Proc. Natl. Acad. Sci. USA Vol. 89, pp. 3869-3873, May 1992 Biochemistry

Ferric reductase of Saccharomyces cerevisiae: Molecular characterization, role in iron uptake, and transcriptional control by iron ANDREW DANCIS*, DRAGOS G. ROMAN*, GREGORY J. ANDERSON*, ALAN G. HINNEBUSCHt, AND RICHARD D. KLAUSNER* *Cell Biology and Metabolism Branch, and tSection on Molecular Genetics of Lower Eukaryotes, Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, MD 20892

Communicated by Helmut Beinert, January 3, 1992 (received for review October 18, 1991)

(9). In this paper we present the structure of the FREI gene and identify DNA sequences that mediate its regulation by

ABSTRACT The principal iron uptake system of Saccharomyces cerevisiae utilizes a reductase activity that acts on ferric iron chelates external to the cell. The FREI gene product is required for this activity. The deduced amino acid sequence of the FRE1 protein exhibits hydrophobic regions compatible with transmembrane domains and has significant similarity to the sequence of the plasma membrane cytochrome b558 (the X-CGD protein), a critical component of a human phagocyte oxidoreductase, suggesting that FREl is a structural component of the yeast ferric reductase. FREI mRNA levels are repressed by iron. Fusion of 977 base pairs of FREI DNA upstream from the translation start site of an Escherichia coil IacZ reporter gene confers iron-dependent regulation on expression of fi-galactosidase in yeast. An 85-base-pair segment of FREI 5' noncoding sequence contains a RAPi binding site and a repeated sequence, TTTTTGCTCAYC; this segment is sufficient to confer iron-repressible transcriptional activity on heterologous downstream promoter elements.

ironA MATERIALS AND METHODS Yeast Strains. The following yeast strains were derived from the wild-type strain F113 (MATa, can), inol-13, ura352): W103 (MATa, can], inol-13, ura3-52,frel-1) was derived by chemical mutagenesis of F113 and selection for a reductase-negative phenotype (9); W126 was derived by transformation of F113 with the plasmid pWDC20, which carries the FREI gene on the high-copy-number vector YEp24 (11). We used one-step gene disruption (12) to create strain N1 (MA Ta, can), inol-13, Afrel:: URA3), in which an 800-base-pair (bp) Xho I fragment internal to the FREI coding region was replaced with a URA3 marker gene, and strain N2 (MATa, can, inol -13, Afrel:: URA3), in which a 2.7-kilobase (kb) Cla I fragment containing the entire FREI coding region was similarly replaced. Disruption/deletion strains of the FREI locus were also constructed in the wild-type strain H1085, yielding strain W218 (MATa, leu2-3,112, Afrel:: URA3) and strain W258 (MATa, leu2-3,112, Afrel:: URA3) by replacement of the FREI Xho I or Cla I fragment, respectively. The structure of the Afrel:: URA3 alleles was confirmed by PCR amplification of genomic sequences (13). The FREI disruption/deletion strains were similar in their ferric reductase activity and sensitivity to iron deprivation. Plasmids. Plasmid pWDC6 conferred reductase activity on the W103 mutant strain. It contains the FREI gene and flanking sequences on a low-copy-number URA3 vector (9). Plasmid pCL4.3 contains the FREI gene on a low-copynumber LEU2 vector. FREI sequences were amplified by PCR with the boundaries specified in Fig. 3A (13) and inserted in-frame 5' of an E. coli lacZ reporter gene on the low-copy-number URA3 plasmid pRS416 (14), yielding pJ105, pJ111, pJ106, and pJ107. The FREI sequence -977 to +9 was similarly amplified and inserted in-frame at the 5' end of lacZ on the high-copy-number plasmid YEp24 (11), yielding pEG2. The low-copy-number URA3 plasmid p1087, generously provided by C. Moehle (15), contains 250 nucleotides of CYC) 5' noncoding sequences and the ATG start codon fused to the E. coli 1acZ gene (16). FREI sequences with the boundaries specified in Fig. 3B were inserted upstream of the CYC) sequence between Bgl II and Xho I restriction sites, creating plasmids pGC7-8, pGC7-8.0, pGC7-8.5, pGC7-9, pGC8-9, pGC6-9, pGCR, and pGCRM. The point mutation in

