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• W1993165787 abstract "Saccharomyces cerevisiaecan accumulate iron through the uptake of siderophore-iron. Siderophore-iron uptake can occur through the reduction of the complex and the subsequent uptake of iron by the high affinity iron transporter Fet3p/Ftr1p. Alternatively, specific siderophore transporters can take up the siderophore-iron complex. The pathogenic fungus Candida albicans can also take up siderophore-iron. Here we identify aC. albicans siderophore transporter, CaArn1p, and characterize its activity. CaARN1 is transcriptionally regulated in response to iron. Through expression studies in S. cerevisiae strains lacking endogenous siderophore transporters, we demonstrate that CaArn1p specifically mediates the uptake of ferrichrome-iron. Iron-ferrichrome and gallium-ferrichrome, but not desferri-ferrichrome, could competitively inhibit the uptake of iron from ferrichrome. Uptake of siderophore-iron resulting from expression of CaARN1 under the control of theMET25-promoter in S. cerevisiae was independent of the iron status of the cells and of Aft1p, the iron-sensing transcription factor. These studies demonstrate that the expression of CaArn1p is both necessary and sufficient for the nonreductive uptake of ferrichrome-iron and suggests that the transporter may be the only required component of the siderophore uptake system that is regulated by iron and Aft1p. Saccharomyces cerevisiaecan accumulate iron through the uptake of siderophore-iron. Siderophore-iron uptake can occur through the reduction of the complex and the subsequent uptake of iron by the high affinity iron transporter Fet3p/Ftr1p. Alternatively, specific siderophore transporters can take up the siderophore-iron complex. The pathogenic fungus Candida albicans can also take up siderophore-iron. Here we identify aC. albicans siderophore transporter, CaArn1p, and characterize its activity. CaARN1 is transcriptionally regulated in response to iron. Through expression studies in S. cerevisiae strains lacking endogenous siderophore transporters, we demonstrate that CaArn1p specifically mediates the uptake of ferrichrome-iron. Iron-ferrichrome and gallium-ferrichrome, but not desferri-ferrichrome, could competitively inhibit the uptake of iron from ferrichrome. Uptake of siderophore-iron resulting from expression of CaARN1 under the control of theMET25-promoter in S. cerevisiae was independent of the iron status of the cells and of Aft1p, the iron-sensing transcription factor. These studies demonstrate that the expression of CaArn1p is both necessary and sufficient for the nonreductive uptake of ferrichrome-iron and suggests that the transporter may be the only required component of the siderophore uptake system that is regulated by iron and Aft1p. open reading frame polyacrylamide gel electrophoresis bathophenanthroline sulfonate synthetic media ferrioxamine B ferrichrome gallium hemagglutinin Iron is an essential element for all eukaryotes and most prokaryotes. Its importance in biology lead to the evolution of multiple uptake mechanisms that would satisfy the requirements for the metal in both single cell and multicellular organisms. Under conditions of iron starvation most microorganisms secrete siderophores, low molecular weight organic molecules that bind extracellular iron (1Neilands J.B. Ann. Rev. Biochem. 1981; 50: 715-731Crossref PubMed Scopus (848) Google Scholar). Siderophores are chemically heterogeneous and there are specific transport systems for different siderophores (2Neilands J.B. J. Biol. Chem. 1995; 270: 26723-26726Abstract Full Text Full Text PDF PubMed Scopus (1215) Google Scholar, 3Neilands J.B. Konopka K. Schwyn B. Coy M. Francis R.T. Paw B.H. Bagg A. Winkelmann G. van der Helm D. Neilands J.B. Iron Transport in Microbes, Plants and Animals. VCH Publishers, Inc., New York1987: 3-34Google Scholar). In some instances a transport system may take up more than one siderophore. Many microorganisms secrete more than one siderophore, and in addition to utilizing their own siderophores they may take up iron complexes of siderophores secreted by other microorganisms. The budding yeast, Saccharomyces cerevisiae, does not secrete siderophores, yet it can use siderophore-iron. Siderophore-iron uptake can be accomplished by either extracellular reduction and subsequent uptake of the iron by the high affinity iron transport system Fet3p/Ftr1p or by the uptake of siderophore-iron complexes by specific transporters belonging to the major facilitator super family (4Lesuisse E. Simon-Casteras M. Labbe P. Microbiology. 