Combinatorial Control of Yeast FET4 Gene Expression by Iron, Zinc, and Oxygen
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m206214200
ISSN1083-351X
Autores Tópico(s)Plant Micronutrient Interactions and Effects
ResumoAcquisition of metals such as iron, copper, and zinc by the yeast Saccharomyces cerevisiae is tightly regulated. High affinity uptake systems are induced under metal-limiting conditions to maintain an adequate supply of these essential nutrients. Low affinity uptake systems function when their substrates are in greater supply. The FET4 gene encodes a low affinity iron and copper uptake transporter. FET4expression is regulated by several environmental factors. In this report, we describe the molecular mechanisms underlying this regulation. First, we found that FET4 expression is induced in iron-limited cells by the Aft1 iron-responsive transcriptional activator. Second, FET4 is regulated by zinc status via the Zap1 transcription factor. We present evidence that FET4 is a physiologically relevant zinc transporter and this provides a rationale for its regulation by Zap1. Finally, FET4expression is regulated in response to oxygen by the Rox1 repressor. Rox1 attenuates activation by Aft1 and Zap1 in aerobic cells. Derepression of FET4 may allow the Fet4 transporter to play an even greater role in metal acquisition under anaerobic conditions. Thus, Fet4 is a multisubstrate metal ion transporter under combinatorial control by iron, zinc, and oxygen. Acquisition of metals such as iron, copper, and zinc by the yeast Saccharomyces cerevisiae is tightly regulated. High affinity uptake systems are induced under metal-limiting conditions to maintain an adequate supply of these essential nutrients. Low affinity uptake systems function when their substrates are in greater supply. The FET4 gene encodes a low affinity iron and copper uptake transporter. FET4expression is regulated by several environmental factors. In this report, we describe the molecular mechanisms underlying this regulation. First, we found that FET4 expression is induced in iron-limited cells by the Aft1 iron-responsive transcriptional activator. Second, FET4 is regulated by zinc status via the Zap1 transcription factor. We present evidence that FET4 is a physiologically relevant zinc transporter and this provides a rationale for its regulation by Zap1. Finally, FET4expression is regulated in response to oxygen by the Rox1 repressor. Rox1 attenuates activation by Aft1 and Zap1 in aerobic cells. Derepression of FET4 may allow the Fet4 transporter to play an even greater role in metal acquisition under anaerobic conditions. Thus, Fet4 is a multisubstrate metal ion transporter under combinatorial control by iron, zinc, and oxygen. Chelex-treated synthetic defined medium δ-aminolevulinic acid bathophenanthroline disulfonic acid Combinatorial control of gene expression occurs when a single gene is regulated in response to different signals (1Latchman D.S. Eukaryotic Transcription Factors. 2nd Ed. Academic Press, New York1995Google Scholar). This control is often mediated by the combination of different transcription factor-binding sites in a promoter of the gene and action on those elements by multiple transcriptional activators and/or repressors. Combinatorial control allows expression of a gene to be modulated by multiple signals and this can provide several advantages to a cell or an organism. For example, activity of a particular gene product may be beneficial under a variety of conditions. Alternatively, a gene product may play different roles under different conditions and combinatorial control can allow this to occur as well. Combinatorial control also allows modulation of expression by more than one signal to optimize the expression of a gene to match a particular combination of factors. In this paper, we describe the combinatorial transcriptional regulation of the yeast FET4 gene. FET4 encodes a metal ion transporter and its regulation by multiple signals allows modulation of expression in response to iron, zinc, and oxygen.Metals ions such as iron and zinc are essential nutrients for cells. However, if accumulated to excessive levels, these same elements can be toxic. Therefore, genes encoding the proteins responsible for uptake of metal ions are highly regulated. Metal uptake systems have been well characterized in the yeast Saccharomyces cerevisiae (2Eide D.J. Adv. Microb. Physiol. 2000; 43: 1-38Crossref PubMed Google Scholar). For example, iron uptake is mediated by several different pathways in this organism. First, yeast can accumulate iron bound to microbial siderophores (3Lesuisse E. Blaiseau P.L. Dancis A. Camadro J.M. Microbiology. 