Iron is an essential nutrient that is required by many enzymes for cellular functions such as DNA synthesis and respiration. Ferric iron forms insoluble ferric hydroxide complexes in the presence of oxygen and water, making iron availability a biological problem under these conditions. Ferrous iron is much more soluble but is rapidly converted to ferric iron in the presence of oxygen (1). Cells have developed two major solutions to the problem of assimilation offerric iron from the environment. In Escherichia coli, specific ferric binding compounds termed siderophores are secreted that bind and solubilize environmental iron and deliver it to specific receptors for transport across the outer and inner membranes (2). In the yeast Saccharomyces cerevisiae, the major iron uptake system under aerobic conditions depends on a transplasma membrane electron transport system (3, 4) that reduces ferric iron external to the cell. Multicellular eukaryotes may also use an externally directed ferric reductase prior to transmembrane movement of ferrous iron (5-8). Our previous results indicated that the FREI gene product in S. cerevisiae is required for external ferric reduction and for ferric iron utilization, thus linking these two processes (9). However, a mutation in FREI did not affect the uptake offerrous iron (9), suggesting that the ferrous transport system is an independent component of the iron utilization apparatus in yeast. Many organisms control their rate of iron assimilation by ascertaining their requirement for iron and expressing a limiting component of the uptake system accordingly (10). Our previous results suggest that during aerobic growth of S. cerevisiae, changes in iron availability are reflected in alterations in the levels of FREI mRNA and of ferric iron uptake

Abbreviations: ORF, open reading frame; UAS, upstream activating sequence. *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M86908).

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.

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Proc. Natl. Acad. Sci. USA 89 (1992)

Biochemistry: Dancis et al.

Genetic and Microbiological Methods. Methods for DNA manipulation were as described (20). The DNA sequence of pWDC6 and its subclones was determined (21) with a Sequenase kit (United States Biochemical). Primer extension of radiolabeled oligonucleotide primers corresponding to the complement of FREI sequences from +8 to -12 and from +60 to -33 was performed by a published protocol (22). Oligonucleotides were synthesized on an Applied Biosystems model 381A DNA synthesizer.

plasmid pGCRM is a C -- A transversion in FREI at position

-307 relative to the ATG. Growth Conditions. Yeast cultures were grown in standard media (SD or YPD) or iron-depleted minimal defined medium (MD) according to published methods (9, 17). For analyses of ferric reductase activity, ferric and ferrous uptake, and f3-galactosidase activity, three individual colonies transformed with each construct were picked from an SD plate and grown to saturation in YPD medium. These cultures were then diluted 1:200 into MD lacking uracil with or without 1 mM added ferric chloride and grown for an additional 12-16 hr. For the analysis of iron-dependent growth, three transformed yeast colonies were initially grown to stationary phase in MD and then diluted 1:1000 into fresh MD medium. Assays. Ferric reductase activity and uptake of ferric and ferrous iron were measured as described (9). The concentration of iron was 20 ,uM for the measurement of ferric uptake and 1 AM for the measurement of ferrous uptake. fB-Galactosidase was measured from washed cells that were permeabilized with 0.1% SDS and chloroform (18, 19).

RESULTS Localization of FREI. The FREI gene was cloned by complementation of the ferric reductase deficiency of afrel-l mutant strain (W103), using a low-copy-number yeast genomic library (9). A 2.7-kb Cla I fragment of plasmid pWDC6 (bp -575 to 2139, Fig. 1) exhibited frel-J complementing activity, while deletion to the EcoRI site at the 5' end (bp 192, Fig. 1) or the BstEII site at the 3' end (bp 1658, Fig. 1) eliminated this activity (data not shown). Sequence analysis attcggtcoatttccct -961

ctcttattggtggcttaggttgatagt tcacaataaatttaggaacctttggtgtgctaacaaattcaagtttttgactggcgactaatgcaggtataggagcacagctttcctcgtcga -841 aagatgacct cacgt tgcagccaccgacac atatcggtccagcgccacat tcat tatacg'gcgagcagcacggccattctgcgggacaagggt tat ttggagaacaagaaatggagt tt t -721 t gggt ttgtat aat tctcccagtgcaaatccgatgtagccaacaaccggcaccagctgtaatattgatattctcatattttitatttcgtgsaaaageccct tgatgatactstaaactaaa -601

cla cgactgttcttgtatt tcgttattaatcgataattattagctggtattgtcttttttttttttttcctacatgatcgtcgcgaggctttacataatt tttgtgacgcctttaaaagtata -481 gtagaacgagcacataagaagtaaatgaaaagt acggcagatgcaat tgacagtaaagagcggtacaatagt tgaagaatacccgataaaaatgtat t taggt tgct tgacgggtt tgca - 361

gcaattgccaagaacactaaciEgtggcaat ct tggtgagaatct actcccaacccaaacattt tcgccgatatttttgctcacct ttt ttt t ttgctcat'cgaaait-tgt tat agcggct

