Proc. Natl. Acad. Sci. USA
Vol. 88, pp. 6112-6116, July 1991 Biochemistry
Isolation of a metal-activated transcription factor gene from Candida 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 metallothionein (MT) genes involves the interaction of metal-responsive trans-acting factors with specific promoter DNA sequence elements. In this report, we present a genetic selection using the baker's yeast, Saccharomyces cerevisiae, to clone a gene from Candida glabrata encoding a metal-activated DNA-binding protein denoted AMT1. This selection is based on the ability of the AMT1 gene product to activate expression of the C. glabrata MT-I gene in a copper-sensitive S. cerevisiae host strain. DNA-binding studies using AMT1 protein expressed in Escherichia coli demonstrate that AMT1 is activated by copper or silver 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 terminal cysteine residues known to be critical for the metal-activated DNA-binding activity of ACEL. In contrast, the carboxylterminal portion of AMT1 bears only slight similarity at the primary structure level to the same region of ACEl known to be important for transcriptional activation. These results suggest that the amino-terminal cysteines, and other conserved residues, play an important role in the ability of AMT1 and ACEl to sense intracellular copper levels and assume a metalactivated DNA-binding structure.
between -105 and -230 with respect to the CUP] transcription initiation site (5). Allelic recessive mutations in a transacting regulatory gene required for copper-inducible transcription, acei-i (cup2), provided a means for cloning the wild type ACEI (CUP2) copper-activated transcription factor by in vivo complementation (6-8). ACEl protein, synthesized either in Escherichia coli or in vitro, binds four distinct regions within UAScup, in a copper- or silver-inducible manner (9-12). Indeed, the ability of copper or silver to activate ACEl binding to UASCup, in vitro corresponds to the exclusive ability of these metals to activate CUP] transcription in vivo (6, 11). Although a large number of DNAbinding motifs have been described (13, 14), the ACEl DNA-binding domain bears no obvious structural relationship to such motifs. Recently, the yeast Candida glabrata was shown to harbor two distinct MT genes; a unique MT-I gene and a tandemly amplified MT-II gene (15, 16). Messenger RNA levels for both MT-I and MT-II were shown to be induced by copper or silver; however, in contrast with mammalian systems, cadmium was shown to be ineffective in enhancing MT-I and MT-II transcription (15). C. glabrata therefore exhibits the MT gene organization typical of higher eukaryotes but a metal specificity for MT biosynthetic regulation similar to that in S. cerevisiae. To begin to understand the structural requirements important for copper-activated DNA-binding proteins in this system, we have isolated a gene encoding a metal-activated, sequence-specific, DNA-binding protein from C. glabrata.t We demonstrate that this protein, denoted AMT1, binds to both members of the C. glabrata MT gene family in a copper- or silver-inducible fashion and bears strong resemblance to the metal-activated DNA-binding domain of ACEL.
The metal copper plays a crucial role in biological systems as a cofactor for enzymes such as cytochrome oxidase, copper, zinc superoxide dismutase, and several other enzymes (1). Due to its proclivity to participate in damaging oxidation reactions, copper is also a potent toxin when allowed to accumulate to high free intracellular concentrations (2). To cope with this apparent nutritional paradox, eukaryotic organisms have evolved a sensory mechanism that triggers the rapid and efficient transcriptional activation of metaldetoxification genes that encode proteins known as metallothioneins (MTs). MTs are low molecular weight, cysteinerich proteins that bind metals such as copper, cadmium, zinc, and mercury efficiently and tenaciously through thiolate bonds. Metal coordination in MTs is achieved through the multiple cysteine residues, in the arrangement Cys-Xaa-Cys or Cys-Xaa-Xaa-Cys (where Xaa represents any amino acid), found in all known MT proteins (3, 4). Through the action of specific cellular metal sensory components, the biosynthesis of MT is activated, at the level of transcription initiation, by many of the same metals that are bound by MT proteins. The baker's yeast, Saccharomyces cerevisiae, has provided one of the most informative model systems for understanding metal-inducible transcription of MT genes. The single S. cerevisiae MT gene, designated CUPi, is transcriptionally activated by copper through a specific promoter region, UAScup, (upstream activation sequence), located
MATERIALS AND METHODS Strains and Growth Conditions. The S. cerevisiae strains used in this work are as follows: 50.L4 (MATa trpl-i leu23,-i12 gall ura3-50 his cupPS (17); DTY89 (MA Ta leu2-3,-112 gall ura3-SO cupi-A6i acei-A225 trpl-i:: MT-I-lacZ:: TRPI); and DTY99 (MA Ta leu2-3,-112:: MT-I:: LEU2 gall his ura3-50 cupi-A61 acei-A225 trpl-l::MT-I-lacZ::TRPI). All strains are isogenic to the parental strain 50.L4. Strain DTY89 was constructed by deletion of the ACE] gene as described (18) and by deleting the entire CUPi coding sequence by using plasmid pSC11-7 (a gift from T. Keng and S. Ushinsky, McGill University). Plasmid pSC11-7 contains CUPi gene flanking sequences interrupted by a hisG-URA3-hisG cassette from pNKY51 (19) and was used to delete the CUPi coding sequence by homologous recombination and to reAbbreviations: 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 the GenBank data base (accession no. M69146).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6112
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Zhou and Thiele cover the ura3 marker by selecting for recombinants on agar containing 5-fluoroorotic acid (20). Strain DTY99 was constructed by integrating the MT-I-lacZ fusion plasmid pTRPMT-I-lacZ at the trpl-J locus and by integration of the MT-I gene in YIpMT-I at the leu2 locus in DTY89. All chromosomal alterations were verified by Southern blotting (20). The C. glabrata strain 85/038 (a gift from P. Magee, University of Minnesota) 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 as described (21) at 30'C. E. coli strains DH5aF' (BRL) and XL1-blue (Stratagene) were used to construct and maintain plasmids by established procedures (20). Plasmids. The C. glabrata MT-I and MT-II genes were isolated from C. glabrata 85/038 genomic DNA by polymerase chain reactions (PCR) (20) using synthetic oligonucleotide primers that hybridize to the extreme termini of published MT-I and MT-II gene sequences (15, 16). PCR products were inserted into pUC19 (New England Biolabs) and pBluescript SK(+) (Stratagene) multiple cloning sites by previously described methods (20). The MT-I-lacZ fusion plasmid, pTRP-MT-I-lacZ, was constructed by inserting a 460-base-pair (bp) EcoRI-Pvu II fragment into the IacZ fusion vector YIp353 (22). A 4.1-kilobase (kb) EcoRI-Stu I fragment from this plasmid was rendered blunt-ended with the Klenow fragment of DNA polymerase I and inserted into the Pvu II site of pRS304 (23) to create the integrating MT-I-lacZ fusion plasmid, pTRPMT-I-lacZ, with TRPI as a selectable marker. The C. glabrata MT-I gene fragment was inserted into the EcoRI-BamHI sites of the S. cerevisiae LEU2-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. coli by using the bacteriophage T7 RNA polymerase system (25). A unique Nco I restriction enzyme site was created in the AMT1 initiation codon by oligonucleotide-directed mutagenesis (26) using the mutagenic primer 5'-GATTACTACCATGGTGCAAATGTGTC-3', which hybridizes to AMTJ gene nucleotide positions -14 to + 12 indicated in Fig. 2B. The resultant 0.9-kb Nco I-BamHI restriction endonuclease fragment was inserted into the corresponding sites in the expres-
6113
sion vector pET-8c, to create plasmid pT7-AMT1, introduced into the E. coli strain BL21 (DE3) pLysE by transformation, and used for expression studies. Cultures containing pET-8c or pT7-AMT1 were grown and induced with 0.5 mM isopropyl 3-D-thiogalactoside and soluble extracts were prepared as described (20). DNA-binding extracts were prepared from C. glabrata 85/038 as described (27) and used in electrophoretic mobility shift assays (20). A 241-bp EcoRI-Xho II fragment from the C. glabrata MT-I promoter and a 335-bp Sma I-Xba I fragment from the MT-II promoter (15, 16) were used as probes in these assays.