1998; 144: 3455-3462Crossref PubMed Scopus (129) Google Scholar, 5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The hydroxamate-type siderophores ferrioxamine B, triacetyl fusarinine C, and ferrichrome are taken up by the S. cerevisiae siderophore-iron transporters Arn1p, Arn2p, and Arn3p (5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 6Yun C.W. Tiedeman J.S. Moore R.E. Philpott C.C. J. Biol. Chem. 2000; 275: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 7Heymann P. Ernst J.F. Winkelmann G. Biometals. 1999; 12: 301-306Crossref PubMed Scopus (79) Google Scholar, 8Heymann P. Ernst J.F. Winkelmann G. FEMS Microbiol. Lett. 2000; 186: 221-227Crossref PubMed Google Scholar). Arn4p was found to be specific for the catecholate-type bacterial siderophore enterobactin (9Heymann P. Ernst J.F. Winkelmann G. Biometals. 2000; 13: 65-72Crossref PubMed Scopus (88) Google Scholar). Iron uptake mechanisms are highly conserved among yeast.Schizosaccharomyces pombe and Candida albicanshave a high affinity uptake system that is homologous to the Fet3p/Ftr1p transport system of S. cerevisiae (10Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Ramanan N. Wang Y. Science. 2000; 288: 1062-1064Crossref PubMed Scopus (253) Google Scholar, 12Eck R. Hundt S. Hartl A. Roemer E. Kunkel W. Microbiology. 1999; 145: 2415-2422Crossref PubMed Scopus (88) Google Scholar). Unlike S. cerevisiae, the pathogenic fungus C. albicans can secrete siderophores (13Ismail A. Lupan D.M. Mycopathologia. 1986; 96: 109-113Crossref PubMed Scopus (15) Google Scholar). To date no specific siderophore uptake system in C. albicans has been identified. Based on studies of siderophore-iron utilization inS. cerevisiae, we hypothesized that C. albicansmay have siderophore transporters. In this paper, we demonstrate the existence of a specific siderophore transporter in C. albicans, CaArn1p, encoded by the ORF1CaYHL040C, 2The nomenclature for the C. albicanssiderophore transporter, CaYHL040C, is based upon homology to theS. cerevisiae ARN1 ORF YHL040C. The C. albicans ORFS have not been assigned to specific chromosomes. which is orthologous to the ARN1 siderophore transporter in S. cerevisiae. Genetic analysis in C. albicans is difficult because it lacks a sexual cycle and exists as a diploid.S. cerevisiae has been used successfully as a tool to characterize C. albicans genes. The elemental iron transport systems from other yeast species retain function when expressed inS. cerevisiae (10Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 11Ramanan N. Wang Y. Science. 2000; 288: 1062-1064Crossref PubMed Scopus (253) Google Scholar, 12Eck R. Hundt S. Hartl A. Roemer E. Kunkel W. Microbiology. 1999; 145: 2415-2422Crossref PubMed Scopus (88) Google Scholar). Characterization ofCaARN1 in S. cerevisiae shows that the C. albicans siderophore transporter has high specificity for ferrichrome and that expression of the transporter is necessary and sufficient for siderophore-iron transport. Yeast strains used in this study are listed in TableI. The following primers were used to construct pmetCaARN1 (5′-CGGGATCCGCATGACATCTTACCAG-3′ and 5′-CGGAATTCCTATTAAACAGCTACTCTTTTCTTC-3′), pmet-flagCaARN1(5′-CGGGATCCGCATGGATTACAAGGATGACGATGACAAGGGTGGTACATCTTACCAG-3′ and 5′-CGGAATTCCTATTAAACAGCTACTCTTTTCTTC-3′), and the pmet-CaARN1-myc(5′-CGGGATCCGCATGACATCTTACCAG-3′ and 5′ CGGAATTCCTATTAATTCAAGTCCTC- TTCAGAAATGAGCTTTTGCTCCATAACAGCTACTCTTTTCTTC-3′).Table IYeast strains used in this studyStrain nameGenotypeReferenceC. albicans C. albicans CAI-4URA3∷imm434/URA3∷imm434(35Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google Scholar)S. cerevisiae Y9Δaft1MATa, ura3–52 lys2–801 (amber) ade2–101 (ochre) trp1-Δ63 his3-Δ200 leu2-Δ1, aft1∷TRP1(6) Y9Δfet3MATa, ura3–52 lys2–801(amber) ade2–101 (ochre) trp1-Δ63 his3-Δ200 leu2-Δ1 Δfet3∷HIS3(6) Δfet3 Δarn1,2,3,4MATa, ura3–52 lys2–801 (amber)ade2–101 (ochre) trp1-Δ63 his3-Δ200 leu2-Δ1 Δfet3∷HIS3 Δarn1∷HISG Δarn3∷HISG Δarn4∷HISG-URA3-HISG Δarn2∷HISG-URA3-HISG(6) Wild type BY4741MATa his3-Δ1 leu2-Δ0 met15-Δo ura3-ΔoA gift from Julian Rutherford, University of Utah BY4741 Δaft1MATα his3-Δ1 leu2-Δ0 met15-Δo ura3-Δ0 aft1∷KanMX4A gift from Julian Rutherford, BY4742 Δaft2MATa his3-Δ1 leu2-Δ0 met15-Δo ura3-Δ0 aft2∷KanMX4A gift from Julian Rutherford, BY4741 Δaft1 Δaft2MATα his3-Δ1 leu2-Δ0 met15-Δo ura3-Δ0 aft1∷KanMX4 aft2∷KanMX4A gift from Julian Rutherford, Open table in a new tab C. albicans (CAI-4) genomic DNA was used as a template to amplify ORF CaYHL040C by PCR using 92 °C, 40 s, 55 °C, 60 s, and 68 °C for 3 min conditions. The PCR products were digested with BamHI and EcoRI and cloned behind a MET25 promoter in the vectors pTF62 (LEU2) or pTF63 (URA3). All constructs were verified by DNA sequencing. pARN1-HA and pmetARN1-HA were as described (6Yun C.W. Tiedeman J.S. Moore R.E. Philpott C.C. J. Biol. Chem. 2000; 275: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Cells were grown in YPD (1.0% yeast extract, 2.0% peptone, 2.0% glucose), CM (a synthetic medium of yeast nitrogen base, amino acids, and glucose), CM deficient in specific amino acids, CM made iron-limited by the addition of 50 μm bathophenanthroline sulfonate (BPS) (14Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (587) Google Scholar) or CM made iron replete by addition of 50 μm FeCl3. The siderophores ferrioxamine B (FOB) (Desferal®), ferrichrome (Fc), and rhodotorulic acid were obtained from Sigma. Siderophore complexes were prepared by overnight incubation of desferri-siderophores with FeCl3 or Ga (NO3)3 at a ratio of 1.0:0.9. All siderophore complexes were filter-sterilized (0.2 μm filter) (15Ardon O. Weizman H. Libman J. Shanzer A. Chen Y. Hadar Y. Microbiology. 1997; 143: 3625-3631Crossref PubMed Scopus (24) Google Scholar,16Ardon O. Nudelman R. Caris C. Libman J. Shanzer A. Chen Y. Hadar Y. J. Bacteriol. 1998; 180: 2021-2026Crossref PubMed Google Scholar). For plate assays, iron-limiting CM was used in which BPS was added to a final concentration of 50 μm, 100 μl of Fe-siderophore complexes were added to a final concentration of 0.5 μm,and the plates allowed to dry at 30 °C overnight. Yeast were grown to mid-log phase and 10-fold dilutions spotted onto plates and grown for 2 or 3 days at 30 °C. Iron transport assays were performed as described (17Davis-Kaplan S.R. Askwith C.C. Bengtzen A.C. Radisky D. Kaplan J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13641-13645Crossref PubMed Scopus (112) Google Scholar) with the following modifications. Briefly, 2 × 107 cells were mixed with 59Fe supplied as59FeCl3 or 59Fe-siderophore. Cells were incubated at 30 °C for 15 min, placed on filters (Whatman GF/C), and washed with EDTA-containing buffer to remove unincorporated iron. The filters were air-dried, and associated radioactivity determined. The uptake activity was expressed as pmol of59Fe/min/106 cells. Cells were grown to mid-log phase, collected by centrifugation, and washed. Spheroplasts were made and Dounce homogenized, and a postnuclear supernatant obtained. A crude membrane fraction was isolated by centrifugation at 15,600 ×g for 30 min. Protein concentrations were determined, and equal amounts run on 10% SDS-polyacrylamide gel electrophoresis. Samples were transferred to nitrocellulose, probed with anti-FLAG (M2 Sigma), (1:10,000) or anti-HA (HA.11Covance) (1:2000) followed by goat anti-mouse horseradish peroxidase (Jackson Immuno Research Laboratories Inc, 1:10,000). Western blots were developed using the chemiluminescence detection reagent Renaissance (PerkinElmer Life Sciences) as per the manufacturer's instructions. Total RNA was isolated using standard techniques (18Ausubel, F. M. (1995) Current Protocols in Molecular Biology, pp. 13:12, John Wiley & Sons, Inc., New York.Google Scholar). All samples were isolated from mid-log phase cultures grown in defined iron media containing either 50 μm FeCl3 (“high iron”) or 50 μm BPS (“low iron”). A biotinylated CaARN1 probe was PCR-generated using a