Cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative-stress-response genes

Abstract

The complete set of viable deletion strains in Saccharomyces cerevisiae was screened for sensitivity of mutants to five oxidants to identify cell functions involved in resistance to oxidative stress. This screen identified a unique set of mainly constitutive functions providing the first line of defense against a particular oxidant; these functions are very dependent on the nature of the oxidant. Most of these functions are distinct from those involved in repair and recovery from damage, which are generally induced in response to stress, because there was little correlation between mutant sensitivity and the reported transcriptional response to oxidants of the relevant gene. The screen identified 456 mutants sensitive to at least one of five different types of oxidant, and these were ranked in order of sensitivity. Many genes identified were not previously known to have a role in resistance to reactive oxygen species. These encode functions including protein sorting, ergosterol metabolism, autophagy, and vacuolar acidification. Only two mutants were sensitive to all oxidants examined, only 12 were sensitive to at least four, and different oxidants had very different spectra of deletants that were sensitive. These findings highlight the specificity of cellular responses to different oxidants: No single oxidant is representative of general oxidative stress. Mitochondrial respiratory functions were overrepresented in mutants sensitive to H(2)O(2), and vacuolar protein-sorting mutants were enriched in mutants sensitive to diamide. Core functions required for a broad range of oxidative-stress resistance include transcription, protein trafficking, and vacuolar function.

Fig. 1.

Determination of oxidant concentrations for mutant screening. The WT and known oxidant-sensitive mutants were inoculated in yeast extract/peptone/dextrose (YEPD) medium in a 96-well plate and grown to stationary phase. The cultures were replicated on plates containing different concentrations of H₂O₂, diamide, CHP, LoaOOH, and menadione. Representative examples of H₂O₂, menadione (Men.), and CHP plates are shown. The first two columns in each panel represent the WT strain.

Fig. 2.

Hierarchical clustering of deletion mutant sensitivity data. (A) Sensitivity data (obtained as described in Methods) were imported into genespring 5 so that highest sensitivity was represented by the most intense red color. Hierarchical clustering was performed by using the Pearson correlation, and clusters were analyzed for an overrepresentation of biological functions. (B) Hierarchical clustering of the microarray data from Gasch et al. (18) and our own data on LoaOOH (N.A., V.J.H., and I.W.D., unpublished data) was combined with the deletion mutant data. Transcriptional data were given a weight of 75, relative to 1 for the sensitivity data. HS, heat shock; C, CHP; L, LoaOOH; M, menadione; H, H₂O₂; D, diamide.

Fig. 3.

Functional grouping of deletion mutant sensitivity data. Sensitive strains were organized in functional groups based on the categories of genes identified in the Munich Information Center for Protein Sequences (MIPS) database and Saccharomyces Genome Database, combined with visual inspection. The relative contribution of each functional group to sensitivity is shown for each oxidative-stress condition examined. The number of sensitive strains is shown next to each condition. More detailed functional grouping is available in Table 2. Mit, mitochondria; Vac, VPS and vacuolar function; Met, metabolism; Pro, mRNA/protein synthesis; CW, cell wall; O/U, other/unknown.