JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Aug. 1999, p. 4774–4779 Vol. 181, No. 16

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Toxicity of Copper, Cobalt, and Nickel Salts Is Dependent onHistidine Metabolism in the Yeast Saccharomyces cerevisiae

DAVID A. PEARCE AND FRED SHERMAN*

Department of Biochemistry and Biophysics, University of Rochester School ofMedicine and Dentistry, Rochester, New York 14642

Received 1 April 1999/Accepted 3 June 1999

The pH-dependent inhibition of 22 metal salts have been systematically investigated for the yeast Saccha-romyces cerevisiae. We have established that the inhibition of growth by Cu, Co, or Ni salts is markedlyenhanced by histidine auxotrophy and by increasing the pH of the medium. Each of the his1-his7 mutantstrains were unable to grow in the presence of elevated levels of Cu, Co, or Ni at nearly neutral pHs, in contrastto His1 strains, which grew under these conditions. The Cu, Co, or Ni inhibition was reversed by the additionof histidine to the medium. Deletion of the high-affinity histidine permease Hip1p in His2 strains resulted ineven greater sensitivity to Cu, Co, and Ni and the requirement of an even higher level of histidine to reversethe inhibition. These results suggest that intracellular histidine, most likely in the vacuole, diminishes thepH-dependent toxicity of Cu, Co, and Ni. Furthermore, the toxicity of many salts is exacerbated in strains witha defective vacuolar H1-ATPase, which abolishes the ability of yeast to maintain an acidic vacuole, a com-partment known to sequester metal compounds. We suggest that the accumulation of histidine in the vacuoleis a normal process used to detoxify Cu, Co, and Ni.

Many metals are essential for all organisms at trace amountsbut can be toxic at higher concentrations. Copper (Cu) is awell-studied important cofactor of a variety of enzymes thatare involved in a variety of biochemical processes, such ascytochrome c oxidase, Cu, Zn superoxide dismutase, lysyl ox-idase, and dopamine-b-monooxygenase (21), and plays a crit-ical role in iron (Fe) assimilation (1, 16). However, accumula-tion of Cu can generate hydroxyl radicals, which cause cellulardamage such as oxidation of proteins, cleavage of DNA andRNA, and membrane destruction by lipid peroxidation (11).Therefore, it is necessary for organisms to have elaboratemechanisms to maintain Cu homeostasis by regulation of up-take of the Cu needed to drive particular biochemical pro-cesses, by detoxification if Cu is accumulated, and by monitor-ing of both of these processes. The importance of this Cuhomeostasis is revealed by the existence of the two humangenetic disorders of Cu homeostasis, Menkes syndrome andWilson’s disease (2, 3, 28, 30).

The yeast Saccharomyces cerevisiae has been extensivelyused to study Cu homeostasis and for genetic screens thatrevealed the genes responsible for Cu uptake, subcellular dis-tribution of Cu, and detoxification of Cu at higher levels (4–6,9, 18, 20, 26, 27, 29, 32). Cu occurs in the environment as theoxidized Cu21 form and is transported as the reduced Cu1

form. In summary, this process is mediated by two membrane-associated high-affinity Cu transporters, Ctr1p and Ctr3p, anda cell surface Cu(II) and Fe(III) reductase, Fre1p (6, 13, 18).In the presence of excess Cu, the Ctr1p, Ctr3p, and Fre1pcomponents are down regulated at the transcriptional levelthrough the action of the metalloregulatory transcription fac-tor Mac1p, essentially abolishing high-affinity uptake of Cu (9,15, 19, 31). Excess levels of Cu are sensed by another tran-scription factor, Ace1p. Through cooperative binding of Cu tospecific cysteine residues of the Ace1p DNA binding domain,

Ace1p binds to metal response elements on the promoters ofgenes, activating such genes as CUP1, CRS5, and SOD1, whichare involved in Cu detoxification and protection against oxida-tive stress (5, 8, 10, 12, 26).

In this study, we demonstrate that the growth inhibition ofthe yeast S. cerevisiae by Cu and other metal salts is pH de-pendent. We also establish that His2 strains containing a le-sion in any one of the seven genes that encode a biosyntheticcomponent for the amino acid histidine are more sensitive toCu, cobalt (Co), or nickel (Ni) salts. Sensitivity to Cu is de-pendent on the pHs of growth media, such that His2 strainscan grow in the presence of 2.4 mM CuSO4 at a pH below 6 butdo not grow at a pH above 6.5. Also, addition of excess histi-

* Corresponding author. Mailing address: Department of Biochem-istry and Biophysics, Box 712, University of Rochester School of Med-icine and Dentistry, Rochester, NY 14642. Phone: (716) 275-6647. Fax:(716) 271-2683. E-mail: Fred_Sherman@urmc.rochester.edu.

