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This review focuses on the role that metal-responsive transcription factors have in …

Biology Articles » Cell biology » Metal-Responsive Transcription Factors That Regulate Iron, Zinc, and Copper Homeostasis in Eukaryotic Cells » Copper

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


A number of homeostatic mechanisms have been identified which ensure that copper is maintained at a level sufficient for, but not toxic to, cell growth. In mammals, posttranslational mechanisms, such as the intracellular trafficking of copper transporters and the copper-stimulated endocytosis and degradation of proteins involved in copper uptake, play a major role in copper homeostasis (109, 112, 113). Although posttranslational control of transporters exists in fungi, copper homeostasis in these organisms is also mediated by the transcriptional regulation of genes involved in copper acquisition, mobilization, and sequestration (105). To date, six copper-responsive fungal factors have been characterized in detail. Ace1 (also known as Cup2), Amt1, and Crf1 activate gene expression in response to elevated copper while Mac1, GRISEA, and Cuf1 activate gene expression in response to copper deficiency. Homeostasis through copper-responsive transcriptional regulation has been observed in insects and plants, as well as in fungi, suggesting that this mechanism of copper control is widespread in nature (69, 126, 169).

Factors that are activated in response to copper regulate the expression of genes encoding proteins involved in copper sequestration and/or protection against copper toxicity (Table 1). In S. cerevisiae, resistance to copper is primarily mediated by the Ace1-dependent induction of the CUP1 gene (Fig. 1B) (139, 150). CUP1 encodes a small, cysteine-rich, copper-binding metallothionein that protects cells by sequestering copper and thereby preventing its toxicity (23, 42, 77, 153). Ace1 also regulates the expression of a second metallothionein gene (CRS5) and the copper and zinc superoxide dismutase gene (SOD1) (29, 55). Functional orthologs of Ace1 confer copper resistance in Candida glabrata (Amt1) and Yarrowia lipolytica (Crf1) (46, 173). Although Amt1 protects cells from copper by regulating the expression of three metallothionein genes, metallothionein expression is still copper responsive in a crf1 mutant strain (46, 141, 172). This latter result suggests that Crf1 guards against copper overload by regulating the expression of a yet-unidentified target gene(s) (46).

Regulatory factors that are active during copper deficiency regulate the expression of genes encoding proteins involved in increasing cytosolic copper (Table 1). In S. cerevisiae, Mac1 protects cells from copper starvation by activating the expression of the high-affinity copper uptake systems encoded by CTR1 and CTR3 (Fig. 1A) (85, 155). Mac1 also regulates the expression of a cell surface Fe3+/Cu2+ reductase (FRE1) and a putative reductase of unknown cellular localization (FRE7) (49, 98). The posttranslational degradation of Ctr1 under conditions of excess copper also requires Mac1. It is possible that an uncharacterized Mac1 target gene (Table 1) encodes a protein that is essential for this regulation or that Mac1 itself functions as a protease or protease-recruiting factor under copper-replete conditions (158).

High-affinity copper uptake is regulated at the transcriptional level in S. pombe and Podospora anserina by the factors Cuf1 and GRISEA, respectively (7, 16, 84). In addition to directly regulating copper uptake, Cuf1 stimulates mobilization of copper from vacuolar stores by regulating the expression of the CTR6 vacuolar efflux system (9). GRISEA activates the expression of P. anserina SOD2, a gene that is not copper regulated in S. pombe or S. cerevisiae (17). P. anserina SOD2 encodes mitochondrial manganese superoxide dismutase. In P. anserina, low intracellular copper levels lead to reduced activity of the copper-requiring enzyme, cytochrome c oxidase. This in turn results in the induction of an iron-dependent pathway that utilizes an alternative terminal oxidase. Induction of P. anserina SOD2 under copper-limiting conditions may therefore be important for protection from oxidative stress inherent in the utilization of the alternate oxidase (16-18).

In addition to activating copper transporter genes, Cuf1 represses the expression of the iron-regulated fip1+, fio1+, and frp1+ genes that encode proteins required for iron uptake (84). Similar to S. cerevisiae, iron uptake in S. pombe requires copper. Cuf1-dependent repression of these genes therefore ensures that iron uptake is inhibited under copper-limiting conditions. As copper levels increase (through Cuf1-dependent copper uptake and copper mobilization), Cuf1 is inactivated, which leads to the loss of Cuf1-mediated repression of fip1+, fio1+, and frp1+ (84). It is currently unknown whether Cuf1 mediates this repression by recruiting a corepressor to these promoters or whether Cuf1 inhibits binding of a transcriptional activator. A similar regulatory pathway may also exist in S. cerevisiae, since the iron-regulated FET3 gene also appears to show Mac1-dependent repression (84). However, it is not clear whether this is a direct result of Mac1 acting as a repressor at the FET3 promoter or whether this is a pleiotropic affect of altered iron homeostasis in strains that lack or express a constitutive allele of MAC1. Thus, the ability of Cuf1, and possibly Mac1, to act as both a repressor and activator allows the coordinated expression of genes involved in both copper and iron homeostasis.

A number of structural domains are conserved between the six known copper regulatory factors (Fig. 2B). Ace1, Amt1, and Crf1 all contain a zinc-binding domain, a conserved (R/K)GRP sequence motif, and eight cysteine residues that are arranged in Cys-X-Cys or Cys-X2-Cys motifs (45, 137, 142). The cysteine-rich motifs form a polycopper cluster that binds 4 Cu(I) ions cooperatively while the zinc-binding domain and (R/K)GRP motif are essential for minor groove site-specific binding (32, 41, 53, 81, 142, 143). Mac1, GRISEA, and Cuf1 share regions of homology to Ace1 but lack all the cysteine-rich motifs required in forming the Ace1-Amt1 N-terminal polycopper cluster. The C terminus of Mac1 contains two Cys-X-Cys-X4-Cys-X-Cys-X2-Cys-X2-His motifs that have been designated C1 and C2 (or REPI and REPII, respectively) (Fig. 2B) (54, 78, 175). The C1 and C2 motifs lie within transactivation domains and bind four Cu(I) ions in a polycopper cluster (20, 73). Similar to Mac1, GRISEA contains two cysteine-rich domains (15). Cuf1 contains only one of these motifs, designated C1 (84).

