Transcriptional Regulation of the Yeast GMP Synthesis Pathway by Its End Products

Abstract

AMP and GMP are synthesized from IMP by specific conserved pathways. In yeast, whereas IMP and AMP synthesis are coregulated, we found that the GMP synthesis pathway is specifically regulated. Transcription of the IMD genes, encoding the yeast homologs of IMP dehydrogenase, was repressed by extracellular guanine. Only this first step of GDP synthesis pathway is regulated, since the latter steps, encoded by the GUA1 and GUK1 genes, are guanine-insensitive. Use of mutants affecting GDP metabolism revealed that guanine had to be transformed into GDP to allow repression of the IMD genes. IMD gene transcription was also strongly activated by mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenase activity. Serial deletions of the IMD2 gene promoter revealed the presence of a negative cis-element, required for guanine regulation. Point mutations in this guanine response element strongly enhanced IMD2 expression, also making it insensitive to guanine and MPA. From these data, we propose that the guanine response element sequence mediates a repression process, which is enhanced by guanine addition, through GDP or a GDP derivative, and abolished in the presence of MPA.Purine nucleotides are involved in many important cellular processes, and therefore a balanced synthesis of AMP and GMP is required. Cells can synthesize purine nucleotides through the de novo pathway, a 10-step pathway that produces IMP, which in turn serves as the common precursor for AMP and GMP nucleotide biosynthesis (Fig. 1). In yeast, the regulation of the ADE genes involved in AMP biosynthesis has been characterized (1, 2). Expression of the ADE genes is repressed by extracellular adenine and activated by the transcription factors Bas1p and Bas2p. However, in Saccharomyces cerevisiae, GMP biosynthesis regulation has not received much attention and is therefore poorly understood. IMP dehydrogenase (IMPDH), 1 catalyzing the first step of de novo guanine nucleotide synthesis, has a key role on growth of many cell types, including lymphocytes and rapidly proliferating cells (see Ref. 3 for a recent review).GMP synthesis and its regulation appear to play a crucial role in cell proliferation, since an increased level of IMPDH activity has been observed in rapidly proliferating cells (4), including human leukemic cell lines (6 -9), solid tumor tissues (10), and B-and T-activated lymphocytes (5). Indeed, substances blocking IMPDH activity, such as mycophenolic acid (MPA), act as immunosuppressive drugs and are used to prevent allograft rejection (11-16; see Ref. 17 for recent reviews). Moreover, it was recently shown that a low level of IMPDH activity is necessary for p53-dependent growth suppression. Indeed, constitutive expression of IMPDH abolishes p53-dependent growth suppression (18), although the exact mechanism is not known.To assess the molecular mechanisms governing guanylic nucleotide synthesis, we have proceeded to study the regulation of GTP synthesis in Saccharomyces cerevisiae. In this yeast, GMP synthetase, GMP kinase, and NDP kin...

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

“…The IMD genes and GUA1 do not respond to adenine as ADE genes do (Escobar-Henriques and Daignan-Fornier 2001). In fact, IMD2/3/4 genes are strongly downregulated when guanine is added to the growth medium, while GUA1 expression is unaffected (Escobar-Henriques and Daignan-Fornier 2001). This effect of guanine is abolished in the fcy2, hpt1, and guk1 guanine utilization mutants, indicating that guanine metabolism is required for GTP to exert its regulatory role (Escobar-Henriques and Daignan-Fornier 2001).…”

“…In fact, IMD2/3/4 genes are strongly downregulated when guanine is added to the growth medium, while GUA1 expression is unaffected (Escobar-Henriques and Daignan-Fornier 2001). This effect of guanine is abolished in the fcy2, hpt1, and guk1 guanine utilization mutants, indicating that guanine metabolism is required for GTP to exert its regulatory role (Escobar-Henriques and Daignan-Fornier 2001). In contrast, inhibitors of IMPDH activity, such as mycophenolic acid (MPA) or 6-azauracil (6AU) that lower GTP pools (Exinger and Lacroute 1992) 2003b; Kuehner and Brow 2008).…”

Actin Bodies in Yeast Quiescent Cells: An Immediately Available Actin Reserve?

“…The YPDA medium was described previously (Martinez et al, 2004). The SD casa medium (Escobar-Henriques and Daignan-Fornier, 2001) was supplemented with 1.8 mM uracil, 0.2 mM tryptophan, and 0.3 mM adenine. The 2% lactate-containing medium was described in Chateaubodeau et al (1976), and the N3 glycerol-containing medium was described in LefebvreLegendre et al (2001) was supplemented with 2% ethanol.…”

Detection of the mycophenolate-inhibited form of IMP dehydrogenase in vivo

“…Only IMD2 provides resistance to the drug mycophenolic acid (MPA), and its expression is tightly regulated by guanine nucleotide levels. It is repressed under guanine-replete conditions and induced when guanine nucleotides are low (3,4).…”

“…For example, bacterial enzymes are highly resistant, whereas human IMPDH is relatively sensitive (6). Yeast attain resistance in part because IMD2 is transcriptionally induced and highly dependent upon efficient transcription elongation (3,4,7). Indeed, many mutations of genes encoding the yeast transcription machinery show sensitivity to MPA due to their inability to up-regulate expression (3).…”