2001; 147: 289-298Crossref PubMed Scopus (93) Google Scholar). Although S. cerevisiae does not produce its own siderophores, a number of different transporters can accumulate iron bound by siderophores secreted by other microbes. S. cerevisiae also uses a reductive mechanism for iron uptake. Extracellular Fe(III) is reduced to Fe(II) by the Fre1 and Fre2 reductases located in the plasma membrane (4Dancis A. Klausner R.D. Hinnebusch A.G. Barriocanal J.G. Mol. Cell. Biol. 1990; 10: 2294-2301Crossref PubMed Scopus (256) Google Scholar, 5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Crossref PubMed Scopus (197) Google Scholar). The Fe(II) product is then the substrate of a high affinity transport system composed of the Fet3 multicopper oxidase and the Ftr1 permease (6Askwith C. Eide D., Ho, A.V. Bernard P.S., Li, L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (582) Google Scholar, 7Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar). Fet3 uses O2 as a substrate to oxidize Fe(II) back to Fe(III) for subsequent transport by Ftr1 (8DeSilva D.M. Askwith C.C. Eide D. Kaplan J. J. Biol. Chem. 1995; 270: 1098-1101Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). High affinity copper uptake is also mediated by a reductive mechanism. Extracellular Cu(II) is reduced to Cu(I) by Fre1 reductase prior to transport across the plasma membrane by the Ctr1 and Ctr3 transporters (9Georgatsou E. Mavrogiannis L.A. Fragiadakis G.S. Alexandraki D. J. Biol. Chem. 1997; 272: 13786-13792Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 10Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 11Dancis A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; 41: 25660-25667Abstract Full Text PDF Google Scholar, 12Knight S.A.B. Labbeá S. Kwon L.F. Kosman D.J. Thiele D.J. Genes Dev. 1996; 10: 1917-1929Crossref PubMed Scopus (219) Google Scholar). Finally, high affinity zinc transport is mediated by the Zrt1 and Zrt2 proteins (13Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar, 14Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar).These high affinity uptake systems are tightly regulated in response to the cellular status of their respective metal substrates. Iron-limiting growth conditions trigger increased expression of the genes encoding Fre1, Fre2, Fet3, Ftr1, and many other genes involved in iron acquisition (2Eide D.J. Adv. Microb. Physiol. 2000; 43: 1-38Crossref PubMed Google Scholar). This induction is mediated by two related transcription factors, Aft1 and Aft2, that share overlapping but nonidentical sets of target genes (15Blaiseau P.L. Lesuisse E. Camadro J.M. J. Biol. Chem. 2001; 276: 34221-34226Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 16Rutherford J.C. Jaron S. Ray E. Brown P.O. Winge D.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14322-14327Crossref PubMed Scopus (127) Google Scholar). In a similar fashion, expression of the ZRT1 and ZRT2 genes is induced under zinc deficiency by the Zap1 transcription factor (17Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (216) Google Scholar) and the copper uptake transporters are regulated by the Mac1 copper-responsive activator protein (18Jungmann J. Reins H. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Crossref PubMed Scopus (229) Google Scholar). Each of these factors controls their respective target genes by binding to specific sequences in their promoters.In addition to the high affinity systems, low affinity uptake systems for these substrates are also present in yeast. This is evident because mutational inactivation of the high affinity systems does not result in nonviable cells. Previous work identified a low affinity iron transporter encoded by the FET4 gene (19Dix D.R. Bridgham J.T. Broderius M.A. Byersdorfer C.A. Eide D.J. J. Biol. Chem. 1994; 269: 26092-26099Abstract Full Text PDF PubMed Google Scholar, 20Dix D. Bridgham J. Broderius M. Eide D. J. Biol. Chem. 1997; 272: 11770-11777Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The Fet4 protein has six predicted membrane spanning regions and is localized to the plasma membrane. In addition to its role in iron transport, Fet4 has also been shown to be a physiologically relevant copper transporter (21Hassett R. Dix D.R. Eide D.J. Kosman D.J. Biochem. J. 2000; 351: 477-484Crossref PubMed Scopus (95) Google Scholar, 22Portnoy M.E. Schmidt P.J. Rogers R.S. Culotta V.C. Mol. Genet. Genomics. 