C. albicans (CAI-4) genomic prep as a template, primers 5′-CGGGATCCGCATGACATCTTACCAG-3′ and 5′-CGGAATTCCTATTAAACAGCTACTCTTTTCTTC-3′ with PCR conditions 92 °C for 40 s, 55 °C for 60 s, and 68 °C for 3 min. A biotinylated C. albicans actin probe was generated using 5′-TTGCCGGTGACGACGCTCC and 5′-GCTCTGAATCTTTCGTTACC. The blots were developed using DNA Detector kit (Kirkegaard & Perry Laboratories, Inc.). The size of the mRNAs on Northern blots correlated with the lengths expected from the calculated sequences. C. albicans has been shown to use different hydroxamate siderophores for its growth on plates (19Minnick A.A. Eizember L.E. McKee J.A. Dolence E.K. Miller M.J. Anal. Biochem. 1991; 194: 223-229Crossref PubMed Scopus (14) Google Scholar). To determine whetherC. albicans has a specific siderophore transport system, we measured the uptake of 59Fe-siderophore complexes inC. albicans. Wild type cells were able to take up59Fe from both 59Fe-Fc and 59Fe-FOB (Fig. 1 a) in a concentration dependent manner. The apparent K m of uptake was 1.88 μm and Vmax 3.20 pmol/min/106 cells for Fc and 5.71 μm and 2.40 pmol/min/106 cells, respectively, for FOB, values that are similar to that seen in S. cerevisiae (6Yun C.W. Tiedeman J.S. Moore R.E. Philpott C.C. J. Biol. Chem. 2000; 275: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Both siderophores were found to promote cell growth (data not shown). In S. cerevisiae siderophore-iron is accumulated by two mechanisms (4Lesuisse E. Simon-Casteras M. Labbe P. Microbiology. 1998; 144: 3455-3462Crossref PubMed Scopus (129) Google Scholar, 5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The first involves reduction of siderophore-iron complexes by cell surface reductases and uptake of iron by the high affinity iron transport system. A second mechanism for siderophore-iron utilization results in the uptake of siderophore-iron complexes by specific transport systems. To determine whether C. albicansalso employs both mechanisms we examined the effect of a non-permeable Fe(II) chelator on siderophore-iron-mediated transport. Reduction of siderophore-iron complexes leads to the formation of Fe(II), which is the substrate for the high affinity iron transport system and chelation of Fe(II) prevents iron uptake. Incubation of C. albicanswith the impermeable Fe(II) chelator BPS results in the complete inhibition of 59Fe uptake when cells are incubated with59Fe(III)Cl3 in citrate buffer (Fig.1 b). This same concentration only effects a 50% reduction in the uptake of 59Fe(III)-Fc. Uptake of59Fe-FOB is completely inhibited by the addition of BPS. These results suggest that a significant component of Fe-Fc uptake does not occur by reduction at the cell surface, whereas uptake of Fe-FOB only occurs by reduction followed by transport of elemental iron. S. cerevisiae has a family of siderophore transporters (5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). We questioned whether C. albicans also had an orthologous siderophore transport system. Inspection of the C. albicansgenome at 5× coverage (sequence.stanford.edu/group/candida/) revealed one ORF that demonstrated a high homology to the S. cerevisiae siderophore transporters (ARN1–4) (Fig.2). This ORF was on the 33,181 base pair contig 4–3057 and was from base pairs 15,417–13,606 on the reverse strand. The amino acid identity between the putative C. albicans ARN, CaYHL040C, and the yeast ARNs ranged between 28–46%, with ARN1 showing the highest identity and homology (46 and 63%, respectively). Primers were designed, and CaYHL040C was amplified using PCR from a C. albicans genomic DNA preparation. Southern analysis of C. albicans genomic DNA, using CaYHL040C as a probe, was performed using non-stringent conditions (2× saline/sodium phosphate/EDTA /0.1% SDS). A single band was detected, consistent with the genome search, strongly suggesting that there is just one ARN homologue in C. albicans. The S. cerevisiae siderophore transporters are regulated by the iron-sensing transcription factor Aft1p (5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). If the C. albicans gene CaYHL040C is a siderophore transporter, then we might expect that its transcription would be iron-regulated, as are other known components of the C. albicans iron transport system (12Eck R. Hundt S. Hartl A. Roemer E. Kunkel W. Microbiology. 