Proc. Natl. Acad. Sci. USAVol. 88, pp. 6112-6116, July 1991Biochemistry

Isolation of a metal-activated transcription factor gene fromCandida glabrata by complementation in Saccharomyces cerevisiae

(metallothionein/copper/silver/DNA-binding protein/promoter)

PENGBO ZHOU AND DENNIS J. THIELE*Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606

Communicated by M. J. Coon, March 25, 1991 (received for review January 17, 1991)

ABSTRACT Metal-inducible transcription of metallothio-nein (MT) genes involves the interaction of metal-responsivetrans-acting factors with specific promoter DNA sequenceelements. In this report, we present a genetic selection using thebaker's yeast, Saccharomyces cerevisiae, to clone a gene fromCandida glabrata encoding a metal-activated DNA-bindingprotein denoted AMT1. This selection is based on the ability oftheAMT1 gene product to activate expression of the C. glabrataMT-I gene in a copper-sensitive S. cerevisiae host strain.DNA-binding studies using AMT1 protein expressed in Esch-erichia coli demonstrate that AMT1 is activated by copper orsilver to bind to both the MT-I and MT-II promoters of C.glabrata. Sequence comparison of AMT1 protein to the S.cerevisiae copper- or silver-activated DNA-binding protein,ACE1, indicates that AMT1 contains the 11 amino terminalcysteine residues known to be critical for the metal-activatedDNA-binding activity of ACEL. In contrast, the carboxyl-terminal portion of AMT1 bears only slight similarity at theprimary structure level to the same region of ACEl known tobe important for transcriptional activation. These results sug-gest that the amino-terminal cysteines, and other conservedresidues, play an important role in the ability of AMT1 andACEl to sense intracellular copper levels and assume a metal-activated DNA-binding structure.

The metal copper plays a crucial role in biological systems asa cofactor for enzymes such as cytochrome oxidase, copper,zinc superoxide dismutase, and several other enzymes (1).Due to its proclivity to participate in damaging oxidationreactions, copper is also a potent toxin when allowed toaccumulate to high free intracellular concentrations (2). Tocope with this apparent nutritional paradox, eukaryotic or-ganisms have evolved a sensory mechanism that triggers therapid and efficient transcriptional activation of metal-detoxification genes that encode proteins known as metal-lothioneins (MTs). MTs are low molecular weight, cysteine-rich proteins that bind metals such as copper, cadmium, zinc,and mercury efficiently and tenaciously through thiolatebonds. Metal coordination in MTs is achieved through themultiple cysteine residues, in the arrangement Cys-Xaa-Cysor Cys-Xaa-Xaa-Cys (where Xaa represents any amino acid),found in all known MT proteins (3, 4). Through the action ofspecific cellular metal sensory components, the biosynthesisofMT is activated, at the level of transcription initiation, bymany of the same metals that are bound by MT proteins.The baker's yeast, Saccharomyces cerevisiae, has pro-

vided one of the most informative model systems for under-standing metal-inducible transcription of MT genes. Thesingle S. cerevisiae MT gene, designated CUPi, is transcrip-tionally activated by copper through a specific promoterregion, UAScup, (upstream activation sequence), located

between -105 and -230 with respect to the CUP] transcrip-tion initiation site (5). Allelic recessive mutations in a trans-acting regulatory gene required for copper-inducible tran-scription, acei-i (cup2), provided a means for cloning thewild type ACEI (CUP2) copper-activated transcription factorby in vivo complementation (6-8). ACEl protein, synthe-sized either in Escherichia coli or in vitro, binds four distinctregions within UAScup, in a copper- or silver-induciblemanner (9-12). Indeed, the ability of copper or silver toactivate ACEl binding to UASCup, in vitro corresponds tothe exclusive ability of these metals to activate CUP] tran-scription in vivo (6, 11). Although a large number of DNA-binding motifs have been described (13, 14), the ACElDNA-binding domain bears no obvious structural relation-ship to such motifs.

