Identification of Genes that Provide Cellular Protection Against a Thiol Cross‐Linking Agent in Saccharomyces cerevisiae

Organic electrophiles represent an important class of xenobiotic and endogenously produced toxins that damage DNA and proteins. The adduction of electrophiles to these macromolecules interrupts biological processes such as DNA replication, protein folding and clearance. We have recently identified an electrophile (diethyl acetylenedicarboxylate, DAD) that is capable of cross‐linking proteins by undergoing reaction with two thiol groups. Because of its unique, electrophilic properties, we sought to determine whether the defense mechanisms that provide protection against DAD are similar to those which protect against electrophiles that cannot cross‐link thiol groups in proteins. To this end, we have screened a panel of Saccharomyces cerevisiae deletion mutants encoding various detoxification enzymes to determine their survival when exposed to DAD. We have identified several cytoprotective genes, including many in the glutathione transferase family, (e.g. GTTI, GTO1, and GTO2 , all of which localize to different cellular compartments). Subsequent studies using N ‐ethylmaleimide (NEM) revealed that a similar group of genes provide protection against monofunctional electrophiles (i.e. electrophiles not capable of cross‐linking). Our results indicate that the defense mechanisms are comparable for monofunctional electrophiles and bifunctional thiol cross‐linkers, and suggest that glutathione metabolic enzymes in multiple cellular compartments protect cells against damage by these electrophiles.