TABLE 1. Yeast strains used in this study

Strain Description

B-7553 ....................MATa his3-D1 leu2-3,112 ura3-52 trp1-289cyc7-D::CYH2 cyh2

B-11842 ..................B-7553 containing plasmid pAB622 (HIS3 CEN6)B-585 ......................MATa his1-1 trp2-1 lys2-1805 ..........................MATa his2 leu1 lys1 met4 ura3 spo11 pet8705 ..........................MATa his3-D1 leu2-3,112 ura3-52 trp1-289 can1-100

steVC9419 ..........................MATa his4-66A4-580a met8-1192 ..........................MATa his5-2 leu1-12 trp5-48 cyc1-73462 ..........................MATa his6 ura2 ura4 arg4 met1 thr1339 ..........................MATa his7 met8 Arg2

W303a ....................MATa his3-11,15 leu2-3,112 ura3-52 trp1-1 ade2-1D273-10B-X ..........MATa his3 ura3 metYPH499 .................MATa his3-D200 leu2-D1 ura3-52 trp1-D63 lys2-801

ade2-100BY4739 ..................MATa leu2-D0 ura3-D0 lys2-D0BY4742 ..................MATa his3-D1 leu2-D0 ura3-D0 lys2-D0BJ6717....................MATa his1 vph1-D::LEU2 ura3-52 trp1 ade6PLY170 ..................MATa leu2-3,112 lys2-D201 ura3-52 ade2-D1PLY171 ..................MATa his3-D200 leu2-3,112 lys2-D201 ura3-52 ade2-D1PLAS112-4B..........MATa his3-D200 hip1-D2::ADE2 leu2-3,112 lys2-D201

ura3-52 ade2-D1PLAS112-4C..........MATa hip1-D2::ADE2 leu2-3,112 lys2-D201 ura3-52

ade2-D1

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dine reverses the Cu sensitivity of His2 strains. Deletion of thehigh-affinity histidine transporter Hip1p in His2 strains causedgreater sensitivity to Cu and a greater requirement for exoge-nous histidine to reverse this Cu sensitivity, suggesting an ab-

solute requirement for histidine in Cu resistance. We alsoreport that sensitivity to a large number of metal salts is de-pendent on the pH of the growth medium and that a functionalvacuolar H1-ATPase is needed to confer resistance to some

FIG. 1. The his3-D strain, but not the HIS31 strain, is sensitive to Cu in a pH-dependent manner. (A) Serial dilutions of B-7553 (his3-D) and B-11842 (HIS31) grownon YPD adjusted to the pH indicated; (B) Same serial dilutions of B-7553 (his3-D) and B-11842 (HIS31) grown on YPD containing CuSO4 at a concentration of 2.4mM and adjusted to the pHs indicated.

TABLE 2. Effect of pH on growth of HIS3, his3, and vph1 strains in the presence of various metal saltsa

Group Metal Concn pH ofYPD

Effect on growth on media between pHs 4.0 and 7.5 in strains with:

HIS3 his3 vph1 his3

A None 6.5 None No growth above pH 6.0 Same as for HIS3 strainPotassium chloride 540 mM 5.8 None Same as for HIS3 strain Poorer growthStrontium chloride 2.25 mM 5.6 None Same as for HIS3 strain Poorer growthMolybdenum trioxide 30 mM 4.8 None Same as for HIS3 strain No growth

B Lithium chloride 40 mM 5.8 Poor growth below pH 5.5 Same as for HIS3 strain Same as for HIS3 strainRubidium chloride 5 mM 5.8 Poor growth at pH 4.0 Same as for HIS3 strain Same as for HIS3 strainChromium chloride 8 mM 3.6 No growth below pH 4.0 Same as for HIS3 strain Same as for HIS3 strainFerrous chloride 7 mM 4.7 No growth below pH 5.5 Same as for HIS3 strain Same as for HIS3 strainFerric chloride 5.5 mM 3.2 No growth below pH 4.0 Same as for HIS3 strain Same as for HIS3 strainSelenium dioxide 0.7 mM 5.4 No growth at pH 5.5 and lower Same as for HIS3 strain Same as for HIS3 strainNickel chloride 2.5 mM 5.5 Poor growth below pH 5.0 No growth below pH 5.0 No growthZinc chloride 4.4 mM 4.6 No growth below pH 5.0 Same as for HIS3 strain No growthAluminum chloride 4.5 mM 3.9 No growth below pH 4.0 Same as for HIS3 strain No growth

