Search

Cancer is most commonly viewed as resulting from somatic mutations enhancing proliferation and invasion. Some hypotheses further propose that these new capacities reveal a breakdown of multicellularity allowing cancer cells to escape proliferation and cooperation control mechanisms that were implemented during evolution of multicellularity. Here we critically review one such hypothesis, named "atavism," which puts forward the idea that cancer results from the re-expression of normally repressed genes forming a program, or toolbox, inherited from unicellular or simple multicellular ancestors. This hypothesis places cancer in an interesting evolutionary perspective that has not been widely explored and deserves attention. Thinking about cancer within an evolutionary framework, especially the major transitions to multicellularity, offers particularly promising perspectives. It is therefore of the utmost important to analyze why one approach that tries to achieve this aim, the atavism hypothesis, has not so far emerged as a major theory on cancer. We outline the features of the atavism hypothesis that, would benefit from clarification and, if possible, unification., (© 2024 Wiley Periodicals LLC.)

Cancers rely on multiple, heterogeneous processes at different scales, pertaining to many biomedical fields. Therefore, understanding cancer is necessarily an interdisciplinary task that requires placing specialised experimental and clinical research into a broader conceptual, theoretical, and methodological framework. Without such a framework, oncology will collect piecemeal results, with scant dialogue between the different scientific communities studying cancer. We argue that one important way forward in service of a more successful dialogue is through greater integration of applied sciences (experimental and clinical) with conceptual and theoretical approaches, informed by philosophical methods. By way of illustration, we explore six central themes: (i) the role of mutations in cancer; (ii) the clonal evolution of cancer cells; (iii) the relationship between cancer and multicellularity; (iv) the tumour microenvironment; (v) the immune system; and (vi) stem cells. In each case, we examine open questions in the scientific literature through a philosophical methodology and show the benefit of such a synergy for the scientific and medical understanding of cancer., (© 2023 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Purines are required for fundamental biological processes and alterations in their metabolism lead to severe genetic diseases associated with developmental defects whose etiology remains unclear. Here, we studied the developmental requirements for purine metabolism using the amphibian Xenopus laevis as a vertebrate model. We provide the first functional characterization of purine pathway genes and show that these genes are mainly expressed in nervous and muscular embryonic tissues. Morphants were generated to decipher the functions of these genes, with a focus on the adenylosuccinate lyase ( ADSL ), which is an enzyme required for both salvage and de novo purine pathways. adsl.L knockdown led to a severe reduction in the expression of the myogenic regulatory factors (MRFs: Myod1, Myf5 and Myogenin), thus resulting in defects in somite formation and, at later stages, the development and/or migration of both craniofacial and hypaxial muscle progenitors. The reduced expressions of hprt1.L and ppat , which are two genes specific to the salvage and de novo pathways, respectively, resulted in similar alterations. In conclusion, our data show for the first time that de novo and recycling purine pathways are essential for myogenesis and highlight new mechanisms in the regulation of MRF gene expression.

The pentose phosphate pathway (PPP) is critical for anabolism and biomass production. Here we show that the essential function of PPP in yeast is the synthesis of phosphoribosyl pyrophosphate (PRPP) catalyzed by PRPP-synthetase. Using combinations of yeast mutants, we found that a mildly decreased synthesis of PRPP affects biomass production, resulting in reduced cell size, while a more severe decrease ends up affecting yeast doubling time. We establish that it is PRPP itself that is limiting in invalid PRPP-synthetase mutants and that the resulting metabolic and growth defect can be bypassed by proper supplementation of the medium with ribose-containing precursors or by the expression of bacterial or human PRPP-synthetase. In addition, using documented pathologic human hyperactive forms of PRPP-synthetase, we show that intracellular PRPP as well as its derived products can be increased in both human and yeast cells, and we describe the ensuing metabolic and physiological consequences. Finally, we found that PRPP consumption appears to take place "on demand" by the various PRPP-utilizing pathways, as shown by blocking or increasing the flux in specific PRPP-consuming metabolic routes. Overall, our work reveals important similarities between human and yeast for both synthesis and consumption of PRPP., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Being able to reproduce and survive is fundamental to all forms of life. In primitive unicellular organisms, the emergence of quiescence as a reversible proliferation arrest has most likely improved cell survival under unfavorable environmental conditions. During evolution, with the repeated appearances of multicellularity, several aspects of unicellular quiescence were conserved while new quiescent cell intrinsic abilities arose. We propose that the formation of a microenvironment by neighboring cells has allowed disconnecting quiescence from nutritional cues. In this new context, non-proliferative cells can stay metabolically active, potentially authorizing the emergence of new quiescent cell properties, and thereby favoring cell specialization. Through its co-evolution with cell specialization, quiescence may have been a key motor of the fascinating diversity of multicellular complexity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Daignan-Fornier, Laporte and Sagot.)