1999; 145: 2415-2422Crossref PubMed Scopus (88) Google Scholar, 20Morrissey J.A. Williams P.H. Cashmore A.M. Microbiology. 1996; 142: 485-492Crossref PubMed Scopus (51) Google Scholar). A Northern analysis was performed on C. albicans RNA isolated from either iron-replete or iron-depleted cultures using CaYHL040C as a probe. A single band was detected in cells grown under iron-limiting conditions (Fig.3). No band was observed under high iron conditions. This finding supports the hypothesis that the C. albicans ORF CaYHL040C, termed CaARN1, may have a role in iron metabolism in this fungus. To study the function of CaARN1, independent of iron conditions, we cloned CaARN1 under the regulation of theMET25 promoter and expressed it in aΔfet3Δarn1–4 strain of S. cerevisiae. These cells are unable to take up siderophore iron either through the high affinity iron transport system or the ARN family of siderophore transporters and were used previously to characterizeS. cerevisiae siderophore transporters (5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). TheΔfet3Δarn1–4 cells can not grow on BPS plates supplemented with siderophores (5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Transformation of this strain with a plasmid containing a MET25-regulated CaARN1, permitted cell growth on media supplemented with Fe-Fc, but not supplemented with Fe-FOB (Fig. 4) or Fe-rhodotorulic acid (data not shown). Both N-terminal and C-terminal epitope-tagged CaARN1 constructs also permitted growth on Fe-Fc. To further characterize the iron uptake activity of cells transformed with CaARN1, uptake studies were conducted using59Fe-Fc. In Δfet3Δarn1–4 cells, the uptake of 59Fe-Fc was significantly higher in cells transformed with the methionine-regulated CaARN1 constructs than in cells transformed with vector alone. The rate of iron accumulation in cells expressing CaArn1p (under the control of theMET25 promoter) was much greater than Δfet3cells expressing the native ARNs under iron-limited conditions, which should result in their maximal induction (Fig.5 a). There were no significant differences in 59Fe-Fc uptake between cells expressing the epitope-tagged protein (5′FLAG or 3′Myc) and the non-tagged CaArn1p. Addition of methionine to the growth media reduced the rate of59Fe-Fc uptake (Fig. 5 b), although not to baseline levels. Western analysis of cells expressing CaArn1p showed regulation of CaARN1 expression (Fig. 5 c). At high levels of methionine (560 μg/ml), no CaArn1p was detected by Western blot, although 59Fe-Fc uptake was still observed. This uptake may be due to the lack of complete repression of theMET25 promoter, particularly in high copy vectors. We utilized pmetCaARN1-expressing cells to define the specificity of Fc recognition and transport. Addition of desferri-Fc to59Fe-Fc-containing media did not inhibit uptake of59Fe-Fc (Fig. 6). This result indicates that the transporter must recognize a structural difference in the Fc once it has bound iron. It has been reported that there is little conformational difference between Fe- and Ga-complexed hydroxamate siderophores (21Emery T. Biochemistry. 1971; 10: 1483-1488Crossref PubMed Scopus (138) Google Scholar). Both metals are bound in the trivalent state, although unlike Fe-Fc, Ga-Fc complexes cannot be reduced. Both Fe-Fc and Ga-Fc exhibited a similar concentration-dependent inhibition of 59Fe-Fc uptake (Fig. 6). Mineral Ga, supplied as Ga(NO3)3 had no effect on the uptake of59Fe-Fc. The inhibition of 59Fe-Fc uptake by Ga-Fc was transient, as cells washed free of Ga-Fc showed no subsequent inhibition of 59Fe-Fc uptake (data not shown). These results suggest that Ga-Fc competes with Fe-Fc for a recognition site on CaArn1p. Ferrichrome synthetic analogues are recognized and taken up by the Ustilago maydis ferrichrome uptake system (15Ardon O. Weizman H. Libman J. Shanzer A. Chen Y. Hadar Y. Microbiology. 1997; 143: 3625-3631Crossref PubMed Scopus (24) Google Scholar, 16Ardon O. Nudelman R. Caris C. Libman J. Shanzer A. Chen Y. Hadar Y. J. Bacteriol. 