`independent of Aft1p
`
`Julian C. Rutherford*, Shulamit Jaron*, Esha Ray†, Patrick O. Brown†, and Dennis R. Winge*‡
`
`*University of Utah Health Sciences Center, Departments of Medicine and Biochemistry, Salt Lake City, Utah 84132; and †Department of Biochemistry,
`Stanford University School of Medicine, Howard Hughes Medical Institute, Stanford, CA 94305-5428
`
`Edited by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, and approved October 19, 2001 (received for review July 23, 2001)
`
`Iron homeostasis in the yeast Saccharomyces cerevisiae is regu-
`lated at the transcriptional level by Aft1p, which activates the
`expression of its target genes in response to low-iron conditions.
`The yeast genome contains a paralog of AFT1, which has been
`designated AFT2. To establish whether AFT1 and AFT2 have over-
`lapping functions, a mutant containing a double aft1⌬aft2⌬ dele-
`tion was generated. Growth assays established that the single
`aft2⌬ strain exhibited no iron-dependent phenotype. However,
`the double-mutant aft1⌬aft2⌬ strain was more sensitive to low-
`iron growth conditions than the single-mutant aft1⌬ strain. A
`mutant allele of AFT2 (AFT2-1up), or overexpression of the wild-
`type AFT2 gene, led to partial complementation of the respiratory-
`deficient phenotype of the aft1⌬ strain. The AFT2-1up allele also
`increased the uptake of 59Fe in an aft1⌬ strain. DNA microarrays
`were used to identify genes regulated by AFT2. Some of the
`AFT2-regulated genes are known to be regulated by Aft1p; how-
`ever, AFT2-1up-dependent activation was independent of Aft1p.
`The kinetics of induction of two genes activated by the AFT2-1up
`allele are consistent with Aft2p acting as a direct transcriptional
`factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex
`containing the Aft1p binding site in vitro. The wild-type allele of
`AFT2 activated transcription in response to growth under low-iron
`conditions. Together, these data suggest that yeast has a second
`regulatory pathway for the iron regulon, with AFT1 and AFT2
`playing partially redundant roles.
`
`metalloregulation 兩 iron homeostasis
`
`Iron is an essential nutrient but it is toxic in the absence of
`
`effective homeostasis. Mechanisms have therefore evolved to
`ensure adequate but not excess levels of reactive intracellular
`iron. In the yeast Saccharomyces cerevisiae, transcriptional reg-
`ulation of the iron regulon is mediated by the transcriptional
`activator Aft1p (1). The Aft1p regulon consists of many genes
`that are involved in the acquisition, compartmentalization, and
`utilization of iron. These include genes involved in iron uptake
`(FET3, FTR1, and FRE1,2), siderophore uptake (ARN1-4 and
`FIT1-3), iron transport across the vacuole membrane (FTH1),
`and iron-sulfur cluster formation (ISU1,2; refs. 1–6). Aft1p binds
`to a conserved promoter sequence in an iron-dependent manner
`and activates transcription under low-iron conditions (2). Mu-
`tants lacking a functional Aft1p grow poorly in iron-limiting
`conditions (1, 7). A Cys291Phe substitution within Aft1p results
`in derepressed transcriptional activation in iron-replete cells (1).
`Because Cys291 is part of a Cys-X-Cys motif, an attractive
`hypothesis is that Aft1p is capable of binding iron; however, the
`mechanism by which Aft1p senses iron levels is not known.
`The S. cerevisiae genome has many duplicated chromosomal
`regions that account for up to 16% of the yeast proteome, and
`it has been proposed that these regions have arisen from an
`ancient duplication of the entire genome (8). AFT1 lies within
`such a region of chromosome VII, with its duplicate ORF
`YPL202c (designated AFT2), located on chromosome XVI (9).
`Characterization of paralogs that have arisen from the genome
`duplication has revealed differences in the extent to which genes
`within a pair have functionally diverged. The transcription
`
`factors Swi5p and Ace2p have nearly identical DNA-binding
`domains, yet they activate the transcription of different genes
`(10). Alternatively, there may be redundancy of function so that
`a clear phenotype is apparent only in a mutant lacking both
`paralogs, as in the case of the Pcl8p and Pcl10p cyclins (11).