Because metabolism is a complex balanced process involving multiple enzymes, understanding how organisms compensate for transient or permanent metabolic imbalance is a challenging task that can be more easily achieved in simpler unicellular organisms. The metabolic balance results not only from the combination of individual enzymatic properties, regulation of enzyme abundance, but also from the architecture of the metabolic network offering multiple interconversion alternatives. Although metabolic networks are generally highly resilient to perturbations, metabolic imbalance resulting from enzymatic defect and specific environmental conditions can be designed experimentally and studied. Starting with a double amd1 aah1 mutant that severely and conditionally affects yeast growth, we carefully characterized the metabolic shuffle associated with this defect. We established that the GTP decrease resulting in an adenylic/guanylic nucleotide imbalance was responsible for the growth defect. Identification of several gene dosage suppressors revealed that TAT1, encoding an amino acid transporter, is a robust suppressor of the amd1 aah1 growth defect. We show that TAT1 suppression occurs through replenishment of the GTP pool in a process requiring the histidine biosynthesis pathway. Importantly, we establish that a tat1 mutant exhibits synthetic sickness when combined with an amd1 mutant and that both components of this synthetic phenotype can be suppressed by specific gene dosage suppressors. Together our data point to a strong phenotypic connection between amino acid uptake and GTP synthesis, a connection that could open perspectives for future treatment of related human defects, previously reported as etiologically highly conserved.

Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.

The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (S N 2) with a mix of S N 1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted S N 2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates., (© 2019 Ozeir et al.)

Purine homeostasis is ensured through a metabolic network widely conserved from prokaryotes to humans. Purines can either be synthesized de novo , reused, or produced by interconversion of extant metabolites using the so-called recycling pathway. Although thoroughly characterized in microorganisms, such as yeast or bacteria, little is known about regulation of the purine biosynthesis network in metazoans. In humans, several diseases are linked to purine metabolism through as yet poorly understood etiologies. Particularly, the deficiency in adenylosuccinate lyase (ADSL)-an enzyme involved both in the purine de novo and recycling pathways-causes severe muscular and neuronal symptoms. In order to address the mechanisms underlying this deficiency, we established Caenorhabditis elegans as a metazoan model organism to study purine metabolism, while focusing on ADSL. We show that the purine biosynthesis network is functionally conserved in C. elegans Moreover, adsl-1 (the gene encoding ADSL in C. elegans ) is required for developmental timing, germline stem cell maintenance and muscle integrity. Importantly, these traits are not affected when solely the de novo pathway is abolished, and we present evidence that germline maintenance is linked specifically to ADSL activity in the recycling pathway. Hence, our results allow developmental and tissue specific phenotypes to be ascribed to separable steps of the purine metabolic network in an animal model., (Copyright © 2019 by the Genetics Society of America.)

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.)

5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP), an intermediate in de novo purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-β Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of KAP123 for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR., (© 2019 Douillet et al.)

Purine nucleotides are involved in a multitude of cellular processes, and the dysfunction of purine metabolism has drastic physiological and pathological consequences. Accordingly, several genetic disorders associated with defective purine metabolism have been reported. The etiology of these diseases is poorly understood and simple model organisms, such as yeast, have proved valuable to provide a more comprehensive view of the metabolic consequences caused by the identified mutations. In this review, we present results obtained with the yeast Saccharomyces cerevisiae to exemplify how a eukaryotic unicellular organism can offer highly relevant information for identifying the molecular basis of complex human diseases. Overall, purine metabolism illustrates a remarkable conservation of genes, functions and phenotypes between humans and yeast.

AICAR is the precursor of ZMP, a metabolite with antiproliferative properties in yeast and human. We aim at understanding how AICAR (and its active form ZMP) affects essential cellular processes. In this work, we found that ZMP accumulation is synthetic lethal with a hypomorphic allele of the ubiquitin-activating enzyme Uba1. A search for gene-dosage suppressors revealed that ubiquitin overexpression was sufficient to restore growth of the uba1 mutant upon AICAR treatment, suggesting that the ubiquitin pool is critical for cells to cope with AICAR. Accordingly, two mutants with constitutive low ubiquitin, ubp6 and doa1, were highly sensitive to AICAR, a phenotype that could be suppressed by ubiquitin overexpression. We established, by genetic means, that these new AICAR-sensitive mutants act in a different pathway from the rad6/bre1 mutants which were previously reported as sensitive to AICAR (Albrecht et al., Genetics 204:1447-1460, 2016). Two ubiquitin-conjugating enzymes (Ubc4 and Cdc34) and a ubiquitin ligase (Cdc4) were found to contribute to the ability of cells to cope with ZMP. This study illustrates the complexity of chemo-genetic interactions and shows how genetic analyses allow deciphering the implicated pathways, the individual gene effects, and their combined phenotypic contribution. Based on additivity and suppression patterns, we conclude that AICAR treatment shows synthetic interactions with distinct branches of the yeast ubiquitin pathway.