1998; 180: 2021-2026Crossref PubMed Google Scholar). These analogues were not taken up by pmet-CaARN1-transformedΔfet3Δarn1–4 cells, as ascertained by growth, radiotracer experiments, and fluorescence microscopy (data not shown). These results demonstrate that the activity of CaArn1p is sensitive to alterations in siderophore structure. The high affinity iron transport system, comprised of Fet3p and Ftr1p, is regulated at the level of transcription by Aft1p (22Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Crossref PubMed Scopus (314) Google Scholar). Expression of FET3 and FTR1 by iron-independent promoters showed that these proteins mediated iron transport even when cells were iron-replete (10Askwith C. Kaplan J. J. Biol. Chem. 1997; 272: 401-405Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). These results indicate that the only two surface proteins required for high affinity iron transport are Fet3p and Ftr1p. Because the S. cerevisiae siderophore transporters are also regulated by Aft1p, we asked whether other proteins regulated by Aft1p are required for siderophore transport. TheS. cerevisiae strain Δfet3Δarn1–4 was transformed with plasmids containing S. cerevisiae ARN1under its own promoter, pmetARN1 orpmetCaARN1. Cultures were grown under high iron or iron-limiting conditions for 6 h and then assayed for59Fe-Fc uptake (Fig.7 a). The 6-h incubation period is long enough to permit expression of Aft1p-regulated genes (14Askwith C. Eide D. Van Ho A. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (587) Google Scholar). As expected, cells expressing Arn1p under the control of its native promoter, showed Fe-Fc uptake when grown under iron-depleted conditions but not under iron-replete conditions. Expression of eitherARN1 or CaARN1 under the control of theMET25 promoter led to a high rate of Fe-Fc uptake regardless of whether cells were grown in high or low iron media. The rate of iron uptake was similar in cells expressing either the S. cerevisiae ARN1 or the C. albicans ARN1. These results suggest that once expressed, the siderophore transporter can function independently of cellular iron or other genes whose transcription is dependent on the Aft1p transcription factor. This conclusion is further supported by measuring 59Fe-Fc uptake in Δaft1cells transformed with pARN1, pmetARN1, or pmetCaARN1 (Fig.7 b. There is little iron taken up by Δaft1cells transformed with a plasmid that has ARN1 under the control of the Aft1p-sensitive promoter. The same cells transformed with the MET25-regulated ARN1 showed high rates of iron accumulation. When the ARNs are expressed from their endogenous promoters there is little measurable siderophore iron transport in the absence ofAFT1. Siderophore iron, however, can support the growth ofS. cerevisiae Δaft1 cells (Fig.8). Recently, a homologue ofAFT1, termed AFT2, has been identified, and it appears to share some functions with AFT1 (23Blaiseau P.L. Lesuisse E. Camadro J.M. J. Biol. Chem. 2001; 276: 34211-34226Abstract Full Text Full Text PDF Scopus (162) Google Scholar). To determine whether AFT2 played a role in siderophore iron transport, we examined a Δaft2 strain for its ability to grow on siderophore iron. Deletion of both AFT1 and AFT2results in a lack of growth on Fe-Fc. In the absence of the Arnp's or Fet3p there is no growth on Fe-Fc (Fig. 8 and Ref. 6Yun C.W. Tiedeman J.S. Moore R.E. Philpott C.C. J. Biol. Chem. 2000; 275: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). These results suggest that in S. cerevisiae AFT2 may permit a low level of expression of ARN1. Siderophore-mediated iron accumulation is an important mechanism of iron uptake for many organisms including bacteria, fungi, and plants (2Neilands J.B. J. Biol. Chem. 1995; 270: 26723-26726Abstract Full Text Full Text PDF PubMed Scopus (1215) Google Scholar, 25Guerinot M.L. Yi Y. Plant Physiol. 1994; 104: 815-820Crossref PubMed Scopus (534) Google Scholar). Numerous studies have shown that siderophore transport systems are virulence factors in microorganisms (26Ratledge C. Dover L.G. Annu. Rev. Microbiol. 