-2 41

taatctaca'gcgatggatacttaaatcat

-121

cgact ttgatt tactaatacacccaat ttctaatatcctc~aggctagatcgttctctcaaggaacttaaagtgcctgatettgcgatgat

gtaaaaatctcagt t ttgaagtcgt ttgc tctct tccatgc ttcagt tcccttt tggaaggtaatataatcatc taatt tetcgcatattacagccgacg'aagaacgagccggatcaat

-1

ATGGTTAGAACCCGTGTATTATTCTGCTTATTTATATCmTGr7rCTACGGTTCAATCGAGTGCTACACTTATTAGCACTTCATGTATTTCCCAAGCTGCGCTATACCAATTTGGATGT M V R T R V L F C L F IS F F _A T V Q S S A T L I S T S C I S Q A A L Y Q F G C Ew TCTAGTAAATCTAAAAGTTGCTACTGTAAAAACATCAATTGGCTGGGTTCAGTGACAGCATGTGCCTATGAGAATTCCAAATCTAACAAAACACTAGACAGCGCCTTAATGAAGTTAGCA

12 0 40

A

240 80

TCCCAATGTTCAAGCATCAAAGTTTATACTTTAGAGGACATGAAGAATATTTATTTAAATG;CGTCAAATTATTTGAGAGCACCTGAGAAAAGTGATAAAAAAACCGT>TAGTCAACCG P

3 60 120

CTCATG&CGAACGAGACAGCGTATCATTATTATTATGAGGAAAATTAT>ATCCATCTTAACCTAATGCGCTCTCAAT>GCGCTT&.GG&TCTCGTCTTCTTCTG.GGT&&CTGT&CTT W V L

480 160

ACTGCAGCCACTATCTTGAACATTCTGAAAAGGGTGTTTGGTAAGAACATCATGGCAAACTCCGTCAAAAAATCACTTATTTATCCTTCTGTTTACAAAGATTATAATGAACGAACTTTT

600 200

S

S L

T

S

Q M

A

K C A A

S S N

T

K

S

E I

S I T

L

C K A

N

Y V

Y

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C Y H

L

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Y

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F

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N

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A

A

N

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N

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A

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TATmATGGAAGCGTCTACCATTTAATTTTACAACTCGAGGCAAGGGTCTCGTCGTATTAATTTTTGblTTATTTTG'ACTATATTATCTCTCAGTTTTGGTCATAATATTAAACTTCCACAC

72 0

H

240

CCATATGATAGGCCCAGAT&GAGAAGAAGTAT&GCCTTTG;TGAGTCGTAGAGCAGACTTGAT&&CCATTGCACTTTTCCCAGTAGTCTATCTATTCGGAATAAGAAATAATCCCTTCATC

84 0 280

Y

P

L

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R

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F

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N

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F

F

G

G

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N

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F

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9 60 320

GTGTTTCAAAGTCTGGTTAGGAAATTTTACTTAGGTr.GGGTATAGTGGCAACGATATTAATGTCTATTATTATTTTCCAAAGTGAAAAAGTATTTAGAAATAGAGGGTATGAGATATTC F

1080 360

CTTCTTATTCATAAAGCGATGAATATTATGTTCATTATTGCCATGTACTACCATTGTCACACCCTGGGCT&GAT&&GGTT&GATTTGGTCAAT&GCTGGTATTTTATGCTTTGATAGATTC F

1200 400

TGCAGGATTGTTrAGAATAATCATGAATGGT&GCTTGAAAACTGCTACTTT&AGTACCACTGATGATTCTAATGTTATTAAAATTTCAGTAAAAAAACCAAAGTTrTTTCAAGTACCAAGTA V K F F K Y

132 0 440

GGAGCTTrrCGCATACATGTAT'TTCTTATCACCAAAAAGTGCATGG&TTCTATAGTTTCCAATCACATCCATTACAGTATTATCGGAACGACACCGTGATCCAAACAATCCAGATCAATTG L N P D

144 0 480

CCTATAACA&GGCTTTCCTTTTCTACATTTAATTTCTATCATAAAT>CT&CCTACGTTTGTTTCATGTTG&CCGTTGTACACTCAATTGTCAT&ACCGCCTCGGGAGTGAAAA&AG&T P V L C