Phosphoribosyltransferases catalyze the displacement of a PRPP α-1'-pyrophosphate to a nitrogen-containing nucleobase. How they control the balance of substrates/products binding and activities is poorly understood. Here, we investigated the human adenine phosphoribosyltransferase (hAPRT) that produces AMP in the purine salvage pathway. We show that a single oxygen atom from the Tyr105 side chain is responsible for selecting the active conformation of the 12 amino acid long catalytic loop. Using in vitro, cellular, and in crystallo approaches, we demonstrated that Tyr105 is key for the fine-tuning of the kinetic activity efficiencies of the forward and reverse reactions. Together, our results reveal an evolutionary pressure on the strictly conserved Tyr105 and on the dynamic motion of the flexible loop in phosphoribosyltransferases that is essential for purine biosynthesis in cells. These data also provide the framework for designing novel adenine derivatives that could modulate, through hAPRT, diseases-involved cellular pathways., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

AICAR (Acadesine) is a pharmacological precursor of purine nucleotide biosynthesis with anti-tumoral properties. Although recognized as an AMP mimetic activator of the protein kinase AMPK, the AICAR monophosphate derivative ZMP was also shown to mediate AMPK-independent effects. In order to unveil these AMPK-independent functions, we performed a transcriptomic analysis in AMPKα1/α2 double knockout murine embryonic cells. Kinetic analysis of the cellular response to AICAR revealed the up-regulation of the large tumor suppressor kinases (Lats) 1 and 2 transcripts, followed by the repression of numerous genes downstream of the transcriptional regulators Yap1 and Taz. This transcriptional signature, together with the observation of increased levels in phosphorylation of Lats1 and Yap1 proteins, suggested that the Hippo signaling pathway was activated by AICAR. This effect was observed in both fibroblasts and epithelial cells. Knockdown of Lats1/2 prevented the cytoplasmic delocalization of Yap1/Taz proteins in response to AICAR and conferred a higher resistance to the drug. These results indicate that activation of the most downstream steps of the Hippo cascade participates to the antiproliferative effects of AICAR., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Identifying synthetic lethal interactions has emerged as a promising new therapeutic approach aimed at targeting cancer cells directly. Here, we used the yeast Saccharomyces cerevisiae as a simple eukaryotic model to screen for mutations resulting in a synthetic lethality with 5-amino-4-imidazole carboxamide ribonucleoside (AICAR) treatment. Indeed, AICAR has been reported to inhibit the proliferation of multiple cancer cell lines. Here, we found that loss of several histone-modifying enzymes, including Bre1 (histone H2B ubiquitination) and Set1 (histone H3 lysine 4 methylation), greatly enhanced AICAR inhibition on growth via the combined effects of both the drug and mutations on G1 cyclins. Our results point to AICAR impacting on Cln3 subcellular localization and at the Cln1 protein level, while the bre1 or set1 deletion affected CLN1 and CLN2 expression. As a consequence, AICAR and bre1/set1 deletions jointly affected all three G1 cyclins (Cln1, Cln2, and Cln3), leading to a condition known to result in synthetic lethality. Significantly, these chemo-genetic synthetic interactions were conserved in human HCT116 cells. Indeed, knock-down of RNF40, ASH2L, and KMT2D/MLL2 induced a highly significant increase in AICAR sensitivity. Given that KMT2D/MLL2 is mutated at high frequency in a variety of cancers, this synthetic lethal interaction has an interesting therapeutic potential., (Copyright © 2016 by the Genetics Society of America.)

Previous genetic analyses showed phenotypic interactions between 5-amino-4-imidazole carboxamide ribonucleotide 5'-phosphate (AICAR) produced from the purine and histidine pathways and methionine biosynthesis. Here, we revisited the effect of AICAR on methionine requirement due to AICAR accumulation in the presence of the fau1 mutation invalidating folinic acid remobilization. We found that this methionine auxotrophy could be suppressed by overexpression of the methionine synthase Met6 or by deletion of the serine hydroxymethyltransferase gene SHM2. We propose that in a fau1 background, AICAR, by stimulating the transcriptional expression of SHM2, leads to a folinic acid accumulation inhibiting methionine synthesis by Met6. In addition, we uncovered a new methionine auxotrophy for the ade3 bas1 double mutant that can be rescued by overexpressing the SHM2 gene. We propose that methionine auxotrophy in this mutant is the result of a competition for 5,10-methylenetetrahydrofolate between methionine and deoxythymidine monophosphate synthesis. Altogether, our data show intricate genetic interactions between one-carbon units, purine and methionine metabolism through fine-tuning of serine hydroxymethyltransferase by AICAR and the transcription factor Bas1.

