Abstract

We will use the yeast Saccharomyces cerevisiae as a model organism to elucidate the consequences of oxidative stress-induced protein aggregate formation. Our recent studies have shown that oxidative stress promotes the formation of both large amorphous protein aggregates as well as heritable prion aggregates and the aim of this project is to understand the processes which drive the formation of these different types of aggregates. Evolutionarily conserved protein deposit sites have been identified which effectively sequester misfolded and aggregated proteins away from their normal productive pathways, protecting against potential cytotoxic effects. However, it is unclear what role the localization of misfolded proteins to these sites plays in amyloid formation, protein heritability and protein degradation. We will use microscopy and biochemical approaches to define the contribution of intracellular protein deposits (IPOD, JUNQ, stress granules) to protein aggregate formation and fate. This will include using mutants that disrupt protein deposit formation to determine whether protein localization is required to facilitate protein-based inheritance. A key question to be addressed is whether such protein localization acts to facilitate autophagic clearance of aggregates versus heritable protein aggregate (prion) formation. These studies will make use of the well-characterized yeast [PSI+] prion to enable us to study the formation and heritability of protein aggregates. We hypothesize that certain protein aggregates may promote stress tolerance and we will use a biochemical approach to isolate the amyloid-like proteins which are formed in response to oxidative stress conditions. We will determine whether protein aggregates can act as a form of memory providing oxidant tolerance. These studies will have broad implications for understanding the role of oxidative protein damage as a trigger of amyloid formation in the eukaryotic cell.