C Sodium chloride 360 mM 5.8 Poor growth above pH 7.0 Same as for HIS3 strain Poorer growthManganese chloride 8.5 mM 5.4 No growth at pH 6.0 and above Same as for HIS3 strain Poorer growthMagnesium chloride 150 mM 5.1 No growth at pH 6.5 and above Same as for HIS3 strain No growthCalcium chloride 100 mM 4.7 No growth at pH 6.5 and above Same as for HIS3 strain No growthVanadium oxide 13 mM 5.4 No growth at pH 6.5 and above Same as for HIS3 strain Same as for HIS3 strainCopper sulfate 2.4 mM 4.6 None No growth above pH 6.5 Same as for his3 strainSilver nitrate 0.35 mM 5.6 No growth at pH 6.5 and above Same as for HIS3 strain Same as for HIS3 strain

D Cadmium chloride 6.3 mM 5.8 Poor growth below pH 5.5 andabove pH 6.0

Same as for HIS3 strain Same as for HIS3 strain

Lead chloride 2.5 mM 4.6 Poor growth below pH 5.5 andno growth above pH 6.0

Same as for HIS3 strain Same as for HIS3 strain

Cobalt chloride 1.2 mM 5.3 No growth above pH 6.5 andbelow pH 5.0

No growth No growth

a The salts have been assigned to four groups, A, B, C, and D, based on their toxicity to HIS3 and his3 strains. Group A contains those metals that had no effect ongrowth at any pH. Group B contains those metals that produced either poor growth or lethality at an acidic pH of 5.5 or lower. Group C contains those metals thatcause poor or no growth at a pH higher than 6. Group D contains those metals that are toxic at both high and low pHs. The three metals Cu, Co, and Ni are moretoxic to his3 strains than to HIS3 strains (Fig. 1 and 6). Mo, Ni, Zn, Al, Mg, Ca, and Co prevented growth of vph1 his3 strains, whereas K, Sr, Na, and Mn reducedgrowth of these strains. The concentrations of metals were at sublethal levels at the unadjusted pH of YPD, as indicated. Growth was assessed at pH 4.0 to 7.5 for eachmetal at these concentrations.

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metal salts. These findings on the effect of pH and histidineauxotrophy should be considered when metal toxicity in yeastis investigated and when His1 plasmids are used to explore thephenotypes of mutations in His2 strains.

MATERIALS AND METHODS

Nomenclature. We used standard genetic nomenclature, including, for exam-ple, the phenotypic symbols His1 and His2 for the independence of and require-ment for histidine, respectively. HIS3 or HIS31, for example, denotes the wild-type allele, whereas his3 denotes any defective recessive allele, his3-D denotesany deleted or disrupted allele, and his3-D1, his3-D200, etc. denote specificdeleted or disrupted alleles.

Cu, for example, denotes a copper salt.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 usedthroughout this study, with the metal salts being added after autoclaving at theconcentrations listed in Table 2. Each medium was either not buffered or buff-ered with 50 mM MES (morpholineethanesulfonic acid) and 50 mM MOPS(morpholinepropanesulfonic acid). Both sets of media were adjusted to the pHvalues indicated in Table 2 with dilute HCl or NaOH. However, growth of yeaston media containing the indicated metal concentrations was the same whetherthe yeast was tested on buffered or nonbuffered media. Histidine and otheramino acids also were added to media after they were autoclaved, as indicated inTable 2.

RESULTS

The growth of His2 strains is sensitive to Cu in a pH-dependent manner. During the course of an investigation ofphenotypes of his3 strains with various disrupted genes andcomplementation with HIS3 plasmids, it became apparent thatthe HIS3 and his3 control strains exhibited different levels ofgrowth on YPD containing CuSO4 at pH 6.5. The B-7553(his3-D) and B-11842 (his3-D p[HIS3]) strains (Table 1) grewidentically on YPD at either pH 4.6 or 6.5, whereas the his3strain 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 moredetailed examination with YPD–2.4 mM CuSO4 media re-vealed that the growth of his3 strains became increasingly moreinhibited as the pH increased past pH 6, until no growth wasobserved past pH 6.3 (Fig. 1). Some of the differential re-sponses of HIS3 and his3 strains are summarized in Table 3.Similar results were seen for the his1, his2, his4, his5, his6, orhis7 strain and related His1 strains (data not presented). Thus,any block in the biosynthetic pathway of the histidine resultedin a pH-dependent sensitivity to Cu.