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside monophosphate (AICAR) is a natural metabolite with potent anti-proliferative and low energy mimetic properties. At high concentration, AICAR is toxic for yeast and mammalian cells, but the molecular basis of this toxicity is poorly understood. Here, we report the identification of yeast purine salvage pathway mutants that are synthetically lethal with AICAR accumulation. Genetic suppression revealed that this synthetic lethality is in part due to low expression of adenine phosphoribosyl transferase under high AICAR conditions. In addition, metabolite profiling points to the AICAR/NTP balance as crucial for optimal utilization of glucose as a carbon source. Indeed, we found that AICAR toxicity in yeast and human cells is alleviated when glucose is replaced by an alternative carbon source. Together, our metabolic analyses unveil the AICAR/NTP balance as a major factor of AICAR antiproliferative effects., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Lesch-Nyhan disease is a rare X-linked neurodevelopemental metabolic disorder caused by a wide variety of mutations in the HPRT1 gene leading to a deficiency of the purine recycling enzyme hypoxanthine-guanine phosphoribosyltransferase (HGprt). The residual HGprt activity correlates with the various phenotypes of Lesch-Nyhan (LN) patients and in particular with the different degree of neurobehavioral disturbances. The prevalence of this disease is considered to be underestimated due to large heterogeneity of its clinical symptoms and the difficulty of diagnosing of the less severe forms of the disease. We therefore searched for metabolic changes that would facilitate an early diagnosis and give potential clues on the disease pathogenesis and potential therapeutic approaches., Methods: Lesch-Nyhan patients were diagnosed using HGprt enzymatic assay in red blood cells and identification of the causal HPRT1 gene mutations. These patients were subsequently classified into the three main phenotypic subgroups ranging from patients with only hyperuricemia to individuals presenting motor dysfunction, cognitive disability and self-injurious behavior. Metabolites from the three classes of patients were analyzed and quantified by High Performance Ionic Chromatography and biomarkers of HGprt deficiency were then validated by statistical analyses., Results: A cohort of 139 patients, from 112 families, diagnosed using HGprt enzymatic assay in red blood cells, was studied. 98 displayed LN full phenotype (86 families) and 41 (26 families) had attenuated clinical phenotypes. Genotype/phenotype correlations show that LN full phenotype was correlated to genetic alterations resulting in null enzyme function, while variant phenotypes are often associated with missense mutations allowing some residual HGprt activity. Analysis of metabolites extracted from red blood cells from 56 LN patients revealed strong variations specific to HGprt deficiency for six metabolites (AICAR mono- and tri-phosphate, nicotinamide, nicotinic acid, ATP and Succinyl-AMP) as compared to controls including hyperuricemic patients without HGprt deficiency., Conclusions: A highly significant correlation between six metabolites and the HGprt deficiency was established, each of them providing an easily measurable marker of the disease. Their combination strongly increases the probability of an early and reliable diagnosis for HGprt deficiency.

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) is the precursor of the active monophosphate form (AICAR), a small molecule with potent anti-proliferative and low energy mimetic properties. The molecular bases for AICAR toxicity at the cellular level are poorly understood. Here, we report the isolation and characterization of several yeast AICAr-hypersensitive mutants. Identification of the cognate genes allowed us to establish that thiamine transporters Thi7 and Thi72 can efficiently take up AICAr under conditions where they are overexpressed. We establish that, under standard growth conditions, Nrt1, the nicotinamide riboside carrier, is the major AICAr transporter in yeast. A study of AICAR accumulation in human cells revealed substantial disparities among cell lines and confirmed that AICAr enters cells via purine nucleoside transporters. Together, our results point to significant differences between yeast and human cells for both AICAr uptake and AICAR accumulation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cell size is a complex quantitative trait resulting from interactions between intricate genetic networks and environmental conditions. Here, taking advantage of previous studies that uncovered hundreds of genes affecting budding yeast cell size homeostasis, we performed a wide pharmaco-epistasis analysis using drugs mimicking cell size mutations. Simple epistasis relationship emerging from this approach allowed us to characterize a new cell size homeostasis pathway comprising the sirtuin Sir2, downstream effectors including the large ribosomal subunit (60S) and the transcriptional regulators Swi4 and Swi6. We showed that this Sir2/60S signaling route acts independently of other previously described cell size controlling pathways and may integrate the metabolic status of the cell through NAD(+) intracellular concentration. Finally, although Sir2 and the 60S subunits regulate both cell size and replicative aging, we found that there is no clear causal relationship between these two complex traits. This study sheds light on a pathway of >50 genes and illustrates how pharmaco-epistasis applied to yeast offers a potent experimental framework to explore complex genotype/phenotype relationships.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine recycling pathway that catalyzes the conversion of 5-phospho-ribosyl-α-1-pyrophosphate and guanine or hypoxanthine to guanosine monophosphate (GMP) or inosine monophosphate (IMP), respectively, and pyrophosphate (PPi). We report the first crystal structure of a fungal 6-oxopurine phosphoribosyltransferase, the Saccharomyces cerevisiae HGPRT (Sc-HGPRT) in complex with GMP. The crystal structures of full length protein with (WT1) or without (WT2) sulfate that mimics the phosphate group in the PPi binding site were solved by molecular replacement using the structure of a truncated version (Δ7) solved beforehand by multiwavelength anomalous diffusion. Sc-HGPRT is a dimer and adopts the overall structure of class I phosphoribosyltransferases (PRTs) with a smaller hood domain and a short two-stranded parallel β-sheet linking the N- to the C-terminal end. The catalytic loops in WT1 and WT2 are in an open form while in Δ7, due to an inter-subunit disulfide bridge, the catalytic loop is in either an open or closed form. The closure is concomitant with a peptide plane flipping in the PPi binding loop. Moreover, owing the flexibility of a GGGG motif conserved in fungi, all the peptide bonds of the phosphate binding loop are in trans conformation whereas in nonfungal 6-oxopurine PRTs, one cis-peptide bond is required for phosphate binding. Mutations affecting the enzyme activity or the previously characterized feedback inhibition by GMP are located at the nucleotide binding site and the dimer interface., (Copyright © 2012 The Protein Society.)

