- Metal-Responsive Transcription Factors That Regulate Iron, Zinc, and Copper Homeostasis in Eukaryotic Cells

Iron is extremely insoluble in the presence of oxygen at physiological pH. Organisms that live in an oxygen environment have therefore evolved specific mechanisms to acquire what would otherwise be an unavailable nutrient. These systems of iron acquisition in many fungi are regulated at the transcriptional level by iron availability. The proteins that mediate this control are, to date, the only known iron-responsive transcription factors within eukaryotes. In the budding yeast Saccharomyces cerevisiae, genes that are involved in the compartmentalization and use of iron are regulated in a similar way to those genes that are involved in iron acquisition. This global regulation of iron metabolism may be established to be the norm in other eukaryotic microorganisms.

The iron metabolism of S. cerevisiae has been the most intensively studied of all the fungi (reviewed in references 83, 114, and 146). This organism can grow in both aerobic and anaerobic environments and can utilize a variety of carbon sources by using both fermentative and respiratory metabolism. This range of growth conditions influences iron availability and the cell requirements for iron. Under anaerobic conditions, iron is in the ferrous form and therefore more readily available. Conversely, cells that are respiring require additional iron for the various iron-containing proteins of the mitochondrial respiratory chain at a time when iron is less soluble. Therefore, mutations that are detrimental to iron metabolism often result in a more severe phenotype when this yeast grows by using a respiratory carbon source.

S. cerevisiae contains a variety of genes that are transcriptionally induced in response to low iron and which encode proteins that are involved in iron acquisition at the cell surface (Table 1; Fig. 1A). Free iron is taken into the cell by both high- and low-affinity transport systems (Fet3, Ftr1, and Fet4) (36, 136). The high-affinity complex contains a ferroxidase (Fet3) that requires copper as a cofactor. Consequently, genes that are involved in the trafficking and transport of copper to this protein (ATX1 and CCC2) are also regulated at the transcriptional level by iron (93, 156, 160). High-affinity iron uptake is therefore compromised when cells experience low copper levels. A cell surface ferric reductase activity is also required for high-affinity iron uptake (33, 34). The majority of this activity is provided by two flavocytochromes (Fre1 and Fre2) that reduce ferric iron to provide ferrous iron as a substrate for the high-affinity transport system (48, 49). S. cerevisiae can also acquire iron through siderophores, which are low-molecular-weight organic molecules that specifically chelate iron. S. cerevisiae does not synthesize its own siderophores, but it is able to utilize those that are produced by other microorganisms (114). A family of transporters (Arn1 to Arn4) that cycle between the cell surface and an endosomal compartment mediate siderophore uptake (162). As an alternative to siderophore uptake, the siderophore iron can be reduced by the cell surface reductases (Fre1 to Fre4) to provide ferrous iron as a substrate for the Fet3/Ftr1 high-affinity uptake system (162). A group of mannoproteins (Fit1 to Fit3) facilitates siderophore iron uptake by sequestering this iron chelate within the cell wall (117). In addition to those genes that are involved in cell surface iron acquisition, a number of genes involved in other aspects of iron metabolism are transcriptionally induced under low-iron conditions (Table 1). These include genes that encode vacuolar transport systems (Fet5, Fth1, and Smf3), a mitochondrial transporter (Mrs4), and proteins involved in the biosynthesis of iron-sulfur clusters (Isu1 and Isu2) (43, 47, 115, 116, 129, 145).

Iron-dependent gene regulation in S. cerevisiae is mediated by two transcription factors. Aft1 and Aft2 (for "activator of ferrous transport") activate gene expression when iron is scarce. Consequently, strains that lack both these factors exhibit reduced expression of the iron regulon (14, 24, 124, 154, 156). The genes that code for these factors are thought to have arisen from a genome duplication event (130). As with many other paralogous genes within S. cerevisiae, AFT1 and AFT2 code for proteins that have significant regions of identity and overlapping functions. The DNA-binding domain of each protein is in a highly conserved N-terminal region, and a conserved cysteine-to-phenylalanine mutation in both proteins generates a factor that activates the high expression of the iron regulon irrespective of iron concentrations (124, 154). There are clear phenotypic differences in strains that separately lack Aft1 and Aft2. An aft1 null strain exhibits low ferrous iron uptake and grows poorly under low-iron conditions or on a respiratory carbon source (24, 154). No phenotype has been attributed to an aft2 null strain. An aft1 aft2 double null strain is, however, more sensitive to low-iron growth than a single aft1 null strain, which is consistent with the functional similarity of these factors (14, 124). The partial redundancy of these factors allows Aft2 to complement an aft1 null strain when it is overexpressed from a plasmid (124).