Histidine reverses the Cu inhibition of His2 strains. It ispertinent to point out that YPD contains sufficient histidinefrom the peptone and yeast extract components to support thegrowth of His2 strains. However, supplementing YPD–2.4 mMCuSO4 media with histidine reversed the Cu inhibition of thehis1 to his7 strains (Fig. 2). This result with exogenous histidineand the lower levels of inhibition of His1 strains suggest thathigher internal concentrations of histidine are required fordiminishing the inhibitory effect of Cu. Furthermore, the ad-

dition of either alanine, leucine, glutamic acid, or adenineinstead of histidine to the YPD–2.4 mM CuSO4 media did notreverse the Cu inhibition of the his1 to his7 strains (data notpresented).

Deletion of the high-affinity histidine transporter Hip1p re-sults in greater Cu sensitivity of His2 strains. The role ofhigher internal concentrations of histidine for diminishing theinhibitory effect of Cu was investigated with mutants lackingthe 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 inthe presence of various concentrations of CuSO4 and over apH range of 4.0 to 7.0. Importantly, the his3 hip1 strain wasmore sensitive to CuSO4 than the his3 HIP1 strain (Fig. 3). Thehis3 hip1 strain was sensitive to the low concentration of 0.012mM CuSO4 and was completely inhibited by 0.06 mM CuSO4,whereas the his3 HIP1 strain grew on media containing up to1.2 mM CuSO4. Interestingly, decreasing the pH of the me-dium, which reversed Cu sensitivity of his HIP1 strains, did notrescue the his3 hip1 strain, even at these lower Cu concentra-tions. Curiously, deletion of HIP1 alone in the HIS3 hip1 strainappeared to result in slightly more tolerance to Cu than wasexhibited by the HIS3 HIP1 strain. These results suggested thatthe intracellular concentration of histidine is the key factor foralleviating the inhibitory effect of Cu. This conclusion wasconfirmed by examining the levels of histidine required toreverse the Cu inhibition of the his3 hip1 and his3 HIP1 strains(Fig. 4). Both his3 hip1 and his3 HIP1 strains were unable togrow on YPD–2.4 mM CuSO4 medium, both could grow onYPD–2.4 mM CuSO4–0.6 mM histidine medium, but only thehis3 HIP1 strain could grow on YPD–2.4 mM CuSO4–0.02 mMhistidine medium (Table 3). These results are most simply

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, whichcan be reversed by the addition of excess histidine to the medium. Serial dilutionsof B-585 (his1), strain 805 (his2), strain 705 (his3), strain 419 (his4), strain 192(his5), strain 462 (his6), strain 339 (his7), and B-11842 (His1) on YPD (pH 7.0)(A), YPD–2.4 mM CuSO4 (pH 7.0) (B), and YPD–2.4 mM CuSO4–2 mMhistidine (pH 7.0) (C) are shown.

TABLE 3. Growth of two sets of isogenic strains on various media

Growth of:

B-11842 and PLY170(HIS3 HIP1)

B-7553 and PLY171(his3 HIP1)

PLAS112-4B(HIS3 hip1)

PLAS112-4C(his3 hip1)

YPD (pH 7.0 or 4.6) 1 1 1 1YPD–2.4 mM CuSO4 (pH 4.6) 1 1 1 2YPD–2.4 mM CuSO4 (pH 7.0) 1 2 1 2YPD–2.4 mM CuSO4 (pH 7.0)–2.0 mM histidine 1 1 1 1YPD–2.4 mM CuSO4 (pH 7.0)–0.02 mM histidine 1 1 1 2YPD–0.06 mM CuSO4 (pH 7.0) 1 1 1 2

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explained by higher internal concentrations of histidine dimin-ishing the inhibitory effect of Cu.