RESULTS Isolation of a Metal-Activated Transcription Factor Gene. Due to a lack of facile genetic analysis in C. glabrata, we designed and implemented a method to clone a C. glabrata metal-activated transcription factor gene by function in S. cerevisiae (Fig. 1A). Strain DTY99 is resistant to approximately 15 ,uM copper sulfate and gives rise to pale blue colonies on solid medium containing X-Gal, due to a low but detectable basal level of transcription from the C. glabrata MT-I promoter in S. cerevisiae. We anticipated that the introduction of a C. glabrata DNA fragment encoding a copper-activated transcription factor should trans-activate both the MT-I gene and the MT-I-lacZ fusion, resulting in copper-resistant colonies with elevated ,3-galactosidase. Total genomic DNA was isolated from C. glabrata strain 85/038, subjected to partial digestion with Sau3A, and ligated into the BamHI site of the yeast-E. coli shuttle vector pEMBLYe24 (28). The ligation mixture was used to directly transform DTY99, and uracil prototrophs were selected on SC minus uracil agar medium. Approximately 35,000 total transformants were obtained, which, given the average library insert size and insertion frequencies (-3 kb, 30%, respectively) and the estimated C. glabrata genome size (1.4 x 107 bp), represents approximately two C. glabrata genome equivalents. The transformants were replica-plated to SC medium containing 50 1.M CuSO4, and after 2 days 30 copper-resistant isolates were recovered. Of these, one isolate gave rise to a dark blue patch of growth when streaked
A S. cerevisiae host sensitive to copper
MT-I lacZ
cup1
I
/
Transform with C. glabrata genomic DNA inserted into high copy S. cerevisiae vector
B
Q :0
I-
Mt 0-
_0
Select for copper resistant transformants Screen for blue colonies on X-gal agar
Rescue plasmid from yeast
0@
C_
aI)
FIG. 1. Isolation of a C. glabrata metal-activated transcription factor gene. (A) Scheme used for the isolation of a C. glabrata gene encoding a protein able to 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 (cupiA61) in which the entire CUP] gene has been deleted; ace IA, a mutant allele (acel-A225) in which the entire ACEI gene has been deleted; and X-Gal, 5-bromo-4chloro-3-indolyl /8-D-galactoside. (B) Plasmid pAMT1 activates the C. glabrata MT-I gene in S. cerevisiae in response to exogenous copper. Total RNA from control (-) and copper-treated (+) cultures of C. glabrata, DTY99(pAMT1), and DTY99(pEMBLYe24) was analyzed by RNA blotting and hybridization with a 32p_ labeled MT-I gene EcoRI-BamHI DNA restriction fragment (20). The arrowhead indicates the position of the MT-I gene-specific RNA species.
6114
Biochemistry: Zhou and Thiele
Proc. Natl. Acad. Sci. USA 88 (1991)
to SC medium containing 25 uM CuS04 and X-Gal. When the plasmid contained within this isolate, designated pAMT1, was rescued in E. coli and reintroduced into DTY99, the recipient strain exhibited both the copper resistance (up to 150 ,iiM CuS04) and activation of the MT-I-lacZ fusion demonstrated by 8-galactosidase assays, as observed in the original transformant; however, pEMBLYe24 did not confer these 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 possibility, RNA-blotting experiments were carried out on control and copper-treated cultures of DTY99 harboring either pAMT1 or the control plasmid pEMBLYe24. In strain DTY99(pAMT1), a copper-inducible MT-I mRNA species was synthesized that is similar in size to that synthesized in copper-treated C. glabrata (Fig. 1B, lanes C. glabrata and pAMT1). MT-I RNA synthesis was not induced by exogenous copper in DTY99(pEMBLYe24), indicating that plasmid pAMT1 contains a DNA fragment encoding a copperresponsive MT-I transcription activation function. Taken together, these results clearly demonstrate that the DNA insert in plasmid pAMT1 encodes a copper-responsive transactivator of the C. glabrata MT-I promoter when both are present simultaneously in S. cerevisiae. Furthermore, the magnitude of MT-I gene activation by pAMT1 in S. cerevisiae suggests that the activity encoded by the pAMT1 insert interacts efficiently with the S. cerevisiae general transcription apparatus. We assign the designation AMTP (activator of MT transcription 1) to that portion of the pAMT1 DNA encoding this activity. Analysis of the AMTI Gene. To identify that portion of the pAMT1 plasmid insert encompassing the C. glabrata AMTJ
gene, the insert (approximately 4 kb) contained within the BamHI site in pAMT1 was characterized by subcloning fragments in pEMBLYe24 and testing for function in S. cerevisiae DTY99. The identification of an approximately 1.4-kb C. glabrata BgI II-Pst I genomic DNA fragment able to direct all phenotypes associated with pAMT1 suggested that this DNA fragment should encompass the functional AMTP gene (data not shown). The existence of a colinear Bgl II-Pst I fragment present in C. glabrata genomic DNA, as ascertained by Southern blotting, suggests that no rearrangements of this genomic fragment have occurred during the cloning process (data not shown). The nucleotide sequence of this DNA fragment was determined for both strands and is shown in Fig. 2. A single extended open reading frame was detected on only one strand of this DNA sequence; it encodes a polypeptide 265 amino acid residues in length with a predicted molecular mass of 29,429 daltons. There is a clear asymmetrical distribution of the 30 basic residues contained within the AMT1 coding region. Of these, 25 basic amino acids are located within the amino-terminal 113 residues, giving this region an overall net charge of + 12. Secondly, the carboxyl-terminal residues 114-265 contain 18 of the total 31 acidic residues, conferring a net charge of -13 to this portion of the AMT1 protein. Furthermore, all 11 cysteine residues within the AMT1 open reading frame are localized within the amino-terminal 100 amino acid residues. These and several other features of AMT1 display close resemblance to ACE1 and suggest functional importance (see Fig. 4 and Discussion). Two features of the AMTI gene sequence suggest that this is expressed in C. glabrata. First, the nucleotide sequence surrounding the ATG that initiates the open reading frame is similar to functional initiator codons proposed by Kozak (29). Second, two blocks of sequence, from -77 to
-510
AGATCTCATTAGTAAAAGTGGGGTGTTCATCAAGTGAGCCAATGCAAGTTTGCGTTTCTCTTACAGGAGCCCGATGAATTACTGGGGTAG
-421
-420
TTGCTGTTGCACATCCCCTTTGCCACCATTCACGTCCTGGTTAGCTACTTGTATCCTGCATTATTTGCGGGCAAGTTTCCAATACTATTT
-331
-330
-241
-240
CTTAAAAGTAGTGGTGGGCGTGATGAGCCCAGTGTTTAGTCCATTTTATGGATAATATTGATCTTTAAAGGCAAGTAGTTGAATTTCTAT
-151
-150
TTGGATAAAAGGTATAAATAGTAGTTGAGAATCCAGACAAAGCACCTTTATATTGAAGATAATAGCGTTACATTATATATAAGAAGCGAT
-61
-60
AACAACAAAAACAATAGGCATATATAAAGCAGAGAGCACAGCAGTACACACATTTGCAGTATGGTAGTAATCAACGGGGTGAAGTATGCC
29 10
M
V V
I
N
G
V
K
Y
A
30 11
C
TGTGATTCATGCATCAAATCACATAAAGCAGCCCAGTGTGAGCATAACGATAGACCCTTAAAGATACTAAAGCCAAGGGGAAGGCCACCG 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
40
120 41
T
209 70
210 71
CAAGAGAAAGGCATAACGATAGAAGAGGATATGTTGATGAGCGGAAACATGGACATGTGCTTGTGTGTTAGAGGTGAGCCTTGTAGGTGT Q 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