Recently, the yeast Candida glabrata was shown to harbortwo distinct MT genes; a unique MT-I gene and a tandemlyamplified MT-II gene (15, 16). Messenger RNA levels forboth MT-I and MT-II were shown to be induced by copper orsilver; however, in contrast with mammalian systems, cad-mium was shown to be ineffective in enhancing MT-I andMT-II transcription (15). C. glabrata therefore exhibits theMT gene organization typical of higher eukaryotes but ametal specificity for MT biosynthetic regulation similar tothat in S. cerevisiae. To begin to understand the structuralrequirements important for copper-activated DNA-bindingproteins in this system, we have isolated a gene encoding ametal-activated, sequence-specific, DNA-binding proteinfrom C. glabrata.t We demonstrate that this protein, denotedAMT1, binds to both members of the C. glabrata MT genefamily in a copper- or silver-inducible fashion and bearsstrong resemblance to the metal-activated DNA-binding do-main of ACEL.

MATERIALS AND METHODSStrains and Growth Conditions. The S. cerevisiae strains

used in this work are as follows: 50.L4 (MATa trpl-i leu2-3,-i12 gall ura3-50 his cupPS (17); DTY89 (MATa leu2-3,-112gall ura3-SO cupi-A6i acei-A225 trpl-i:: MT-I-lacZ:: TRPI);and DTY99 (MATa leu2-3,-112:: MT-I::LEU2 gall his ura3-50cupi-A61 acei-A225 trpl-l::MT-I-lacZ::TRPI). All strainsare isogenic to the parental strain 50.L4. Strain DTY89 wasconstructed by deletion of the ACE] gene as described (18)and by deleting the entire CUPi coding sequence by usingplasmid pSC11-7 (a gift from T. Keng and S. Ushinsky,McGill University). Plasmid pSC11-7 contains CUPi geneflanking sequences interrupted by a hisG-URA3-hisG cas-sette from pNKY51 (19) and was used to delete the CUPicoding sequence by homologous recombination and to re-

Abbreviations: MT, metallothionein; X-Gal, 5-bromo-4-chloro-3-indolyl f8-D-galactoside.*To whom reprint requests should be addressed.tThe sequence reported in this paper has been deposited in theGenBank data base (accession no. M69146).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 88 (1991) 6113

cover the ura3 marker by selecting for recombinants on agarcontaining 5-fluoroorotic acid (20). Strain DTY99 was con-structed by integrating the MT-I-lacZ fusion plasmid pTRP-MT-I-lacZ at the trpl-J locus and by integration of the MT-Igene in YIpMT-I at the leu2 locus in DTY89. All chromo-somal alterations were verified by Southern blotting (20). TheC. glabrata strain 85/038 (a gift from P. Magee, University ofMinnesota) was the sole C. glabrata strain used in this work.Yeast strains were grown in rich (YPD), synthetic complete(SC), or SC media lacking specific indicated nutrients asdescribed (21) at 30'C. E. coli strains DH5aF' (BRL) andXL1-blue (Stratagene) were used to construct and maintainplasmids by established procedures (20).

Plasmids. The C. glabrata MT-I and MT-II genes wereisolated from C. glabrata 85/038 genomic DNA by polymer-ase chain reactions (PCR) (20) using synthetic oligonucleo-tide primers that hybridize to the extreme termini of pub-lished MT-I and MT-IIgene sequences (15, 16). PCR productswere inserted into pUC19 (New England Biolabs) andpBluescript SK(+) (Stratagene) multiple cloning sites bypreviously described methods (20). The MT-I-lacZ fusionplasmid, pTRP-MT-I-lacZ, was constructed by inserting a460-base-pair (bp) EcoRI-Pvu II fragment into the IacZfusion vector YIp353 (22). A 4.1-kilobase (kb) EcoRI-Stu Ifragment from this plasmid was rendered blunt-ended withthe Klenow fragment ofDNA polymerase I and inserted intothe Pvu II site of pRS304 (23) to create the integratingMT-I-lacZ fusion plasmid, pTRPMT-I-lacZ, with TRPI as aselectable marker. The C. glabrata MT-I gene fragment wasinserted into the EcoRI-BamHI sites of the S. cerevisiaeLEU2-based integrating plasmid YIp32 (24).

