Yeast AMP pathway genes respond to adenine through regulated synthesis of a metabolic intermediate.

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 the ADE 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.

Highly conserved features of DNA binding between two divergent members of the Myb family of transcription factors.

Bas1p, a divergent yeast member of the Myb family of transcription factors, shares with the proteins of this family a highly conserved cysteine residue proposed to play a role in redox regulation. Substitutions of this residue in Bas1p (C153) allowed us to establish that, despite its very high conservation, it is not strictly required for Bas1p function: its substitution with a small hydrophobic residue led to a fully functional protein in vitro and in vivo. C153 was accessible to an alkylating agent in the free protein but was protected by prior exposure to DNA. The reactivity of cysteines in the first and third repeats was much lower than in the second repeat, suggesting a more accessible conformation of repeat 2. Proteolysis protection, fluorescence quenching and circular dichroism experiments further indicated that DNA binding induces structural changes making Bas1p less accessible to modifying agents. Altogether, our results strongly suggest that the second repeat of the DNA-binding domain of Bas1p behaves similarly to its Myb counterpart, i.e. a DNA-induced conformational change in the second repeat leads to formation of a full helix-turn-helix-related motif with the cysteine packed in the hydrophobic core of the repeat.

Signaling through regulated transcription factor interaction: mapping of a regulatory interaction domain in the Myb-related Bas1p.

Gene activation in eukaryotes is inherently combinatorial depending on cooperation between different transcription factors. An example where this cooperation seems to be directly exploited for regulation is the Bas1p/Bas2p couple in yeast. Bas1p is a Myb-related transcription factor that acts together with the homeodomain-related Bas2p (Pho2p) to regulate purine and histidine biosynthesis genes in response to extracellular purine limitation. We show that fusion of the two factors abolished adenine repression, suggesting that what is regulated by adenine is the Bas1p-Bas2p interaction. Analysis of Bas1p deletions revealed a critical domain (Bas1p interaction and regulatory domain, BIRD) acting in two-hybrid assays as an adenine-dependent Bas1p-Bas2p interaction domain. BIRD had a dual function, as an internal repressor of a centrally located Bas1p transactivation domain on the ADE1 promoter and as a Bas2p-dependent activator on the HIS4 promoter. This promoter-dependent behavior reflected a differential binding to the two promoters in vivo. On ADE1 Bas1p bound the promoter efficiently by itself, but required adenine limitation and Bas2p interaction through BIRD for derepression. On HIS4 efficient promoter binding and derepression required both factors and adenine limitation. We propose a promoter-dependent model for adenine regulation in yeast based on controlled Bas1p-Bas2p interactions through BIRD and exploited differentially by the two promoters.

The isolation and characterization of Saccharomyces cerevisiae mutants that constitutively express purine biosynthetic genes.

In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes. To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression. These mutants were named bra (for bypass of repression by adenine). BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase. BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase. These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes. We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway. Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression. Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.

Yeast GMP kinase mutants constitutively express AMP biosynthesis genes by phenocopying a hypoxanthine-guanine phosphoribosyltransferase defect.

We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase. The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs. The bra3 locus also corresponds to the previously described pur5 locus. Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene. First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance. Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants. Third, we show that purified yeast HGPRT is inhibited by GMP in vitro. Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo. We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT. The possible occurrence of a similar phenomenon in humans is discussed.

In vivo functional characterization of a yeast nucleotide sequence: construction of a mini-Mu derivative adapted to yeast.

We have constructed a derivative of the bacteriophage Mu (called MudIIZZ1), which contains the lacZ gene coding for beta-galactosidase (beta Gal) and markers suited for yeast transformation (2 mu circle replication origin and LEU2). This new transposon is an efficient tool for studying the expression of cloned yeast nucleotide sequences through beta Gal-protein fusions. It is also adapted for one-step disruption experiments so that a functional map of the same sequence can be drawn. We have used this MudIIZZ1 transposon to study a 5-kb DNA fragment which had been cloned by complementation of a cold-sensitive respiration-deficient phenotype. By testing the expression of the beta Gal fusions and the disruption phenotype, we have confirmed the presence of a gene required for mitochondrial functions, and revealed another two open reading frames in the same fragment; one of these also interferes with mitochondrial biogenesis. The method is fast and reliable, and has potential for more general purposes which are discussed.

