Yeast AMP Pathway Genes Respond to Adenine through Regulated Synthesis of a Metabolic Intermediate

ABSTRACT In Saccharomyces cerevisiae, AMP biosynthesis genes (ADE genes) are transcriptionally activated in the absence of extracellular purines by the Bas1p and Bas2p (Pho2p) transcription factors. We now show that expression of theADE genes is low in mutant strains affected in the first seven steps of the pathway, while it is constitutively derepressed in mutant strains affected in later steps. Combined with epistasy studies, these results show that 5′-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR), an intermediate metabolite of the pathway, is needed for optimal activation of the ADE genes. Two-hybrid studies establish that SAICAR is required to promote interaction between Bas1p and Bas2p in vivo, while in vitro experiments suggest that the effect of SAICAR on Bas1p-Bas2p interaction could be indirect. Importantly, feedback inhibition by ATP of Ade4p, catalyzing the first step of the pathway, appears to regulate SAICAR synthesis in response to adenine availability. Consistently, both ADE4 dominant mutations and overexpression of wild-type ADE4 lead to deregulation of ADE gene expression. We conclude that efficient transcription of yeast AMP biosynthesis genes requires interaction between Bas1p and Bas2p which is promoted in the presence of a metabolic intermediate whose synthesis is controlled by feedback inhibition of Ade4p acting as the purine nucleotide sensor within the cell.

[1]  J. Sze,et al.  In vitro transcriptional activation by a metabolic intermediate: activation by Leu3 depends on alpha-isopropylmalate. , 1992, Science.

[2]  P. Flynn,et al.  Activation of Transcription by Metabolic Intermediates of the Pyrimidine Biosynthetic Pathway , 1999, Molecular and Cellular Biology.

[3]  S. Henikoff.  The Saccharomyces cerevisiae ADE5,7 protein is homologous to overlapping Drosophila melanogaster Gart polypeptides. , 1986, Journal of molecular biology.

[4]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[5]  B. Daignan-Fornier,et al.  The isolation and characterization of Saccharomyces cerevisiae mutants that constitutively express purine biosynthetic genes. , 1997, Genetics.

[6]  D. Hardie,et al.  Glucose repression/derepression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP:ATP ratio , 1996, Current Biology.

[7]  M. Künzler,et al.  Amino Acid and Adenine Cross-pathway Regulation Act through the Same 5′-TGACTC-3′ Motif in the Yeast HIS7 Promoter* , 1996, The Journal of Biological Chemistry.

[8]  G. Fink,et al.  BAS1 has a Myb motif and activates HIS4 transcription only in combination with BAS2. , 1989, Science.

[9]  A. Hinnebusch,et al.  Evidence that complex formation by Bas1p and Bas2p (Pho2p) unmasks the activation function of Bas1p in an adenine-repressible step of ADE gene transcription , 1997, Molecular and cellular biology.

[10]  T. Satyanarayana,et al.  Regulation of the purine pathway in bakers yeast: activity and feedback inhibition of phosphoribosyl-pyrophosphate amidotransferase. , 1971, Archives of biochemistry and biophysics.

[11]  H. Zalkin,et al.  Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties. , 1979, The Journal of biological chemistry.

[12]  Roger Brent,et al.  C dil, a Human Gl and S Phase Protein Phosphatase That Associates with Cdk2 , 2003 .

[13]  S. Gianì,et al.  Cloning and transcriptional analysis of the ADE6 gene of Saccharomyces cerevisiae. , 1991, Gene.

[14]  S. Hino,et al.  Feedback Inhibition of Amidophosphoribosyltransferase Regulates the Rate of Cell Growth via Purine Nucleotide, DNA, and Protein Syntheses* , 2001, The Journal of Biological Chemistry.

[15]  B. Daignan-Fornier,et al.  Signaling through regulated transcription factor interaction: mapping of a regulatory interaction domain in the Myb-related Bas1p. , 2000, Nucleic acids research.