Expression of AMT1 in E. coli and DNA-Binding Assays.The entire AMT1 open reading frame was expressed in E. coliby using the bacteriophage T7 RNA polymerase system (25).A unique Nco I restriction enzyme site was created in theAMT1 initiation codon by oligonucleotide-directed mutagen-esis (26) using the mutagenic primer 5'-GATTACTACCATG-GTGCAAATGTGTC-3', which hybridizes to AMTJ genenucleotide positions -14 to + 12 indicated in Fig. 2B. Theresultant 0.9-kb Nco I-BamHI restriction endonuclease frag-ment was inserted into the corresponding sites in the expres-

sion vector pET-8c, to create plasmid pT7-AMT1, introducedinto the E. coli strain BL21 (DE3) pLysE by transformation,and used for expression studies. Cultures containing pET-8cor pT7-AMT1 were grown and induced with 0.5 mM isopro-pyl 3-D-thiogalactoside and soluble extracts were prepared asdescribed (20). DNA-binding extracts were prepared from C.glabrata 85/038 as described (27) and used in electrophoreticmobility shift assays (20). A 241-bp EcoRI-Xho II fragmentfrom the C. glabrata MT-I promoter and a 335-bp Sma I-XbaI fragment from the MT-II promoter (15, 16) were used asprobes in these assays.

RESULTSIsolation of a Metal-Activated Transcription Factor Gene.

Due to a lack of facile genetic analysis in C. glabrata, wedesigned and implemented a method to clone a C. glabratametal-activated transcription factor gene by function in S.cerevisiae (Fig. 1A). Strain DTY99 is resistant to approxi-mately 15 ,uM copper sulfate and gives rise to pale bluecolonies on solid medium containing X-Gal, due to a low butdetectable basal level of transcription from the C. glabrataMT-I promoter in S. cerevisiae. We anticipated that theintroduction of a C. glabrata DNA fragment encoding acopper-activated transcription factor should trans-activateboth the MT-I gene and the MT-I-lacZ fusion, resulting incopper-resistant colonies with elevated ,3-galactosidase.

Total genomic DNA was isolated from C. glabrata strain85/038, subjected to partial digestion with Sau3A, and ligatedinto the BamHI site of the yeast-E. coli shuttle vectorpEMBLYe24 (28). The ligation mixture was used to directlytransform DTY99, and uracil prototrophs were selected onSC minus uracil agar medium. Approximately 35,000 totaltransformants were obtained, which, given the average li-brary insert size and insertion frequencies (-3 kb, 30%,respectively) and the estimated C. glabrata genome size (1.4x 107 bp), represents approximately two C. glabrata genomeequivalents. The transformants were replica-plated to SCmedium containing 50 1.M CuSO4, and after 2 days 30copper-resistant isolates were recovered. Of these, one iso-late gave rise to a dark blue patch of growth when streaked

A

MT-I lacZ

cup1 /

S. cerevisiae hostsensitive to copper

Transform with C. glabratagenomic DNA inserted into

I high copy S. cerevisiae vector B

Q:0

_0

Select for copper resistant transformants

Screen for blue colonies on X-gal agar

I-

0-MtC_aI)