Identification of nuclear genes which participate to mitochondrial translation in Saccharomyces cerevisiae.

The mitochondrial protein synthesis presents specific features and uses specific components different from their cytoplasmic counterparts. Since most genes which code for these components are localized in the chromosomes and only a small number are encoded by the mitochondrial DNA, it is important to identify and characterize the nuclear genes involved in this process. In order to do this, we have used a genetic screening which implies the selection and study of nuclear suppressors of mitochondrial mutations (or the reverse situation) which affect the mitochondrial protein synthesis. Three mutations have been used for this purpose. Two of them (ts 1398, cs 909) impair the mitochondrial ribosome; they were used to characterize new interacting components as well as two genes, MBR1 and MBR2, which control the assembly or the regulation of other genes involved in mitochondrial protein synthesis. The third mutation (ts 932), blocks the 3'-end maturation of the mitochondrial aspartyl tRNA. A nuclear suppressor has been obtained which presents all the characteristics of a mutation in the gene encoding the enzyme responsible for this process.

Coregulation of purine and histidine biosynthesis by the transcriptional activators BAS1 and BAS2.

We have found cross-pathway regulation between purine and histidine biosynthesis in yeast. The transcription factors BAS1 and BAS2/PHO2, which are also regulators of the histidine pathway, participate in the regulation of the purine biosynthetic pathway. Analysis of four genes of the purine pathway (ADE1, ADE2, ADE5,7, and ADE8) shows that their expression is repressed by adenine. The maximal basal and induced expression of these purine genes requires the presence of both BAS1 and BAS2. The factor BAS1 has been shown to bind at a site containing the TGACTC hexanucleotide motif in the ADE2 and ADE5,7 promoters. This motif is required for both basal and induced activation of the ADE2 gene by BAS1 and BAS2.

Mutational study of the rRNA in yeast mitochondria: functional importance of T1696 in the large rRNA gene.

Four intragenic suppressors of a mitochondrial mutation in the 21S rRNA gene have been characterized in S. cerevisiae. The determination of the nature of the nucleotide changes in the suppressor strains showed that a T at position 1696 in the large rRNA gene is essential for correct function of the mitoribosome. The importance of this specific nucleotide and the fact that this mitochondrial mutation can also be suppressed by a mutation in a nuclear gene are in good agreement with a rRNA-r protein interaction in this part of domain IV, which functional importance is demonstrated in vivo by our results.

Synthesis of glutamine, glycine and 10-formyl tetrahydrofolate is coregulated with purine biosynthesis in Saccharomyces cerevisiae.

Glutamine, glycine and 10-formyl tetrahydrofolate are consumed during de novo purine biosynthesis. We have found that, in Saccharomyces cerevisiae, synthesis of these cosubstrates is coregulated with synthesis of enzymes of the purine biosynthetic pathway. Analysis of three genes required for synthesis of glutamine, glycine and 10-formyl tetrahydrofolate (GLN1, SHM2 and MTD1, respectively) shows that their expression is repressed by adenine and requires the transcription factors Baslp and Bas2p. Northern analysis reveals that regulation of SHM2 and MTD1 expression by adenine takes place at the transcriptional level. We also show that Bas1p and Bas2p bind in vitro to the promoters of the SHM2 and MTD1 genes, and that mutations in the consensus Bas1p binding sequences strongly affect expression of these genes in vivo. Finally, we have found that a SHM2-lacZ fusion is expressed at a significantly higher level in a bas2-2 disrupted strain than in bas1-2 or bas1-2 bas2-2 mutant strains. The BAS1-dependent, BAS2-independent expression of SHM2-lacZ suggests that, in the absence of Bas2p, Bas1p can interact with another protein partner to activate SHM2 expression.

Transcriptional regulation of the yeast gmp synthesis pathway by its end products.

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.

Functional exploration of the yeast (Saccharomyces cerevisiae) genome: use of a mini-Mu transposon to analyse randomly cloned DNA sequences.