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.

AICAR (5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate) is a natural metabolic intermediate of purine biosynthesis that is present in all organisms. In yeast, AICAR plays important regulatory roles under physiological conditions, notably through its direct interactions with transcription factors. In humans, AICAR accumulates in several metabolic diseases, but its contribution to the symptoms has not yet been elucidated. Further, AICAR has highly promising properties which have been recently revealed. Indeed, it enhances endurance of sedentary mice. In addition, it has antiproliferative effects notably by specifically inducing apoptosis of aneuploid cells. Some of the effects of AICAR are due to its ability to stimulate the AMP-activated protein kinase but some others are not. It is consequently clear that AICAR affects multiple targets although only few of them have been identified so far. This review proposes an overview of the field and suggests future directions.

Ever since the beginning of biochemical analysis, yeast has been a pioneering model for studying the regulation of eukaryotic metabolism. During the last three decades, the combination of powerful yeast genetics and genome-wide approaches has led to a more integrated view of metabolic regulation. Multiple layers of regulation, from suprapathway control to individual gene responses, have been discovered. Constitutive and dedicated systems that are critical in sensing of the intra- and extracellular environment have been identified, and there is a growing awareness of their involvement in the highly regulated intracellular compartmentalization of proteins and metabolites. This review focuses on recent developments in the field of amino acid, nucleotide, and phosphate metabolism and provides illustrative examples of how yeast cells combine a variety of mechanisms to achieve coordinated regulation of multiple metabolic pathways. Importantly, common schemes have emerged, which reveal mechanisms conserved among various pathways, such as those involved in metabolite sensing and transcriptional regulation by noncoding RNAs or by metabolic intermediates. Thanks to the remarkable sophistication offered by the yeast experimental system, a picture of the intimate connections between the metabolomic and the transcriptome is becoming clear.

5-Amino-4-imidazolecarboxamide ribonucleotide 5'-phosphate (AICAR) is a monophosphate metabolic intermediate of the de novo purine synthesis pathway that has highly promising metabolic and antiproliferative properties. Yeast mutants unable to metabolize AICAR are auxotroph for histidine. A screening for suppressors of this phenotype identified recessive and dominant mutants that result in lowering the intracellular AICAR concentration. The recessive mutants affect the adenosine kinase, which is shown here to catalyze the phosphorylation of AICAR riboside in yeast. The dominant mutants strongly enhance the capacity of the alkaline phosphatase Pho13 to dephosphorylate 5-amino-4-imidazole N-succinocarboxamide ribonucleotide 5'-phosphate(SAICAR) into its non-toxic riboside form. By combining these mutants with transcriptomics and metabolomics analyses, we establish that in yeast responses to AICAR and SAICAR are clearly linked to the concentration of the monophosphate forms, whereas the derived nucleoside moieties have no effect even at high intracellular concentration. Finally, we show that AICAR/SAICAR concentrations vary under physiological conditions known to modulate transcription of the purine and phosphate pathway genes.

Quiescence is defined as a temporary arrest of proliferation, yet it likely encompasses various cellular situations. Our knowledge about this widespread cellular state remains limited. In particular, little is known about the molecular determinants that orchestrate quiescence establishment and exit. Here we show that upon carbon source exhaustion, budding yeast can enter quiescence from all cell cycle phases. Moreover, using cellular structures that are candidate markers for quiescence, we found that the first steps of quiescence exit can be triggered independently of cell growth and proliferation by the sole addition of glucose in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Importantly, glucose needs to be internalized and catabolized all the way down to glycolysis to mobilize quiescent cell specific structures, but, strikingly, ATP replenishment is apparently not the key signal. Altogether, these findings strongly suggest that quiescence entry and exit primarily rely on cellular metabolic status and can be uncoupled from the cell cycle.