299 100
300 101
CACGCTAGGAGGAAAAGGACACAGAAATCAAACAAAAAAGATAACTTAAGCATTAATTCGCCCACAAATAATTCTCCTTCACCAGCGCTC H 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
389 130
390 131
TCTGTGAATATTGGAGGGATGGTGGTGGCTAATGATGACATCCTAAAATCATTGGGACCAATTCAAAATGTCGATCTAACGGCTCCACTA S 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
479 160
480 161
D
570 191 660 221
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
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
GATCATCTAATGAATATGCAAATGAGGAATGATAACTCCCTTTCATTTCCTATGTCTGCAAACCAGAATGAAGTCGGTTATCAATTTAAT D
H
N E
L M N M Q M R N
D
N
S
659 220
L H D
749 250
S
F
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
ATAGACGAAATTCTCAATAACGGTATTGAACTTGGTAATGTAAATTGAAGATCGTAAATCTGTAAAACAGTTTGTCGGATATTAGGCTGT
840
ATCTCCTCAAAGCGTATTCGAATATCATTGAGAAGCTGCAG
D
E
I
L
N
N
G
I
E
L
G
N
V
569 190
P M S A N Q N E V G Y Q F N
L
750 251
I
119
839
N
880
FIG. 2. Sequence of 1391 nucleotides encompassing the C. glabrata AMTP gene. Nucleotide positions are numbered relative to the first nucleotide in the AMTJ sequence, which begins the 265-codon open reading frame (deduced amino acid residues are shown in the single-letter code). Two potential binding sites for transcription factor TFIID are underlined.
Biochemistry: Zhou and Thiele
Proc. Natl. Acad. Sci. USA 88 (1991)
-69 and from -40 to -33, with respect to the ATG that initiates this open reading frame, strongly resemble TATA box elements, which serve as binding sites for the conserved eukaryotic 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 ability of the pAMT1 plasmid to activate MT-I expression in S. cerevisiae in a copper-inducible manner, and the striking similarity between the amino acid sequences in the amino termini of AMT1 and ACE1, strongly suggest that AMTJ encodes a metal-activated MT transcription factor. To test this hypothesis, we expressed AMT1 protein in E. coli at a high level and in a soluble form by using the bacteriophage 17 promoter system (ref. 25; data not shown). Total soluble E. coli extracts containing full-length AMT1 protein were used in electrophoretic mobility shift assays to examine the interaction of AMT1 with the C. glabrata MT-I and MT-II promoters. These promoter fragments, which cover the 5' 241 bp of the MT-I promoter and the 5' 335 bp of the MT-II promoter (15, 16), were used in mobility shift assays employing C. glabrata extracts and the E. coli extracts described above (Fig. 3). These assays demonstrated that C. glabrata extracts contain a copper-inducible DNA-binding activity that gives rise to a major and other less prominent DNAprotein complexes of faster mobility that could represent complexes with proteolyzed DNA-binding proteins (Fig. 3, Candida lanes). We also noted the existence of a copperindependent complex, formed with the MT-Il promoter fragment; the nature or specificity of this complex has not been investigated. At high concentrations of extract we observed copper-inducible DNA-protein complexes of slower mobility than the major complexes, suggesting that this activity may recognize multiple target sites within the MT-I and MT-II promoter fragments. Similar DNA-protein complexes were also observed when silver was added to C. glabrata DNAbinding extracts (data not shown). MT-I and MT-lI promoter DNA-protein complexes, of similar mobility to the major species observed in C. glabrata extracts, were formed with 50 ,uM copper or silver-treated E. coli extracts expressing high levels of AMT1, but not with the 17 expression vector control extracts (Fig. 3). Major complexes were formed when low levels (0.1 ,g) of copper- or silver-treated AMT1 extract were used, and slower-migrating complexes, similar in mobility to those observed with high levels of C. glabrata extracts, were observed when 0.25 ,ug of copper-treated AMT1 extract was used. These results demonstrate that the C. glabrata AMT1 protein synthesized in E. coli binds to both Candida --_+-_
+
AMT1
_
0 20 20 30 30 40 40 .1
_-+ _-+-_
'we.~~
iS
I
is
+
the MT-I and the MT-lI promoters in the presence of copper or silver, two metals known to activate expression of both genes in vivo (15).
DISCUSSION In this work we present an approach for the use of a genetic selection in S. cerevisiae to clone genes encoding DNA-
binding transcription activation proteins from heterologous organisms. This approach relies on the known ability of several sequence-specific DNA-binding activation factors to interact productively with the basic transcription machinery from different organisms such as yeast and humans (30). Utilizing this approach, we isolated a gene for a metalactivated DNA-binding protein, AMT1, on the basis of its ability to bind to and activate from putative cognate promoter elements of the C. glabrata MT-I gene. Although we have used the entire MT-I promoter and structural gene in this work, in principle, synthetic DNA-binding sites from any organism could be fused to a 5'-terminally truncated MT-I gene (containing sequences from the TATA box through the structural gene). Selections for the presence of DNA fragments encoding proteins capable of binding to the target site and activating MT-I transcription are then carried out by identifying copper-resistant S. cerevisiae transformants. The use of a secondary screen, such as the promoter-lacZ gene fusion used in this work, allows the facile identification of trans-acting transcription factors rather than metal-binding proteins. Additionally, successful utilization of this selection requires that a single polypeptide bind to the target DNA and activate transcription, and furthermore, that a genomic or cDNA 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 in response to exogenous copper, encodes a copper- or silveractivated sequence-specific DNA-binding protein. Importantly, these same metals are the only ones known to induce C. glabrata MT-I and MT-II gene transcription in vivo (15), strongly implicating AMT1 as a metal-activated C. glabrata MT gene transcription factor in vivo. Definitive evidence for this function of the AMTP gene awaits the results of AMTP gene disruption experiments in C. glabrata. Our observations in this report, however, suggest that in C. glabrata, both classes of MT genes are transcriptionally activated by the same metal-responsive factor. Mobility-shift experiments suggest that AMT1 protein forms multiple distinct complexes with both the MT-I and the MT-II promoter fragments. This Candida
T7
.1 .25 .25 .25.25 .25.25
_-__-
6115
AMT1
T7
0 20 20 50 50 .1 .1 .25 .25.25 .25.25.25 extract -
-
+
-
+
-
+
-
+
-
+
-
-
AgNO3 -
+
CuSO4
*
-
i-U A Ai
.
..-
MT-I
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 by electrophoretic mobility shift on 5% polyacrylamide gels were carried out with a 32P-labeled MT-I promoter fragment (Left) or with an MT-Il promoter 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 of AgNO3 or CUSO4 (at a final concentration of 50 ,uM each) is indicated by a + or -, respectively. The arrowhead on the right side of each panel indicates the location of the major metal-induced complex formed between the probe fragments and AMT1 or C. glabrata extracts.