`Previous work relating to the function of AFT1 has suggested
`that there is an AFT1-independent pathway for iron regulation
`in S. cerevisiae (1, 7). We were interested to learn whether such
`a pathway involves AFT2. We have used a combination of
`phenotypic analysis, functional complementation, and analysis
`of global gene expression to identify the relationship between
`AFT1 and AFT2. We present evidence that AFT2 codes for a
`transcription factor that activates gene expression in response to
`low-iron conditions. Although Aft1p and Aft2p are functionally
`similar, and have overlapping functions, comparison of gene
`activation by both transcriptional factors suggests that they also
`have distinct functions.
`
`Materials and Methods
`Yeast Strains and Culture Conditions. The following S. cerevisiae
`strains were purchased from Research Genetics (Huntsville,
`AL) and used in this study: BY4741 (MATa his3⌬1 leu2⌬0
`met15⌬0 ura3⌬0) as wild type; BY4741aft1⌬ (MATa his3⌬1
`leu2⌬0 met15⌬0 ura3⌬0 aft1::kanMX4); BY4742aft1⌬ (MAT␣
`his3⌬1 leu2⌬0 lys2⌬0 ura3⌬0 aft1::kanMX4); and BY4741aft2⌬
`(MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 aft2::kanMX4). A hap-
`loid aft1⌬aft2⌬ strain (MAT␣ his3⌬1 leu2⌬0 lys2⌬0 met15⌬0
`ura3⌬0 aft1::kanMX4 aft2::kanMX4) was isolated after the mat-
`ing of BY4741aft2⌬ and BY4742aft1⌬, and its allele status was
`verified by using PCR and DNA sequencing. Cells were grown
`in either 1% yeast extract, 2% peptone medium (YP), complete-
`synthetic medium (CM), or, when appropriate, complete-
`synthetic medium lacking uracil [CM(⫺Ura)]. These media were
`supplemented with either 2% glucose, 2% raffinose, or 3%
`glycerol as indicated. For plate phenotypes, 10-fold serial dilu-
`tions of cells were spotted onto agar plates and grown at 30°C.
`For anaerobic growth, agar plates were incubated in a GasPak
`(Becton Dickinson) anaerobic chamber.
`
`Vectors. Yeast-genomic DNA from strain BY4741 was isolated
`and used as template for PCR to amplify a 2.6-kb fragment
`containing AFT2, with both upstream and downstream se-
`quences, which was ligated into the BamHI兾XbaI site of the YCp
`vector pRS416 (12) to create pAFT2. QuikChange (Stratagene)
`mutagenesis was used to generate an allele of AFT2 that codes
`for a Cys187Phe mutation by using pAFT2 as template to create
`pAFT2-1up. To place the AFT2-1up allele under the control of the
`GAL10 promoter, pAFT2-1up was used as template for PCR to
`
`This paper was submitted directly (Track II) to the PNAS office.
`
`Abbreviations: BPS, bathophenanthroline disulfonate; EMSA, electrophoretic mobility-
`shift assay; YPD, yeast extract兾peptone兾dextrose; CM, complete medium.
`‡To whom reprint requests should be addressed. E-mail: dennis.winge@hsc.utah.edu.
`
`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.
`
`14322–14327 兩 PNAS 兩 December 4, 2001 兩 vol. 98 兩 no. 25
`
`www.pnas.org兾cgi兾doi兾10.1073兾pnas.261381198
`
`
`
`BIOCHEMISTRY
`
`amplify a 1.3-kb fragment containing AFT2-1up, which was
`ligated into the BamHI兾ClaI site of GFP-pYeF2, a derivative of
`pYeF2 (13), to generate pGAL-AFT2-1up. Plasmid pAFT1-1up
`contains a 3-kb fragment, consisting of AFT1 with both upstream
`and downstream sequences, that originated from pCM16 (ref.
`14; a kind gift from Enrique Herrero, Universitat de Lleida,
`Lleida, Spain), which was ligated into the XhoI兾SacI site of
`pRS316 (12). Site-directed mutagenesis by using the pALTER
`mutagenesis system (Promega) was used to generate an allele of
`AFT1 that codes for a Cys291Phe mutation. To generate plasmids
`that express truncated forms of Aft1p (pAft1-313) and Aft2p
`(pAft2-214), PCR was used to amplify the 5⬘ ends of AFT1 and
`AFT2 containing the first 313 codons of Aft1p and the first 214
`codons of Aft2p, which were then ligated into pET20 and pET3
`(Novagen), respectively. All of the AFT1, AFT2, AFT1-1up, and
`AFT2-1up sequences were confirmed by DNA sequencing. All
`yeast transformations were performed by using the lithium
`acetate procedure. In the case of transformations using the
`aft1⌬aft2⌬ strain, cells were pregrown in yeast extract兾pep-
`tone兾dextrose (YPD) under nitrogen, and the agar plates were
`supplemented with FeCl2 (100 M).