Mitochondrial biogenesis is a complex process. It necessitates the participation of both the nuclear and the mitochondrial genomes. This process is highly regulated, and mitochondrial content within a cell varies according to energy demand. In the yeast Saccharomyces cerevisiae, the cAMP pathway is involved in the regulation of mitochondrial biogenesis. An overactivation of this pathway leads to an increase in mitochondrial enzymatic content. Of the three yeast cAMP protein kinases, we have previously shown that Tpk3p is the one involved in the regulation of mitochondrial biogenesis. In this paper, we investigated the molecular mechanisms that govern this process. We show that in the absence of Tpk3p, mitochondria produce large amounts of reactive oxygen species that signal to the HAP2/3/4/5 nuclear transcription factors involved in mitochondrial biogenesis. We establish that an increase in mitochondrial reactive oxygen species production down-regulates mitochondrial biogenesis. It is the first time that a redox sensitivity of the transcription factors involved in yeast mitochondrial biogenesis is shown. Such a process could be seen as a mitochondria quality control process.

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.

Cells use strategic metabolites to sense the metabolome and accordingly modulate gene expression. Here, we show that the purine and phosphate pathways are positively regulated by the metabolic intermediate AICAR (5'-phosphoribosyl-5-amino-4-imidazole carboxamide). The transcription factor Pho2p is required for up-regulation of all AICAR-responsive genes. Accordingly, the binding of Pho2p to purine and phosphate pathway gene promoters is enhanced upon AICAR accumulation. In vitro, AICAR binds both Pho2p and Pho4p transcription factors and stimulates the interaction between Pho2p and either Bas1p or Pho4p in vivo. In contrast, SAICAR (succinyl-AICAR) only affects Pho2p-Bas1p interaction and specifically up-regulates purine regulon genes. Together, our data show that Bas1p and Pho4p compete for Pho2p binding, hence leading to the concerted regulation of cellular nucleotide synthesis and phosphate consumption.

We found previously that inactivation of the FCY2 gene, encoding a purine-cytosine permease, or the HPT1 gene, encoding the hypoxanthine guanine phosphoribosyl transferase, enhances cisplatin resistance in yeast cells. Here, we report that in addition to fcy2Delta and hpt1Delta mutants in the salvage pathway of purine nucleotide biosynthesis, mutants in the de novo pathway that disable the feedback inhibition of AMP and GMP biosynthesis also enhanced cisplatin resistance. An activity-enhancing mutant of the ADE4 gene, which constitutively synthesizes AMP and excretes hypoxanthine, and a GMP kinase mutant (guk1), which accumulates GMP and feedback inhibits Hpt1 function, both enhanced resistance to cisplatin. In addition, overexpression of the ADE4 gene in wild-type cells, which increases de novo synthesis of purine nucleotides, also resulted in elevated cisplatin resistance. Cisplatin cytotoxicity in wild-type cells was abolished by low concentration of extracellular purines (adenine, hypoxanthine, and guanine) but not cytosine. Inhibition of cytotoxicity by exogenous adenine was accompanied by a reduction of DNA-bound cisplatin in wild-type cells. As a membrane permease, Fcy2 may mediate limited cisplatin transport because cisplatin accumulation in whole cells was slightly affected in the fcy2Delta mutant. However, the fcy2Delta mutant had a greater effect on the amount of DNA-bound cisplatin, which decreased to 50 to 60% of that in the wild-type cells. Taken together, our results indicate that dysregulation of the purine nucleotide biosynthesis pathways and the addition of exogenous purines can modulate cisplatin cytotoxicity in Saccharomyces cerevisiae.

Background: Polarity establishment and maintenance are crucial for morphogenesis and development. In budding yeast, these two intricate processes involve the superposition of regulatory loops between polarity landmarks, RHO GTPases, actin-mediated vesicles transport and endocytosis. Deciphering the chronology and the significance of each molecular step of polarized growth is therefore very challenging., Principal Findings: We have taken advantage of the fact that yeast quiescent cells display actin bodies, a non polarized actin structure, to evaluate the role of F-actin in bud emergence. Here we show that upon exit from quiescence, actin cables are not required for the first steps of polarized growth. We further show that polarized growth can occur in the absence of actin patch-mediated endocytosis. We finally establish, using latrunculin-A, that the first steps of polarized growth do not require any F-actin containing structures. Yet, these structures are required for the formation of a bona fide daughter cell and cell cycle completion. We propose that upon exit from quiescence in the absence of F-actin, secretory vesicles randomly reach the plasma membrane but preferentially dock and fuse where polarity cues are localized, this being sufficient to trigger polarized growth.

The 26S proteasome is responsible for the controlled proteolysis of a vast number of proteins, including crucial cell cycle regulators. Accordingly, in Saccharomyces cerevisiae, 26S proteasome function is mandatory for cell cycle progression. In budding yeast, the 26S proteasome is assembled in the nucleus, where it is localized throughout the cell cycle. We report that upon cell entry into quiescence, proteasome subunits massively relocalize from the nucleus into motile cytoplasmic structures. We further demonstrate that these structures are proteasome cytoplasmic reservoirs that are rapidly mobilized upon exit from quiescence. Therefore, we have named these previously unknown structures proteasome storage granules (PSGs). Finally, we observe conserved formation and mobilization of these PSGs in the evolutionary distant yeast Schizosaccharomyces pombe. This conservation implies a broad significance for these proteasome reserves.