G

I F L R

A

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Q I I

F

G

S H V

A

L

L K R

Y

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V A I

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ACGATGTACGTAAAGGCAAATAAAG&TATCAC TCGAG TTTTGTTATCGAAAGTTC TAAGTGCTCCAAATCATACTGTTGATTGTAAAATATTCCTTGAAGGCCCATAT>GTAACGGTT 1560 520 T M Y V K A N K G I T R V L L S K V L S A P N H T V D C K I F L E G P Y G V T V Bst

CCACATATCGCTAAGCTAAAAAGAAATCTGGTAGGTGTAGCCGCTGGTTTGGGTGTTGCGGCTATTATCCGCACTTTGTCGAATGTTACGGTTACCATCTACTGATCAACTTCAGCAT P

H

I

A

X

L

K

R

N

L

V

G

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A

A

G

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A

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F

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C

L

R

L

P

S

T

D

Q

L

Q

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1680 560

AAA7=TACTGGATTG TTAATGAC CTATCC CATTTGAAATG&TTTAAAATGAATTGCAATGG TTAAAGGAGAAAAGTTGTGAAG TCTC AGTCATATATACTGGTTCC AGTGTTGAGGAC 180 0 600 K F Y W I V N D L S H L K W F E N E L Q W L K E K S C E V S V I Y T G S S V E D

l192 0 ACAAATTCAGATGAGAGTACAAAAGGTTTTGMAT&ATAAAGAAGAAAGCGAAATCACTGTTGAATGTCTCAATAAAAGACCTGATTTGMAAAGAACTAGTGCGCTCGGAAATAAAACTCTCA S 640 T

N

S

D

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G

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R

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GAACTAGAGAATAATAATATTAC CTTTTATTCCTGCGGG CCAGCAACGTTAACGACGATTTTAGAAATGCAGTGGTCCAAGGTATAGACTCTTC CTTGAAGATTGACGTTGAACTAGAA 2 04 0 680 E L E N N N I T F Y S C G P A T F N D D F R N A V V Q G I D S S L K I D V E L E

GAAGAAAGTTTTACATGGTAAggcccc t tgt taataat tc ttgcacgcat actt ttgt tat tttgt tgc t ttatcggataaaagt taatataaatcgatgtaaataatt t tatataatca 2160 686 E E S F T W *

tacaacagggtaataaaaagacgaatatat tcaaatgcgggt tctaaatctaagtttcat tctcactatcactgctttcttcctttgcctgttgagagagtagt tctt tccattt ttgag 2280 2287 tgagctc

FIG. 1. DNA sequence of FREI. Nucleotides are numbered with respect to the translation start so that the A of the initiator ATG is +1. Restriction enzyme sites for Cla I, EcoRI, and BstEII are indicated by the enzyme name over the first nucleotide of the enzyme recognition sequence. The transcription start is shown by an arrow over the A at position -49. The sequence of the 85-bp fragment (-339 to -255) able to confer iron-repressible transcription on downstream elements of a CYCJ-IacZ fusion is enclosed in brackets. The coding region is shown in uppercase letters. Amino acids, indicated in one-letter code below each codon, are numbered beginning with the initiator methionine. A candidate leader peptide is underlined. Potential sites for N-linked glycosylation are underlined. Hydrophobic stretches of amino acids that are candidates for transmembrane domains occur at positions 147-169, 216-236, 258-277, 2%-316, 329-348, 369-397, and 529-550. Figure annotations were made with the DNADRAW (23) program.

Biochemistry: Dancis et al. 150

160

Proc. Natl. Acad. Sci. USA 89 (1992) 180

170

E SY L NFA R KR I KN PE GG LY LA VT L LA GI TGVVIT

FREl

IRNNPF IP ITGLSFSTFNFYHKWSAYVCFM.LAVVHSIV 280

290

300

220

210

G':

FREl

310

230

AT:ILM:1:IDFQSL

330

320

240

340

350

1fK1TIV1.GPM

X-CGD

tKV 13NRY

250

260

340

MY F

350

L:SI M3SAW F Y:SFO:P:QS

450

V1V

40

X-CGD

IY

A

530

I.V N I

500

KON

FRE1

I TI620 5 60

510 I T G LI

520

550

560

480

490

.::'.YLMVM

I

580

50 0 430

tSWYKYCNRX-TNIKL1 : .: Q Hu

540 470

NEM

570

X-CGD

F

f'A::MJ[EQQRNNX

GMCn

4 90

420

9

460

380

370

*:A C G OO

LF

4 80

DYFSYS

450

O M

FREl

360

P0TTMY V:K'A NXZ.:TRV L L:S.:K VLS APN H T

410

520

510 440

.P N N

400

AVDMF

KLP

S E R HR 4 70

390 X-CGD

380

J-:r:EE :R

VK

FRE1

370

R

LGw.--

-AM Y Y HL

F:WR-S:QQKV:V:IDKVVUH PF:K~~TD.~E-~LQ~M -1GMEd :K S:Vff P F M YQj~jAF'AI 43 410 420 04440

400

330

X-CGD

M NI MFDI

360

:L:MEWL:

