Phenotypic Consequences of Purine Nucleotide Imbalance inSaccharomyces cerevisiae

Abstract

Coordinating homeostasis of multiple metabolites is a major task for living organisms, and complex interconversion pathways contribute to achieving the proper balance of metabolites. AMP deaminase (AMPD) is such an interconversion enzyme that allows IMP synthesis from AMP. In this article, we show that, under specific conditions, lack of AMPD activity impairs growth. Under these conditions, we found that the intracellular guanylic nucleotide pool was severely affected. In vivo studies of two AMPD homologs, Yjl070p and Ybr284p, indicate that these proteins have no detectable AMP, adenosine, or adenine deaminase activity; we show that overexpression of YJL070c instead mimics a loss of AMPD function. Expression of the yeast transcriptome was monitored in a AMPD-deficient mutant in a strain overexpressing YJL070c and in cells treated with the immunosuppressive drug mycophenolic acid, three conditions that lead to severe depletion of the guanylic nucleotide pool. These three conditions resulted in the up-or downregulation of multiple transcripts, 244 of which are common to at least two conditions and 71 to all three conditions. These transcriptome results, combined with specific mutant analysis, point to threonine metabolism as exquisitely sensitive to the purine nucleotide balance.T HE purine nucleotides, ATP and GTP, are involved in almost all aspects of cellular life. In addition to their role as building blocks of nucleic acids, adenylic and guanylic nucleotides have specific roles. For example, GTP is critical for translation and for signaling through GTPases, while ATP is the major energyproviding molecule in the cell. In yeast, intracellular concentrations of ATP and GTP are clearly different ($5 and 1.5 mm, respectively; Breton et al. 2008;Gauthier et al. 2008), most probably as the result of regulatory processes that maintain homeostasis. In most eukaryotic cells, including yeast, adenylic and guanylic nucleotides either are synthesized from a common precursor (IMP) or are recycled from preformed bases or nucleosides (Figure 1). While most enzymes involved in these processes have been identified, the physiological consequences of purine nucleotide imbalance are far from being understood. Interestingly, drugs specifically inhibiting GTP synthesis, such as mycophenolic acid (MPA), have a strong immunosuppressive effect and are now widely used to limit allograft rejection. We have previously established the effect of MPA on the yeast proteome and have identified numerous yeast mutants hypersensitive to this drug (Desmoucelles et al. 2002). MPA effects are due to GTP shortage, since they are reversed by exogenous guanine, allowing replenishment of the GTP pool. However, drugs often have secondary effects and can be detoxified and/or diluted during cell growth. As an alternative, consequences of purine nucleotide imbalance can be investigated using yeast mutants. In two previous studies, we have used specific mutants to increase the GTP pool or decrease the ATP pool (Breton et al. 2008;Gauthier et al. 20...

Guanine Nucleotide Pool Imbalance Impairs Multiple Steps of Protein Synthesis and Disrupts GCN4 Translational Control in Saccharomyces cerevisiae

“…In light of our data, we suggest that GCN4 mRNA translation was derepressed by the GDP/GTP shortages evoked by the different conditions examined by Saint-Marc et al (2009) and it would be interesting to determine whether tRNA i Met containing complexes accumulate under those conditions. We found that reduced synthesis of G nucleotides in gua1-G388D cells impairs the processing of pre-tRNA i Met and precursors of certain elongator tRNA species (Figure 7).…”

“…Partial guanine deprivation results in a drastic reduction of the internal GTP pool in a yeast gua1 mutant, inducing meiosis and sporulation in homothallic strains (Varma et al 1985) and leads to concomitant reductions of the methionine and S-adenosyl-methionine (SAM) pools (Freese et al 1984). Recently the effects on the yeast transcriptome of a marked depletion of guanine nucleotides have been reported (Saint-Marc et al 2009). Remarkably, 71 genes are affected under conditions that lead to GDP/GTP shortage, most of which are involved in amino acid metabolism and regulated by Gcn4 (Natarajan et al 2001).…”

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…What happens if the nucleotide balance is disturbed? To tackle this question, specific mutations disrupting the purine nucleotide balance that result in lower ATP or GTP concentration (Gauthier et al 2008;Saint-Marc et al 2009;Iglesias-Gato et al 2011) or that strongly increase the GTP pool (Breton et al 2008) were constructed. While these mutations had a strong impact on yeast growth and resulted in general transcriptional and translational responses, no evidence for a common specific cellular response to defective nucleotide balance emerged.…”

Adaptive Response and Tolerance to Weak Acids inSaccharomyces cerevisiae: A Genome-Wide View

“…Sweetner; preservation of beer and diary products; precursor in the synthesis of polyesters and acrylates Desmoucelles et al, 2002;Escobar-Henriques et al, 2001;Saint-Marc et al, 2009 A list of references is also provided in which the mechanisms of adaptive response and resistance to these weak acids in yeast cells are addressed.…”