2001; 265: 873-882Crossref PubMed Scopus (93) Google Scholar). Other studies suggested that Fet4 may be capable of transporting cobalt, manganese, and zinc as well (23Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These observations lead us to the hypothesis that Fet4 plays a central role in the accumulation of a number of metal ions in yeast.Various studies have indicated that FET4 expression and/or activity is influenced by a number of environmental factors. First, levels of Fet4 were ∼4-fold higher in iron-deficient cells than in iron-replete cells (20Dix D. Bridgham J. Broderius M. Eide D. J. Biol. Chem. 1997; 272: 11770-11777Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). A recent microarray study (24Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (250) Google Scholar) from our laboratory revealed that FET4 transcription was increased under zinc deficiency and that FET4 may be a Zap1 target gene. Finally, another microarray study comparing yeast cells grown in aerobic and anaerobic conditions indicated that FET4mRNA levels were greatly elevated under anaerobiosis (25ter Linde J.J. Liang H. Davis R.W. Steensma H.Y. van Dijken J.P. Pronk J.T. J. Bacteriol. 1999; 181: 7409-7413Crossref PubMed Google Scholar). These results suggested that FET4 transcription is under combinatorial control and regulated by multiple environmental factors. However, the underlying mechanisms for this regulation were unknown. In this report, we provide an understanding of FET4 regulation at a molecular level. FET4 transcription is subject to regulation by iron status via the Aft1 transcription factor and zinc status via Zap1. We present evidence that Fet4 is a physiologically relevant zinc transporter in yeast and this is likely the reason for its regulation by zinc status. Finally, repression of FET4expression in aerobic cells is mediated by the Rox1 repressor in response to O2 levels. These different regulatory proteins integrate FET4 gene expression with several environmental factors.DISCUSSIONDuring the preparation of this manuscript, Jensen and Culotta (41Jensen L.T. Culotta V.C. J. Mol. Biol. 2002; 318: 251-260Crossref PubMed Scopus (52) Google Scholar) reported a related analysis of FET4 gene expression. Our studies confirm and extend on their work. We demonstrate here that theFET4 gene is subject to transcriptional regulation in response to iron and zinc status and the presence or absence of O2. These studies also provide an understanding of this regulation at a molecular level. In response to iron- or zinc-limiting growth conditions, FET4 expression is activated by Aft1 or Zap1, respectively. The role of Rox1 is to attenuate iron and zinc responsive expression in aerobic cells and allow derepression in anaerobic conditions. We have not provided evidence here that these proteins directly interact with the FET4 promoter. However, the effects of mutations in the genes encoding these transcription factors on FET4 expression plus the presence and importance of promoter elements similar to their consensus binding sites strongly support a direct role of these factors in regulating FET4transcription.Regulation of FET4 by iron status is easily understood given the importance of the Fet4 protein in iron accumulation (19Dix D.R. Bridgham J.T. Broderius M.A. Byersdorfer C.A. Eide D.J. J. Biol. Chem. 1994; 269: 26092-26099Abstract Full Text PDF PubMed Google Scholar, 20Dix D. Bridgham J. Broderius M. Eide D. J. Biol. Chem. 1997; 272: 11770-11777Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Both our results and those of Jensen and Culotta (41Jensen L.T. Culotta V.C. J. Mol. Biol. 2002; 318: 251-260Crossref PubMed Scopus (52) Google Scholar) indicate thatFET4 is regulated in response to iron status by Aft1. This conclusion is also supported by the work of Li and Kaplan (23Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) who showed that FET4 expression is up-regulated in afet3Δ mutant. Our results indicate that this increase is because of iron limitation imposed by loss of the high affinity iron uptake system. These observations were surprising given that two previous studies addressing FET4 regulation had discounted a role of Aft1. Casas et al. (42Casas C. Aldea M. Espinet C. Gallego C. Gil R. Herrero E. Yeast. 1997; 13: 621-637Crossref PubMed Scopus (75) Google Scholar) observed thatFET4 mRNA levels were not altered in cells treated with the iron chelator ferrozine, whereas other Aft1 target genes were induced. One possible explanation is that this treatment regimen was not sufficiently iron limiting to elicit a detectable response fromFET4. Also of note in their study (42Casas C. Aldea M. Espinet C. Gallego C. Gil R. Herrero E. Yeast. 