Transcriptome profiling of aSaccharomyces cerevisiaemutant with a constitutively activated Ras/cAMP pathway

Abstract

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2⌬ mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of ϳ11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway. constitutive activation of PKA by PDE2 deletion; Ras/cAMP pathway; cell wall integrity THE RAS/CAMP PATHWAY is a highly conserved signal transduction pathway operating via the second messenger, cAMP (6). In Saccharomyces cerevisiae, it controls cell-cycle progression, cell growth and proliferation (3, 81), reprogramming of transcription at the diauxic transition (8), mating (1), pseudohyphal morphogenesis (27, 55), metabolism (9), and stress responses. The synthesis of cAMP is catalyzed by adenylate cyclase (40), which is regulated by Ras proteins (18,24,80), the G protein ␣-subunit homolog, Gpa2 (48), and the adenylate-cyclase-associated protein, Cap1p (23,25). The only known biochemical role of cAMP is to activate protein kinase A (PKA) (12,78,79). High activity of PKA in yeast leads to low levels of the storage carbohydrates trehalose and glycogen, low stress resistance due to reduced expression of STRE ("stress response element")-controlled genes, aberrant G 0 arrest, poor growth on nonfermentable and weakly fermentable carbon sources, and failure of sporulation in diploid cells. Low activity yields de-repression of STRE-controlled genes leading to high stress resistance, constitutive expression of heat-shock genes, and sporulation of diploid cells in rich media (for reviews, see Refs. 10,64,[74][75][76]. Two trans-acting factors (Msn2p and Msn4p), negatively regulated by PKA, have been shown to be involved in STRE-mediated gene expression (47,67).Intracellular levels of cAMP, and hence the state of the Ras/cAMP pathway, are also contr...

Deletion of PDE2, the gene encoding the high‐affinity cAMP phosphodiesterase, results in changes of the cell wall and membrane in Candida albicans

“…These results suggest a possible relationship between β-glucan synthesis and the cAMP pathway. These observations correlate well with findings of reduced β-glucan levels in the S. cerevisiae pde2 null mutant and differential expression of genes such as FKS1, GSC2, RHO1 and ROM2, responsible for β-glucan synthesis (Jones et al, 2003). However, unlike S. cerevisiae pde2 null mutant, which showed slightly increased chitin levels (Jones et al, 2003), there was no significant difference in chitin levels between the C. albicans pde2 null mutant and the control strains during growth in the yeast form (Table 4, last column).…”

“…These observations correlate well with findings of reduced β-glucan levels in the S. cerevisiae pde2 null mutant and differential expression of genes such as FKS1, GSC2, RHO1 and ROM2, responsible for β-glucan synthesis (Jones et al, 2003). However, unlike S. cerevisiae pde2 null mutant, which showed slightly increased chitin levels (Jones et al, 2003), there was no significant difference in chitin levels between the C. albicans pde2 null mutant and the control strains during growth in the yeast form (Table 4, last column). This suggests that the slightly higher sensitivity to Calcofluor white ( Figure 1A) in the mutant could be due to other reasons, one being the Hog1 MAPkinase pathway, which in S. cerevisiae has been demonstrated to be another target for Calcofluor white or, alternatively, to an increased permeability to the drug (Garcia-Rodriguez et al, 2000).…”

“…These defects manifest themselves in the form of increased sensitivity to SDS, Calcofluor white, some antifungals, reduced thickness of the cell wall and alterations of the ergosterol and glucan composition. Changes in expression of cell wall-related genes and associated cell wall defects have previously been demonstrated in S. cerevisiae pde2 mutants (Jones et al, 2003). Our results suggest that a similar effect might also exist in C. albicans, since recent reports have demonstrated, first, that EFG1 is significantly downregulated in the C.albicans pde2 mutant (Jung and Stateva, 2003) and second, that EFG1 regulates transcription of several cell wall proteinencoding genes such as HWP1, HWP2, YWP1 and RBE1, which are significantly downregulated in an efg1 mutant (Sohn et al, 2003).…”

“…Intracellular levels of cAMP dramatically increase upon addition of exogenous cAMP to pde2 mutants, suggesting that Pde2p is responsible for breaking down exogenous cAMP (Wilson et al, 1993). Significant changes in the transcriptome have recently been described for the S. cerevisiae pde2 mutant (Jones et al, 2003). These changes which are representative of a constitutive activation of the cAMP pathway manifest themselves in a range of cell wall-related phenotypes, supporting the role for PDE2 (and/or cAMP) in the maintenance of cell wall integrity in S. cerevisiae, as previously suggested (Heale et al, 1994;Tomlin et al, 2000).…”

The Sensitivity of Yeast Mutants to Oleic Acid Implicates the Peroxisome and Other Processes in Membrane Function

“…Data for two of these, pde2D and sac1D, are also presented in Figure 5. Deletion of PDE2, which encodes the yeast high-affinity cAMP phosphodiesterase, has major effects on cell-wall integrity ( Jones et al 2003). Sac1, one of several yeast phosphoinositide phosphatases, influences the cell wall by controlling chitin synthase trafficking (Schorr et al 2001).…”