`
`Electrophoretic Mobility-Shift Assay (EMSA). The identification of
`specific protein-DNA complexes was analyzed as described with
`the following modifications (15). The DNA probe consisted of
`oligonucleotide 5⬘-ATCTTCAAAAGTGCACCCATTTG-
`CAGGTGC-3⬘ and its reverse complement, which contains the
`Aft1p-binding site within the FET3 promoter.
`Crude protein extracts were isolated from Escherichia coli
`cells (BL21-CodonPlus(DE3)-RIL from Stratagene) that had
`been transformed separately with pET3, pAft1-313, and pAft2-
`214. Cells were grown to mid-log phase at 37°C with ampicillin
`and chloramphenicol, protein expression was induced, and the
`cells were incubated for an additional 2 h before harvesting. The
`cells were pelleted and resuspended in 1兾50th vol of lysis buffer
`(20 mM Tris䡠HCl, pH 7.5兾1 mM DTT) and sonicated, and the
`cell debris was pelleted. The standard EMSA binding reaction
`consisted of equal volumes of an E. coli cell extract and the
`hybridization mix with the addition of poly(dI-dC)䡠poly(dI-dC)
`to a final concentration of 4 ng䡠l⫺1. The binding reaction was
`incubated for 15 min and applied to a 6% polyacrylamide
`nondenaturing gel and electrophoresed by using Tris-borate-
`EDTA buffer.
`
`mRNA Quantification by S1 Nuclease Analysis. Total RNA was
`isolated from mid-log cells and was hybridized with 32P-labeled,
`single-stranded DNA oligonucleotides that were complementary
`to the candidate gene (between 50 and 69 nt) and the control
`gene CMD1 (40 nt). After digestion with S1 nuclease, the
`samples were electrophoresed through an 8% polyacrylamide兾5
`M urea gel.
`
`Microarray Analysis. Cells were harvested from 300 ml of culture
`at an A at 600 nm of 0.4. Total RNA was isolated by the hot
`phenol method. mRNA was isolated from total RNA by using
`the PolyATtract mRNA Isolation System IV kit from Promega
`following the manufacturer’s instructions. Cy3-dUTP or Cy5-
`dUTP (Amersham Pharmacia) was incorporated during reverse
`transcription of the polyadenylated RNA. The fluorescently
`labeled product was recovered and hybridized to microarrays,
`which were washed and scanned as described (16).
`
`GF兾C filters, which were then washed, and the level of 59Fe was
`measured. In some cases, the Fe(II) chelator bathophenanthro-
`line disulfonate (BPS) was added to the cell cultures 4 h before
`the cells being harvested.
`
`Results
`S.cerevisiaeContains a Paralog of AFT1. Analysis of the S. cerevisiae
`genome has revealed two similar regions of chromosomes VII
`and XVI that contain the paralogs AFT1 and AFT2 (8). The
`predicted product of AFT2 exhibits strong identity to Aft1p from
`residues 38 to 285 (39% identity; Fig. 1). The homologous region
`includes the N-terminal basic region of Aft1p, which is predicted
`to represent the Aft1p DNA-binding domain, and the region that
`contains the Cys-X-Cys motif that confers iron sensitivity. The
`two histidine-rich regions of the extreme N terminus and C
`terminus of Aft1p are not conserved in Aft2p. It has been
`proposed that these regions may be involved in iron binding (1).
`The high degree of identity within the N terminus of both
`proteins encourages the prediction that AFT2 codes for a
`DNA-binding protein.