Biochemistry: Zhou and Thiele
6116 AMT1
1 MVVINGVKY
ACEl
1
4CSI KSHKAAH CHNDRPLKILKPRGRPPT1
Proc. Nad. Acad. Sci. USA 88 (1991) CDMR E50
111i111111111 :.jIj::1:II11I.1.1 11..:::.:111.I11 I :III1:.:1 RGHRAA HTDGPLQMIRRKGRPS TC3SELR 50 MVVINGVKYA
51 KTKNVNPSGSC KLEKIRQEKGITIEEDMLMSGNMDCVRGEC 51 RTKNFNPSGG
CCSARR .... PAVGSKED ...... ET
100
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 in length (8)] proteins using programs from the University of Wisconsin Genetics Computer Group. A vertical line indicates identical amino acids; a colon indicates amino acids with an evolutionary comparison value of 0.5 or greater; and a period indicates an evolutionary comparison of 0.1-0.5, according to the analysis of Gribskov and Burgess (31). Rectangular outlines indicate the 11 amino-terminal cysteine residues conserved between ACEl and AMT1.
resembles the interaction of ACE1 with UAScup1 (9-12) and suggests that, like all MT promoters studied to date, the MT-I and MT-II genes may contain multiple metal-responsive promoter elements (3). Future experiments should define the AMT1 binding sites in the MT-I and MT-II promoters and determine the role these sites play in the regulation of C. glabrata MT gene transcription. The sequence of the C. glabrata AMT1 polypeptide displays striking homology to the amino-terminal 100 amino acids of the S. cerevisiae ACEl protein (Fig. 4). This region of AMT1 is highly positively charged and contains 11 cysteine residues in arrangements (Cys-Xaa-Cys or Cys-XaaXaa-Cys) known to be important for metal binding in both MT proteins and ACE1 (4, 12). Interestingly, the first 7 cysteine residues of ACE1 and AMT1 are exactly conserved with respect to their position in the amino termini of the proteins. Although AMT1 contains 10 more amino acids than ACE1 in the region encompassing the amino-terminal 11 cysteines, the position of the remaining 4 cysteines is conserved when two gaps are introduced into the alignment (Fig. 4). On the basis of the analysis of in vivo and in vitro generated ACEl mutants, which indicates that the 11 amino-terminal cysteines are important in copper binding and UAScup, DNA binding (6, 10, 12), we predict that the conserved cysteine residues in AMT1 may also play an important role in these functions. Indeed, given the high level of homology between the first 63 amino acids of ACE1 and AMT1 (65% identity, 85.7% similarity), it is likely that these portions of the proteins adopt similar structures when coordinated with metal. We note that all of the amino acid substitutions in ACEl previously demonstrated to abolish or significantly diminish DNA-binding activity are either precisely conserved or maintain the same charge in AMT1 (6, 10, 12). In addition to the cysteine residues, these include the following in ACE1: Gly-37, Arg-51 (Lys in AMT1), Lys-53, His-91 (His-101 in AMT1), and Arg-94 (Arg-104 in AMT1). A clearer understanding of the importance of these residues should emerge from structural analyses of the ACEl and AMT1
amino-terminal cysteine-rich domains. The isolation of a gene for a second member of a copper- or silver-activated DNA-binding transcription factor class of proteins should greatly facilitate studies aimed at understanding the structure-function relationships important for metal-activated DNA-binding proteins. Furthermore, C. glabrata provides a convenient model system for studying metal-regulated transcription in an organism containing an MT gene family. We thank D. Engelke and members of the Thiele laboratory for critically reading the manuscript and for valuable suggestions, G. Butler and M. Szczypka for assistance with PCR, S. Ghosh for assistance with C. glabrata DNA-binding extracts, B. Magee and P. T. Magee for the C. glabrata strain, and S. Ushinsky and T. Keng for plasmid pSC11-7. This work was supported by Grant GM41840 from the National Institutes of Health and in part by National Institutes of Health Grant M01 RR00042. 1. Underwood, E. J. (1977) Trace Elements in Human andAnimal Nutrition (Academic, New York), 4th Ed. 2. Halliwell, B. & Gutteridge, J. M. C. (1984) Biochem. J. 219, 1-14. 3. Hamer, D. H. (1986) Annu. Rev. Biochem. 55, 913-951. 4. Kagi, J. H. R. & Kojima, Y. (1987) in Metallothionein II, eds. KA-gi, J. H. R. & Kojima, Y. (Birkhiuser, Basel), pp. 25-61. 5. Thiele, D. J. & Hamer, D. H. (1986) Mol. Cell. Biol. 6, 11581163. 6. Thiele, D. J. (1988) Mol. Cell. Biol. 8, 2745-2752. 7. Welch, J., Fogel, S., Buchman, C. & Karin, M. (1989) EMBO J. 8, 255-260. 8. Szczypka, M. S. & Thiele, D. J. (1989) Mol. Cell. Biol. 9, 421-429. 9. Evans, C. F., Engelke, D. R. & Thiele, D. J. (1990) Mol. Cell. Biol. 10, 426-429. 10. Buchman, C., Skroch, P., Dixon, W., Tullius, T. D. & Karin,
M. (1990) Mol. Cell. Biol. 10, 4778-4787. 11. Furst, P., Hu, S., Hackett, R. & Hamer, D. (1988) Cell 55, 705-717. 12. Hu, S., Furst, P. & Hamer, D. (1990) New Biologist 2, 1-13.
13. Mitchell, P. J. & Tjian, R. (1989) Science 245, 371-378. 14. Struhl, K. (1989) Trends Biochem. Sci. 14, 137-140. 15. Mehra, R. K., Garey, J. R., Butt, T. R., Gray, W. R. & Winge, D. R. (1989) J. Biol. Chem. 264, 19747-19753. 16. Mehra, R. K., Garey, J. R. & Winge, D. R. (1990) J. Biol. Chem. 265, 6369-6375.
17.
Thiele, D. J., Walling, M. J. & Hamer, D. H. (1986) Science
231, 854-856. 18. Butler, G. & Thiele, D. J. (1991) Mol. Cell. Biol. 11, 476-485. 19. Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541-545. 20. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. O., Seidman, J. G., Smith, J. A. & Struhl, K. (1987) Current Protocols in Molecular Biology (Wiley, New York). 21. Sherman, F., Fink, G. R. & Hicks, J. B. (1983) Methods in Yeast Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 22. Myers, A. M., Tzagoloff, A., Kinney, D. M. & Lusty, C. J.
(1986) Gene 45, 299-310.
23. Sikorski, R. S. & Hieter, P. (1989) Genetics 122, 19-27. 24. Orr-Weaver, T. L., Szostak, J. W. & Rothstein, R. J. (1981) Proc. Nat!. Acad. Sci. USA 78, 6354-6358. 25. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89. 26. Su, T. & El-Gewely, M. R. (1988) Gene 69, 81-89. 27. Company, M., Adler, C. & Errede, B. (1988) Mol. Cell. Biol.
8, 2545-2554. 28. Baldari, C. & Cesarini, G. (1982) Gene 35, 27-32. 29. Kozak, M. (1983) Microbiol. Rev. 47, 1-45. 30. Ptashne, M. & Gann, A. A. F. (1990) Nature (London) 346, 329-331. 31. Gribskov, M. & Burgess, R. R. (1986) Nucleic Acids Res. 14, 6745-6763.