The development of mega-sequencing techniques requires new methods for global functional analysis of cloned DNA fragments. We have developed a mini-Mu transposon adapted to yeast cloned DNA fragment analysis. This transposon allows us to do the following in a single construction: (i) to probe yeast cells for the presence of expressed open reading frames (ORFs) in the cloned DNA fragment; (ii) to localize these ORFs in the fragment and determine their transcription orientation; (iii) to use beta-galactosidase protein fusions to study regulation of these ORFs; and (iv) to disrupt the corresponding chromosomal genes. On a 5-kb yeast DNA sequence, we have verified the reliability of this new tool by comparing the data obtained with the mini-Mu transposon to those obtained by classical methods. This transposon should be of immediate use in the yeast genome sequencing programme.

Identification of genes affecting selenite toxicity and resistance in Saccharomyces cerevisiae.

Recent studies associating dietary selenium with reduced cancer susceptibility have aroused interest in this substance. In the millimolar range, selenite is toxic and slightly mutagenic for yeast. We show that selenite-treated yeast cells tend to arrest as large budded cells and that this arrest is abolished in a rad9 mutant that is significantly sensitive to selenite. Interestingly, a rev3 mutant affected in the error-prone repair pathway is also sensitive to selenite, whereas mutations in the other DNA repair pathways do not strongly affect resistance to selenite. We propose that selenite treatment leads to DNA damage inducing the RAD9-dependent cell cycle arrest. Selenite-induced DNA damage could be converted to mutations by the Rev3p-dependent lesion bypass system, thus allowing the cell cycle to progress. We have also investigated the selenite detoxification mechanisms and identified three genes involved in this process. In the present study, we show that lack of the cadmium glutathione-conjugate vacuolar pump Ycf1p or overexpression of the sulphite resistance membrane protein Ssu1p enhance the capacity of yeast cells to resist selenite treatment. Finally, we show that overexpression of the glutathione reductase Glr1p increases resistance to selenite, suggesting that selenite toxicity in yeast is closely linked to its oxidative capacity.

Role of the myb-like protein bas1p in Saccharomyces cerevisiae: a proteome analysis.

The effect of extracellular adenine and the role of the transcriptional activator Bas1p on expression of the yeast genome was assessed by two-dimensional (2D) analysis of the yeast proteome. These data combined with LacZ fusions and northern blot analysis allow us to show that synthesis of enzymes for all 10 steps involved in purine de novo synthesis is repressed in the presence of adenine and requires BAS1 and BAS2 for optimal expression. We also show that expression of ADE12 and ADE13, the two genes required for synthesis of AMP from inosine 5'monophosphate (IMP), is co-regulated with the de novo pathway genes. The same combined approach, used to study histidine biosynthesis gene expression, showed that HIS1 and HIS4 expression is co-regulated with purine biosynthesis genes whereas HIS2, HIS3, HIS5 and HIS6 expression is not. This work, together with previously published data, gives the first comprehensive overview of the regulation of purine and histidine pathways in a eukaryotic organism. Finally, the expression of two pyrimidine biosynthesis genes URA1 and URA3 was found to be severely affected by bas1 and bas2 mutations in the absence of adenine, establishing a regulatory link between the two nucleotide biosynthesis pathways.

Role of adenosine kinase in Saccharomyces cerevisiae: identification of the ADO1 gene and study of the mutant phenotypes.

Sequencing of the Saccharomyces cerevisiae genome revealed an open reading frame (YJR105w) encoding a putative protein highly similar to adenosine kinases from other species. Disruption of this gene (renamed ADO1) affected utilization of S-adenosyl methionine (AdoMet) as a purine source and resulted in a severe reduction of adenosine kinase activity in crude extracts. Furthermore, knock-out of ADO1 led to adenosine excretion in the medium and resistance to the toxic adenosine analogue cordycepin. From these data we conclude that ADO1 encodes yeast adenosine kinase. We also show that ADO1 does not play a major role in adenine utilization in yeast and we propose that the physiological role of adenosine kinase in S. cerevisiae could primarily be to recycle adenosine produced by the methyl cycle.

Epitope-tagging vectors designed for yeast.

In order to facilitate the process of epitope-tagging of yeast proteins, we have constructed two Saccharomyces cerevisiae-Escherichia coli shuttle vectors that allow fusion of a sequence encoding an epitope of the human c-myc protein at the 3' end of any gene. An example of the use of this technique is presented.

