Reactive oxygen species production and the influence of exogenous copper in yeast mitochondria

Mitochondria are essential organelles within eukaryotic cells due to their role in metabolism, ATP production via the oxidative phosphorylation (OXPHOS) pathway, and other regulatory cellular processes. Our aim is to understand how copper in the mitochondria of yeast affects the production of reactive oxygen species due to its influence on the electron transport chain (ETC) and the localization of reactive oxygen species production during yeast lifespan. Reactive oxygen species (ROS) provide signaling in the cells of organisms which is beneficial for cells to maintain homeostasis. However, when there is a surplus of ROS this can lead to harmful and/or lethal results for the cell. To detect ROS, yeast cultures were grown for one to three days and treated with the fluorescent ROS indicator dihydroethidium (DHE), as well as the fluorescent indicators SYTO and DAPI which stain mitochondrial DNA and adenine-thymine rich regions in the DNA respectively. The ROS generated from the yeast was then analyzed via a fluorescence plate reader, fluorescent activated cell sorting (FACS), and fluorescent microscopy to compare methods of determining ROS in the samples. To investigate mitochondrial ROS production, cells were grown in the presence of antimycin A (a Complex III inhibitor), oligomycin (a Complex V inhibitor), or menadione (a futile redox cycling molecule). Detectable increases in ROS were observed through the inhibition of Complex III and in the presence of menadione. We are now using these methods in yeast with a superoxide dismutase 1 (SOD1) gene deletion, to understand the influence of exogenous copper on the generation of ROS. Copper is worth investigating in conjunction with ROS because the mitochondria in mammals and yeast cells contain a labile copper pool located in the matrix, which is used in the metalation of the copper containing enzymes Complex IV, Cytochrome C Oxidase(CcO), and superoxide dismutase (Sod1). It has been established that supplementing cultures with copper increases CcO and Sod1 activity, supporting previous results that exogenous copper contributes to a labile copper pool within the mitochondria to increase enzyme activity. Our results show that small amounts exogenous copper (0.25 mM copper sulfate in restricted nutrient media) extend yeast chronological lifespan, but a dramatic reduction in lifespan is observed when copper levels are increased to 2.0 mM copper sulfate or higher. These data indicates that low levels of exogenous copper in the media is beneficial for yeast in restricted media conditions. Our current work assesses how exogenous copper changes ETC protein complex expression, specifically looking at CcO subunits, and the production of ROS under these conditions.