Many laboratory strains show pH-dependent Cu toxicity.We wish to emphasize that many yeast strains used by re-searchers are auxotrophic for histidine, as they bear his3 mark-ers used in plasmid and other manipulations. The levels ofgrowth of the following strains were tested on YPD–2.4 mMCuSO4 (pH 7.0) medium: W303a (his3-11,15), D273-10B-X(his3), YPH499 (his3-D200), and the S288C derivatives BY4739(His1) and BY4742 (his3-D1), which have been chosen fordeletion of all yeast genes (23). The two His1 strains B-11842and BY4739 grew on the YPD–2.4 mM CuSO4 (pH 7.0) me-dium, whereas the his3 strains W303a and YPH499 did notgrow and the his3 strains D273-10B-X and BY4742 grewpoorly on this medium (Fig. 5). In addition, all of these his3strains grew on the lower-pH medium YPD–2.4 mM CuSO4(pH 6.0) and Cu sensitivity was rescued by the addition ofhistidine to the medium (data not presented).

pH dependency of metal toxicity. The effect of pH on thetoxicity of other metal salts, listed in Table 2, were investigatedwith the B-11842 (HIS3) and B-7553 (his3) strains. The tests

were carried out with sublethal concentrations of each of themetal salts, which were determined by simply increasing theconcentration until the HIS3 strain ceased to grow (Table 2).Thus, the working concentration for each metal was just belowthe toxicity level, allowing a sensitive means to evaluate theeffect of pH. Not surprisingly, as with CuSO4, addition of metalsalts altered the pH of YPD (Table 2). The metal salts wereassigned to the following four groups based on the pH-depen-dent 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; groupB, containing lithium (Li), rubidium (Rb), chromium (Cr), iron(Fe), Nickel (Ni), selenium (Se), and aluminum (Al), in whicha pH of 5.5 or lower prevented or caused poor growth; groupC, 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 5.0and below prevented or caused poor growth, thus allowinggrowth only with a narrow range of pHs.

FIG. 3. Deletion of the high-affinity transporter for histidine (HIP1) results in increased sensitivity to Cu. Serial dilutions of PLAS112-4C (HIS31 hip1-D),PLAS112-4B (his3-D hip1-D), PLY171 (his3-D HIP11), and PLY170 (HIS31 HIP11) on YPD containing the indicated concentrations of CuSO4 (pH 7.0) are shown.

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 toallow for growth in the presence of Cu. Serial dilutions of PLAS112-4B (his3-D hip1-D) and PLY171 (his3-D HIP11) on YPD–2.4 mM CuSO4 (pH 7.0) with the indicatedconcentrations of histidine are shown.

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The results for HIS3 and his3 strains were essentially thesame except with media containing Cu, Ni, and Co. Serialdilution of HIS3 and his3 strains on YPD containing either 2.5mM NiCl2 or 1.2 mM CoCl2 over a pH range of 5.3 to 6.7 (Fig.6) revealed that the HIS3 strains are resistant to Ni at andabove pH 6 but that his3 strains do not grow in the presence ofNi at any pH. Similarly, the HIS3 strain is resistant to Co at andabove pH 6.7 whereas his3 strains do not grow in the presenceof Co at pH 6.7. Also similar to the results with Cu, theaddition of histidine alleviated the toxicity of Ni and Co withthe his3 strain (data not presented).

Metal toxicity is enhanced with a defective vacuolar H1-ATPase. It has been reported that a functional vacuole is re-quired for resistance to many metal salts and that many metalsalts are actually sequestered in the vacuole (14). Altered pH-dependent metal toxicity was investigated with a vph1 straincontaining a defective vacuole. The VPH1 gene encodes the100-kDa V0 subunit of the vacuolar H1-ATPase, and vph1strains contain vacuoles that are defective in vacuolar acidifi-cation. The results of the growth of a normal VPH1 strain anda vph1 strain on YPD containing all of the previously testedmetal salts, over a pH range of 4.0 to 7.5, is summarized inTable 3. First, it is well documented that vph1 strains do notgrow at a pH above 6.5 on normal YPD. As all of the metalstested dropped the pH of YPD to well below pH 6.0, we areessentially reporting the effect of this metal on growth, al-though observations on growth at pHs between 4.0 and 6.0should also be considered. Mg, Ca, Co, Ni, Zn, Al, and Moinhibited the growth of the vph1 strains at the unadjusted pHsof 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 theunadjusted pHs of the media whereas the VPH1 strain grewnormally. In the same way, the vph1 strain grew poorly onmedia with these metal salts over the entire pH range. Inter-estingly, the normal and vph1 strains grew similarly on mediawith Li, Rb, V, Cr, Fe, Cu, Se, Ag, Cd, and Pb between pHs 4.0and 7.5.

DISCUSSION

The results presented here demonstrate that growth inhibi-tion of the yeast S. cerevisiae by metal salts is generally pHdependent. To our knowledge this is the most thorough inves-tigation of the spectrum of metal toxicity in yeast and takesinto account the previously unknown fact that the effect ofthese metal salts depends on the pH of the medium. Becausethe addition of metal compounds alters the pH of the growthmedium, the responses to each metal salt were determinedover a range of pH values.