0@

Rescue plasmid from yeast

FIG. 1. Isolation of a C. glabrata metal-activatedtranscription factor gene. (A) Scheme used for theisolation of a C. glabrata gene encoding a protein ableto trans-activate MT-I and MT-I-lacz fusion genes in S.cerevisiae strain DTY99. Abbreviations: MT-I, the C.glabrata metallothionein I gene; lacZ, the E. coli,3-galactosidase gene; cuplA, a mutant allele (cupi-A61) in which the entire CUP] gene has been deleted;aceIA, a mutant allele (acel-A225) in which the entireACEI gene has been deleted; and X-Gal, 5-bromo-4-chloro-3-indolyl /8-D-galactoside. (B) Plasmid pAMT1activates the C. glabrata MT-I gene in S. cerevisiae inresponse to exogenous copper. Total RNA from con-trol (-) and copper-treated (+) cultures of C. glabrata,DTY99(pAMT1), and DTY99(pEMBLYe24) was ana-lyzed by RNA blotting and hybridization with a 32p_labeled MT-I gene EcoRI-BamHI DNA restrictionfragment (20). The arrowhead indicates the position ofthe MT-I gene-specific RNA species.

Biochemistry: Zhou and Thiele

6114 Biochemistry: Zhou and Thiele

to SC medium containing 25 uM CuS04 and X-Gal. When theplasmid contained within this isolate, designated pAMT1,was rescued in E. coli and reintroduced into DTY99, therecipient strain exhibited both the copper resistance (up to150 ,iiM CuS04) and activation of the MT-I-lacZ fusiondemonstrated by 8-galactosidase assays, as observed in theoriginal transformant; however, pEMBLYe24 did not conferthese phenotypes.The results described above suggest that pAMT1 contains

a gene whose product activates transcription from the C.glabrata MT-I promoter in S. cerevisiae. To test this possi-bility, RNA-blotting experiments were carried out on controland copper-treated cultures of DTY99 harboring eitherpAMT1 or the control plasmid pEMBLYe24. In strainDTY99(pAMT1), a copper-inducible MT-I mRNA specieswas synthesized that is similar in size to that synthesized incopper-treated C. glabrata (Fig. 1B, lanes C. glabrata andpAMT1). MT-I RNA synthesis was not induced by exoge-nous copper in DTY99(pEMBLYe24), indicating that plas-mid pAMT1 contains a DNA fragment encoding a copper-responsive MT-I transcription activation function. Takentogether, these results clearly demonstrate that the DNAinsert in plasmid pAMT1 encodes a copper-responsive trans-activator of the C. glabrata MT-I promoter when both arepresent simultaneously in S. cerevisiae. Furthermore, themagnitude ofMT-Igene activation by pAMT1 in S. cerevisiaesuggests that the activity encoded by the pAMT1 insertinteracts efficiently with the S. cerevisiae general transcrip-tion apparatus. We assign the designation AMTP (activator ofMT transcription 1) to that portion of the pAMT1 DNAencoding this activity.

Analysis of the AMTI Gene. To identify that portion of thepAMT1 plasmid insert encompassing the C. glabrata AMTJ

Proc. Natl. Acad. Sci. USA 88 (1991)

gene, the insert (approximately 4 kb) contained within theBamHI site in pAMT1 was characterized by subcloningfragments in pEMBLYe24 and testing for function in S.cerevisiae DTY99. The identification of an approximately1.4-kb C. glabrata BgI II-Pst I genomic DNA fragment ableto direct all phenotypes associated with pAMT1 suggestedthat this DNA fragment should encompass the functionalAMTP gene (data not shown). The existence of a colinear BglII-Pst I fragment present in C. glabrata genomic DNA, asascertained by Southern blotting, suggests that no rearrange-ments of this genomic fragment have occurred during thecloning process (data not shown). The nucleotide sequence ofthis DNA fragment was determined for both strands and isshown in Fig. 2. A single extended open reading frame wasdetected on only one strand of this DNA sequence; it encodesa polypeptide 265 amino acid residues in length with apredicted molecular mass of 29,429 daltons. There is a clearasymmetrical distribution of the 30 basic residues containedwithin the AMT1 coding region. Of these, 25 basic aminoacids are located within the amino-terminal 113 residues,giving this region an overall net charge of + 12. Secondly, thecarboxyl-terminal residues 114-265 contain 18 of the total 31acidic residues, conferring a net charge of -13 to this portionof the AMT1 protein. Furthermore, all 11 cysteine residueswithin the AMT1 open reading frame are localized within theamino-terminal 100 amino acid residues. These and severalother features of AMT1 display close resemblance to ACE1and suggest functional importance (see Fig. 4 and Discus-sion). Two features of the AMTI gene sequence suggest thatthis is expressed in C. glabrata. First, the nucleotide se-quence surrounding the ATG that initiates the open readingframe is similar to functional initiator codons proposed byKozak (29). Second, two blocks of sequence, from -77 to