1997; 13: 621-637Crossref PubMed Scopus (75) Google Scholar) was thatFET4 mRNA was detected in an aft1Δ mutant, indicating that other factors were involved in its expression. We now know that at least one other positive factor, i.e. Zap1, activates FET4 expression. Intriguingly, Dix et al. (20Dix D. Bridgham J. Broderius M. Eide D. J. Biol. Chem. 1997; 272: 11770-11777Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) noted that the Fet4 protein level and activity were induced ∼4-fold in response to iron limitation. However, a constitutive allele of AFT1,AFT1–1up, did not cause increased Fet4 uptake activity in iron-replete cells. Given that theAFT1–1up allele greatly increasedFET4 mRNA levels in iron-replete cells (41Jensen L.T. Culotta V.C. J. Mol. Biol. 2002; 318: 251-260Crossref PubMed Scopus (52) Google Scholar), it now seems likely that some form of post-transcriptional regulation of Fet4 activity may occur in response to iron status. Post-translational control of copper (43Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (178) Google Scholar), manganese (44Liu X.F. Culotta V.C. J. Biol. Chem. 1999; 274: 4863-4868Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and zinc (45Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 46Gitan R.S. Eide D.J. Biochem. J. 2000; 346: 329-336Crossref PubMed Scopus (144) Google Scholar) uptake transporters has already been observed in yeast so this seems a likely prospect.Previous microarray analyses from our laboratory suggested the zinc-responsive control of FET4 transcription by Zap1 (24Lyons T.J. Gasch A.P. Gaither L.A. Botstein D. Brown P.O. Eide D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7957-7962Crossref PubMed Scopus (250) Google Scholar). Here we confirm that FET4 expression is increased under zinc-deficient conditions. Most of the increase in FET4expression under zinc limitation was lost in the zap1Δmutant or when the ZRE in the FET4 promoter was mutated. However, there was a residual 2-fold response to zinc deficiency observed in a zap1Δ mutant strain and with aFET4 reporter where the ZRE was mutated. This residual zinc responsiveness may be because of loss of some Rox1 repression in zinc-deficient cells resulting from a possible decrease in heme levels. The second enzyme in heme biosynthesis, ALA dehydratase, is zinc-dependent (47Senior N.M. Brocklehurst K. Cooper J.B. Wood S.P. Erskine P. Shoolingin-Jordan P.M. Thomas P.G. Warren M.J. Biochem. J. 1996; 320: 401-412Crossref PubMed Scopus (55) Google Scholar), and in zinc-deficient conditions, this enzyme could conceivably become limiting for heme biosynthesis. It is also attractive to speculate that the iron status of aerobic cells may be communicated to FET4 transcription through effects of iron depletion on heme levels with a resultant decrease in Rox1 repression. However, this does not appear to occur; all iron responsiveness we observed in aerobic cells required Aft1. Iron-deficient aft1Δ mutants did not show residual iron responsiveness as we would expect if heme levels were also used as an indicator of iron status.Regulation of FET4 by Zap1 is now understandable given our new appreciation of the role of Fet4 in zinc uptake. Several of our observations indicate that Fet4 is a relevant pathway for zinc uptake in wild-type cells. Most zinc uptake in aerobically grown yeast is mediated by the Zrt1 and Zrt2 transporters (14Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). However, azrt1Δzrt2Δ double mutant is viable indicating that additional systems of zinc uptake are present and our results suggest that Fet4 is one of those systems. A zrt1Δzrt2Δfet4Δmutant, whereas requiring more zinc to grow than the double mutant, is also viable indicating that still other mechanisms of zinc uptake are present. One possible pathway for zinc accumulation is by fluid-phase endocytosis to the vacuole where the Zrt3 zinc transporter could mediate transport of zinc into the cytoplasm (32MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (301) Google Scholar).Microarray analysis indicated that FET4 expression was greatly increased in anaerobic cells when compared with cells grown aerobically (25ter Linde J.J. Liang H. Davis R.W. Steensma H.Y. van Dijken J.P. Pronk J.T. J. Bacteriol. 1999; 181: 7409-7413Crossref PubMed Google Scholar). Our results are in agreement with this previous study and we further showed that Rox1 is responsible for repressingFET4 expression in aerobic conditions. Loss of Rox1 repression under anaerobiosis results in increased expression ofFET4. In aerobic cells, Rox1 serves to attenuate activation by both of the metal-responsive activators, Aft1 and Zap1, that controlFET4 expression. Rox1 functions by recruiting the Ssn6 and Tup1 repressors to promoters where they block activation by positive factors (38Zitomer R.S. Limbach M.P. Rodriguez-Torres A.M. Balasubramanian B. Deckert J. Snow P.M. Methods Companion Methods Enzymol. 1997; 11: 279-288Crossref Scopus (28) Google Scholar). It is interesting to note that in Candida albicans, Tup1 regulates iron uptake genes in response to iron status (48Knight S.A.B. Lesuisse E. Stearman R. Klausner R.D. Dancis A. Microbiology. 