 2. Each medium was either not buffered or buffered with 50 mM MES (morpholineethanesulfonic acid) and 50 mM MOPS (morpholinepropanesulfonic acid). Both sets of media were adjusted to the pH values indicated in Table 2 with dilute HCl or NaOH. However, growth of yeast on media containing the indicated metal concentrations was the same whether the yeast was tested on buffered or nonbuffered media. Histidine and other amino acids also were added to media after they were autoclaved, as indicated in Table 2.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The growth of His strains is sensitive to Cu in a pH-dependent manner. During the course of an investigation of phenotypes of his3 strains with various disrupted genes and complementation with HIS3 plasmids, it became apparent that the HIS3 and his3 control strains exhibited different levels of growth on YPD containing CuSO₄ at pH 6.5. The B-7553 (his3-) and B-11842 (his3- p[HIS3]) strains (Table 1) grew identically on YPD at either pH 4.6 or 6.5, whereas the his3 strain grew on YPD-2.4 mM CuSO₄ media only at pH 4.6 and, in contrast to HIS3 strains, did not grow at pH 6.5. A more detailed examination with YPD-2.4 mM CuSO₄ media revealed that the growth of his3 strains became increasingly more inhibited as the pH increased past pH 6, until no growth was observed past pH 6.3 (Fig. 1). Some of the differential responses of HIS3 and his3 strains are summarized in Table 3. Similar results were seen for the his1, his2, his4, his5, his6, or his7 strain and related His⁺ strains (data not presented). Thus, any block in the biosynthetic pathway of the histidine resulted in a pH-dependent sensitivity to Cu.

View larger version (79K): [in this window] [in a new window]   FIG. 1.   The his3- strain, but not the HIS3+ strain, is sensitive to Cu in a pH-dependent manner. (A) Serial dilutions of B-7553 (his3-) and B-11842 (HIS3+) grown on YPD adjusted to the pH indicated; (B) Same serial dilutions of B-7553 (his3-) and B-11842 (HIS3+) grown on YPD containing CuSO4 at a concentration of 2.4 mM and adjusted to the pHs indicated.

Histidine reverses the Cu inhibition of His strains. It is pertinent to point out that YPD contains sufficient histidine from the peptone and yeast extract components to support the growth of His strains. However, supplementing YPD-2.4 mM CuSO₄ media with histidine reversed the Cu inhibition of the his1 to his7 strains (Fig. 2). This result with exogenous histidine and the lower levels of inhibition of His⁺ strains suggest that higher internal concentrations of histidine are required for diminishing the inhibitory effect of Cu. Furthermore, the addition of either alanine, leucine, glutamic acid, or adenine instead of histidine to the YPD-2.4 mM CuSO₄ media did not reverse the Cu inhibition of the his1 to his7 strains (data not presented).

View larger version (51K): [in this window] [in a new window]   FIG. 2.   Mutation of any of the histidine biosynthetic genes HIS1, HIS2, HIS3, HIS4, HIS5, HIS6, and HIS7 causes increased sensitivity to Cu at pH 7.0, which can be reversed by the addition of excess histidine to the medium. Serial dilutions of B-585 (his1), strain 805 (his2), strain 705 (his3), strain 419 (his4), strain 192 (his5), strain 462 (his6), strain 339 (his7), and B-11842 (His+) on YPD (pH 7.0) (A), YPD-2.4 mM CuSO4 (pH 7.0) (B), and YPD-2.4 mM CuSO4-2 mM histidine (pH 7.0) (C) are shown.

Deletion of the high-affinity histidine transporter Hip1p results in greater Cu sensitivity of His strains. The role of higher internal concentrations of histidine for diminishing the inhibitory effect of Cu was investigated with mutants lacking the high-affinity transporter for histidine, encoded by HIP1 (25). An isogenic series of strains, PLAS112-4C (HIS3 hip1), PLAS112-4B (his3 hip1), PLY171 (his3 HIP1), and PLY170 (HIS3 HIP1) (Table 1), was examined for growth on YPD in the presence of various concentrations of CuSO₄ and over a pH range of 4.0 to 7.0. Importantly, the his3 hip1 strain was more sensitive to CuSO₄ than the his3 HIP1 strain (Fig. 3). The his3 hip1 strain was sensitive to the low concentration of 0.012 mM CuSO₄ and was completely inhibited by 0.06 mM CuSO₄, whereas the his3 HIP1 strain grew on media containing up to 1.2 mM CuSO₄. Interestingly, decreasing the pH of the medium, which reversed Cu sensitivity of his HIP1 strains, did not rescue the his3 hip1 strain, even at these lower Cu concentrations. Curiously, deletion of HIP1 alone in the HIS3 hip1 strain appeared to result in slightly more tolerance to Cu than was exhibited by the HIS3 HIP1 strain. These results suggested that the intracellular concentration of histidine is the key factor for alleviating the inhibitory effect of Cu. This conclusion was confirmed by examining the levels of histidine required to reverse the Cu inhibition of the his3 hip1 and his3 HIP1 strains (Fig. 4). Both his3 hip1 and his3 HIP1 strains were unable to grow on YPD-2.4 mM CuSO₄ medium, both could grow on YPD-2.4 mM CuSO₄-0.6 mM histidine medium, but only the his3 HIP1 strain could grow on YPD-2.4 mM CuSO₄-0.02 mM histidine medium (Table 3). These results are most simply explained by higher internal concentrations of histidine diminishing the inhibitory effect of Cu.