-510 AGATCTCATTAGTAAAAGTGGGGTGTTCATCAAGTGAGCCAATGCAAGTTTGCGTTTCTCTTACAGGAGCCCGATGAATTACTGGGGTAG

-420

-330

-240

-150

-60

3011

12041

21071

300101

390131

480161

570191

660221

750251

840

TTGCTGTTGCACATCCCCTTTGCCACCATTCACGTCCTGGTTAGCTACTTGTATCCTGCATTATTTGCGGGCAAGTTTCCAATACTATTT

CTTAAAAGTAGTGGTGGGCGTGATGAGCCCAGTGTTTAGTCCATTTTATGGATAATATTGATCTTTAAAGGCAAGTAGTTGAATTTCTAT

TTGGATAAAAGGTATAAATAGTAGTTGAGAATCCAGACAAAGCACCTTTATATTGAAGATAATAGCGTTACATTATATATAAGAAGCGAT

AACAACAAAAACAATAGGCATATATAAAGCAGAGAGCACAGCAGTACACACATTTGCAGTATGGTAGTAATCAACGGGGTGAAGTATGCCM V V I N G V K Y A

TGTGATTCATGCATCAAATCACATAAAGCAGCCCAGTGTGAGCATAACGATAGACCCTTAAAGATACTAAAGCCAAGGGGAAGGCCACCGC D S C I K S H K A A Q C E H N D R P L K I L K P R G R P P

T T C D H C K D M R K T K N V N P S G S C N C S K L E K I R

CAAGAGAAAGGCATAACGATAGAAGAGGATATGTTGATGAGCGGAAACATGGACATGTGCTTGTGTGTTAGAGGTGAGCCTTGTAGGTGTQ E K G I T I E E D M L M S G N M D M C L C V R G E P C R C

CACGCTAGGAGGAAAAGGACACAGAAATCAAACAAAAAAGATAACTTAAGCATTAATTCGCCCACAAATAATTCTCCTTCACCAGCGCTCH A R R K R T OK S N K K D N L S I N S P T N N S P S P A L

TCTGTGAATATTGGAGGGATGGTGGTGGCTAATGATGACATCCTAAAATCATTGGGACCAATTCAAAATGTCGATCTAACGGCTCCACTAS V N I G G M V V A N D D I L K S L G P I Q N V D L T A P L

D F P P N G I D N K P M E S F Y T Q T S K S D A V D S L E F

GATCATCTAATGAATATGCAAATGAGGAATGATAACTCCCTTTCATTTCCTATGTCTGCAAACCAGAATGAAGTCGGTTATCAATTTAATD H L M N M Q M R N D N S L S F P M S A N Q N E V G Y Q F N

N E G N N S M N S T M K N T I T Q M D Q G N S H S M T L H D

ATAGACGAAATTCTCAATAACGGTATTGAACTTGGTAATGTAAATTGAAGATCGTAAATCTGTAAAACAGTTTGTCGGATATTAGGCTGTI D E I L N N G I E L G N V N

ATCTCCTCAAAGCGTATTCGAATATCATTGAGAAGCTGCAG 880

-421

-331

-241

-151

-61

2910

11940

20970

299100

389130

479160

569190

659220

749250

839

FIG. 2. Sequence of 1391 nucleotides encompassing the C. glabrata AMTP gene. Nucleotide positions are numbered relative to the firstnucleotide in the AMTJ sequence, which begins the 265-codon open reading frame (deduced amino acid residues are shown in the single-lettercode). Two potential binding sites for transcription factor TFIID are underlined.