`
`The Effect of Iron on the aft Mutants. To determine whether AFT2
`plays a role in iron homeostasis, the growth of strains with either
`a single deletion of AFT1 or AFT2, or the double deletion of both
`genes, was compared. The growth of a strain lacking AFT1 is
`impaired under low-iron conditions, and this defect is exacer-
`bated when the medium contains a nonfermentable carbon
`source (7). As expected, the aft1⌬ strain failed to grow on
`medium containing glycerol, whereas the aft2⌬ strain grew
`equivalently to the wild-type strain (Fig. 2). The growth defect
`of the aft1⌬ strain was partially reversed in an aft1⌬ strain
`harboring low-copy plasmids containing either the wild-type
`AFT2 allele (pAFT2) or the AFT2-1up allele (pAFT2-1up). The
`AFT2-1up allele contains a mutation that codes for a Cys187Phe
`substitution, which corresponds to the mutation within the
`AFT1-1up allele that codes for a Cys291Phe substitution.
`The growth defect of the aft1⌬ strain is less pronounced on
`medium containing a fermentable carbon source, and it can be
`suppressed in three ways (Fig. 3A). The aft1⌬ strain grows
`normally on complex YPD medium containing glucose, in the
`absence of oxygen. Alternatively, full aerobic growth can be
`attained by pregrowth of the aft1⌬ strain in liquid medium
`supplemented with Fe(II) or by supplementing the agar with
`Fe(II) (Fig. 3A). The aft1⌬aft2⌬ strain exhibits a more exagger-
`ated iron-deficient phenotype under aerobic conditions (Fig.
`3A). This growth defect is reversed only by supplementing both
`the preculture and the agar with exogenous Fe(II). As in the case
`of the aft1⌬ strain, the aft1⌬aft2⌬ strain grows fully under
`anaerobic conditions.
`the
`The exaggerated iron-deficient growth defect of
`aft1⌬aft2⌬ strain relative to the aft1⌬ strain is also apparent on
`minimal medium (Fig. 3B). The addition of the Fe(II) chelator
`BPS to the agar markedly attenuates the aerobic growth of the
`aft1⌬aft2⌬ strain even when it is pregrown in liquid medium
`supplemented with Fe(II). The addition of BPS to cultures is
`known to lower iron availability, resulting in Aft1p activation (1,
`18). In contrast to growth on YPD, supplementation of the
`minimal medium agar with Fe(II) fully restores growth of the
`aft1⌬aft2⌬ strain. The growth defect of the aft1⌬aft2⌬ strain is
`also reversed when it is transformed with either pAFT2 or
`pAFT2-1up (data not shown).
`
`59Fe Uptake Analysis. Iron uptake was analyzed as described (17).
`Briefly, mid-log phase cells were grown in complete synthetic
`medium and harvested, and the cells were washed and resus-
`pended in low-iron medium plus 1 mM ascorbate. 59Fe (0.5 M)
`was added to the cells, which were then incubated at 30°C for 10
`min. The cell cultures were then filtered through Whatman
`
`The Effect of the AFT2-1up Allele on Gene Expression Within S.
`cerevisiae. The exacerbated growth phenotype of the aft1⌬aft2⌬
`strain and the observed sequence similarity between Aft1p and
`Aft2p are consistent with both proteins having partial overlap-
`ping functions. We hypothesized that Aft2p may be a transcrip-
`tional activator and that the AFT2-1up allele may result in the
`
`Rutherford et al.
`
`PNAS 兩 December 4, 2001 兩 vol. 98 兩 no. 25 兩 14323
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`
`
`Sequence comparison of Aft1p and Aft2p. The amino acid sequences of Aft1p and Aft2p of S. cerevisiae were aligned by using the CLUSTALW program.
`Fig. 1.
`The conserved CXC motif is underlined and in bold (see text).
`
`derepressed transcriptional activation of genes regulated by
`Aft2p. DNA microarray experiments were conducted to identify
`candidate target genes regulated by Aft2p. We compared the
`differential gene expression of the aft2⌬ strain harboring either
`pAFT2-1up or a control vector. These microarray experiments
`were conducted at both the Stanford Microarray Facility and the
`Huntsman Cancer Institute Microarray Facility at the University
`of Utah. A number of genes were reproducibly elevated in their
`expression in the strain containing the AFT2-1up allele (Table 1).
`These include genes that are known to be regulated by Aft1p
`(FIT1, FIT3, FTR1, FTH1, FRE1, and TIS11) and by the zinc-
`responsive transcription factor Zap1p (ZRT1 and YOL154w;
`refs. 1, 4, and 19). Genes that are not known to be metal-
`loregulated by either Aft1p or Zap1p were also identified
`(MRS4, UBC8, PRB1, and ECM4) (Caroline Philpott and Dave
`Eide, personal communications).
