Copper Modulation and Reactive Oxygen Species Production Across Yeast Lifespan

Abstract

The mitochondrial electron transport chain (ETC) machinery is the primary site of superoxide formation, a reactive oxygen species (ROS) associated with premature cell aging. A primary defense against ROS in yeast (Saccharomyces cerevisiae) is the enzyme superoxide dismutase (Sod1p). This enzyme requires copper and zinc as metal cofactors in the conversion of superoxide to hydrogen peroxide, a less harmful ROS. How mitochondrial function contributes to yeast chronological lifespan (CLS) is particularly of interest, specifically how essential metals for mitochondrial function and their role in the production of ROS drive the aging process of yeast cells. Our aim is to determine the effect exogenous copper treatment on the CLS of yeast strains lacking Sod1p and its copper chaperone, Lys7p. Our results indicate that low levels of exogenous copper (0.25 mM copper sulfate or less in restricted nutrient media) extend yeast chronological lifespan, especially in cells lacking Lys7p. All cell types see a dramatic reduction in lifespan when exogenous copper levels are increased to 2.0 mM copper sulfate or higher. This indicates that a small increase in the amount of copper in the media is beneficial for yeast in these restricted media conditions. Our current studies are to assess protein levels by western blot and gene expression by qRT‐PCR of various components the yeast ETC machinery, specifically Cox2 and Cox4, to mechanistically understand how exogenous copper in a small dose is contributing to lifespan extension. ROS levels are also being assessed via fluorescent staining. By using biochemical dyes such as Dihydroethidium (DHE) for cytosolic ROS, and MitoSox and mitochondria superoxide production, we are able to monitor ROS production and show localization of these metabolic byproducts with both fluorescent microscopy and FACS at various points of the chronological lifespan yeast.