Copper Uptake In Yeast Cells

Two genes encoding high-affinity copper transporters, CTR1 and CTR3, have been identified in the yeast S. cerevisiae (44,45). In most commonly used S. cerevisiae laboratory strains, the CTR3 gene is disrupted by a transposon. Inactivation of the CTR1 gene in such a strain eliminates high-affinity copper uptake and makes the strain resistant to high external concentrations of copper. The defective copper uptake furthermore results in deficiency of Cu,Zn superoxide dismutase activity and the intracellular copper concentration appears to be reduced as judged from the reduced expression of a lacZ reporter gene fused to the copper-inducible CUP1 promoter. On the other hand, overexpression of the CTR1 gene leads to increased uptake of copper (44,46).

High-affinity copper uptake is not severely impaired by the CTR1 deletion in yeast strains where the CTR3 gene is functional, reflecting the functional redundancy of the two genes. However, strains in which both genes are functional have a higher rate of copper uptake compared to strains mutated in either of the two genes (45).

Both Ctrl and Ctr3 are integral membrane proteins and have been localized to the cell surface, by microscopy of yeast strains transformed with plasmids expressing epitope-tagged Ctrl or Ctr3 fusion proteins (44,47). Both proteins are predicted to have similar membrane topologies with three transmembranal segments, an extracellular N-terminal domain, and an intracellular C-terminal domain (44,47,48). By immunoprecipitation, Ctrl has been shown to exist at least as a homodimer (46), whereas Ctr3 apparently forms a trimer at the plasma membrane (47). It is speculated that such homomultimeric complexes, having from 6-12 transmembrane domains might form a pore through which the copper substrate could be translocated.

In spite of their similar topology and function in copper uptake, the Ctrl and Ctr3 proteins have limited primary sequence homology. The Ctrl protein consists of 406 amino acid residues and has a relatively large N-terminal domain containing ll potential copper-binding motifs of the type MXXM (44). The Ctr3 protein consists of only 24l amino acid residues and lacks the numerous methionine-rich motifs found in Ctrl, but it contains several cysteine residues, some of which might be involved in metal binding (45). Ctrl is heavy glycosylated with O-linked sugars (46), whereas Ctr3 migrates in sodium dodecyl sulfate (SDS)-polyacrylamide gels as a 27-kDa monomeric protein consistent with its predicted mass, suggesting little or no glycosylation (47).

In addition to the high-affinity transporters Ctrl and Ctr3, several putative low-affinity copper-uptake systems have been identified in the yeast. The Ctr2 protein is l89 amino acid residues long and has some homology to Ctrl and Ctr3, and like these, it is predicted to have three membrane-spanning segments (49). Ctr2 has been proposed to be a low-affinity uptake system for copper, as a mutation in the CTR2 gene increased resistance to toxic copper concentrations and overproduction of Ctr2 provided increased resistance to copper starvation. However, overproduction of Ctr2 was not sufficient to complement a ctr1 ctr3 mutant with respect to copper uptake (49).

Fig. 1. Uptake of 64Cu, by human fibroblast cultures. The culture designated WT is a primary cell line derived from a healthy individual. The remaining cultures were obtained from the same primary fibroblast culture that had been immortalized and transfected with plasmids as indicated. The amount of 64Cu accumulation (ng/mg protein) during 20 h incubation in the presence of 10 |lM 64Cu is shown. (Data from ref. 53.)

Fig. 1. Uptake of 64Cu, by human fibroblast cultures. The culture designated WT is a primary cell line derived from a healthy individual. The remaining cultures were obtained from the same primary fibroblast culture that had been immortalized and transfected with plasmids as indicated. The amount of 64Cu accumulation (ng/mg protein) during 20 h incubation in the presence of 10 |lM 64Cu is shown. (Data from ref. 53.)

Recently, the low-affinity Fe(II) permease Fet4p from S. cerevisiae has been demonstrated to function as a low-affinity copper permease (50). Furthermore, three proteins, Smf1, Smf2, and Smf3, belonging to the Nramp family of metal transporters, have also been implicated in copper uptake, although they appear to have a higher affinity for manganese. It was found that EGTA arrested the growth of yeast strains in which one or more of the three SMF genes had been disrupted, and this growth arrest could be reversed by addition of 5 |M Cu(II) (51).

0 0

Post a comment