Adenylate kinase (Adk1p) is a pivotal enzyme in both energetic and adenylic nucleotide metabolisms. In this paper, using a transcriptomic analysis, we show that the lack of Adk1p strongly induced expression of the PHO and ADE genes involved in phosphate utilization and AMP de novo biosynthesis respectively. Isolation and characterization of adk1 point mutants affecting PHO5 expression revealed that all these mutations also severely affected Adk1p catalytic activity, as well as PHO84 and ADE1 transcription. Furthermore, overexpression of distantly related enzymes such as human adenylate kinase or yeast UMP kinase was sufficient to restore regulation. These results demonstrate that adenylate kinase catalytic activity is critical for proper regulation of the PHO and ADE pathways. We also establish that adk1 deletion and purine limitation have similar effects on both adenylic nucleotide pool and PHO84 or ADE17 expression. Finally, we show that, in the adk1 mutant, upregulation of ADE1 depends on synthesis of the previously described effector(s) (S)AICAR ((N-succinyl)-5-aminoimidazol-4-carboxamide ribotide), while upregulation of PHO84 necessitates the Spl2p positive regulator. This work reveals that adenylic nucleotide availability is a key signal used by yeast to co-ordinate phosphate utilization and purine synthesis.

Guanylic nucleotide biosynthesis is a conserved and highly regulated process. Drugs reducing GMP synthesis affect the immunological response and mutations enabling guanylic-derivative recycling lead to severe mental retardation. While the effects of decreased GMP synthesis have been well documented, the consequences of GMP overproduction in eukaryotes are poorly understood. In this work, we selected and characterized several mutations making yeast hypoxanthine-guanine phosphoribosyltransferase insensitive to feedback inhibition by GMP. In these mutants, accumulation of guanylic nucleotides can be triggered by addition of extracellular guanine. We show that such an accumulation is highly toxic for yeast cells and results in arrest of proliferation and massive cell death. This growth defect could be partially suppressed by overexpression of Rfx1p, a transcriptional repressor of the DNA damage response pathway. Importantly, neither guanylic nucleotide toxicity nor its suppression by Rfx1p was associated with an alteration of forward mutation frequency.

When yeast cells enter into quiescence in response to nutrient limitation, the adenine deaminase Aah1p is specifically degraded via a process requiring the F-box protein Saf1p and components of the Skp1-Cullin-F-box complex. In this paper, we show that Saf1p interacts with both Aah1p and Skp1p. Interaction with Skp1p, but not with Aah1p, requires the F-box domain of Saf1p. Based on deletion and point mutations, we further demonstrate that the F-box domain of Saf1p is critical for degradation of Aah1p. We also establish that overexpression of Saf1p in proliferating cells is sufficient to trigger the degradation of Aah1p. Using this property and a two-dimensional protein gel approach, we found that Saf1p has a small number of direct targets. Finally, we isolated and characterized several point mutations in Aah1p, which increase its stability during quiescence. The majority of the mutated residues are located in two distinct exposed regions in the Aah1p three-dimensional model structure. Two hybrid experiments strongly suggest that these domains are directly involved in interaction with Saf1p. Importantly, we obtained a mutation in Aah1p that does not affect the protein interaction with Saf1p but abolishes Aah1p degradation. Because this mutated residue is an exposed lysine in the Aah1p three-dimensional model, we propose that it is likely to be a major ubiquitylation site. All together, our data strongly argue for Saf1p being a bona fide Skp1-Cullin-F-box subunit.

Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named "actin bodies." We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle.

In response to nutrient limitation, Saccharomyces cerevisiae cells enter into a non-proliferating state termed quiescence. This transition is associated with profound changes in gene expression patterns. The adenine deaminase encoding gene AAH1 is among the most precociously and tightly downregulated gene upon entry into quiescence. We show that AAH1 downregulation is not specifically due to glucose exhaustion but is a more general response to nutrient limitation. We also found that Aah1p level is tightly correlated to RAS activity indicating thus an important role for the protein kinase A pathway in this regulation process. We have isolated three deletion mutants, srb10, srb11 and saf1 (ybr280c) affecting AAH1 expression during post-diauxic growth and in early stationary phase. We show that the Srb10p cyclin-dependent kinase and its cyclin, Srb11p, regulate AAH1 expression at the transcriptional level. By contrast, Saf1p, a previously uncharacterized F-box protein, acts at a post-transcriptional level by promoting degradation of Aah1p. This post-transcriptional regulation is abolished by mutations affecting the proteasome or constant subunits of the SCF (Skp1-Cullin-F-box) complex. We propose that Saf1p targets Aah1p for proteasome-dependent degradation upon entry into quiescence. This work provides the first direct evidence for active degradation of proteins in quiescent yeast cells.