`To confirm the microarray data, S1 analysis was performed to
`
`AFT2 partially complements the aft1⌬ mutant strain. Overnight
`Fig. 2.
`cultures of each strain were grown in CM(⫺Ura) medium containing glucose.
`The cultures were washed, and 10-fold serial dilutions were spotted onto
`CM(⫺Ura) agar plates containing either glucose or glycerol, which were then
`incubated at 30°C.
`
`quantify the expression of selected AFT2-1up induced genes. The
`expression of FIT3, FIT1, and MRS4 was clearly activated in the
`aft2⌬ strain containing pAFT2-1up but not in the control strain
`or the strain containing pAFT2 (Fig. 4A). The AFT2-1up allele
`also activated the expression of FIT3, FIT1, and MRS4 in an
`aft1⌬ strain, demonstrating that the AFT2-1up allele activates
`transcription in an Aft1p-independent manner (Fig. 4B). The
`ZRT1 and YOL154w genes were induced in only a subset of the
`S1 experiments (data not shown). Activation of these two genes
`was not observed in a zap1⌬ strain harboring pAFT2-1up, sug-
`gesting that their induction was the indirect result of fluctuating
`concentrations of intracellular zinc.
`The AFT2-1up allele was placed under the control of a
`galactose-inducible promoter to test whether it directly regulates
`its target genes. The aft2⌬ strain containing the
`two of
`GAL10兾AFT2-1up construct was precultured in raffinose. Ga-
`lactose was then added to induce the expression of AFT2-1up,
`which was maximal within 15 min (data not shown). Both FET5
`and UBC8 were significantly induced within 15 min of the
`addition of galactose (Fig. 5A). No induction was observed in the
`aft2⌬ strain lacking the GAL10兾AFT2-1up construct. The coin-
`duction of AFT2-1up, FET5, and UBC8 is consistent with Aft2p
`being a direct transcriptional regulator.
`Aft1p is activated in iron-deficient cells and inhibited in
`iron-replete cells (1). To determine whether wild-type Aft2p is
`similarly iron regulated, the aft1⌬aft2⌬ strain containing either
`pAFT2 or pAFT2-1up was cultured under iron-deficient and
`iron-replete conditions (Fig. 5B). The AFT2-1up allele induced
`the expression of FIT3 in cells cultured in either BPS or Fe(II),
`demonstrating that the mutation in this allele results in dere-
`pressed activity (Fig. 5B). In contrast, the wild-type AFT2 allele
`induced the expression of FIT3 only in iron-deficient conditions.
`Therefore, Aft2p induces gene expression in iron-deficient cells.
`
`Aft1p and Aft2p Bind to the Same Region of the FET3 Promoter. To
`determine whether the level of transcriptional activation by
`
`14324 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.261381198
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`Rutherford et al.
`
`
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`BIOCHEMISTRY
`
`Table 1. Genes activated by the AFT2-1up allele
`Target gene Cy5兾Cy3 ratio
`
`Function
`
`FIT3
`YOL154w
`ZRT1
`FIT1
`ECM4
`YOL083w
`UBC8
`YJR078w
`MRS4
`TIS11
`FTR1
`FTH1
`LAP4
`PRB1
`GRX4
`AHP1
`HCR1
`FET5
`PEP4
`YDL124w
`FRE1
`SMF3
`
`8.8
`8.8
`6.2
`5.7
`5.0
`4.2
`4.1
`3.6
`3.5
`3.3
`3.3
`3.2
`3.0
`2.9
`2.8
`2.7
`2.6
`2.6
`2.6
`2.5
`2.2
`2.0
`
`Cell wall protein
`Zn-metalloprotease-like protein
`High affinity zinc transporter
`Cell wall protein
`Cell wall structure兾biosynthesis
`Unknown
`Ubiquitin-conjugating enzyme
`Similar to indoleamine 2,3-dioxygenase
`Member of mitochondrial carrier family
`Similar to mammalian TIS11 family
`High affinity iron transporter
`Vacuolar iron transporter
`Vacuolar aminopeptidase I
`Vacuolar protease B
`Glutaredoxin
`Alkyl hydroperoxide reductase
`Putative component of eIF3
`Vacuolar multicopper oxidase
`Vacuolar proteinase A
`Unknown
`Cell surface iron reductase
`Vacuolar iron transporter
`
`Transcripts of these genes were at least 2-fold more abundant in the strain
`containing the AFT2-1up allele compared with the control strain. The value
`shown is the Cy5兾Cy3 ratio (which reflects the ratio of the transcripts’ abun-
`dance in the two strains) measured in one experiment at the Stanford Mi-
`croarray Facility. The same genes were identified in two similar experiments
`conducted at the University of Utah Microarray Facility.
