Abstract
The LYS7 gene in Saccharomyces cerevisiae encodes a protein (yCCS) that delivers copper to the active site of copper-zinc superoxide dismutase (CuZn-SOD, a product of the SOD1 gene). In yeast lacking Lys7 (lys7Delta), the SOD1 polypeptide is present but inactive. Mutants lacking the SOD1 polypeptide (sod1Delta) and lys7Delta yeast show very similar phenotypes, namely poor growth in air and aerobic auxotrophies for lysine and methionine. Here, we demonstrate certain phenotypic differences between these strains: 1) lys7Delta cells are slightly less sensitive to paraquat than sod1Delta cells, 2) EPR-detectable or "free" iron is dramatically elevated in sod1Delta mutants but not in lys7Delta yeast, and 3) although sod1Delta mutants show increased sensitivity to extracellular zinc, the lys7Delta strain is as resistant as wild type. To restore the SOD catalytic activity but not the zinc-binding capability of the SOD1 polypeptide, we overexpressed Mn-SOD from Bacillus stearothermophilus in the cytoplasm of sod1Delta yeast. Paraquat resistance was restored to wild-type levels, but zinc was not. Conversely, expression of a mutant CuZn-SOD that binds zinc but has no SOD activity (H46C) restored zinc resistance but not paraquat resistance. Taken together, these results strongly suggest that CuZn-SOD, in addition to its antioxidant properties, plays a role in zinc homeostasis.
Highlights
With the appearance of molecular oxygen (O2) in the earth’s atmosphere, all aerobic organisms evolved methods to utilize O2 for energy production
Because the SOD1 polypeptide in lys7⌬ mutants lacks copper in its active site, it is incompetent to catalyze the removal of O2.. lys7⌬ and sod1⌬ strains exhibit very similar phenotypic defects, but they are not identical, as we report here
These dissimilarities strongly suggest an in vivo role for SOD1 in metal metabolism, and we have provided further evidence that the SOD1 polypeptide may play a role in cellular resistance to elevated levels of zinc ions
Summary
MAT␣ leu his trp ura MAT␣ leu his trp ura sod1⌬ϻURA3 MAT␣ leu his trp ura lys7⌬ϻLEU2 MAT␣ leu his trp ura lys7⌬ϻLEU2 sod1⌬ϻURA3 MATa his leu met ura MATa his leu met ura sod1⌬ϻKANR MATa his leu met ura lys7⌬ϻKANR JW201 with pEMBLyex4-URA3 JW201 with pSALSOD-URA3 EG118 with YEp351 EG118 with YEp600 EG118 with YEp351-yH46C SOD. Ref. 3 Ref. 3 This work This work Purchased Purchased Purchased This work This work This work This work This work stasis of transition metal ions: copper, manganese, or iron Out of this scientific endeavor has come the discovery of the copper chaperone for SOD1 (CCS). Certain differences exist between these two mutant derivatives, most notably the following two characteristics: 1) sod1⌬ mutants display a 5-fold increase in the levels of EPR-detectable iron relative to lys7⌬ and wild-type yeast and 2) sod1⌬ mutants are sensitive to zinc while lys7⌬ yeast are not These dissimilarities strongly suggest an in vivo role for SOD1 in metal metabolism, and we have provided further evidence that the SOD1 polypeptide may play a role in cellular resistance to elevated levels of zinc ions
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