[16]  P. Linder,et al.  The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors. , 1990, Gene.

[17]  B. Ames,et al.  ZTP (5-amino 4-imidazole carboxamide riboside 5′-triphosphate): A proposed alarmone for 10-formyl-tetrahydrofolate deficiency , 1982, Cell.

[18]  Roger Brent,et al.  DNA specificity of the bicoid activator protein is determined by homeodomain recognition helix residue 9 , 1989, Cell.

[19]  D. Kinney,et al.  Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. , 1986, Gene.

[20]  B. Daignan-Fornier,et al.  Role of the Myb‐like protein Bas1p in Saccharomyces cerevisiae: a proteome analysis , 1998, Molecular microbiology.

[21]  H. Zalkin,et al.  Identification of the Bacillus subtilis pur operon repressor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Coleman,et al.  Purine metabolism abnormalities in a hyperuricosuric subclass of autism. , 2000, Biochimica et biophysica acta.

[23]  B. Daignan-Fornier,et al.  Mutations in the yeast Myb-like protein Bas1p resulting in discrimination between promoters in vivo but notin vitro. , 1998, Nucleic acids research.

[24]  D. Appling,et al.  Saccharomyces cerevisiae expresses two genes encoding isozymes of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase. , 1997, Archives of biochemistry and biophysics.

[25]  L. Gustafsson,et al.  Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions , 1997, Journal of bacteriology.

[26]  Jack E. Dixon,et al.  A mutation in adenylosuccinate lyase associated with mental retardation and autistic features , 1992, Nature Genetics.

[27]  M Aldea,et al.  A Set of Vectors with a Tetracycline‐Regulatable Promoter System for Modulated Gene Expression in Saccharomyces cerevisiae , 1997, Yeast.

[28]  B. Daignan-Fornier,et al.  Coregulation of purine and histidine biosynthesis by the transcriptional activators BAS1 and BAS2. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[29]  H. Zalkin,et al.  Glutamine nucleotide sequence of Saccharomyces cerevisiae ADE4 encoding phosphoribosylpyrophosphate amidotransferase. , 1984, The Journal of biological chemistry.

[30]  F. Kippert.  A rapid permeabilization procedure for accurate quantitative determination of beta-galactosidase activity in yeast cells. , 1995, FEMS microbiology letters.

[31]  J. Broach,et al.  Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. , 1979, Gene.

[32]  R. A. Woods,et al.  Purine-excreting mutants of Saccharomyces cerevisiae. I. Isolation and genetic analysis. , 1970, Genetical research.

[33]  H. Ronne,et al.  Yeast MIG1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. , 1990, The EMBO journal.

[34]  R. A. Woods,et al.  Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae. , 1983, Canadian journal of microbiology.

[35]  G. Fink,et al.  Multiple global regulators control HIS4 transcription in yeast. , 1987, Science.

[36]  R. Rolfes,et al.  Purification of the Escherichia coli purine regulon repressor and identification of corepressors , 1990, Journal of bacteriology.

[37]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[38]  D. Kaback,et al.  Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the ADE1 gene , 1984, Journal of bacteriology.

[39]  A. Hinnebusch,et al.  The Transcriptional Activators BAS1, BAS2, and ABF1 Bind Positive Regulatory Sites as the Critical Elements for Adenine Regulation of ADE5,7 * , 1997, The Journal of Biological Chemistry.

[40]  V. Kushnirov.  Rapid and reliable protein extraction from yeast , 2000, Yeast.

[41]  B. Daignan-Fornier,et al.  Yeast GMP kinase mutants constitutively express AMP biosynthesis genes by phenocopying a hypoxanthine-guanine phosphoribosyltransferase defect. , 2000, Genetics.

[42]  R. Brent,et al.  Fused protein domains inhibit DNA binding by LexA , 1992, Molecular and cellular biology.