2000; 54: 881-941Crossref PubMed Scopus (1166) Google Scholar, 27Wooldridge K.G. Williams P.H. FEMS Microbiol. Rev. 1993; 12: 325-348Crossref PubMed Google Scholar, 28Howard D.H. Clin. Microbiol. Rev. 1999; 12: 394-404Crossref PubMed Google Scholar). S. cerevisiae, which does not secrete siderophores, has multiple transport systems that can utilize siderophores secreted by other organisms (5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 6Yun C.W. Tiedeman J.S. Moore R.E. Philpott C.C. J. Biol. Chem. 2000; 275: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 7Heymann P. Ernst J.F. Winkelmann G. Biometals. 1999; 12: 301-306Crossref PubMed Scopus (79) Google Scholar, 8Heymann P. Ernst J.F. Winkelmann G. FEMS Microbiol. Lett. 2000; 186: 221-227Crossref PubMed Google Scholar, 9Heymann P. Ernst J.F. Winkelmann G. Biometals. 2000; 13: 65-72Crossref PubMed Scopus (88) Google Scholar, 29De Luca N.G. Wood P.M. Adv. Microb. Physiol. 2000; 43: 40-74Google Scholar). C. albicans, a pathogenic fungus of major medical importance, can secrete siderophores of the hydroxamate- and possibly phenolate-type (30Ismail A. Bedell G.W. Lupan D.M. Biochem. Biophys. Res. Commun. 1985; 130: 885-891Crossref PubMed Scopus (45) Google Scholar). We took advantage of the sequence ofS. cerevisiae siderophore transporters to identify a siderophore transporter in C. albicans. A gene, homologous to the S. cerevisiae ARNs was shown to be iron-regulated inC. albicans, consistent with a role in iron homeostasis. Proof that this gene, CaARN1, could mediate iron transport was shown by its ability to complement a strain of S. cerevisiae that was unable to grow on siderophores due to the deletion of both the high affinity iron transport system and the native siderophore iron transporters. Through both growth assays and direct measurement of iron transport, we demonstrated that ectopic expression of the C. albicans gene permitted 59Fe-Fc uptake. A data base search of 5× DNA sequence coverage of the C. albicans genome identified a single C. albicanshomologue of the S. cerevisiae ARN gene family. Both Southern and PCR analysis also showed a single C. albicans ARN homologue. This gene, at least as expressed in S. cerevisiae, was highly specific for ferrichrome. This result leads to the suggestion that C. albicans may only have a transporter specific to the siderophores it secretes. S. cerevisiae, which does not secrete siderophores, has multiple transporters that permit utilization of a broad range of siderophores. Perhaps the ferrichrome transporter is an ancestral transporter, which under evolutionary pressure and gene duplication led to the generation of the other siderophore transporter genes. C. albicans, because of its ability to secrete siderophores, may not have been subject to the same pressure and consequently retained a single siderophore transporter gene. This hypothesis could be tested by examination of the fission yeast S. pombe. There is no evidence that this yeast secretes siderophores, yet it has three homologues of the ARN family. If these homologues can be shown to be functional siderophore transporters, then it would strengthen the argument that a lack of siderophore synthesis is the driving force behind the diversity in siderophore transporter gene evolution. We took advantage of the expression of CaArn1p in S. cerevisiae to examine some of the characteristics of siderophore transport. Based on competition studies, CaArn1p recognized metallo-Fc but not desferri-Fc. Ga-Fc, which can not be reduced, blocked the uptake of 59Fe-Fc at concentrations comparable with that of Fe-Fc. This competition was transient as removal of Ga-Fc alleviated the block in 59Fe-Fc uptake. Analogues of ferrichrome were previously shown to be biologically active in the ferrichrome-producing fungus U. maydis (15Ardon O. Weizman H. Libman J. Shanzer A. Chen Y. Hadar Y. Microbiology. 1997; 143: 3625-3631Crossref PubMed Scopus (24) Google Scholar, 16Ardon O. Nudelman R. Caris C. Libman J. Shanzer A. Chen Y. Hadar Y. J. Bacteriol. 1998; 180: 2021-2026Crossref PubMed Google Scholar). While it has been suggested thatS. cerevisiae can accumulate these siderophores and their fluorescently labeled derivatives (31Lesuisse E. Blaiseau P.L. Dancis A. Camadro J.M. Microbiology. 