View larger version (41K): [in this window] [in a new window]   FIG. 3.   Deletion of the high-affinity transporter for histidine (HIP1) results in increased sensitivity to Cu. Serial dilutions of PLAS112-4C (HIS3+ hip1-), PLAS112-4B (his3- hip1-), PLY171 (his3- HIP1+), and PLY170 (HIS3+ HIP1+) on YPD containing the indicated concentrations of CuSO4 (pH 7.0) are shown.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Deletion of the gene encoding the high-affinity transporter for histidine (HIP1) results in a greater requirement for exogenous histidine in the medium to allow for growth in the presence of Cu. Serial dilutions of PLAS112-4B (his3- hip1-) and PLY171 (his3- HIP1+) on YPD-2.4 mM CuSO4 (pH 7.0) with the indicated concentrations of histidine are shown.

Many laboratory strains show pH-dependent Cu toxicity. We wish to emphasize that many yeast strains used by researchers are auxotrophic for histidine, as they bear his3 markers used in plasmid and other manipulations. The levels of growth of the following strains were tested on YPD-2.4 mM CuSO₄ (pH 7.0) medium: W303a (his3-11,15), D273-10B-X (his3), YPH499 (his3-200), and the S288C derivatives BY4739 (His⁺) and BY4742 (his3-1), which have been chosen for deletion of all yeast genes (23). The two His⁺ strains B-11842 and BY4739 grew on the YPD-2.4 mM CuSO₄ (pH 7.0) medium, whereas the his3 strains W303a and YPH499 did not grow and the his3 strains D273-10B-X and BY4742 grew poorly on this medium (Fig. 5). In addition, all of these his3 strains grew on the lower-pH medium YPD-2.4 mM CuSO₄ (pH 6.0) and Cu sensitivity was rescued by the addition of histidine to the medium (data not presented).

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Commonly used strains exhibit different levels of growth on YPD-2.4 mM CuSO4, depending on the his3 marker. Comparative levels of growth of the following strains on YPD (pH 7.0) (bottom) and YPD-2.4 mM CuSO4 (pH 7.0) (top) are shown. (A) B-11842 (HIS3+); (B) W303a (his3); (C) D273-10B-X (his3); (D) YPH499 (his3); (E) BY4739 (HIS3+); (F) BY4742 (his3).

pH dependency of metal toxicity. The effect of pH on the toxicity of other metal salts, listed in Table 2, were investigated with the B-11842 (HIS3) and B-7553 (his3) strains. The tests were carried out with sublethal concentrations of each of the metal salts, which were determined by simply increasing the concentration until the HIS3 strain ceased to grow (Table 2). Thus, the working concentration for each metal was just below the toxicity level, allowing a sensitive means to evaluate the effect of pH. Not surprisingly, as with CuSO₄, addition of metal salts altered the pH of YPD (Table 2). The metal salts were assigned to the following four groups based on the pH-dependent effect on growth of the HIS3 strain (Table 2): group A, containing potassium (K), strontium (Sr), and molybdenum (Mo), in which pH did not change the growth responses; group B, containing lithium (Li), rubidium (Rb), chromium (Cr), iron (Fe), Nickel (Ni), selenium (Se), and aluminum (Al), in which a pH of 5.5 or lower prevented or caused poor growth; group C, containing sodium (Na), magnesium (Mg), calcium (Ca), vanadium (V), manganese (Mn), copper (Cu), and silver (Ag), in which a pH of 6 or higher prevented or caused poor growth; and group D, containing cobalt (Co), cadmium (Cd), and lead (Pb), in which pHs of 6.0 to 6.5 and above and pHs of 5.5 to 5.0 and below prevented or caused poor growth, thus allowing growth only with a narrow range of pHs.

View larger version (56K): [in this window] [in a new window]   FIG. 6.   Resistance of a HIS3+ strain to Ni and Co is pH dependent. Serial dilutions of B-7553 (his3-) and B-11842 (HIS3+) grown on YPD (A), YPD-2.5 mM NiCl2 (B), YPD-1.2 mM CoCl2 (C), adjusted to the pHs indicated, are shown.

