To further exploit yeast as a model for cellular aging we have modified the replicative life span assay to force respiration, by replacing glucose with the non-fermentable carbon source glycerol. The growth rates of several different strains varied greatly, with doubling times ranging from 2.7 to 7 hours. Life spans of all strains were lower on media containing glycerol than on media containing glucose. However, supplementation of glycerol-containing media with copper resulted in increases in life span of between 17 and 72%; life spans equivalent to or beyond those obtained on glucose media. Addition of copper to glucose medium had no effect on life span. Microarray analysis showed that genes responsible for high affinity import of copper display reduced expression upon addition of copper, while most genes showed no change in expression. No differences in growth rate, oxygen uptake, or the levels of subunit II of the copper-containing cytochrome c oxidase were found between cultures of yeast grown with or without copper supplementation. Copper supplementation greatly extended the life span of sod1 and sod2 strains, suggesting that addition of copper may reduce the generation of superoxide. Forcing yeast to respire places an emphasis on mitochondrial function and may aid in the identification of factors involved in aging in other respiratory-dependent organisms.
We have previously shown that copper supplementation extends the replicative life span of Saccharomyces cerevisiae when grown under conditions forcing cells to respire. We now show that copper’s effect on life span is through Fet3p, a copper containing enzyme responsible for high affinity transport of iron into yeast cells. Life span extensions can also be obtained by supplementing the growth medium with 1mM ferric chloride. Extension by high iron levels is still dependent on the presence of Fet3p. Life span extension by iron or copper requires growth on media containing glycerol as the sole carbon source, which forces yeast to respire. Yeast grown on glucose containing media supplemented with iron show no extension of life span. The iron associated with cells grown in media supplemented with copper or iron is 1.4–1.8 times that of cells grown without copper or iron supplementation. As with copper supplementation, iron supplementation partially rescues the life span of superoxide dismutase mutants. Cells grown with copper supplementation display decreased production of superoxide as measured by dihydroethidium staining.
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