Abstract
Copper isotopic composition is altered in cancerous compared to healthy tissues. However, the rationale for this difference is yet unknown. As a model of Cu isotopic fractionation, we monitored Cu uptake in Saccharomyces cerevisiae, whose Cu import is similar to human. Wild type cells are enriched in 63Cu relative to 65Cu. Likewise, 63Cu isotope enrichment in cells without high-affinity Cu transporters is of slightly lower magnitude. In cells with compromised Cu reductase activity, however, no isotope fractionation is observed and when Cu is provided solely in reduced form for this strain, copper is enriched in 63Cu like in the case of the wild type. Our results demonstrate that Cu isotope fractionation is generated by membrane importers and that its amplitude is modulated by Cu reduction. Based on ab initio calculations, we propose that the fractionation may be due to Cu binding with sulfur-rich amino acids: methionine and cysteine. In hepatocellular carcinoma (HCC), lower expression of the STEAP3 copper reductase and heavy Cu isotope enrichment have been reported for the tumor mass, relative to the surrounding tissue. Our study suggests that copper isotope fractionation observed in HCC could be due to lower reductase activity in the tumor.
Highlights
To test this hypothesis, we used the yeast model-organism Saccharomyces cerevisiae, a long-recognized genetic model for Cu metabolism in eukaryotes, which transmembrane Cu transport mechanisms (Fig. 1) are very similar to those of human cells, and which is prone to genetic manipulation
Four strains of S. cerevisiae were used in this study: a wild type (DTY165), a Δctr1Δctr[3] mutant (MPY17), a Δctr1Δmac[1] mutant (SKY34) and a SKY34 strain where FRE1 was expressed under the constitutive promoter of ADH
We have demonstrated that the main mechanism responsible for the enrichment in light Cu isotope in the model organism S. cerevisiae is the import of Cu via high- (Ctr1p and Ctr3p) and low-affinity transporters
Summary
We used the yeast model-organism Saccharomyces cerevisiae, a long-recognized genetic model for Cu metabolism in eukaryotes, which transmembrane Cu transport mechanisms (Fig. 1) are very similar to those of human cells (review by Nevitt et al.3), and which is prone to genetic manipulation. In S. cerevisiae, Cu is first reduced by reductases such as the Fre[1] and Fre[2] proteins (Fre1p and Fre2p)[14,15]. Reduced Cu enters the cell through two high-affinity importers Ctr1p16 and Ctr3p17 as well as through low-affinity transporters such as Fet proteins[18]. The expression of the FRE1, FRE2, CTR1 and CTR3 genes is controlled by the Mac1p transcription factor[19]. Under low Cu, Mac1p induces the expression of both FRE1 and the high-affinity Cu transporters. Under high Cu conditions, the expression of the MAC1 gene and the activity of Mac1p is inhibited, leading to the lower expression of CTR1, CTR3, and FRE114,20–22. Our study demonstrates that copper uptake leads to isotopic fractionation between cells and growth-medium, and that this fractionation is generated by Cu importers and modulated by Cu reductases
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