Proc. Natl. Acad. Sci. USA 88 (1991) 6115

-69 and from -40 to -33, with respect to the ATG thatinitiates this open reading frame, strongly resemble TATAbox elements, which serve as binding sites for the conservedeukaryotic transcription initiation factor TFIID (12) (Fig. 2).AMT1 Produced in E. coli Binds the C. glabrata MT-I and

MT-Il Promoters in Response to Silver or Copper. The abilityof the pAMT1 plasmid to activate MT-I expression in S.cerevisiae in a copper-inducible manner, and the strikingsimilarity between the amino acid sequences in the aminotermini of AMT1 and ACE1, strongly suggest that AMTJencodes a metal-activated MT transcription factor. To testthis hypothesis, we expressed AMT1 protein in E. coli at ahigh level and in a soluble form by using the bacteriophage 17promoter system (ref. 25; data not shown). Total soluble E.coli extracts containing full-length AMT1 protein were usedin electrophoretic mobility shift assays to examine the inter-action of AMT1 with the C. glabrata MT-I and MT-IIpromoters. These promoter fragments, which cover the 5'241 bp of the MT-I promoter and the 5' 335 bp of the MT-IIpromoter (15, 16), were used in mobility shift assays employ-ing C. glabrata extracts and the E. coli extracts describedabove (Fig. 3). These assays demonstrated that C. glabrataextracts contain a copper-inducible DNA-binding activitythat gives rise to a major and other less prominent DNA-protein complexes of faster mobility that could representcomplexes with proteolyzed DNA-binding proteins (Fig. 3,Candida lanes). We also noted the existence of a copper-independent complex, formed with the MT-Il promoter frag-ment; the nature or specificity of this complex has not beeninvestigated. At high concentrations of extract we observedcopper-inducible DNA-protein complexes ofslower mobilitythan the major complexes, suggesting that this activity mayrecognize multiple target sites within the MT-I and MT-IIpromoter fragments. Similar DNA-protein complexes werealso observed when silver was added to C. glabrata DNA-binding extracts (data not shown). MT-I and MT-lI promoterDNA-protein complexes, of similar mobility to the majorspecies observed in C. glabrata extracts, were formed with50 ,uM copper or silver-treated E. coli extracts expressinghigh levels of AMT1, but not with the 17 expression vectorcontrol extracts (Fig. 3). Major complexes were formed whenlow levels (0.1 ,g) of copper- or silver-treated AMT1 extractwere used, and slower-migrating complexes, similar in mo-bility to those observed with high levels of C. glabrataextracts, were observed when 0.25 ,ug of copper-treatedAMT1 extract was used. These results demonstrate that theC. glabrata AMT1 protein synthesized in E. coli binds to both

Candida _ AMT1 T70 20 20 30 30 40 40 .1 .1 .25 .25 .25.25 .25.25

--_+-_ + _-+ _-+-_ + _-__-

iS *'we.~~Iis -

MT-I

the MT-I and the MT-lI promoters in the presence of copperor silver, two metals known to activate expression of bothgenes in vivo (15).