2001; 147: 289-298Crossref PubMed Scopus (94) Google Scholar), we have not seen uptake or growth promotion by the fluorescent ferrichrome analogues in cells expressing either the CaARN1p or the endogenous S. cerevisiae ARN1p. This result suggests that there may be significant differences in the recognition of ferrichrome by different fungal siderophore transporters. The ability to regulate the synthesis of siderophore transporters independent of cellular iron status also provided data on the role ofAFT1 and iron in siderophore transporter expression and function. Deletion of AFT1 dramatically reduces the transcription of genes encoding the elemental iron transporters or proteins required for their assembly. Genes that encode siderophore transporters (ARNs) are also highly transcriptionally regulated (5Yun C.W. Ferea T. Rashford J. Ardon O. Brown P.O. Botstein D. Kaplan J. Philpott C.C. J. Biol. Chem. 2000; 275: 10709-10715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). While the recently identified AFT2 (23Blaiseau P.L. Lesuisse E. Camadro J.M. J. Biol. Chem. 2001; 276: 34211-34226Abstract Full Text Full Text PDF Scopus (162) Google Scholar) may share some functions with AFT1, it is clear that AFT1 is the major iron-sensing transcriptional factor in S. cerevisiae. Expression of either the endogenous ferrichrome transporter (ARN1) or the C. albicans ferrichrome transporter (CaARN1) by iron-independent promoters resulted in a functional siderophore transport system. Under high iron conditions and in the absence of AFT1, Fe-Fc uptake occurred. This result suggests that the transporter is the only iron-regulated gene that is a structural component of the siderophore uptake system. C. albicans is an important pathogen. The ability to study siderophore transport in a well defined system (i.e.genetically characterized S. cerevisiae) offers an opportunity to study the recognition properties of the pathogen's siderophore transporter. This may help in the development of siderophore analogs with antimicrobial properties (32Nudelman R. Ardon O. Hadar Y. Chen Y. Libman J. Shanzer A. J. Med. Chem. 1998; 41: 1671-1678Crossref PubMed Scopus (96) Google Scholar, 33Diarra M.S. Lavoie M.C. Jacques M. Darwish I. Dolence E.K. Dolence J.A. Ghosh A. Ghosh M. Miller M.J. Malouin F. Antimicrob. Agents Chemother. 1996; 40: 2610-2617Crossref PubMed Google Scholar). While this development may be done empirically by screening analogs, knowledge of the mechanism of siderophore-iron uptake would accelerate this approach. It is still unresolved whether the iron-siderophore complex is transported into the cell or whether iron is released from the siderophore as a consequence of binding to the transporter. Studies suggest that iron can be released from siderophores by either reduction through cell surface reductases, by intracellular-reducing agents, or as shown recently through ligand exchange with endogenous siderophores (34Stintzi A. Barnes C. Xu J. Raymond K.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10691-10696Crossref PubMed Scopus (190) Google Scholar). A major question remaining is the mechanism of siderophore iron release. An answer to this question will affect the development of toxic-siderophores. A release of siderophore iron while the siderophore is bound to the surface of the transporter, as opposed to being transported into the cell prior to iron release, would affect the classes of toxic compounds that might be made. Our results suggest that whatever the mechanism of siderophore iron release, it is highly similar for both C. albicans and S. cerevisiae, and perhaps for other fungi as well. We thank our colleagues in the Kaplan laboratory for their help in preparing this manuscript. We thank Julian Rutherford, University of Utah for sharing unpublished data. We would like to acknowledge the Stanford Genome Technology Center for the availability of the genome sequence of Candida albicans." @default.
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• W1993165787 title "Identification of a Candida albicans Ferrichrome Transporter and Its Characterization by Expression inSaccharomyces cerevisiae" @default.
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