2002; 148: 29-40Crossref PubMed Scopus (115) Google Scholar). Thus, ours is not the first example of these multifunctional transcription repressors acting to control metal ion uptake in fungi.Given that FET4 can contribute to the accumulation of iron and zinc in aerobic cells, the question arises as to whyFET4 is repressed under these conditions. One possible explanation for this repression is that, because Fet4 is relatively nonselective in its metal substrates, metal toxicity may occur by expressing FET4 at high levels in aerobic conditions. Indeed, such an effect was observed in experiments withfet3Δ mutants where FET4 is more highly expressed; fet3Δ mutants have increased sensitivity to cobalt, copper, manganese, and zinc (23Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). For cobalt, this increased sensitivity was shown to be dependent on FET4expression. Moreover, Jensen and Culotta (41Jensen L.T. Culotta V.C. J. Mol. Biol. 2002; 318: 251-260Crossref PubMed Scopus (52) Google Scholar) showed that cells overexpressing FET4 aerobically because of mutation ofROX1 are hypersensitive to cadmium. Thus, repression of the relatively nonspecific Fet4 transporter allows aerobic cells to rely on the more specific high affinity metal uptake systems.If high level Fet4 activity is so dangerous to cells, why then isFET4 expression derepressed in anaerobic cells? One possible explanation is that Fet4 is required under anaerobic conditions to compensate for the loss of Fet3 activity. The high affinity iron uptake system is composed of the Ftr1 transporter and the Fet3 multicopper oxidase. Fet3 activity is oxygen-dependent and the high affinity iron uptake system does not function under anaerobic conditions (8DeSilva D.M. Askwith C.C. Eide D. Kaplan J. J. Biol. Chem. 1995; 270: 1098-1101Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). 3D. Kosman, personal communication.Up-regulation of Fet4 in anaerobic cells may be a necessary risk taken by cells to maintain adequate iron accumulation and predicts that Fet4 will play a predominant role in iron accumulation under anaerobic conditions. In support of this hypothesis, Jensen and Culotta (41Jensen L.T. Culotta V.C. J. Mol. Biol. 2002; 318: 251-260Crossref PubMed Scopus (52) Google Scholar) observed that anaerobically grown fet4Δ mutants accumulated much less iron than wild-type cells. Furthermore, expression of many iron uptake genes including FET3,FTR1, SIT1, and FIT2 was found to be repressed in anaerobic conditions (25ter Linde J.J. Liang H. Davis R.W. Steensma H.Y. van Dijken J.P. Pronk J.T. J. Bacteriol. 1999; 181: 7409-7413Crossref PubMed Google Scholar, 49Hassett R.F. Romeo A.M. Kosman D.J. J. Biol. Chem. 1998; 273: 7628-7636Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). This repression, which is mediated through Aft1 (49Hassett R.F. Romeo A.M. Kosman D.J. J. Biol. Chem. 1998; 273: 7628-7636Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), may result from changes in iron status because of FET4 derepression. An additional reason underlying the increased expression of FET4 under anaerobic conditions may be related to changes in metal toxicity. Under anaerobic conditions, metal ions such as iron and copper are less likely to participate in metal-catalyzed Fenton chemistry that contributes to their toxicity (50Avery S.V. Adv. Appl. Microbiol. 2001; 49: 111-142Crossref PubMed Scopus (124) Google Scholar). Thus, higher levels of some metals may be more tolerable under conditions of low O2. One scenario that illustrates this point is that zinc-limited anaerobic cells may accumulate more iron and/or copper through Fet4 than would be tolerable under aerobic conditions.Early studies of metal ion transport suggested the existence of a general divalent metal ion uptake system that was responsible for accumulation of many different substrates (51Cooper T.G. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982: 399-461Google Scholar). With the subsequent characterization of the high affinity systems responsible for the specific uptake of copper (Ctr1 and Ctr3), iron (Fet3/Ftr1), manganese (Smf1), and zinc (Zrt1 and Zrt2), this hypothesis fell from favor. However, with our growing appreciation of the function of Fet4 in the uptake of multiple metal ion substrates, this model gains renewed credibility. We now know that Fet4 is a relevant transporter of iron, copper, and zinc in aerobic cells. Given the even higher levels ofFET4 expression in anaerobic cells, our data and those of others (41Jensen L.T. Culotta V.C. J. Mol. Biol. 2002; 318: 251-260Crossref PubMed Scopus (52) Google Scholar) argue that Fet4 plays an even greater role in the uptake of multiple metal ions under these conditions. Thus, combinatorial control of FET4 expression allows this protein to function in iron and/or zinc uptake depending on the metal status of the cell. Combinatorial control also allows modulation of activity of the Fet4 in aerobic cells to prevent overaccumulation of toxic metal ions and compensate for lost high affinity transporter activity under these conditions. Combinatorial control of gene expression occurs when a single gene is regulated in response to different signals (1Latchman D.S. Eukaryotic Transcription Factors. 