DISCUSSIONIn this work we present an approach for the use of a geneticselection in S. cerevisiae to clone genes encoding DNA-binding transcription activation proteins from heterologousorganisms. This approach relies on the known ability ofseveral sequence-specific DNA-binding activation factors tointeract productively with the basic transcription machineryfrom different organisms such as yeast and humans (30).Utilizing this approach, we isolated a gene for a metal-activated DNA-binding protein, AMT1, on the basis of itsability to bind to and activate from putative cognate promoterelements of the C. glabrata MT-I gene. Although we haveused the entire MT-I promoter and structural gene in thiswork, in principle, synthetic DNA-binding sites from anyorganism could be fused to a 5'-terminally truncated MT-Igene (containing sequences from the TATA box through thestructural gene). Selections for the presence of DNA frag-ments encoding proteins capable of binding to the target siteand activating MT-I transcription are then carried out byidentifying copper-resistant S. cerevisiae transformants. Theuse of a secondary screen, such as the promoter-lacZ genefusion used in this work, allows the facile identification oftrans-acting transcription factors rather than metal-bindingproteins. Additionally, successful utilization of this selectionrequires that a single polypeptide bind to the target DNA andactivate transcription, and furthermore, that a genomic orcDNA library be used that is expressed in S. cerevisiae.The C. glabrata AMTI gene, cloned on the basis of its

ability to activate MT-I transcription in S. cerevisiae inresponse to exogenous copper, encodes a copper- or silver-activated sequence-specific DNA-binding protein. Impor-tantly, these same metals are the only ones known to induceC. glabrata MT-I and MT-II gene transcription in vivo (15),strongly implicating AMT1 as a metal-activated C. glabrataMT gene transcription factor in vivo. Definitive evidence forthis function of the AMTP gene awaits the results of AMTPgene disruption experiments in C. glabrata. Our observationsin this report, however, suggest that in C. glabrata, bothclasses of MT genes are transcriptionally activated by thesame metal-responsive factor. Mobility-shift experimentssuggest that AMT1 protein forms multiple distinct complexeswith both the MT-I and the MT-II promoter fragments. This

Candida AMT1 T 70 20 20 50 50 .1 .1 .25 .25.25 .25.25.25 extract

- + AgNO3- - + - + - + - + - - - + CuSO4

i-U..-A Ai .

MT-l1

FIG. 3. E. coli-produced AMT1 is activated by copper or silver to bind both the MT-I and the MT-II promoters. DNA-binding assays byelectrophoretic mobility shift on 5% polyacrylamide gels were carried out with a 32P-labeled MT-I promoter fragment (Left) or with an MT-Ilpromoter fragment (Right). Extracts used in the binding assays were from C. glabrata (Candida), E. coli cells expressing the pT7-AMT1 plasmid(AMT1), or E. coli cells harboring the T7 vector without insert (T7). The amount of each extract used in the binding reactions is indicated in,ug. The presence or absence ofAgNO3 or CUSO4 (at a final concentration of 50 ,uM each) is indicated by a + or -, respectively. The arrowheadon the right side of each panel indicates the location of the major metal-induced complex formed between the probe fragments and AMT1 orC. glabrata extracts.