The properties of Aft1 and Aft2 that distinguish them from each other have not been fully identified. Both factors mediate gene regulation via an iron-responsive element that contains the core sequence 5'-CACCC-3' (125, 156). It is likely that sequences adjacent to this element influence the ability of each factor to mediate regulation via a particular iron-responsive element (125). The differential regulation of individual genes by Aft1 and Aft2 results in each factor generating a distinct global transcriptional profile (125). Aft1 autoregulation, which is consistent with the in vivo binding of Aft1 to its own promoter, may influence Aft1 control of the iron regulon (89). Critical to the function of Aft1 is its ability to shuttle between the nucleus and cytoplasm in response to iron levels. Nuclear export is dependent on a leucine rich N-terminal nuclear export signal (NES), mutation of which results in the nuclear retention of Aft1 and constitutive expression of Aft1-regulated genes (157). Aft1 nuclear import is mediated by a direct interaction with the nuclear import factor Pse1 (144). Aft1 contains two distinct basic nuclear localization signals, which together are sufficient and necessary to direct Aft1 to the nucleus (144).

Aft1 function is regulated in response to glucose levels independently of iron. Certain genes in the iron regulon are induced immediately following entry into the diauxic shift when cells adapt from fermentative to respiratory metabolism. This control is dependent on both the global regulatory complex Snf1/Snf4 and on Aft1 (64). The iron regulon also responds to glucose levels via the cyclic AMP-dependent protein kinase A. TPK2 encodes a protein kinase A catalytic subunit, and a tpk2 null strain shows derepressed expression of the iron regulon (123). Tpk2-dependent and Snf1/Snf4-dependent regulation of the iron regulon is consistent with an increased requirement for iron during respiratory metabolism (64, 123). Direct phosphorylation of Aft1 that is independent of Snf1 and cyclic AMP levels occurs when cells undergo a transient or permanent cell cycle arrest. Conditions that result in this modification of Aft1 include the change or removal of a carbon source, traversal of the diauxic shift, and the shifting of temperature-sensitive cdc28 or cdc25 mutants to a nonpermissive temperature (24, 64). The functional consequences of this cell cycle-dependent phosphorylation of Aft1 are not clear.

The mechanisms(s) by which the Aft1 and Aft2 factors sense iron are not fully understood. Nucleocytoplasmic shuttling of Aft1 in response to iron is fundamental to the ability of a cell to sense iron. However, the signal that shifts the equilibrium of Aft1 localization that results in nuclear translocation is not known. It is also not clear if that signal acts at the level of Aft1 nuclear export or import. The phosphorylation of Aft1 and the effect of the various phosphorylation pathways on the iron regulon described above are not involved in iron sensing per se. These regulatory pathways integrate Aft1 function with other aspects of cellular metabolism such as carbon source utilization. However, phosphorylation by an unknown signaling pathway could be the triggering event for Aft1 nuclear localization. Alternatively, Aft1 may bind iron directly and the loss of iron binding could initiate the Aft1 response. Direct metal binding of iron as a signal of iron status has been demonstrated with various prokaryotic iron-responsive transcription factors (reviewed in reference 6). Consistent with this hypothesis, the iron regulon in S. cerevisiae is sensitive to the intracellular chelation of iron and mutants that accumulate iron in the mitochondria exhibit enhanced expression of the iron regulon (44, 63). In addition, iron-responsive gene regulation is significantly compromised in cells that are unable to synthesize heme (28). The Fe(II)/Fe(III) redox equlibrium in the cell may also influence iron metabolism (63). This is supported by the phenotype of a sod1 null strain that lacks superoxide dismutase activity. This mutant is sensitive to oxidative stress and has higher than wild-type levels of Fe(III) with a concurrent increase in the expression of an Aft1/2-regulated gene (35, 134).

