Genome‐wide analysis of the effects of heat shock on a Saccharomyces cerevisiae mutant with a constitutively activated cAMP‐dependent pathway

Dawn L Jones,Simon J Gaskell,Stephen G Oliver,Lubomira Stateva,David C Hoyle,June Petty,Andrew Hayes,Isabel Riba-Garcia

doi:10.1002/cfg.415

Dawn L Jones, Simon J Gaskell + Show 6 more

Open Access

https://doi.org/10.1002/cfg.415

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Abstract

We have used DNA microarray technology and 2-D gel electrophoresis combined with mass spectrometry to investigate the effects of a drastic heat shock from 30℃ to 50℃ on a genome-wide scale. This experimental condition is used to differentiate between wild-type cells and those with a constitutively active cAMP-dependent pathway in Saccharomyces cerevisiae. Whilst more than 50% of the former survive this shock, almost all of the latter lose viability. We compared the transcriptomes of the wildtype and a mutant strain deleted for the gene PDE2, encoding the high-affinity cAMP phosphodiesterase before and after heat shock treatment. We also compared the two heat-shocked samples with one another, allowing us to determine the changes that occur in the pde2Δ mutant which cause such a dramatic loss of viability after heat shock. Several genes involved in ergosterol biosynthesis and carbon source utilization had altered expression levels, suggesting that these processes might be potential factors in heat shock survival. These predictions and also the effect of the different phases of the cell cycle were confirmed by biochemical and phenotypic analyses. 146 genes of previously unknown function were identified amongst the genes with altered expression levels and deletion mutants in 13 of these genes were found to be highly sensitive to heat shock. Differences in response to heat shock were also observed at the level of the proteome, with a higher level of protein degradation in the mutant, as revealed by comparing 2-D gels of wild-type and mutant heat-shocked samples and mass spectrometry analysis of the differentially produced proteins.

Highlights

High activity of protein kinase A (PKA) in yeast leads to low levels of the storage carbohydrates trehalose and glycogen, low stress resistance due to reduced expression of STRE-controlled genes, aberrant G0 arrest, poor growth on non-fermentable and weakly fermentable carbon sources and failure of sporulation in diploid cells
HWT1–4, HXT6 and HXT7 are the major hexose transporters in yeast transporting glucose, mannose and fructose (Reifenberger et al, 1995, 1997). These results suggested that the carbon source might have an effect on survival after heat shock
Mutants with a constitutively active cAMP-dependent pathway, such as those arising from deletion of PDE2, or a constitutively activated RAS2 allele (RAS2 Val19), are highly sensitive to a brief heat shock treatment at 50 ◦C (Sass et al, 1986; Toda et al, 1985)

Summary

Introduction

We have used this technology to determine the changes in the cellular transcriptome after a more drastic heat shock from 30 ◦C to 50 ◦C This experimental condition differentiates between wildtype cells and those with a constitutively activated cAMP pathway, as the former survive, whilst cells of the latter lose almost all viability upon this type of shock (Toda et al, 1985). Of 146 orphan genes (yeast genes of previously unknown function) identified amongst the genes with altered expression levels, 13 were found to be linked to the cAMP pathway, as suggested by the high heat-shock sensitivity phenotype of their respective deletion mutants. There was little if any correlation between transcriptome and proteome expression data

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