Vol. 88, pp. 6112-6116, July 1991 Biochemistry
Isolation of a metal-activated transcription factor gene from Candida 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 metallothionein (MT) genes involves the interaction of metal-responsive trans-acting factors with specific promoter DNA sequence elements. In this report, we present a genetic selection using the baker's yeast, Saccharomyces cerevisiae, to clone a gene from Candida glabrata encoding a metal-activated DNA-binding protein denoted AMT1. This selection is based on the ability of the AMT1 gene product to activate expression of the C. glabrata MT-I gene in a copper-sensitive S. cerevisiae host strain. DNA-binding studies using AMT1 protein expressed in Escherichia coli demonstrate that AMT1 is activated by copper or silver 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 terminal cysteine residues known to be critical for the metal-activated DNA-binding activity of ACEL. In contrast, the carboxylterminal portion of AMT1 bears only slight similarity at the primary structure level to the same region of ACEl known to be important for transcriptional activation. These results suggest that the amino-terminal cysteines, and other conserved residues, play an important role in the ability of AMT1 and ACEl to sense intracellular copper levels and assume a metalactivated DNA-binding structure.
between -105 and -230 with respect to the CUP] transcription initiation site (5). Allelic recessive mutations in a transacting regulatory gene required for copper-inducible transcription, acei-i (cup2), provided a means for cloning the wild type ACEI (CUP2) copper-activated transcription factor by in vivo complementation (6-8). ACEl protein, synthesized either in Escherichia coli or in vitro, binds four distinct regions within UAScup, in a copper- or silver-inducible manner (9-12). Indeed, the ability of copper or silver to activate ACEl binding to UASCup, in vitro corresponds to the exclusive ability of these metals to activate CUP] transcription in vivo (6, 11). Although a large number of DNAbinding motifs have been described (13, 14), the ACEl DNA-binding domain bears no obvious structural relationship to such motifs. Recently, the yeast Candida glabrata was shown to harbor two distinct MT genes; a unique MT-I gene and a tandemly amplified MT-II gene (15, 16). Messenger RNA levels for both MT-I and MT-II were shown to be induced by copper or silver; however, in contrast with mammalian systems, cadmium was shown to be ineffective in enhancing MT-I and MT-II transcription (15). C. glabrata therefore exhibits the MT gene organization typical of higher eukaryotes but a metal specificity for MT biosynthetic regulation similar to that in S. cerevisiae. To begin to understand the structural requirements important for copper-activated DNA-binding proteins in this system, we have isolated a gene encoding a metal-activated, sequence-specific, DNA-binding protein from C. glabrata.t We demonstrate that this protein, denoted AMT1, binds to both members of the C. glabrata MT gene family in a copper- or silver-inducible fashion and bears strong resemblance to the metal-activated DNA-binding domain of ACEL.
The metal copper plays a crucial role in biological systems as a cofactor for enzymes such as cytochrome oxidase, copper, zinc superoxide dismutase, and several other enzymes (1). Due to its proclivity to participate in damaging oxidation reactions, copper is also a potent toxin when allowed to accumulate to high free intracellular concentrations (2). To cope with this apparent nutritional paradox, eukaryotic organisms have evolved a sensory mechanism that triggers the rapid and efficient transcriptional activation of metaldetoxification genes that encode proteins known as metallothioneins (MTs). MTs are low molecular weight, cysteinerich proteins that bind metals such as copper, cadmium, zinc, and mercury efficiently and tenaciously through thiolate bonds. Metal coordination in MTs is achieved through the multiple cysteine residues, in the arrangement Cys-Xaa-Cys or Cys-Xaa-Xaa-Cys (where Xaa represents any amino acid), found in all known MT proteins (3, 4). Through the action of specific cellular metal sensory components, the biosynthesis of MT is activated, at the level of transcription initiation, by many of the same metals that are bound by MT proteins. The baker's yeast, Saccharomyces cerevisiae, has provided one of the most informative model systems for understanding metal-inducible transcription of MT genes. The single S. cerevisiae MT gene, designated CUPi, is transcriptionally activated by copper through a specific promoter region, UAScup, (upstream activation sequence), located
MATERIALS AND METHODS Strains and Growth Conditions. The S. cerevisiae strains used in this work are as follows: 50.L4 (MATa trpl-i leu23,-i12 gall ura3-50 his cupPS (17); DTY89 (MA Ta leu2-3,-112 gall ura3-SO cupi-A6i acei-A225 trpl-i:: MT-I-lacZ:: TRPI); and DTY99 (MA Ta leu2-3,-112:: MT-I:: LEU2 gall his ura3-50 cupi-A61 acei-A225 trpl-l::MT-I-lacZ::TRPI). All strains are isogenic to the parental strain 50.L4. Strain DTY89 was constructed by deletion of the ACE] gene as described (18) and by deleting the entire CUPi coding sequence by using plasmid pSC11-7 (a gift from T. Keng and S. Ushinsky, McGill University). Plasmid pSC11-7 contains CUPi gene flanking sequences interrupted by a hisG-URA3-hisG cassette from pNKY51 (19) and was used to delete the CUPi coding sequence by homologous recombination and to reAbbreviations: 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 the GenBank data base (accession no. M69146).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6112
Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemistry: Zhou and Thiele cover the ura3 marker by selecting for recombinants on agar containing 5-fluoroorotic acid (20). Strain DTY99 was constructed by integrating the MT-I-lacZ fusion plasmid pTRPMT-I-lacZ at the trpl-J locus and by integration of the MT-I gene in YIpMT-I at the leu2 locus in DTY89. All chromosomal alterations were verified by Southern blotting (20). The C. glabrata strain 85/038 (a gift from P. Magee, University of Minnesota) 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 as described (21) at 30'C. E. coli strains DH5aF' (BRL) and XL1-blue (Stratagene) were used to construct and maintain plasmids by established procedures (20). Plasmids. The C. glabrata MT-I and MT-II genes were isolated from C. glabrata 85/038 genomic DNA by polymerase chain reactions (PCR) (20) using synthetic oligonucleotide primers that hybridize to the extreme termini of published MT-I and MT-II gene sequences (15, 16). PCR products were inserted into pUC19 (New England Biolabs) and pBluescript SK(+) (Stratagene) multiple cloning sites by previously described methods (20). The MT-I-lacZ fusion plasmid, pTRP-MT-I-lacZ, was constructed by inserting a 460-base-pair (bp) EcoRI-Pvu II fragment into the IacZ fusion vector YIp353 (22). A 4.1-kilobase (kb) EcoRI-Stu I fragment from this plasmid was rendered blunt-ended with the Klenow fragment of DNA polymerase I and inserted into the Pvu II site of pRS304 (23) to create the integrating MT-I-lacZ fusion plasmid, pTRPMT-I-lacZ, with TRPI as a selectable marker. The C. glabrata MT-I gene fragment was inserted into the EcoRI-BamHI sites of the S. cerevisiae LEU2-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. coli by using the bacteriophage T7 RNA polymerase system (25). A unique Nco I restriction enzyme site was created in the AMT1 initiation codon by oligonucleotide-directed mutagenesis (26) using the mutagenic primer 5'-GATTACTACCATGGTGCAAATGTGTC-3', which hybridizes to AMTJ gene nucleotide positions -14 to + 12 indicated in Fig. 2B. The resultant 0.9-kb Nco I-BamHI restriction endonuclease fragment was inserted into the corresponding sites in the expres-
6113
sion vector pET-8c, to create plasmid pT7-AMT1, introduced into the E. coli strain BL21 (DE3) pLysE by transformation, and used for expression studies. Cultures containing pET-8c or pT7-AMT1 were grown and induced with 0.5 mM isopropyl 3-D-thiogalactoside and soluble extracts were prepared as described (20). DNA-binding extracts were prepared from C. glabrata 85/038 as described (27) and used in electrophoretic mobility shift assays (20). A 241-bp EcoRI-Xho II fragment from the C. glabrata MT-I promoter and a 335-bp Sma I-Xba I fragment from the MT-II promoter (15, 16) were used as probes in these assays.