Redox regulation of AMP synthesis in yeast: a role of the Bas1p and Bas2p transcription factors.

Expression of yeast AMP synthesis genes (ADE genes) was severely affected when cells were grown under oxidative stress conditions. To get an insight into the molecular mechanisms of this new transcriptional regulation, the role of the Bas1p and Bas2p transcription factors, known to activate expression of the ADE genes, was investigated. In vitro, DNA-binding of Bas1p was sensitive to oxidation. However, this sensitivity could not account for the regulation of the ADE genes because we showed, using a BAS1-VP16 chimera, that Bas1p DNA-binding activity was not sensitive to oxidation in vivo. Consistently, a triple cysteine mutant of Bas1p (fully resistant to oxidation in vitro) was unable to restore transcription of the ADE genes under oxidative conditions. We then investigated the possibility that Bas2p could be the oxidative stress responsive factor. Interestingly, transcription of the PHO5 gene, which is dependent on Bas2p but not on Bas1p, was found to be severely impaired by oxidative stress. Nevertheless, a Bas2p cysteine-free mutant was not sufficient to confer resistance to oxidative stress. Finally, we found that a Bas1p-Bas2p fusion protein restored ADE gene expression under oxidative conditions, thus suggesting that redox sensitivity of ADE gene expression could be due to an impairment of Bas1p/Bas2p interaction. This hypothesis was further substantiated in a two hybrid experiment showing that Bas1p/Bas2p interaction is affected by oxidative stress.

A genetic screen to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p.

We have developed a screening method to isolate yeast genes regulated by a specific transcription activator. The screen is based on the use of expression libraries in which the lacZ reporter gene is placed under control of yeast regulatory elements. Two partially representative libraries, constructed by different methods, were used to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p. Among 26 fusions shown to be regulated by Hap2p only CYT1 was known to be regulated by this activator. Sequence analysis revealed that most of the remaining regulated fusions are in new yeast genes, while some are in previously characterized yeast genes (PTP1, RPM2, SDH1). Optimal expression of these three genes also requires Hap3p and Hap4p and is regulated by carbon source. Hap2p was known to regulate expression of genes involved in Krebs cycle, electron transport and heme biosynthesis. Our results suggest that Hap2p could play a more general role by regulating other mitochondrial processes such as protein import and phosphate transport (PTP1) or maturation of mitochondrial tRNAs (RPM2). Among the remaining regulated fusions, two of them correspond to open reading frames (ORFs) on chromosomes III and XI whose nucleotide sequences have been entirely determined. The use of this approach to functionally analyse ORFs of unknown function is discussed.

Structure and regulation of SDH3, the yeast gene encoding the cytochrome b560 subunit of respiratory complex II.

Using an expression library, we have isolated yeast genes activated in the presence of the yeast CCAAT box-binding protein HAP2. One of these genes, SDH3, encodes the cytochrome b560 subunit of respiratory complex II. The SDH3 protein contains three potential transmembrane domains and is more than 30% identical to bovine cytochrome b560 and to a mitochondrially encoded protein from Marchantia polymorpha. Disruption of SDH3 shows that this gene is required for growth on non-fermentable carbon sources. Expression of SDH1, SDH3, and SDH4 is activated in the presence of the HAP2 transcriptional activator.

MBR1 and MBR3, two related yeast genes that can suppress the growth defect of hap2, hap3 and hap4 mutants.

Two new yeast genes, named MBR1 and MBR3, were isolated as multicopy suppressors of the growth defect of a strain lacking the HAP2 transcriptional activator. Both genes when overexpressed can also suppress the growth defect of hap3 and hap4 null mutants. However, overexpression of MBR1 cannot substitute for the HAP2/3/4 complex in activation of the CYC1 gene. Nucleotide sequencing of MBR1 and MBR3 revealed that these two genes encode serine-rich, hydrophilic proteins with regions of significant homology. The functional importance of one of these conserved regions was shown by mutagenesis. Disruption of MBR1 leads to a partial growth defect on glycerol medium. Disruption of MBR3 has no major effect but the double disruptant shows a synthetic phenotype suggesting that the MBR1 and MBR3 gene products participate in common function.