Regulation of protein synthesis by oxidative stress in yeast

All organisms must respond to changes in their external environment. With the availability of genome sequences much attention has focused on analyzing the changes in gene expression/transcription profiles during these adaptive responses. The translation of mRNA into protein is a fundamental component of the gene expression pathway. However, relatively little is known regarding the role of translational control mechanisms in the response to stress conditions, which is the focus of this proposed study. Our preliminary data have shown that protein synthesis in yeast is rapidly and reversibly inhibited in response to hydrogen peroxide stress. Paradoxically however, certain mRNAs are resistant to inhibition and continue to be translated during stress conditions. Our data show that translation initiation factor 2 (eIF2) is phosphorylated in response to oxidative stress. eIF2 serves as a focus for translational regulation in all eukaryotic species. In the past, most work has focused on the kinases which mediate phosphorylation of eIF2. In this current work programme, we will study both the phosphorylation and dephosphorylation of eIF2. This is critical in order to understand the co-ordinated regulation of protein synthesis that is observed in response to oxidative stress.

Despite the importance of oxidative stress in eukaryotic cell growth and survival, little is known regarding the translational responses that are invoked in response to this stress. Our preliminary data indicate that the eukaryotic translation initiation factor eIF2, is phosphorylated by the Gcn2 kinase in response to hydrogen peroxide. The phosphorylation of Ser51 on eIF2 is a common effecter step in response to diverse stress conditions in many organisms. Phosphorylation of eIF2 converts it from a substrate to a competitive inhibitor of the guanine nucleotide exchange factor eIF2B, and the resulting decrease in eIF2B activity inhibits translation initiation. We will characterize the inhibition and recovery of protein synthesis in response to oxidative stress induced by reactive oxygen species as part of this work programme. A central hypothesis to be tested is that the Gcn2 kinase and the Sit4 phosphatase moderate the levels of eIF2 phosphorylation to control the rate of translation initiation in response to oxidative stress. Most protein phosphatases are thought to posses little intrinsic substrate specificity in the absence of additional regulatory proteins. Previous biochemical studies have shown that the Sit4 phosphatase physically associates with a number of high-molecular-weight proteins known as Sit4-associated proteins (Sap4, Sap155, Sap185 and Sap190). Our data show that hydrogen peroxide induces SAP185 translation and we will determine the role of Sap proteins in Sit4 complex formation and phosphatase activity during oxidative stress. Our aim is to determine the role of Sap proteins in regulating eIF2 phosphorylation during oxidative stress and recovery conditions. Another important aim is to determine how the SAP185 mRNA is translated during oxidative stress conditions which inhibit global protein synthesis. We will identify the features of the SAP185 mRNA which allow it to overcome the block imposed by this global inhibitory response mechanism.