An important facet of copper homeostasis is that copper regulates the activity of each transcription factor. Evidence to date indicates that the conserved structural domains within each class of copper regulatory factors are important for copper sensing. Copper-dependent DNA-binding activity primarily regulates Ace1 and Amt1 activity. Both factors bind as a monomer, in a copper-dependent manner, to upstream activating sequences (22, 45, 70, 140, 172). The copper-induced gene activation is thought to be mediated by the formation of the N-terminal polycopper cluster in response to copper, which converts Ace1 and Amt1 from a nonactive form to an active DNA-binding form (reviewed in reference 152). Haa1, a transcriptional activator in S. cerevisiae, contains the eight conserved cysteine residues that are required for polycopper cluster formation in Ace1, yet is not regulated by copper. Amino acids present in the Haa1 N-terminal region but not present in Ace1 or Amt1 may disrupt polycopper cluster formation and prevent this domain from being used as a copper regulatory domain (79). Additional regulatory mechanisms control both Amt1 and Crf1 activity. Amt1 autoregulates its own expression, a critical factor in copper resistance, since cells that are unable to autoactivate AMT1 are sensitive to growth on copper (174). Crf1 activity is subject to copper-dependent nuclear translocation (46).

Mac1 mediates the response to copper limitation by binding as a homodimer, in a site-specific manner, to copper-responsive cis-acting elements (CuREs) that are located in the promoter regions of target genes (71, 76, 85, 131, 155). In vivo, activation of gene expression requires multiple CuREs, which are arranged in tandem or as inverted repeats and have a synergistic effect on gene expression (74, 85, 155). On exposure to copper, repression of Mac1 is primarily mediated by a copper-dependent interaction between the C1 domain and the DNA-binding domain. This interaction inhibits both transactivation domain function and DNA-binding activity (54, 74, 85). In support of this model, mutations that substitute single cysteine residues in the Mac1 C1 motif lead to a total loss of copper-responsive regulation (78, 155, 175).

Cuf1 activity is similarly controlled by a copper-dependent interaction between the DNA-binding domain and the C1 domain (8). However, a number of differences between Mac1 and Cuf1 regulation have been observed. The N-terminal region of Cuf1 exhibits a higher percentage of sequence identity with the corresponding Ace1 domain than with the N-terminal domain of its functional homolog Mac1. Indeed, Cuf1 can activate Ace1 target gene expression when introduced into an ace1 null S. cerevisiae strain (7, 8). A second difference between Mac1 and Cuf1 is that the substitution of cysteine residues in the Cuf1 C1 domain only leads to a partial loss of regulation. This result suggests that the cysteine residues of Cuf1 are not equally involved in copper coordination (8). Similarities and differences between the regulation of Mac1 and GRISEA by copper are also observed. For example, copper-responsive repression of GRISEA is mediated by an interaction between the DNA-binding domain and the C2 domain rather than the C1 domain (15).

While the copper-dependent control of Mac1 activity via an intramolecular interaction remains undisputed, more-recent studies of Mac1 have superimposed a number of additional regulatory mechanisms. Mac1 must be phosphorylated to bind to CuREs, and homodimerization of Mac1 is essential for maximal in vivo activity (65, 76, 131). It is noteworthy that overexpression of Mac1 constructs that lack the homodimerization domain are fully functional proteins (74, 148). Homodimerization of Mac1 may therefore allow a protein that is normally expressed at a low level to achieve maximal activation of target gene expression. A third additional level of regulation is that in the presence of excess copper, C-terminally tagged Mac1 undergoes a rapid copper-dependent degradation. However, this effect is lost when Mac1 is overexpressed (74, 175). Finally, the C2 transactivation domain may have a role in modulating the activity of Mac1 (78, 148). Further studies that identify other proteins required for Mac1 regulation may reveal the finer details of how Mac1 responds to copper.

In addition to Mac1 and its functional homologs, novel copper regulatory factors may also exist in the plant kingdom. Copper is an essential cofactor of a number of the enzymes required for photosynthesis. During copper deficiency, the photosynthetic algae Chlamydomonas reinhardtii can bypass this copper requirement by decreasing its reliance on copper-requiring enzymes and using alternative enzymes that utilize heme cofactors. At the transcriptional level, this transition is mediated by the Crr1-dependent induction of CPX1 and CYC6 (120, 121). These genes encode the enzyme coproporphyrinogen oxidase that is required for heme biosynthesis and cytochrome c6, a heme-containing electron transfer catalyst. Crr1 also reciprocally regulates the expression of the partially redundant genes CRD1 and CTH1, which are required for the maintenance of photosystem I under copper-limiting and -replete conditions, respectively (100, 101). Although the precise genetic locus of Crr1 has yet to be identified, a number of observations suggest that Crr1 is not simply an ortholog of Mac1. First, one CuRE is both necessary and sufficient to mediate copper-responsive regulation (119). Second, the consensus sequence of GTAC, rather than the Mac1 consensus 5'-TTTGC(T/G)C(A/G)-3', is found in all known Crr1 target genes and is essential for copper-responsive transcription (119). Finally, Crr1 possibly responds to Cu2+ not Cu+, since Hg2+ and not Ag+ will also repress Crr1 target gene transcription (119).

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