RESULTS Isolation of a Metal-Activated Transcription Factor Gene. Due to a lack of facile genetic analysis in C. glabrata, we designed and implemented a method to clone a C. glabrata metal-activated transcription factor gene by function in S. cerevisiae (Fig. 1A). Strain DTY99 is resistant to approximately 15 ,uM copper sulfate and gives rise to pale blue colonies on solid medium containing X-Gal, due to a low but detectable basal level of transcription from the C. glabrata MT-I promoter in S. cerevisiae. We anticipated that the introduction of a C. glabrata DNA fragment encoding a copper-activated transcription factor should trans-activate both the MT-I gene and the MT-I-lacZ fusion, resulting in copper-resistant colonies with elevated ,3-galactosidase. Total genomic DNA was isolated from C. glabrata strain 85/038, subjected to partial digestion with Sau3A, and ligated into the BamHI site of the yeast-E. coli shuttle vector pEMBLYe24 (28). The ligation mixture was used to directly transform DTY99, and uracil prototrophs were selected on SC minus uracil agar medium. Approximately 35,000 total transformants were obtained, which, given the average library insert size and insertion frequencies (-3 kb, 30%, respectively) and the estimated C. glabrata genome size (1.4 x 107 bp), represents approximately two C. glabrata genome equivalents. The transformants were replica-plated to SC medium containing 50 1.M CuSO4, and after 2 days 30 copper-resistant isolates were recovered. Of these, one isolate gave rise to a dark blue patch of growth when streaked
A S. cerevisiae host sensitive to copper
MT-I lacZ
cup1
I
/
Transform with C. glabrata genomic DNA inserted into high copy S. cerevisiae vector
B
Q :0
I-
Mt 0-
_0
Select for copper resistant transformants Screen for blue colonies on X-gal agar
Rescue plasmid from yeast
0@
C_
aI)
FIG. 1. Isolation of a C. glabrata metal-activated transcription factor gene. (A) Scheme used for the isolation of a C. glabrata gene encoding a protein able to 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 (cupiA61) in which the entire CUP] gene has been deleted; ace IA, a mutant allele (acel-A225) in which the entire ACEI gene has been deleted; and X-Gal, 5-bromo-4chloro-3-indolyl /8-D-galactoside. (B) Plasmid pAMT1 activates the C. glabrata MT-I gene in S. cerevisiae in response to exogenous copper. Total RNA from control (-) and copper-treated (+) cultures of C. glabrata, DTY99(pAMT1), and DTY99(pEMBLYe24) was analyzed by RNA blotting and hybridization with a 32p_ labeled MT-I gene EcoRI-BamHI DNA restriction fragment (20). The arrowhead indicates the position of the MT-I gene-specific RNA species.
6114
Biochemistry: Zhou and Thiele
Proc. Natl. Acad. Sci. USA 88 (1991)
to SC medium containing 25 uM CuS04 and X-Gal. When the plasmid contained within this isolate, designated pAMT1, was rescued in E. coli and reintroduced into DTY99, the recipient strain exhibited both the copper resistance (up to 150 ,iiM CuS04) and activation of the MT-I-lacZ fusion demonstrated by 8-galactosidase assays, as observed in the original transformant; however, pEMBLYe24 did not confer these 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 possibility, RNA-blotting experiments were carried out on control and copper-treated cultures of DTY99 harboring either pAMT1 or the control plasmid pEMBLYe24. In strain DTY99(pAMT1), a copper-inducible MT-I mRNA species was synthesized that is similar in size to that synthesized in copper-treated C. glabrata (Fig. 1B, lanes C. glabrata and pAMT1). MT-I RNA synthesis was not induced by exogenous copper in DTY99(pEMBLYe24), indicating that plasmid pAMT1 contains a DNA fragment encoding a copperresponsive MT-I transcription activation function. Taken together, these results clearly demonstrate that the DNA insert in plasmid pAMT1 encodes a copper-responsive transactivator of the C. glabrata MT-I promoter when both are present simultaneously in S. cerevisiae. Furthermore, the magnitude of MT-I gene activation by pAMT1 in S. cerevisiae suggests that the activity encoded by the pAMT1 insert interacts efficiently with the S. cerevisiae general transcription apparatus. We assign the designation AMTP (activator of MT transcription 1) to that portion of the pAMT1 DNA encoding this activity. Analysis of the AMTI Gene. To identify that portion of the pAMT1 plasmid insert encompassing the C. glabrata AMTJ
gene, the insert (approximately 4 kb) contained within the BamHI site in pAMT1 was characterized by subcloning fragments in pEMBLYe24 and testing for function in S. cerevisiae DTY99. The identification of an approximately 1.4-kb C. glabrata BgI II-Pst I genomic DNA fragment able to direct all phenotypes associated with pAMT1 suggested that this DNA fragment should encompass the functional AMTP gene (data not shown). The existence of a colinear Bgl II-Pst I fragment present in C. glabrata genomic DNA, as ascertained by Southern blotting, suggests that no rearrangements of this genomic fragment have occurred during the cloning process (data not shown). The nucleotide sequence of this DNA fragment was determined for both strands and is shown in Fig. 2. A single extended open reading frame was detected on only one strand of this DNA sequence; it encodes a polypeptide 265 amino acid residues in length with a predicted molecular mass of 29,429 daltons. There is a clear asymmetrical distribution of the 30 basic residues contained within the AMT1 coding region. Of these, 25 basic amino acids are located within the amino-terminal 113 residues, giving this region an overall net charge of + 12. Secondly, the carboxyl-terminal residues 114-265 contain 18 of the total 31 acidic residues, conferring a net charge of -13 to this portion of the AMT1 protein. Furthermore, all 11 cysteine residues within the AMT1 open reading frame are localized within the amino-terminal 100 amino acid residues. These and several other features of AMT1 display close resemblance to ACE1 and suggest functional importance (see Fig. 4 and Discussion). Two features of the AMTI gene sequence suggest that this is expressed in C. glabrata. First, the nucleotide sequence surrounding the ATG that initiates the open reading frame is similar to functional initiator codons proposed by Kozak (29). Second, two blocks of sequence, from -77 to
-510
AGATCTCATTAGTAAAAGTGGGGTGTTCATCAAGTGAGCCAATGCAAGTTTGCGTTTCTCTTACAGGAGCCCGATGAATTACTGGGGTAG
-421
-420
TTGCTGTTGCACATCCCCTTTGCCACCATTCACGTCCTGGTTAGCTACTTGTATCCTGCATTATTTGCGGGCAAGTTTCCAATACTATTT
-331
-330
-241
-240
CTTAAAAGTAGTGGTGGGCGTGATGAGCCCAGTGTTTAGTCCATTTTATGGATAATATTGATCTTTAAAGGCAAGTAGTTGAATTTCTAT
-151
-150
TTGGATAAAAGGTATAAATAGTAGTTGAGAATCCAGACAAAGCACCTTTATATTGAAGATAATAGCGTTACATTATATATAAGAAGCGAT
-61
-60
AACAACAAAAACAATAGGCATATATAAAGCAGAGAGCACAGCAGTACACACATTTGCAGTATGGTAGTAATCAACGGGGTGAAGTATGCC
29 10
M
V V
I
N
G
V
K
Y
A
30 11
C
TGTGATTCATGCATCAAATCACATAAAGCAGCCCAGTGTGAGCATAACGATAGACCCTTAAAGATACTAAAGCCAAGGGGAAGGCCACCG 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
40
120 41
T
209 70
210 71
CAAGAGAAAGGCATAACGATAGAAGAGGATATGTTGATGAGCGGAAACATGGACATGTGCTTGTGTGTTAGAGGTGAGCCTTGTAGGTGT Q 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