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

The assignment to these four groups cannot be attributedsimply to the chemical properties of the metal salts. Mostlikely, the pH dependency of toxicity reflects complex interac-tions with a variety of physiological components. Interestingly,most, but not all, metal salts that equally affect the normal andvph1 strains are mainly from groups B and D, producing poorgrowth at pH 4.0 to 5.5. Also, most, but not all metal salts thatwere more toxic to the vph1 strain than to the normal strainwere assigned mainly to groups A, C, and D, in which thenormal strain grew or grew poorly at and above pH 6. The vph1defect has been shown to alter the vacuolar pH from thenormal 6.1 to 6.9 (22), an elevation that may play a role in thepreferential 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

FIG. 5. Commonly used strains exhibit different levels of growth on YPD–2.4mM CuSO4, depending on the his3 marker. Comparative levels of growth of thefollowing strains on YPD (pH 7.0) (bottom) and YPD–2.4 mM CuSO4 (pH 7.0)(top) are shown. (A) B-11842 (HIS31); (B) W303a (his3); (C) D273-10B-X(his3); (D) YPH499 (his3); (E) BY4739 (HIS31); (F) BY4742 (his3).

FIG. 6. Resistance of a HIS31 strain to Ni and Co is pH dependent. Serial dilutions of B-7553 (his3-D) and B-11842 (HIS31) grown on YPD (A), YPD–2.5 mMNiCl2 (B), YPD–1.2 mM CoCl2 (C), adjusted to the pHs indicated, are shown.

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produces a greater inhibition of His2 strains. Thus, the inhi-bition by these three salts have different pH requirements.

The fact that sensitivity to Cu, Co, and Ni of His2 strains canbe reversed by the addition of excess histidine to the mediumsuggests that the ability to synthesize this amino acid somehowconfers resistance to Cu, Co, and Ni. Furthermore, hip1 mu-tants, which lack the high-affinity permease for histidine (7),increased the sensitivity of His2 strains to Cu, Co, and Ni.Also, it was previously reported that the growth on syntheticmedia of His2 hip1 mutants was more sensitive to Cu, Ni, Co,and Zn and that this inhibition could be reversed by the addi-tion of histidine to the medium (7). We also demonstrated thata higher level of histidine was required to reverse the Cuinhibition of his3 hip1 strains. These results indicate that theintracellular histidine alleviates the toxicity of Cu, Co, and Ni,probably by direct interaction. The fact that histidine bindsdivalent metals has long been known, and this fact is routinelyexploited by insertion of polyhistidine tracts into proteins, sothat the protein can be bound to resins with bound divalentmetals ions such as Co21 and Ni21. It is curious that thenormal amount of histidine in YPD is unable to confer theresistance of His2 strains to Cu, Co, and Ni and that His1

strains, having the ability to synthesize histidine, effectivelyproduce sufficient histidine to match the addition used to re-verse the inhibition of His2 strains. Only a small addition of0.02 mM histidine to the YPD was necessary to reverse theinhibition of His2 strains. We have determined that intracel-lular levels of histidine, both cytosolic and vacuolar, in His1

and His2 strains grown on YPD do not show a significantdifference (not shown). A study of additions of a variety ofamino acids to growth media, and the fates of these aminoacids in the cell, revealed that histidine increased 42-fold in thevacuolar pool of histidine but that the cytosolic pool did notchange (17). Histidine accumulated far more than any of theother amino acids, strongly suggesting that an excess of thisamino acid preferentially accumulates in the vacuole. It waspreviously reported that mutation of either PEP3, PEP5, orVMA3, which encode proteins involved in vacuole assembly oracidification, is required for normal Cu and Fe metal ion ho-meostasis and that PEP3 and PEP5 mutants are hypersensitiveto Cu (24). We suggest that vacuolar accumulation of histidinemay be a normal cellular process for detoxification of Cu, Co,and Ni.

ACKNOWLEDGMENTS

We thank Carrie J. Carr, Seth A. Nosel, and Michael D. Latourellefor technical assistance. We thank P. O. Ljungdahl (Ludwig Institutefor Cancer Research, Stockholm, Sweden) for yeast strains PLY170,PLY171, PLAS112-4B, and PLAS112-4C and E. W. Jones (CarnegieMellon University, Pittsburgh, Pa.) for yeast strain BJ6717.

This work was supported by NIH grants RO1 GM12702 and RO1NS36610.

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