L:Y:-

&''F'

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390

I: L:LIHKA

H

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H:LF.V.::T..:F F.OLA DHGAMWRV GOQ.T.:AWS't..:A:VHN:I:TVCEQK USEWG:~KIK~ELPI:P Q.F.A GN P P

X-CGD

590

600

530

540

A~~

A

V

H.HD0:EW

DD1(-:E:E 610

550

BQK.TIIYGR PNWD.N::F:K~T:I-A:$:Q:HP NTR UG:ViFL WEAlE:A:A:E:T:L:$.'K:QS:I~SN-S-EMGP

JE C L N

IZP 1RPKD

EL V R

630

SS

KLS:

AT

640

650

660

670

57 0

kG V H F IF7:NKW2NU K -:I6V E L::E:E

X-CGD FREl

of the

200

190

LCLIIT'!S'STKTIR~RW1YFEVEIV8NWT

X-CGD

Cla

fragment revealed a single long open reading (bp 1-2061, Fig. 1). To confirm the correct reading frame and location of the ORF, we fused a fragment including FREI 5' noncoding sequences and the first three codons to an E. coli lacZ reporter gene (bp -977 to +9, Fig. 1). When lacZ was fused in the same reading frame as the predicted FREI ORF, the construct (pEG2) gave rise to f8-galactosidase activity that was regulated by iron in yeast (see below), whereas constructs with lacZ fused at nearly the same position in each of the other two reading frames gave no detectable enzyme activity. An iron-regulated 3-kb transcript was detected previously by RNA blot-hybridization analysis (9). The 2-kb EcoRI-Sac I fragment used as the probe includes the ORF from bp 192 to its 3' end (Fig. 1). The size of this transcript is sufficient to encode the FREl protein. The major transcription start of FREI was mapped by primer I

frame (ORF)

extension to the A nucleotide at -49 with respect to the ATG

initiation codon (data not shown),

predicting

a

49-nucleotide

region of FREI mRNA that is devoid of AUG triplets. Homology Between FREl and the Large Subunit of Human Cytochrome b5m. The predicted FREl protein is 686 amino acids long with a calculated molecular mass of 78.8 kDa. There are six potential sites for the addition of N-linked sugars (24) (Fig. 1). The first 22 amino acids conform to the von Heijne consensus for the leader peptide of a membrane or secreted protein (25) (Fig. 1). Hydrophobicity analysis (26) revealed two amino-terminal hydrophobic regions that are untranslated

strong candidates for transmembrane domains and five other

hydrophobic regions that may also cross the membrane (coordinates in Fig. 1 legend). Comparison of the FREl sequence with the protein data bank (National Biomedical Research Foundation, June 1991) by the FA5TA algorithm (27, 28) revealed similarity to the large subunit of the human cytochrome b558, also known as

3871

FIG. 2. Homology of FRE1 and X-CGD protein. The FRE1 and the X-CGD protein amino acid sequences were aligned by using the FASTA program (27). The optimal alignment score of 171 is > 16 standard deviations above the mean optimal score determined by aligning the FRE1 sequence with 500 randomly generated amino acid sequences of the same amino acid composition as FREl (28). The DNADRAW program (23) was used to depict the alignment of the FRE1 and the X-CGD proteins, with black background indicating identical residues and shading indicating conserved residues.