`
`induction of genes involved in iron uptake (FET3, FTR1 and
`FRE1) suggested that iron uptake would be enhanced in cells
`containing AFT2-1up. To determine whether AFT2 stimulates
`
`S1 nuclease protection assays to quantify mRNA levels of genes
`Fig. 4.
`activated by the AFT2-1up allele. RNA was isolated from (A) the aft2⌬ strain
`and (B) the aft1⌬ strain transformed with pRS416 (⫺), pAFT2 (⫹), and pAFT2-
`1up (up) and grown in CM(⫺Ura) with glucose medium to mid-log phase. The
`upper band for each sample is the specified gene, and the lower band the
`calmodulin loading control (CMD1).
`
`The effect of iron on the growth of the aft mutant strains. Overnight
`Fig. 3.
`cultures of the wild-type strain and each of the aft-mutant strains were grown
`in liquid CM(⫺Ura) with glucose medium. Each strain was grown in duplicate,
`with one of each pair being supplemented with FeCl2 (100 M). Iron supple-
`mentation in the pregrowth medium is indicated by ‘‘⫹ Fe.’’ The cells were
`washed, and 10-fold serial dilutions were spotted onto (A) complex medium
`(YPD) agar plates with or without supplemented FeCl2 (100 M) or (B) syn-
`thetic medium (CMD) agar plates with or without supplemental FeCl2 (100
`M) or BPS (10 M). Plates were incubated at 30°C and, in the case of one YPD
`plate, under anaerobic conditions.
`
`Aft1p and Aft2p is the same, the expression of FET3, FET5, and
`MRS4 was analyzed in an aft1⌬aft2⌬ strain containing either the
`AFT1-1up allele or the AFT2-1up allele (Fig. 6A). In the strain
`carrying the AFT1-1up allele, FET3 is more highly expressed than
`in the strain carrying the AFT2-1up allele. This pattern of
`expression is reversed with respect to MRS4, which is more highly
`expressed in the strain carrying the AFT2-1up allele. The level of
`activation of FET5 is the same in the strains containing either the
`AFT1-1up allele or the AFT2-1up allele. Therefore, the AFT1-1up
`and the AFT2-1up alleles differentially activate gene expression.
`To address whether Aft2p can bind to the same DNA site as
`Aft1p, EMSAs were performed using a 30-bp fragment that
`included the Aft1p-binding site within the FET3 promoter. The
`Aft1p consensus binding site is PyPuCACCCPu and was iden-
`tified by DNase1 footprinting of the FET3 promoter and se-
`quence comparison of Aft1p-regulated genes (2). Truncated
`forms of Aft1p (Aft1p313) and of Aft2p (Aft2p214) containing
`the conserved putative DNA-binding domains of each protein
`and the conserved Cys-X-Cys motif were used. A specific single
`complex was formed between the FET3 promoter fragment and
`lysate from E. coli cells that contained plasmids that separately
`expressed the truncated forms of Aft1p and Aft2p (Fig. 6B). No
`complex was formed by using lysate from control E. coli cells that
`did not express Aft1p313 or Aft2p214. Aft1p and Aft2p are
`therefore capable of binding to the same DNA fragment in vitro.
`
`Iron Uptake by the aftMutants. Iron uptake is markedly attenuated
`in a strain lacking a functional AFT1 (1). The observed AFT2-1up
`
`Rutherford et al.
`
`PNAS 兩 December 4, 2001 兩 vol. 98 兩 no. 25 兩 14325
`
`
`
`Fig. 5. Aft2p is a direct transcriptional activator that responds to low iron.
`S1 nuclease protection assays were carried out by using RNA isolated from (A)
`the aft2⌬ strain transformed with either pGAL-AFT2-1up or pYeF2 (control),
`which were grown in CM(⫺Ura) with raffinose to mid-log phase, at which time
`2% galactose was added to induce the transcription of the AFT2-1up allele.
