Fre1p Cu2+ reduction and Fet3p Cu1+ oxidation modulate copper toxicity in Saccharomyces cerevisiae

Fre1p is a metalloreductase in the yeast plasma membrane that is essential to uptake of environmental Cu2+ and Fe3+. Fet3p is a multicopper oxidase in this membrane essential for high affinity iron uptake. In the uptake of Fe3+, Fre1p produces Fe2+ that is a substrate for Fet3p; the Fe3+ 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 ftr1delta 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 ftr1delta strain. A ferroxidase-negative Fet3p did not suppress the copper sensitivity in a fet3delta strain, although it supported the plasma membrane localization of the Fet3p.Ftr1p complex. Thus, loss of membrane-associated Fet3p oxidase activity correlated with copper sensitivity. Furthermore, in vitro Cu1+ was shown to be an excellent substrate for Fet3p. Last, the copper sensitivity of the fet3delta strain was suppressed by co-deletion of FRE1, suggesting that the cytotoxic species was Cu1+. In contrast, deletion of CTR1 or of FET4 did not suppress the copper sensitivity in the fet3delta 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.     

The yeast FET5 gene encodes a FET3 -related multicopper oxidase implicated in iron transport

The yeast FET3 gene encodes an integral membrane multicopper oxidase required for high-affinity iron uptake. The FET4 gene encodes an Fe(II) transporter required for low-affinity uptake. To identify other yeast genes involved in iron uptake, we isolated genes that could, when overexpressed, suppress the iron-limited growth defect of a fet3 fet4 mutant. The FET5 gene was isolated in this screen and it encodes a multicopper oxidase closely related to Fet3p. Several observations indicate that Fet5p plays a role analogous to Fet3p in iron transport. Suppression of the fet3 fet4 mutant phenotype by FET5 overexpression required the putative FTR1 transporter subunit of the high-affinity system. Fet5p is an integral membrane protein whose oxidase domain is located on the cell surface or within an intracellular compartment. Oxidase activity measured in cells with altered levels of FET5 expression suggested that Fet5p is a functional oxidase. FET5 overexpression increased the rate of iron uptake by a novel uptake system. Finally, FET5 mRNA levels are regulated by iron and are increased in cells grown in iron-limited media. These results suggest that Fet5p normally plays a role in the transport of iron.     

The yeast FET5 gene encodes a FET3 -related multicopper oxidase implicated in iron transport

The yeast FET3 gene encodes an integral membrane multicopper oxidase required for high-affinity iron uptake. The FET4 gene encodes an Fe(II) transporter required for low-affinity uptake. To identify other yeast genes involved in iron uptake, we isolated genes that could, when overexpressed, suppress the iron-limited growth defect of a fet3 fet4 mutant. The FET5 gene was isolated in this screen and it encodes a multicopper oxidase closely related to Fet3p. Several observations indicate that Fet5p plays a role analogous to Fet3p in iron transport. Suppression of the fet3 fet4 mutant phenotype by FET5 overexpression required the putative FTR1 transporter subunit of the high-affinity system. Fet5p is an integral membrane protein whose oxidase domain is located on the cell surface or within an intracellular compartment. Oxidase activity measured in cells with altered levels of FET5 expression suggested that Fet5p is a functional oxidase. FET5 overexpression increased the rate of iron uptake by a novel uptake system. Finally, FET5 mRNA levels are regulated by iron and are increased in cells grown in iron-limited media. These results suggest that Fet5p normally plays a role in the transport of iron. Received: 12 May 1997 / Accepted: 4 July 1997