(29). The carboxyl-terminal 402 the X-CGD protein share 17.9o identity and 62.2% similarility. In addition, there are several clusters of much higher i4 lentity, such as KK-K-FK--VG beginning at amino acid pi)osition 431, HPFT at 462, KI--GP--G at 509, and V-AG-C 'V at 533 of the FREl sequence. Characterization of FREII Disruption/Deletion Strains. We used one-step gene replac,ement (12) to construct haploid strains in which the FREI girenomic sequence was interrupted and/or deleted. FREI is niot required for viability, and the rate of growth of the haplo~ )id strains in iron-replete medium indistinguishable fronfn that of the isogenic wild-type strain (compare H1085 wit] Ih W218 in Table 1). The deletion of FREI led to growth retaardation in iron-depleted medium (Table 1) and impaired ferricc reductase activity and ferric iron describted previously for thefrel- allele uptake (Table 2), (9). The presence of nparable ferrous iron uptake in the X-CGD

protein (Fig.

amino acids of FREl

21

and

was

as

con

wild-type and AfreJ]:: URA3i strains indicates that the ferrous uptake system is independlent of the FREI gene. Furthermore, expression of FREI) from the high-copy-number plasmid pWDC2O in strain WE426 led to ferric reductase activity 3-old higher than that seein in the parental wild-type cells, whereas the rate of iron the wild type

ulptake was indistinguishable from

(Table 2). The

strains carrying the AfreJ:: URA3 of wild-type)

disruption/deletion retaine d residual (5-20% ferric reductase activity an(Id ferric iron uptake residual ferric reductase

activity. This regulated by the medium, being repressed about

acttivity was clearly

iron content of the growth

by iron (Table 1). Transcriptional Regulatio)n of FREI bp of the 5' noncoding regi ion and the

20-fold

by Iron. We fused 977

first three codons of coli lacZ gene on the high-copynumber plasmid pEG2. Thilis construct gave rise to P-galactosidase activity in wild-tylpe cells that was regulated by the iron content of the growth [medium, showing 55-fold higher

FREI

in-frame with the E.

Table 1. Effect of FREI deletion on ferric reductase activity and iron-dependent growth Iron-depleted medium Iron-containing medium Relevant Ferric reductase, Ferric reductase, Doubling Doubling Strain genotype nmol/hr per 106 cells time, min nmol/hr per 106 cells time, min H1085 FREI 12 + 0.3 271 ± 11 0.32 ± 0.01 274 ± 5.0 W218 2.1 ± 0.17 449 ± 8.5 0.11 ± 0.01 295 ± 3.0 AfreI::URA3 13 ± 0.12 282 ± 20 0.28 ± 0.02 293 ± 20 W218/pCL4.3 Afrel::URA3/FREJ Cells were grown in iron-depleted medium or iron-supplemented MD medium prior to assay of ferric reductase activity.

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Biochemistry: Dancis et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

by deletion to -279 (pJ107). Examination of the FREI sequence in the interval between -341 and -279 revealed a sequence (-311 to -298) that matches at 11 of 14 positions with the consensus binding site for RAPi (30), a multifunctional yeast protein known to serve as a transcriptional activator in some sequence contexts (31). While the expression of (3-galactosidase activity in pJ106 was significantly repressed by iron, the loss of expression with deletion to -279 precluded the ability to assess whether an ironregulatory element is present between -341 and -279. We therefore asked whether FREI sequences from this region could serve as an upstream activating sequence (UAS) in a CYCI-lacZ fusion from which the endogenous UAS was deleted. The parental construct, which contained 250 bp of CYCI 5' noncoding sequence (including the transcription start site and the ATG start codon) fused in-frame to the E. coli lacZ gene, gave negligible (6. These results suggest that RAP1 activates FREI transcription but is not sufficient for iron-mediated regulation.

Table 2. Effect of overexpression and disruption of FREI on femc reductase activity and iron uptake Ferrous Ferric Ferric uptake, uptake, reductase, fmol/hr fmol/hr nmol/hr per 106 per 106 per 106 Relevant cells cells cells Strain genotype 2200 ± 540 79 ± 7.6 30 ± 1.1 F113 FREI 2200 ± 320 79 ± 16 93 ± 7.5 W126 FREI (high-copy) 120 ± 0.2 120 ± 3.7 3.5 ± 0.24 N1 4frel::URA3 Cells were grown in iron-depleted MD medium prior to assay.

expression in MD medium depleted of iron than in MD containing iron (420 units and 7.6 units, respectively). The range of regulation of 83-galactosidase activity from this construct was similar to that seen for ferric reductase. These results suggest that regulation of FREI expression by iron occurs at the level of transcription. To locate more precisely the regulatory sequences, we examined the effects of 5' deletions of FREI sequences beginning at position -977 in the low-copy-number plasmid pJ105 (Fig. 3A). For reasons not well understood, the range ofregulation by pJ105 was less than that by pEG2. The high activity present under low-iron conditions in the construct pJ105 was essentially unaffected by removal of sequences 5' to -341 (pJ106) but was abolished