Iron-regulated gene expression in fungi other than S. cerevisiae is mediated by a group of GATA-type transcription factors. These include Fep1 from Schizosaccharomyces pombe, SREA from Aspergillus nidulans, SRE from Neurospora crassa, SreP from Penicillium chrysogenum, and Urbs1 from Ustilago maydis (60, 61, 107, 147, 171). These factors regulate, or are predicted to regulate, the expression of genes involved in siderophore production, siderophore transport, and free iron transport (Table 1). GATA factors are a group of transcription factors that are characterized by conserved zinc finger motifs and their ability to bind to a core 5'-GATA-3' element. The fungal iron-responsive GATA factors contain two zinc finger Cys-X2-Cys-X17-Cys-X2-Cys motifs that flank a region that contains four conserved cysteine residues. Adjacent to the C-terminal zinc finger of each factor is a basic region that is conserved in other eukaryotic GATA factors (Fig. 2A). The iron-responsive GATA-type factors repress the transcription of their target genes in response to high iron. Therefore, although the iron-responsive GATA factors are transcriptional repressors and the Aft factors are transcriptional activators, both classes of factors ensure that their target genes are induced when the relevant organism senses that iron is limiting.

The phenotypes of strains that lack an iron-responsive GATA factor are consistent with deregulation of iron metabolism. Wild-type siderophore production is repressed under high iron conditions but derepressed in strains that lack Urbs1, SREA, and SRE (61, 147, 170). Uptake of 59Fe(III) is higher in an sreA null strain than in a wild-type strain, and this confers sensitivity to the iron-dependent antibiotics phleomycin and streptonigrin (61). A fep1 null strain exhibits constitutive cell surface metalloreductase activity and is also sensitive to phleomycin (107). Consistent with these phenotypes, mutant strains that lack the relevant GATA factor exhibit constitutive expression of genes involved in the acquisition of iron from the environment (2, 103, 107, 108, 161). Fep1-dependent repression also requires Tup11 and Tup12 that may act as corepressors in a complex with Fep1 (108). A similar role in the regulation of the iron regulon in Candida albicans has also been proposed for the homologous Tup1, although the factor that recruits it to the relevant promoters has not been identified (80).

The number of functional 5'-GATA-3' elements differs in the target promoters of the iron-responsive GATA factors. Fep1 regulates gene expression via two adjacent sites or a single site depending on the gene in question (107, 108). The full Urbs1-dependent regulation of a gene involved in siderophore synthesis requires two adjacent 5'-GATA-3' elements, although these are not in themselves sufficient to confer repression of a reporter gene. Therefore, Urbs1 may interact with other GATA sites in this particular promoter region (2). The in vitro affinity of SRE for two adjacent 5'-GATA-3' elements is dependent on the spacing between those two sites (62). Urbs1 and Fep1 have a higher in vitro affinity for one site when the target DNA contains two adjacent 5'-GATA-3' elements. The loss of this high-affinity site results in a greater loss of in vivo repression of a reporter gene than does loss of the low-affinity site (2, 107). In addition, the phenotypes of various Urbs1 mutants suggest that, of the two zinc fingers, the C-terminal finger is more important for DNA binding (3).

Evidence to date suggests that the iron-responsive GATA factors bind iron. Recombinant SRE is reddish brown in color and gives a spectrum that is characteristic of iron-binding proteins, which is lost when the protein is reduced (62). The diagnostic spectrum of the wild-type protein is lost with proteins that contain cysteine-to-serine mutations in the conserved region between the zinc fingers. In vivo, these substitutions result in a constitutive repressor that does not respond to iron (62). Furthermore, the in vitro DNA-binding ability and stability of recombinant Fep1 is dependent on the protein being expressed in cells that are grown in high-iron medium before the protein is purified (107). In addition to the conserved cysteines between the zinc fingers, it has also been proposed that a conserved RXXE motif in the C-terminal zinc finger is a potential iron-binding site (107). An arginine-to-leucine mutation in the same motif in Urbs1 renders it unable to respond to iron (3). As with the iron-responsive factors from S. cerevisiae, further work is required to determine the precise mechanism of iron sensing by these factors.

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