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Metabolism is a highly integrated process resulting in energy and biomass production. While individual metabolic routes are well characterized, the mechanisms ensuring crosstalk between pathways are poorly described, although they are crucial for homeostasis. Here, we establish a co-regulation of purine and pyridine metabolism in response to external adenine through two separable mechanisms. First, adenine depletion promotes transcriptional upregulation of the de novo NAD + biosynthesis genes by a mechanism requiring the key-purine intermediates ZMP/SZMP and the Bas1/Pho2 transcription factors. Second, adenine supplementation favors the pyridine salvage route resulting in an ATP-dependent increase of intracellular NAD + . This control operates at the level of the nicotinic acid mononucleotide adenylyl-transferase Nma1 and can be bypassed by overexpressing this enzyme. Therefore, in yeast, pyridine metabolism is under the dual control of ZMP/SZMP and ATP, revealing a much wider regulatory role for these intermediate metabolites in an integrated biosynthesis network., Competing Interests: BP, JC, CS, BD No competing interests declared, (© 2019, Pinson et al.)

The yeast YLR209c (PNP1) gene encodes a protein highly similar to purine nucleoside phosphorylases. This protein specifically metabolized inosine and guanosine. Disruption of PNP1 led to inosine and guanosine excretion in the medium, thus showing that PNP1 plays an important role in the metabolism of these purine nucleosides in vivo.

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.

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.

Bas1p is a yeast transcription factor that activates expression of purine and histidine biosynthesis genes in response to extracellular purine limitation. The N-terminal part of Bas1p contains an Myb-like DNA binding domain composed of three tryptophan-rich imperfect repeats. We show that mutating the conserved tryptophan residues in the DNA binding domain of Bas1p severely impairs in vivo activation of target genes and in vitro DNA binding of Bas1p. We also found that two mutations (H34L and W42A) in the first repeat make Bas1p discriminate between promoters in vivo . These two BAS1 mutants are able to activate expression of an HIS4-lacZ fusion but not that of ADE1-lacZ or ADE17-lacZ fusions. Surprisingly, these mutant proteins bind equally well to the three promoters in vitro , suggesting that the mutations affect the interaction of Bas1p with some promoter-specific factor(s) in vivo . By mutating a potential nucleotide binding site in the DNA binding domain of Bas1p, we also show that this motif does not play a major role in purine regulation of Bas1p activity. Finally, using a green fluorescence protein (GFP)-Bas1p fusion, we establish the strict nuclear localization of Bas1p and show that it is not affected by extracellular adenine.

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.

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.

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.

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.

Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20 degrees C). At 28 degrees C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20 degrees C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.