`Cells were harvested at the specified times (B), the aft1⌬aft2⌬ strain was
`transformed with pRS416 (control), pAFT2, and pAFT2-1up, which were pr-
`egrown overnight with 10 M FeCl2, harvested, washed, and resuspended in
`CMD(⫺Ura) medium with or without supplemental FeCl2 (100 M) or BPS (100
`M) and then grown exponentially for 8 h. The Upper band for each sample
`is the specified gene, and the Lower band the calmodulin loading control
`(CMD1).
`
`iron uptake, the level of 59Fe uptake was quantified in all of the
`aft mutant strains. The level of 59Fe uptake was similar between
`the wild-type and the aft2⌬ strain but was significantly reduced
`in the aft1⌬ and the aft1⌬aft2⌬ strains (Fig. 7A). This result
`suggests that the chromosomally encoded Aft2p is not a signif-
`icant factor in regulating iron uptake in exponential cells grown
`in synthetic glucose medium. However, expression of the AFT2-
`1up allele in the aft1⌬ strain significantly increased 59Fe uptake
`(Fig. 7B). The 59Fe uptake data are therefore consistent with the
`growth phenotypes of each strain.
`
`Discussion
`We found that Aft2p, like its paralog, Aft1p, is a functional
`transcriptional activator that responds to iron. A strain that lacks
`both Aft1p and Aft2p is more sensitive to growth in low-iron
`conditions than a strain lacking only Aft1p. An allele of AFT2
`that contains a mutation analogous to the AFT1-1up mutation
`activates the transcription of a subset of the Aft1p regulon in an
`AFT1-independent manner. The expression profiles of two
`Aft2p target genes after the induction of the AFT2-1up allele is
`consistent with Aft2p directly regulating these genes. The wild-
`type AFT2 allele activates transcription of its target genes in
`response to treatment with the iron-chelator BPS. In addition,
`truncated forms of both Aft1p and Aft2p bind to the same DNA
`fragment in vitro, consistent with Aft2p being a direct transcrip-
`tional regulator. There are, however, differences in the extent to
`which Aft1p and Aft2p regulate the iron regulon. The aft1⌬
`strain and the aft2⌬ strain exhibit different iron-deficient and
`respiratory-deficient phenotypes. The AFT2-1up allele only par-
`tially suppresses the respiratory-deficient phenotype of the aft1⌬
`strain and partially stimulates iron uptake in the aft1⌬ strain.
`Furthermore, the AFT1-1up allele and the AFT2-1up allele dif-
`ferentially regulate gene expression.
`Microarray experiments identified a subset of the Aft1 regulon
`as being activated by the AFT2-1up allele. Those genes that were
`
`Fig. 6. Aft1p and Aft2p bind to the same promoter fragment in vitro but
`differentially regulate gene expression. (A) S1 nuclease protection assays to
`quantify mRNA levels of genes activated by the AFT1-1up and AFT2-1up alleles.
`RNA was isolated from the aft1⌬aft2⌬ strain transformed with a control
`plasmid (⫺) (pRS316 in the case of pAFT1-1up and pRS416 in the case of
`pAFT2-1up), pAFT1-1up (1up), and pAFT2-1up (2up) and grown in CMD(⫺Ura)
`medium to mid-log phase. The Upper band for each sample is the specified
`gene, and the Lower band the calmodulin loading control (CMD1). (B) Gel
`retardation assays were performed by using a 32P-labeled 30-bp duplex of the
`FET3 promoter as probe with no protein (lane 1), and lysates from E. coli
`containing pET3 (lane 2), pAft1-313 (lane 3), and pAft2-214 (lane 4).
`
`not identified include FET3 and ARN1-4. However, S1 analysis
`clearly shows that the AFT2-1up allele is able to activate FET3
`expression in the absence of Aft1p. The microarray experiments
`were carried out in an AFT1 wild-type strain, and S1 analysis
`showed that FET3 is expressed in the control strain to such a
`level that no differential expression was observed in the strain
`carrying the AFT2-1up allele (data not shown). This result implies
`that there are different thresholds of activation of genes within
`the iron regulon by Aft1p. Therefore, AFT2 may be capable of
`
`Iron uptake in the aft mutants. The level of 59Fe uptake was analyzed
`Fig. 7.
