The related transcription factors ACE1 of Saccharomyces cerevisiae and AMT1 of Candida glabrata are involved in copper metabolism by activating the transcription of copper metallothionein genes. ACE1 and AMT1 are ‘copper‐fist’ transcription factors which possess a conserved cysteine‐rich copper binding domain required for DNA binding. Here we report the identification of a nuclear protein from S. cerevisiae, MAC1, whose N‐terminal region is highly similar to the copper and DNA binding domains of ACE1 and AMT1. Loss‐of‐function mutants of MAC1 have a defect in the plasma membrane Cu(II) and Fe(III) reductase activity, are slow growing, respiratory deficient, and hypersensitive to heat and exposure to cadmium, zinc, lead and H2O2. Conversely, a dominant gain‐of‐function mutant of MAC1 shows an elevated reductase activity and is hypersensitive to copper. We have identified two target genes of MAC1 whose altered expression in mutants of MAC1 can account for some of the observed mutant phenotypes. First, MAC1 is involved in basal level transcription of FRE1, encoding a plasma membrane component associated with both Cu(II) and Fe(III) reduction. Second, MAC1 is involved in the H2O2‐induced transcription of CTT1, encoding the cytosolic catalase. This suggests that MAC1 may encode a novel metal‐fist transcription factor required for both basal and regulated transcription of genes involved in Cu/Fe utilization and the stress response.

No abstract

High affinity iron uptake in Saccharomyces cerevisiae requires a metal reductase, a multicopper ferroxidase, and an iron permease. Fet3, the apparent ferroxidase, is proposed to facilitate iron uptake by catalyzing the oxidation of reductase-generated Fe(II) to Fe(III) by O 2 ; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1 (Kaplan, J., and O'Halloran, T. V. (1996) Science 271, 1510 -1512). We show here that dioxygen also plays an essential role in the expression of these iron uptake activities. Cells grown anaerobically exhibited no Fe(III) reductase or high affinity iron uptake activity, even if assayed for these activities under air. Northern blot analysis showed that the amount of those mRNAs encoding proteins associated with this uptake was repressed in anaerobic cultures but was rapidly induced by exposure of the culture to dioxygen. The anaerobic repression was reduced in cells expressing an iron-independent form of the trans-activator, Aft1, a protein that regulates the expression of these proteins. Thus, the effect of oxygenation on this expression appeared due at least in part to the state or distribution of iron in the cells. In support of this hypothesis, the membrane-permeant Fe(II) chelator, 2,2-bipyridyl, in contrast to the impermeant chelator bathophenanthroline disulfonate, caused a strong and rapid induction of these transcripts under anaerobic conditions. An increase in the steady-state levels of iron-regulated transcripts upon oxygenation or 2,2-bipyridyl addition occurred within 5 min, indicating that a relatively small, labile intracellular pool of Fe(II) regulates the expression of these activities. The strength of the anaerobic repression was dependent on the low affinity, Fe(II)-specific iron transporter, encoded by FET4, suggesting that this Fe(II) pool was linked in part to iron brought into the cell via Fet4 protein. The data suggest a model in which dioxygen directly or indirectly modulates the Fe(III)/Fe(II) ratio in an iron pool linked to Aft1 protein while bipyridyl increases this ratio by chelating Fe(II). These results indicate that dioxygen both modulates the sensitivity to iron-dependent transcriptional regulation and acts as substrate for Fet3 in the ferroxidase reaction catalyzed by this ceruloplasmin homologue.

Fre1p is a metalloreductase in the yeast plasma membrane that is essential to uptake of environmental Cu 2؉ and Fe 3؉ . Fet3p is a multicopper oxidase in this membrane essential for high affinity iron uptake. In the uptake of Fe 3؉ , Fre1p produces Fe 2؉ that is a substrate for Fet3p; the Fe 3؉ produced by Fet3p is a ligand for the iron permease, Ftr1p. Deletion of FET3 leads to iron deficiency; this deletion also causes a copper sensitivity not seen in wild type. Deletion of FTR1 leads to copper sensitivity also. Production in the ftr1⌬ strain of an iron-uptake negative Ftr1p mutant, Ftr1p(RAGLA), suppressed this copper sensitivity. This Ftr1p mutant supported the plasma membrane targeting of active Fet3p that is blocked in the parental ftr1⌬ strain. A ferroxidase-negative Fet3p did not suppress the copper sensitivity in a fet3⌬ strain, although it supported the plasma membrane localization of the Fet3p⅐Ftr1p complex. Thus, loss of membrane-associated Fet3p oxidase activity correlated with copper sensitivity. Furthermore, in vitro Cu 1؉ was shown to be an excellent substrate for Fet3p. Last, the copper sensitivity of the fet3⌬ strain was suppressed by co-deletion of FRE1, suggesting that the cytotoxic species was Cu 1؉ . In contrast, deletion of CTR1 or of FET4 did not suppress the copper sensitivity in the fet3⌬ strain; these genes encode the two major copper transporters in laboratory yeast strains. This result indicated that the apparent cuprous ion toxicity was not due to excess intracellular copper. These biochemical and physiologic results indicate that at least with respect to cuprous and ferrous ions, Fet3p can be considered a metallo-oxidase and appears to play an essential role in both iron and copper homeostasis in yeast. Its functional homologs, e.g. ceruloplasmin and hephaestin, could play a similar role in mammals.There is a well recognized link between copper and iron metabolism. This link is illustrated by the copper dependence of high affinity iron uptake in the yeast Saccharomyces cerevisiae (1). This dependence is due to the role that copper plays as cofactor for the ferroxidase, Fet3p (2). Fet3p and the iron permease, Ftr1p, form a complex in the yeast plasma membrane (3). Genetic and biochemical studies show that in high affinity iron uptake Fet3p catalyzes the oxidation of Fe 2ϩ to Fe 3ϩ ;Ftr1p then transports the Fe 3ϩ into the cell (3-7). In humans, the Fet3p ortholog, ceruloplasmin, also catalyzes the oxidation of Fe 2ϩ for iron release into circulation (see Ref. 8 and references therein). Hephaestin, a membrane-bound ceruloplasmin paralog, is required for iron efflux from the placenta and enterocytes in mammals (9). Thus, the copper-dependent ferroxidase activity characteristic of these members of the multicopper oxidase family has a conserved role in iron metabolism.Studies of FET3 suggest that this ferroxidase might also play a role in cellular defense against copper toxicity; that is, a yeast mutant carrying a disruption of the FET3 gene was found to be sensitive to high concentrati...