A

FREl SEQUENCE

PLASMID

P-Galactosidase (U) Ratio -Fe +Fe

pJ 105

-977

pJ111

-600

pJ 106

-341

-900

B

-800

-700

-600

i0 -500

I

-400

-

7.4

7.9 1.9

4.1

+3

10

1.4

7.2

0.3 0.4

0.8

-0 - 200

1 -

100

I3-Galactosidase (U) Ratio - Fe/+ Fe -Fe +Fe

-280

-317

-280

-317 X

-278

-339

pGC7-8 pGC7-8.0

-255

-339

pGC7-8.5 pGC7-9 pGC8-9

-176

-254

pGC6-9

R -

-400

-176

E

-2

4 -4

-440

3

30

5.9 0.8

+3

FREl SEQUENCE

PLASMID pGCR pGCRM

C

rnn -0

+3

+3

-279 R E

pJ107

-360

280

- Fe/+ Fe

240

9.8 13

0.74

1.5 1.9

0.74

13

13

1.0

99

29

3.4

141

26

5.4

88

12

6.9

0.3

29

0.7

0.43

4.4

6.5

- 200 -200

-25 -39 AAACATT CGCCGATATTTTTGCTCACCTTTTTTTTTTGCTCATCGAAAA

TGTGGCAATCTTGGTGAGAATCTACTC

AA

ACACCGTTAGAACCACTCTTAGATGAG

TT

a

TTTGTAA

GCGGCTATAAAAACGAGTGGAAAAAAAAAACGAGTAGCTTTT

FIG. 3. Localization of iron-responsive transcriptional regulatory elements in the FREI promoter. (A) Deletion of 5' noncoding sequences from FREJ-IacZ constructs. pJ105, pJ111, pJ106, and pJ107 are low-copy-number plasmids containing FREJ-4acZ fusions. FREI sequences are represented by solid lines, and the 5' and 3' boundaries of these sequences are indicated by the numbers adjacent to the lines. The scale indicates the position of these sequences relative to the A of the ATG codon of the FREI ORF. The locations of the RAPi consensus binding site (R) and repeat element TTTTTGCTCAYC (E) are shown on this scale. The FREJ-4acZ constructs were introduced into strain F113 and assayed for 3-galactosidase expression (units, U) in iron-depleted (-Fe) or iron-supplemented (+Fe) medium. (B) FREI sequences confer iron regulation upon CYCJ-4acZ expression. The plasmids are low-copy-number plasmids containing FREI sequences fused to downstream elements of a CYCIJ-acZ fusion from which the UAS has been deleted. The boundaries of these FRE1 sequences are indicated by the numbers adjoining the solid lines. The X represents the location of a point mutation in the RAPi consensus binding site. The scale indicates the position of these sequences relative to the A of the ATG codon of the FREI ORF. The RAPi consensus binding site (R) and repeat (E) are indicated. Plasmids were introduced by transformation into wild-type strain F113 and analyzed as in A. (C) Minimal FREI sequence able to confer iron-repressible transcription on a UAS-deleted CYCJ-IacZ fusion. The FREI sequence contained in pGC7-8.0 is depicted. The RAP1 consensus binding site is boxed. The point mutation in this site shown to impair function is shaded. The repeat is indicated by arrows.

Biochemistry: Dancis et al. We next studied the effect of adding flanking FREI sequences to the pGCR construct containing the RAP1 binding site from FREI. Addition of FREI sequence from between -317 and -339 resulted in negligible changes in derepressed activity or repression by iron (PGC7-8 in Fig. 3B). However, further addition of FRE1 sequence, -277 to -255, led to a 7.5-fold increase in activity under low-iron conditions and the acquisition of iron-repressible expression (compare pGC7-8 and pGC7-8.0 in Fig. 3B). The 85-bp sequence in pGC7-8.0 that confers iron-repressible transcription on downstream elements of a CYCI-lacZ fusion contains a 12-bp direct repeat, TlTIT'GCTCAYC, located 3' to the RAPI binding site. A search of sequence motifs for the recognition sites of DNA-binding proteins failed to reveal any match with this repeated sequence (32). Inserting sequences to positions -208 or -176 resulted in an increased range of regulation by iron similar to that seen for the larger promoter fragments in pGC6-9 and pJ105, and thus a second iron-control element may be present 3' of -255.