Metal toxicity is enhanced with a defective vacuolar H⁺-ATPase. It has been reported that a functional vacuole is required for resistance to many metal salts and that many metal salts are actually sequestered in the vacuole (14). Altered pH-dependent metal toxicity was investigated with a vph1 strain containing a defective vacuole. The VPH1 gene encodes the 100-kDa V₀ subunit of the vacuolar H⁺-ATPase, and vph1 strains contain vacuoles that are defective in vacuolar acidification. The results of the growth of a normal VPH1 strain and a vph1 strain on YPD containing all of the previously tested metal salts, over a pH range of 4.0 to 7.5, is summarized in Table 3. First, it is well documented that vph1 strains do not grow at a pH above 6.5 on normal YPD. As all of the metals tested dropped the pH of YPD to well below pH 6.0, we are essentially reporting the effect of this metal on growth, although observations on growth at pHs between 4.0 and 6.0 should also be considered. Mg, Ca, Co, Ni, Zn, Al, and Mo inhibited the growth of the vph1 strains at the unadjusted pHs of the media, whereas the VPH1 strain grew normally. In fact, the vph1 strain was unable to grow in the presence of these metal salts over the entire pH range tested. Similarly, Na, K, Sr, and Mn caused poor growth of the vph1 strains at the unadjusted pHs of the media whereas the VPH1 strain grew normally. In the same way, the vph1 strain grew poorly on media with these metal salts over the entire pH range. Interestingly, the normal and vph1 strains grew similarly on media with Li, Rb, V, Cr, Fe, Cu, Se, Ag, Cd, and Pb between pHs 4.0 and 7.5.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The results presented here demonstrate that growth inhibition of the yeast S. cerevisiae by metal salts is generally pH dependent. To our knowledge this is the most thorough investigation of the spectrum of metal toxicity in yeast and takes into account the previously unknown fact that the effect of these metal salts depends on the pH of the medium. Because the addition of metal compounds alters the pH of the growth medium, the responses to each metal salt were determined over a range of pH values.

We have divided the effects of the metal salts on yeast growth with respect to pH into the following four groups: group A, containing metal salts which had no effect on growth in media with pHs from 4.0 to 7.5; group B, containing metal salts which caused defective growth at low pHs (pH 4.0 to 5.5); group C, containing metal salts which caused defective growth at nearly neutral pHs (pH 6.0 to 7.0); and group D, containing metal salts which allowed growth through a narrow range of pHs and caused defective growth at both high and low pHs.

The assignment to these four groups cannot be attributed simply to the chemical properties of the metal salts. Most likely, the pH dependency of toxicity reflects complex interactions with a variety of physiological components. Interestingly, most, but not all, metal salts that equally affect the normal and vph1 strains are mainly from groups B and D, producing poor growth at pH 4.0 to 5.5. Also, most, but not all metal salts that were more toxic to the vph1 strain than to the normal strain were assigned mainly to groups A, C, and D, in which the normal strain grew or grew poorly at and above pH 6. The vph1 defect has been shown to alter the vacuolar pH from the normal 6.1 to 6.9 (22), an elevation that may play a role in the preferential toxicity of group C and D metal salts.

Cu, Co, and Ni, which were assigned to groups C, D, and B, respectively, represent an important subset that preferentially produces a greater inhibition of His strains. Thus, the inhibition by these three salts have different pH requirements.

The fact that sensitivity to Cu, Co, and Ni of His strains can be reversed by the addition of excess histidine to the medium suggests that the ability to synthesize this amino acid somehow confers resistance to Cu, Co, and Ni. Furthermore, hip1 mutants, which lack the high-affinity permease for histidine (7), increased the sensitivity of His strains to Cu, Co, and Ni. Also, it was previously reported that the growth on synthetic media of His hip1 mutants was more sensitive to Cu, Ni, Co, and Zn and that this inhibition could be reversed by the addition of histidine to the medium (7). We also demonstrated that a higher level of histidine was required to reverse the Cu inhibition of his3 hip1 strains. These results indicate that the intracellular histidine alleviates the toxicity of Cu, Co, and Ni, probably by direct interaction. The fact that histidine binds divalent metals has long been known, and this fact is routinely exploited by insertion of polyhistidine tracts into proteins, so that the protein can be bound to resins with bound divalent metals ions such as Co²⁺ and Ni²⁺. It is curious that the normal amount of histidine in YPD is unable to confer the resistance of His strains to Cu, Co, and Ni and that His⁺ strains, having the ability to synthesize histidine, effectively produce sufficient histidine to match the addition used to reverse the inhibition of His strains. Only a small addition of 0.02 mM histidine to the YPD was necessary to reverse the inhibition of His strains. We have determined that intracellular levels of histidine, both cytosolic and vacuolar, in His⁺ and His strains grown on YPD do not show a significant difference (not shown). A study of additions of a variety of amino acids to growth media, and the fates of these amino acids in the cell, revealed that histidine increased 42-fold in the vacuolar pool of histidine but that the cytosolic pool did not change (17). Histidine accumulated far more than any of the other amino acids, strongly suggesting that an excess of this amino acid preferentially accumulates in the vacuole. It was previously reported that mutation of either PEP3, PEP5, or VMA3, which encode proteins involved in vacuole assembly or acidification, is required for normal Cu and Fe metal ion homeostasis and that PEP3 and PEP5 mutants are hypersensitive to Cu (24). We suggest that vacuolar accumulation of histidine may be a normal cellular process for detoxification of Cu, Co, and Ni.