2nd Ed. Academic Press, New York1995Google Scholar). This control is often mediated by the combination of different transcription factor-binding sites in a promoter of the gene and action on those elements by multiple transcriptional activators and/or repressors. Combinatorial control allows expression of a gene to be modulated by multiple signals and this can provide several advantages to a cell or an organism. For example, activity of a particular gene product may be beneficial under a variety of conditions. Alternatively, a gene product may play different roles under different conditions and combinatorial control can allow this to occur as well. Combinatorial control also allows modulation of expression by more than one signal to optimize the expression of a gene to match a particular combination of factors. In this paper, we describe the combinatorial transcriptional regulation of the yeast FET4 gene. FET4 encodes a metal ion transporter and its regulation by multiple signals allows modulation of expression in response to iron, zinc, and oxygen. Metals ions such as iron and zinc are essential nutrients for cells. However, if accumulated to excessive levels, these same elements can be toxic. Therefore, genes encoding the proteins responsible for uptake of metal ions are highly regulated. Metal uptake systems have been well characterized in the yeast Saccharomyces cerevisiae (2Eide D.J. Adv. Microb. Physiol. 2000; 43: 1-38Crossref PubMed Google Scholar). For example, iron uptake is mediated by several different pathways in this organism. First, yeast can accumulate iron bound to microbial siderophores (3Lesuisse E. Blaiseau P.L. Dancis A. Camadro J.M. Microbiology. 2001; 147: 289-298Crossref PubMed Scopus (93) Google Scholar). Although S. cerevisiae does not produce its own siderophores, a number of different transporters can accumulate iron bound by siderophores secreted by other microbes. S. cerevisiae also uses a reductive mechanism for iron uptake. Extracellular Fe(III) is reduced to Fe(II) by the Fre1 and Fre2 reductases located in the plasma membrane (4Dancis A. Klausner R.D. Hinnebusch A.G. Barriocanal J.G. Mol. Cell. Biol. 1990; 10: 2294-2301Crossref PubMed Scopus (256) Google Scholar, 5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Crossref PubMed Scopus (197) Google Scholar). The Fe(II) product is then the substrate of a high affinity transport system composed of the Fet3 multicopper oxidase and the Ftr1 permease (6Askwith C. Eide D., Ho, A.V. Bernard P.S., Li, L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Abstract Full Text PDF PubMed Scopus (582) Google Scholar, 7Stearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (574) Google Scholar). Fet3 uses O2 as a substrate to oxidize Fe(II) back to Fe(III) for subsequent transport by Ftr1 (8DeSilva D.M. Askwith C.C. Eide D. Kaplan J. J. Biol. Chem. 1995; 270: 1098-1101Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). High affinity copper uptake is also mediated by a reductive mechanism. Extracellular Cu(II) is reduced to Cu(I) by Fre1 reductase prior to transport across the plasma membrane by the Ctr1 and Ctr3 transporters (9Georgatsou E. Mavrogiannis L.A. Fragiadakis G.S. Alexandraki D. J. Biol. Chem. 1997; 272: 13786-13792Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 10Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 11Dancis A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; 41: 25660-25667Abstract Full Text PDF Google Scholar, 12Knight S.A.B. Labbeá S. Kwon L.F. Kosman D.J. Thiele D.J. Genes Dev. 1996; 10: 1917-1929Crossref PubMed Scopus (219) Google Scholar). Finally, high affinity zinc transport is mediated by the Zrt1 and Zrt2 proteins (13Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (445) Google Scholar, 14Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). These high affinity uptake systems are tightly regulated in response to the cellular status of their respective metal substrates. Iron-limiting growth conditions trigger increased expression of the genes encoding Fre1, Fre2, Fet3, Ftr1, and many other genes involved in iron acquisition (2Eide D.J. Adv. Microb. Physiol. 2000; 43: 1-38Crossref PubMed Google Scholar). This induction is mediated by two related transcription factors, Aft1 and Aft2, that share overlapping but nonidentical sets of target genes (15Blaiseau P.L. Lesuisse E. C
Referência(s)