Biochemistry: Zhou and Thiele

6116 Biochemistry: Zhou and Thiele

AMT1 1 MVVINGVKY 4CSI KSHKAAHCHNDRPLKILKPRGRPPT1 C EDMR 50111i111111111 :.jIj::1:II11I.1.1 11..:::.:111.I11 I :III1:.:1

ACEl 1 MVVINGVKYA RGHRAA HTDGPLQMIRRKGRPSTC3SELR 50

51 KTKNVNPSGSC KLEKIRQEKGITIEEDMLMSGNMDCVRGEC 100

51 RTKNFNPSGG CCSARR .... PAVGSKED...... ET DEGEPC 90

101 HARRKRTQKSNKKDNLSINSPTNNSPSPALSVNIGGMVVANDDILKSL.G 149

91 HTKRKSSRKSK... GGSCHRRANDEAA..... HVNGLGIADLDVLLGLNG 132

150 PIQNVDLTAPLDFPPNGIDNKPMESFYTQTSKSDAVDSLEFDHLMNMQMR 199

133 RSSDVDMTTTLPSLKPPLQNGEI ........ KADSIDNLDLASLDPLEQS 174

200 NDNSLSFPMSANQNEVGYQFNNEGNNSMN ..... STMKNTITQMDQGNSH 244

175 PSISME . PVSINETGSAYTTTNTALNDIDIPFSINELNELYKQVSSHNSH 223

245 SMTLHDIDEILNNGIELGNVN* 265

224 SO*,................... 225

FIG. 4. Sequence comparison between the C. glabrata AMT1(265 residues in length) and S. cerevisiae ACEl [225 residues inlength (8)] proteins using programs from the University of WisconsinGenetics Computer Group. A vertical line indicates identical aminoacids; a colon indicates amino acids with an evolutionary comparisonvalue of 0.5 or greater; and a period indicates an evolutionarycomparison of 0.1-0.5, according to the analysis of Gribskov andBurgess (31). Rectangular outlines indicate the 11 amino-terminalcysteine residues conserved between ACEl and AMT1.

resembles the interaction of ACE1 with UAScup1 (9-12) andsuggests that, like allMT promoters studied to date, the MT-Iand MT-II genes may contain multiple metal-responsivepromoter elements (3). Future experiments should define theAMT1 binding sites in the MT-I and MT-II promoters anddetermine the role these sites play in the regulation of C.glabrata MT gene transcription.The sequence of the C. glabrata AMT1 polypeptide dis-

plays striking homology to the amino-terminal 100 aminoacids of the S. cerevisiae ACEl protein (Fig. 4). This regionof AMT1 is highly positively charged and contains 11 cys-teine residues in arrangements (Cys-Xaa-Cys or Cys-Xaa-Xaa-Cys) known to be important for metal binding in bothMT proteins and ACE1 (4, 12). Interestingly, the first 7cysteine residues ofACE1 and AMT1 are exactly conservedwith respect to their position in the amino termini of theproteins. Although AMT1 contains 10 more amino acids thanACE1 in the region encompassing the amino-terminal 11cysteines, the position of the remaining 4 cysteines is con-served when two gaps are introduced into the alignment (Fig.4). Onthe basis ofthe analysis ofin vivo and in vitro generatedACEl mutants, which indicates that the 11 amino-terminalcysteines are important in copper binding and UAScup,DNAbinding (6, 10, 12), we predict that the conserved cysteineresidues in AMT1 may also play an important role in thesefunctions. Indeed, given the high level of homology betweenthe first 63 amino acids of ACE1 and AMT1 (65% identity,85.7% similarity), it is likely that these portions of theproteins adopt similar structures when coordinated withmetal. We note that all of the amino acid substitutions inACEl previously demonstrated to abolish or significantlydiminish DNA-binding activity are either precisely con-served or maintain the same charge in AMT1 (6, 10, 12). Inaddition to the cysteine residues, these include the followingin ACE1: Gly-37, Arg-51 (Lys in AMT1), Lys-53, His-91(His-101 in AMT1), and Arg-94 (Arg-104 in AMT1). A clearerunderstanding of the importance of these residues shouldemerge from structural analyses of the ACEl and AMT1

amino-terminal cysteine-rich domains. The isolation of agene for a second member of a copper- or silver-activatedDNA-binding transcription factor class of proteins shouldgreatly facilitate studies aimed at understanding the struc-ture-function relationships important for metal-activatedDNA-binding proteins. Furthermore, C. glabrata provides aconvenient model system for studying metal-regulated tran-scription in an organism containing an MT gene family.

We thank D. Engelke and members of the Thiele laboratory forcritically reading the manuscript and for valuable suggestions, G.Butler and M. Szczypka for assistance with PCR, S. Ghosh forassistance with C. glabrata DNA-binding extracts, B. Magee andP. T. Magee for the C. glabrata strain, and S. Ushinsky and T. Kengfor plasmid pSC11-7. This work was supported by Grant GM41840from the National Institutes of Health and in part by NationalInstitutes of Health Grant M01 RR00042.

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