299 100
300 101
CACGCTAGGAGGAAAAGGACACAGAAATCAAACAAAAAAGATAACTTAAGCATTAATTCGCCCACAAATAATTCTCCTTCACCAGCGCTC H 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
389 130
390 131
TCTGTGAATATTGGAGGGATGGTGGTGGCTAATGATGACATCCTAAAATCATTGGGACCAATTCAAAATGTCGATCTAACGGCTCCACTA S 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
479 160
480 161
D
570 191 660 221
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
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
GATCATCTAATGAATATGCAAATGAGGAATGATAACTCCCTTTCATTTCCTATGTCTGCAAACCAGAATGAAGTCGGTTATCAATTTAAT D
H
N E
L M N M Q M R N
D
N
S
659 220
L H D
749 250
S
F
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
ATAGACGAAATTCTCAATAACGGTATTGAACTTGGTAATGTAAATTGAAGATCGTAAATCTGTAAAACAGTTTGTCGGATATTAGGCTGT
840
ATCTCCTCAAAGCGTATTCGAATATCATTGAGAAGCTGCAG
D
E
I
L
N
N
G
I
E
L
G
N
V
569 190
P M S A N Q N E V G Y Q F N
L
750 251
I
119
839
N
880
FIG. 2. Sequence of 1391 nucleotides encompassing the C. glabrata AMTP gene. Nucleotide positions are numbered relative to the first nucleotide in the AMTJ sequence, which begins the 265-codon open reading frame (deduced amino acid residues are shown in the single-letter code). Two potential binding sites for transcription factor TFIID are underlined.
Biochemistry: Zhou and Thiele
Proc. Natl. Acad. Sci. USA 88 (1991)
-69 and from -40 to -33, with respect to the ATG that initiates this open reading frame, strongly resemble TATA box elements, which serve as binding sites for the conserved eukaryotic 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 ability of the pAMT1 plasmid to activate MT-I expression in S. cerevisiae in a copper-inducible manner, and the striking similarity between the amino acid sequences in the amino termini of AMT1 and ACE1, strongly suggest that AMTJ encodes a metal-activated MT transcription factor. To test this hypothesis, we expressed AMT1 protein in E. coli at a high level and in a soluble form by using the bacteriophage 17 promoter system (ref. 25; data not shown). Total soluble E. coli extracts containing full-length AMT1 protein were used in electrophoretic mobility shift assays to examine the interaction of AMT1 with the C. glabrata MT-I and MT-II promoters. These promoter fragments, which cover the 5' 241 bp of the MT-I promoter and the 5' 335 bp of the MT-II promoter (15, 16), were used in mobility shift assays employing C. glabrata extracts and the E. coli extracts described above (Fig. 3). These assays demonstrated that C. glabrata extracts contain a copper-inducible DNA-binding activity that gives rise to a major and other less prominent DNAprotein complexes of faster mobility that could represent complexes with proteolyzed DNA-binding proteins (Fig. 3, Candida lanes). We also noted the existence of a copperindependent complex, formed with the MT-Il promoter fragment; the nature or specificity of this complex has not been investigated. At high concentrations of extract we observed copper-inducible DNA-protein complexes of slower mobility than the major complexes, suggesting that this activity may recognize multiple target sites within the MT-I and MT-II promoter fragments. Similar DNA-protein complexes were also observed when silver was added to C. glabrata DNAbinding extracts (data not shown). MT-I and MT-lI promoter DNA-protein complexes, of similar mobility to the major species observed in C. glabrata extracts, were formed with 50 ,uM copper or silver-treated E. coli extracts expressing high levels of AMT1, but not with the 17 expression vector control extracts (Fig. 3). Major complexes were formed when low levels (0.1 ,g) of copper- or silver-treated AMT1 extract were used, and slower-migrating complexes, similar in mobility to those observed with high levels of C. glabrata extracts, were observed when 0.25 ,ug of copper-treated AMT1 extract was used. These results demonstrate that the C. glabrata AMT1 protein synthesized in E. coli binds to both Candida --_+-_
+
AMT1
_
0 20 20 30 30 40 40 .1
_-+ _-+-_
'we.~~
iS
I
is
+
the MT-I and the MT-lI promoters in the presence of copper or silver, two metals known to activate expression of both genes in vivo (15).
DISCUSSION In this work we present an approach for the use of a genetic selection in S. cerevisiae to clone genes encoding DNA-
binding transcription activation proteins from heterologous organisms. This approach relies on the known ability of several sequence-specific DNA-binding activation factors to interact productively with the basic transcription machinery from different organisms such as yeast and humans (30). Utilizing this approach, we isolated a gene for a metalactivated DNA-binding protein, AMT1, on the basis of its ability to bind to and activate from putative cognate promoter elements of the C. glabrata MT-I gene. Although we have used the entire MT-I promoter and structural gene in this work, in principle, synthetic DNA-binding sites from any organism could be fused to a 5'-terminally truncated MT-I gene (containing sequences from the TATA box through the structural gene). Selections for the presence of DNA fragments encoding proteins capable of binding to the target site and activating MT-I transcription are then carried out by identifying copper-resistant S. cerevisiae transformants. The use of a secondary screen, such as the promoter-lacZ gene fusion used in this work, allows the facile identification of trans-acting transcription factors rather than metal-binding proteins. Additionally, successful utilization of this selection requires that a single polypeptide bind to the target DNA and activate transcription, and furthermore, that a genomic or cDNA 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 in response to exogenous copper, encodes a copper- or silveractivated sequence-specific DNA-binding protein. Importantly, these same metals are the only ones known to induce C. glabrata MT-I and MT-II gene transcription in vivo (15), strongly implicating AMT1 as a metal-activated C. glabrata MT gene transcription factor in vivo. Definitive evidence for this function of the AMTP gene awaits the results of AMTP gene disruption experiments in C. glabrata. Our observations in this report, however, suggest that in C. glabrata, both classes of MT genes are transcriptionally activated by the same metal-responsive factor. Mobility-shift experiments suggest that AMT1 protein forms multiple distinct complexes with both the MT-I and the MT-II promoter fragments. This Candida
T7
.1 .25 .25 .25.25 .25.25
_-__-
6115
AMT1
T7
0 20 20 50 50 .1 .1 .25 .25.25 .25.25.25 extract -
-
+
-
+
-
+
-
+
-
+
-
-
AgNO3 -
+
CuSO4
*
-
i-U A Ai
.
..-
MT-I
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 by electrophoretic mobility shift on 5% polyacrylamide gels were carried out with a 32P-labeled MT-I promoter fragment (Left) or with an MT-Il promoter 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 of AgNO3 or CUSO4 (at a final concentration of 50 ,uM each) is indicated by a + or -, respectively. The arrowhead on the right side of each panel indicates the location of the major metal-induced complex formed between the probe fragments and AMT1 or C. glabrata extracts.