DISCUSSION The mechanism(s) by which eukaryotic cells acquire iron from the environment has remained one of the unsolved problems in the biology of this essential nutrient. Although an externally oriented ferric reductase has been proposed to be important for iron uptake by many eukaryotes, until recently, no molecular evidence for, or characterization of, such a system had been reported. We initially provided genetic evidence suggesting that the FREI gene of S. cerevisiae is required for high-level ferric reductase activity, ferric iron uptake, and the ability to grow in iron-limited environments. Here we characterize that gene at the molecular level. Complete deletion of FREI in wild-type cells led to reduced ferric reductase activity, deficient ferric iron uptake, and impaired growth on iron-depleted medium. Ferrous assimilation was unimpaired in strains containing a disruption or deletion of FREI, demonstrating that the ferrous uptake system is independent of FREI. The DNA sequence of FREI suggests that it encodes a structural component of the externally directed reductase. The deduced FRE1 protein sequence of 686 amino acids contains a potential leader sequence and several hydrophobic regions consistent with transmembrane domains. In addition, the FRE1 amino acid sequence shows significant similarity to the large subunit of cytochrome b558, also known as the X-CGD protein, an essential component of a multisubunit reductase present in the plasma membranes of human phagocytic cells. Cytochrome b558 is thought to be the terminal component of a respiratory chain that transfers single electrons from cytoplasmic NADPH across the plasma membrane to molecular oxygen on the exterior (29). FREI may not encode the only reductase used by S. cerevisiae in iron reduction and iron uptake, as suggested by the residual ferric reductase and ferric iron uptake activities of the Afrel:: URA3 strains. Ferric iron in the growth medium represses the residual reductase activity of these strains, a feature consistent with an activity involved in iron uptake. The existence of an alternative uptake system, perhaps one of lower affinity than that involving FRE1, is also suggested by the ability of high concentrations of ferric iron in the medium to completely correct the growth deficiency of the strains carrying the FREI disruptions/deletions. As with other iron uptake systems, in S. cerevisiae, increased availability of iron leads to reduced expression of an essential component of the uptake system. FREI expression is controlled by alterations in the rate of transcription of its mRNA. This was demonstrated by the ability of FREI 5' nontranscribed sequences to transfer this regulation to the E. coli lacZ gene. The range of iron regulation by the complete FREI-lacZ fusion in the high-copy-number plasmid pEG2

Proc. Natl. Acad. Sci. USA 89 (1992)

3873

closely reflects the iron-induced changes in ferric reductase. An 85-bp FREI sequence element sufficient for transferring iron-dependent activity to a CYCJ-IacZ construct lacking its native UAS includes a consensus binding site for RAP1 and the repeated sequence, T-`TTTTGCTCAYC. This repeated sequence constitutes a candidate binding site for an ironresponsive regulatory protein. The RAP1 binding site by itself is not sufficient for this regulatory effect, but a protein mediating the iron-dependent changes in transcriptional initiation might interact with RAP1. M. Shapiro assisted with the design of Figs. 1 and 2. P. Fitzgerald, V. Zenger, and R. Blasco patiently answered questions related to computer sequence analysis. C. Moehle provided plasmid p1087 and helpful advice and discussions. G.J.A. is supported by a C. J. Martin Fellowship from the National Health and Medical Research Council of Australia. 1. Williams, R. J. P. (1990) in Iron Transport and Storage, eds. Ponka, P., Schulman, H. M. & Woodworth, R. C. (CRC, Cleveland), pp. 2-15. 2. Nielands, J. B. (1981) Annu. Rev. Nutr. 1, 27-46. 3. Crane, F. L., Roberts, H., Linnane, A. W. & Low, H. (1982) J. Bioenerg. Biomembr. 14, 191-205. 4. Yamashoji, S. & Kajimoto, G. (1986) Biochim. Biophys. Acta 852, 25-29. 5. Chaney, R. L., Brown, J. C. & Tiffin, L. 0. (1972) Plant Physiol. 50, 208-213. 6. Bienfait, F. (1987) in Iron Transport in Microbes, Plants and Animals, eds. Winkelmann, B., van der Helm, D. & Nielands, J. B. (VCH, Weinheim, F.R.G.), pp. 337-349. 7. Buckhout, T. J., Bell, P. F., Luster, D. G. & Chaney, R. L. (1989) Plant Physiol. 90, 151-156. 8. Seligman, P. A., Klausner, R. D. & Huebers, H. A. (1987) in The Molecular Basis of Blood Diseases, eds. Stamatoyannopoulos, G., Nienhuis, A. W., Leder, P. & Majerus, P. W. (Saunders, Philadelphia), pp. 219-244. 9. Dancis, A., Klausner, R., Hinnebusch, A. G. & Barriocanal, J. G.

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