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 ftr1Δ 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 ftr1Δ strain. A ferroxidase-negative Fet3p did not suppress the copper sensitivity in a fet3A 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 Cu ¹⁺ was shown to be an excellent substrate for Fet3p. Last, the copper sensitivity of the fet3Δ 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 fet3Δ 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.