Biochemistry: Zhou and Thiele
6116 AMT1
1 MVVINGVKY
ACEl
1
4CSI KSHKAAH CHNDRPLKILKPRGRPPT1
Proc. Nad. Acad. Sci. USA 88 (1991) CDMR E50
111i111111111 :.jIj::1:II11I.1.1 11..:::.:111.I11 I :III1:.:1 RGHRAA HTDGPLQMIRRKGRPS TC3SELR 50 MVVINGVKYA
51 KTKNVNPSGSC KLEKIRQEKGITIEEDMLMSGNMDCVRGEC 51 RTKNFNPSGG
CCSARR .... PAVGSKED ...... ET
100
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 in length (8)] proteins using programs from the University of Wisconsin Genetics Computer Group. A vertical line indicates identical amino acids; a colon indicates amino acids with an evolutionary comparison value of 0.5 or greater; and a period indicates an evolutionary comparison of 0.1-0.5, according to the analysis of Gribskov and Burgess (31). Rectangular outlines indicate the 11 amino-terminal cysteine residues conserved between ACEl and AMT1.
resembles the interaction of ACE1 with UAScup1 (9-12) and suggests that, like all MT promoters studied to date, the MT-I and MT-II genes may contain multiple metal-responsive promoter elements (3). Future experiments should define the AMT1 binding sites in the MT-I and MT-II promoters and determine the role these sites play in the regulation of C. glabrata MT gene transcription. The sequence of the C. glabrata AMT1 polypeptide displays striking homology to the amino-terminal 100 amino acids of the S. cerevisiae ACEl protein (Fig. 4). This region of AMT1 is highly positively charged and contains 11 cysteine residues in arrangements (Cys-Xaa-Cys or Cys-XaaXaa-Cys) known to be important for metal binding in both MT proteins and ACE1 (4, 12). Interestingly, the first 7 cysteine residues of ACE1 and AMT1 are exactly conserved with respect to their position in the amino termini of the proteins. Although AMT1 contains 10 more amino acids than ACE1 in the region encompassing the amino-terminal 11 cysteines, the position of the remaining 4 cysteines is conserved when two gaps are introduced into the alignment (Fig. 4). On the basis of the analysis of in vivo and in vitro generated ACEl mutants, which indicates that the 11 amino-terminal cysteines are important in copper binding and UAScup, DNA binding (6, 10, 12), we predict that the conserved cysteine residues in AMT1 may also play an important role in these functions. Indeed, given the high level of homology between the first 63 amino acids of ACE1 and AMT1 (65% identity, 85.7% similarity), it is likely that these portions of the proteins adopt similar structures when coordinated with metal. We note that all of the amino acid substitutions in ACEl previously demonstrated to abolish or significantly diminish DNA-binding activity are either precisely conserved or maintain the same charge in AMT1 (6, 10, 12). In addition to the cysteine residues, these include the following in ACE1: Gly-37, Arg-51 (Lys in AMT1), Lys-53, His-91 (His-101 in AMT1), and Arg-94 (Arg-104 in AMT1). A clearer understanding of the importance of these residues should emerge from structural analyses of the ACEl and AMT1
amino-terminal cysteine-rich domains. The isolation of a gene for a second member of a copper- or silver-activated DNA-binding transcription factor class of proteins should greatly facilitate studies aimed at understanding the structure-function relationships important for metal-activated DNA-binding proteins. Furthermore, C. glabrata provides a convenient model system for studying metal-regulated transcription in an organism containing an MT gene family. We thank D. Engelke and members of the Thiele laboratory for critically reading the manuscript and for valuable suggestions, G. Butler and M. Szczypka for assistance with PCR, S. Ghosh for assistance with C. glabrata DNA-binding extracts, B. Magee and P. T. Magee for the C. glabrata strain, and S. Ushinsky and T. Keng for plasmid pSC11-7. This work was supported by Grant GM41840 from the National Institutes of Health and in part by National Institutes of Health Grant M01 RR00042. 1. Underwood, E. J. (1977) Trace Elements in Human andAnimal Nutrition (Academic, New York), 4th Ed. 2. Halliwell, B. & Gutteridge, J. M. C. (1984) Biochem. J. 219, 1-14. 3. Hamer, D. H. (1986) Annu. Rev. Biochem. 55, 913-951. 4. Kagi, J. H. R. & Kojima, Y. (1987) in Metallothionein II, eds. KA-gi, J. H. R. & Kojima, Y. (Birkhiuser, Basel), pp. 25-61. 5. Thiele, D. J. & Hamer, D. H. (1986) Mol. Cell. Biol. 6, 11581163. 6. Thiele, D. J. (1988) Mol. Cell. Biol. 8, 2745-2752. 7. Welch, J., Fogel, S., Buchman, C. & Karin, M. (1989) EMBO J. 8, 255-260. 8. Szczypka, M. S. & Thiele, D. J. (1989) Mol. Cell. Biol. 9, 421-429. 9. Evans, C. F., Engelke, D. R. & Thiele, D. J. (1990) Mol. Cell. Biol. 10, 426-429. 10. Buchman, C., Skroch, P., Dixon, W., Tullius, T. D. & Karin,
M. (1990) Mol. Cell. Biol. 10, 4778-4787. 11. Furst, P., Hu, S., Hackett, R. & Hamer, D. (1988) Cell 55, 705-717. 12. Hu, S., Furst, P. & Hamer, D. (1990) New Biologist 2, 1-13.
13. Mitchell, P. J. & Tjian, R. (1989) Science 245, 371-378. 14. Struhl, K. (1989) Trends Biochem. Sci. 14, 137-140. 15. Mehra, R. K., Garey, J. R., Butt, T. R., Gray, W. R. & Winge, D. R. (1989) J. Biol. Chem. 264, 19747-19753. 16. Mehra, R. K., Garey, J. R. & Winge, D. R. (1990) J. Biol. Chem. 265, 6369-6375.
17.
Thiele, D. J., Walling, M. J. & Hamer, D. H. (1986) Science
231, 854-856. 18. Butler, G. & Thiele, D. J. (1991) Mol. Cell. Biol. 11, 476-485. 19. Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541-545. 20. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. O., Seidman, J. G., Smith, J. A. & Struhl, K. (1987) Current Protocols in Molecular Biology (Wiley, New York). 21. Sherman, F., Fink, G. R. & Hicks, J. B. (1983) Methods in Yeast Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 22. Myers, A. M., Tzagoloff, A., Kinney, D. M. & Lusty, C. J.
(1986) Gene 45, 299-310.
23. Sikorski, R. S. & Hieter, P. (1989) Genetics 122, 19-27. 24. Orr-Weaver, T. L., Szostak, J. W. & Rothstein, R. J. (1981) Proc. Nat!. Acad. Sci. USA 78, 6354-6358. 25. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89. 26. Su, T. & El-Gewely, M. R. (1988) Gene 69, 81-89. 27. Company, M., Adler, C. & Errede, B. (1988) Mol. Cell. Biol.
8, 2545-2554. 28. Baldari, C. & Cesarini, G. (1982) Gene 35, 27-32. 29. Kozak, M. (1983) Microbiol. Rev. 47, 1-45. 30. Ptashne, M. & Gann, A. A. F. (1990) Nature (London) 346, 329-331. 31. Gribskov, M. & Burgess, R. R. (1986) Nucleic Acids Res. 14, 6745-6763.