`in the following cultures. (A) The wild-type strain and the aft-deletion strains
`grown to mid-log phase in synthetic medium (CMD) with BPS (100 M). (B) The
`wild-type strain and the aft1⌬ strain containing either pRS416 (WT, 1⌬) or
`pAFT2-1up (WT兾up, 1⌬兾up) grown to mid-log phase in CMD(⫺Ura) medium.
`
`14326 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.261381198
`
`Rutherford et al.
`
`
`
`activating the entire Aft1p regulon in the absence of Aft1p.
`Identification of the extent to which the Aft1p and Aft2p
`regulons overlap will require more detailed analysis. It is clear
`however, that there is differential expression of FET3 and MRS4,
`depending on whether the AFT1-1up allele or the AFT2-1up allele
`is present. This result is consistent with both Aft1p and Aft2p
`having distinct effects on the cell and therefore distinct func-
`tions. Within 500 bp of the start codon of MRS4, there are
`AFT1-like binding sites that contain the conserved central
`CACCC motif but that differ in the flanking bases. Identification
`of the ability of Aft1p and Aft2p to bind these sites will provide
`insight into what determines Aft1p- or Aft2p-specific gene
`expression. During the drafting of this manuscript, Blaiseau et al.
`published work relating to AFT2 that shows that AFT2 activates
`the expression of FET3 in an iron-dependent manner (20).
`Furthermore, a double mutant lacking both functional AFT1 and
`AFT2 has a more severe iron-dependent phenotype and is more
`sensitive to oxidative stress than either of the single-mutant
`strains. Blaiseau et al. conclude that Aft2p is an iron-responsive
`transcriptional regulator. Our data relating to the phenotype of
`the aft-mutant strains is in agreement with Blaiseau et al., and
`our microarray data strengthens the conclusions that both
`groups have made.
`Aft2p may mediate a regulatory program of the iron regulon
`different from that mediated by Aft1p. Microarray experiments
`analyzing global gene expression in S. cerevisiae in response to
`different stress conditions show that the expression of AFT2 is
`elevated during stationary phase, under nitrogen starvation
`conditions, and after treatment with the alkylating agent methyl
`methanesulfonate (21, 22). Therefore, there may be environ-
`mental conditions where one factor is the predominant regulator
`of the iron regulon. Alternatively, Aft1p and Aft2p may both be
`activated under iron-deficient conditions, but the mechanism by
`
`which each senses the levels of reactive iron may be different.
`The activity of the two mammalian translational iron regulators
`IRP1 and IRP2 is inhibited in iron-replete cells but through
`different mechanisms (23). If Aft2p is the predominant iron-
`responsive regulator of a group of genes, then it may be
`significant that many of the apparently Aft2p-specific genes
`identified by the microarray analysis are involved in vacuole
`function. Aft2p may play a special role in mobilizing internal iron
`stores. Yeast vacuoles are an important site of iron storage and
`reutilization using a transport system that involves Fet5p and
`Fth1p (6, 24), both of which were found to be induced by the
`AFT2-1up allele. The iron-deficient phenotype of the aft1⌬aft2⌬
`strain was significantly alleviated when it was pregrown with
`supplemental
`iron. Similar observations were made with a
`fth1⌬fet5⌬ strain (6). Pregrowth of this strain in iron-deficient
`medium compromised the ability of that strain to make a growth
`transition from fermentative to respiratory metabolism, which
`was not alleviated by the addition of exogenous iron (6). Thus,
`a specialized role of AFT2 in regulating vacuolar iron storage,
`particularly under conditions of starvation, warrants further
`investigation.
`
`We acknowledge the excellent technical assistance of the University of
`Utah Microarray Core Facility led by Dr. Brian Dalley and supported by
`the Huntsman Cancer Foundation. We thank Jerry Kaplan’s group for
`assistance in radioiron uptake. This research was supported by a grant
`(CA 61286) from the National Cancer Institute, National Institutes of
`Health (to D.R.W.) and a microarray supplemental award (ES03817)
`from the National Institutes of Environmental Health Sciences (to
`D.R.W.). P.O.B. acknowledges support of HG00983 and the Howard
`Hughes Medical Institute. P.O.B. is an associate investigator of the
`Howard Hughes Medical Institute. We acknowledge support from the
`National Institutes of Health (5P30-CA 42014) to the Biotechnology
`Core Facility for DNA synthesis at the University of Utah.
`
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