In S. cerevisiae, high-affinity copper uptake is mediated by the CTR1 and CTR3 gene products (Ctr1p and Ctr3p, respectively) (2-5) and is associated with copper reductase activity of Fre1p and Fre2p to reduce Cu2+ to Cu*+ prior to uptake (6,7). CTR1, CTR3, and FRE1 are transcriptionally activated in response to copper deficiency by the copper-dependent transcriptional activator Mac1p (2,8-10). Copper-dependent endocytosis of Ctr1p at low copper concentrations and degradation of Ctr1p in copper-replete cells provides an additional posttranslational level of regulation (11). Three putative low-affinity copper-transport systems also have been described: Fet4p (12), Smf1/Smf2 (13,14), and Ctr2 (15).
2.1.2. Distribution, Storage, and Regulation
Inside the cell, copper is bound by cytoplasmic copper chaperones and delivered to specific destinations within the cell. These chaperones include Cox17p, Lys7p, and Atx1p, for copper delivery to cytochrome oxidase in the mitochondria, cytosolic and mitochondrial superoxide dismutase, and the copper transporter Ccc2p in the secretory pathway, respectively (2,16-20). Ccc2p is required for loading copper onto Fet3p, a multicopper oxidase, required for high-affinity iron transport across the plasma membrane (21). The chloride-ion channel Gef1p, located in the same compartment, is also required for copper loading onto Fet3p (22,23). Sco1p and Sco2p were identified as integral membrane proteins within the inner mitochondrial membrane and may be involved in the transfer of copper from Cox17p to cytochrome oxidase (5,24,25).
With increased intracellular copper levels, copper-storage proteins such as the metallothioneins encoded by CUP1 and CRS5 are induced and sequester the copper (8). Storage of copper in the vacuole may be an additional mechanism of preventing copper toxicity (5).
Mac1p and Ace1p are the two copper-responsive transcriptional activators in S. cerevisiae (2,9,10). Under copper-deficient conditions, Mac1p activates the expression of six genes, CTR1, CTR3, FRE1, FRE7, YFR055w, and YJL217w. With elevated copper levels, Ace1p activates the expression of copper-detoxification proteins, Cup1p and Crs5p.
Iron uptake into S. cerevisiae involves several different assimilatory pathways depending on the chemical source of iron and its concentration. In general, all of the pathways require reduction of Fe3+ to Fe2+ by one or more products of the FRE1-FRE7 genes (14,26). Under iron-limiting conditions, high-affinity uptake is mediated by the inducible Fet3p/Ftr1p complex in the plasma membrane (1,14,27). Fet3p is a multicopper oxidase (28) whose active sites are related to multicopper oxidases such as laccase, ascorbate oxidase, and ceruloplasmin. Fet3p functions to oxidize Fe2+ to Fe3+ at the cell surface; Fe3+ is then delivered from Fet3p to the associated Fe3+ permease, Ftr1p, for transport into the cell (14,27). Highly related pathways operate in other fungi. In Schizosaccharomyces pombe, Frp1 is related to the Fre reductases, and Fio1/Fip1 forms an iron-uptake complex related to Fet3p/Ftr1p (29). The fungal pathogen Candida albicans also uses a similar pathway, involving a surface-associated reductase, Cfl1p (30), two Ftr1p-related permeases, CaFtr1 and CaFtr2 (31), and a Fet3p-like component (30). Expression of the permeases and their associated oxidases is regulated by iron nutritional status (27,29), whereas some reductases are regulated by iron status and others by both copper and iron—the latter category functioning in both copper and iron assimilation (10,14). Under iron-replete conditions, low-affinity systems, such as Fet4p, with broad transition metal specificity (12,32), or Smf1/Smf2, which were originally identified as manganese transporters (13), operate. When iron is available in complex with siderophores, it can enter cells either through the Fet3p/Ftr1p complex after reduction by the Fre proteins and subsequent release of the iron from the siderophore, or if the Fet3p/Ftr1p is inoperable, by the ARN family of siderophore transporters through an endocytic pathway (33-38).
2.2.2. Distribution, Storage, and Regulation
Whereas the Fet3p component of iron assimilation is conserved from yeast to man, the mechanism of iron storage appears diverse. S. cerevisiae lacks genetic information for ferritin (8); rather, iron (with other ions) is stored in the vacuole (39,40). A homolog of the oxidase-permease complex Fet3p/Ftr1p, designated Fet5p/Fth1, was localized to the vacuole and proposed to function under iron starvation conditions to mobilize stored iron from the vacuole (41).
Another key organelle in iron metabolism is the mitochondrion. Not only is it an important target site for iron utilization, but it is also a major player in the maintenance of cellular iron homeostasis. Yfhlp regulates mitochondrial iron accumulation, possibly at the level of efflux (42,43), Ccclp limits mitochondrial iron uptake (44), Nfslp is involved in maintaining or synthesizing cytosolic Fe-S proteins (45,46), Atmlp is a transporter of the inner mitochondrial membrane (47), and Ssc2p is a protein of the Hsp70 class of chaperones with a role in Yfhlp maturation (48). Although there are as yet no defined chaperones for the delivery of iron to specific intracellular targets, the IscA family of proteins are candidates for iron delivery to sites of iron-sulfur cluster assembly (49).
Iron uptake in S. cerevisiae is regulated in response to cellular iron levels via the iron-responsive transcriptional activator Aftlp. Under iron-limiting conditions, Aftlp-mediated activation leads to induction of FRE1, FRE2, FTR1, FET3, FET5, and FTH1, as well as the induction of expression of the genes involved in copper transport, CCC2 and ATX1 (8). A further level of regulation of iron uptake exists that is mediated through Tpk2, a catalytic subunit of the yeast A kinases (PKA) (50). According to the proposed model, during fermentative growth on glucose Tpk2, activated by cAMP, represses genes involved in iron uptake, but as the glucose is depleted, Tpk2 activity is inhibited, thus relieving the repression of the iron-transport genes. This derepression allows iron transport into the cell for incorporation into respiratory enzymes and permits growth on nonfermentable carbon sources.
In S. cerevisiae, the copper-iron link is evident at both the level of protein biosynthesis and function and at the level of gene regulation. Copper is required for the assembly and function of the high-affinity Fet3p/Ftr1p iron-uptake complex, and thus for high-affinity iron uptake. Therefore, S. cerevisiae cells that are copper deficient are also iron deficient. The iron-uptake genes are regulated as a function of iron availability, so that the expression of these genes is activated in cells that are starved for iron. Because the Frel reductase functions in copper and iron uptake, its expression is induced by both copper and iron deficiency, whereas the copper-transport genes (CCC2 and ATX1), whose activity is critical for the production of functional iron-uptake proteins, are induced by iron deficiency, but not by copper deficiency (51-54). In addition, in S. pombe, a copper-responsive transcription factor, Cufl, activates CTR4 gene expression for copper uptake under copper-starvation conditions while mediating repression of the iron-uptake genes under these same conditions to prevent futile expression of these genes when there is insufficient copper cofactor available to produce functional iron-uptake proteins (55). Therefore, an intricate system of interplay among transport, storage, chaperone and regulatory proteins exists to maintain copper and iron homeostasis in S. cerevisiae and S. pombe.
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