Background: Guanylic nucleotides are both macromolecules constituents and crucial regulators for a variety of cellular processes. Therefore, their intracellular concentration must be strictly controlled. Consistently both yeast and mammalian cells tightly correlate the transcription of genes encoding enzymes critical for guanylic nucleotides biosynthesis with the proliferation state of the cell population., Results: To gain insight into the molecular relationships connecting intracellular guanylic nucleotide levels and cellular proliferation, we have studied the consequences of guanylic nucleotide limitation on Saccharomyces cerevisiae cell cycle progression. We first utilized mycophenolic acid, an immunosuppressive drug that specifically inhibits inosine monophosphate dehydrogenase, the enzyme catalyzing the first committed step in de novo GMP biosynthesis. To approach this system physiologically, we next developed yeast mutants for which the intracellular guanylic nucleotide pools can be modulated through changes of growth conditions. In both the pharmacological and genetic approaches, we found that guanylic nucleotide limitation generated a mother-daughter separation defect, characterized by cells with two unseparated daughters. We then showed that this separation defect resulted from cell wall perturbations but not from impaired cytokinesis. Importantly, cells with similar separation defects were found in a wild type untreated yeast population entering quiescence upon nutrient limitation., Conclusion: Our results demonstrate that guanylic nucleotide limitation slows budding yeast cell cycle progression, with a severe pause in telophase. At the cellular level, guanylic nucleotide limitation causes the emergence of cells with two unseparated daughters. By fluorescence and electron microscopy, we demonstrate that this phenotype arises from defects in cell wall partition between mother and daughter cells. Because cells with two unseparated daughters are also observed in a wild type population entering quiescence, our results reinforce the hypothesis that guanylic nucleotide intracellular pools contribute to a signal regulating both cell proliferation and entry into quiescence.

Because some metabolic intermediates are involved in more than one pathway, crosstalk between pathways is crucial to maintaining homeostasis. AMP and histidine biosynthesis pathways are coregulated at the transcriptional level in response to adenine availability. 5'-Phosphoribosyl-4-carboxamide-5-aminoimidazole (AICAR), a metabolic intermediate at the crossroads between these two pathways, is shown here to be critical for activation of the transcriptional response in the absence of adenine. In this study, we show that both AMP and histidine pathways significantly contribute to AICAR synthesis. Furthermore, we show that upregulation of the histidine pathway clearly interferes with regulation of the AMP pathway, thus providing an explanation for the regulatory crosstalk between these pathways. Finally, we revisit the histidine auxotrophy of ade3 or ade16 ade17 mutants. Interestingly, overexpression of PMU1, encoding a potential phosphomutase, partially suppresses the histidine requirement of an ade3 ade16 ade17 triple mutant, most probably by reducing the level of AICAR in this mutant. Together our data clearly establish that AICAR is not just a metabolic intermediate but also acts as a true regulatory molecule.

Purine salvage is a complex pathway allowing a correct balance between adenylic and guanylic derivatives. In this paper, we show that GUD1 (YDL238c) encodes guanine deaminase, a catabolic enzyme producing xanthine and ammonia from guanine. Importantly, Gud1p activity was higher during post-diauxic growth, suggesting that a decrease of the guanylic nucleotide pool could be required when cells shift from proliferation to quiescence.

Phosphate is an essential nutrient that must be taken up from the growth medium through specific transporters. In Saccharomyces cerevisiae, both high and low affinity orthophosphate carriers allow this micro-organism to cope with environmental variations. Intriguingly, in this study we found a tight correlation between selenite resistance and expression of the high affinity orthophosphate carrier Pho84p. Our work further revealed that mutations in the low affinity orthophosphate carrier genes (PHO87, PHO90, and PHO91) cause deregulation of phosphate-repressed genes. Strikingly, the deregulation due to pho87Delta, pho90Delta, or pho91Delta mutations was neither correlated to impaired orthophosphate uptake capacity nor to a decrease of the intracellular orthophosphate or polyphosphate pools, as shown by (31)P NMR spectroscopy. Thus, our data clearly establish that the low affinity orthophosphate carriers affect phosphate regulation independently of intracellular orthophosphate concentration through a new signaling pathway that was found to strictly require the cyclin-dependent kinase inhibitor Pho81p. We propose that phosphate-regulated gene expression is under the control of two different regulatory signals as follows: the sensing of internal orthophosphate by a yet unidentified protein and the sensing of external orthophosphate by low affinity orthophosphate transporters; the former would be required to maintain phosphate homeostasis, and the latter would keep the cell informed on the medium phosphate richness.

No gene coding for an adenine deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that adenine deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the adenine deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A.nidulans gene was expressed in Escherichia coli and the purified enzyme shows adenine but not adenosine deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine deaminases rather than to the bacterial adenine deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal adenine deaminases and the homologous adenosine deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that adenine deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the alpha/beta barrel scaffold.

The yeast IMD2 to IMD4 and GUA1 genes, involved in GMP synthesis, are highly expressed in exponentially growing cells but are shut off when cells cease to grow upon nutrient limitation. We show for the IMD2 gene that this effect is not specific to certain carbon sources or to growth rate. Strikingly, the cis elements responsible for this nutritional response are contained within a 23-nucleotide sequence in the coding region of the IMD2 gene. Despite its very unusual location, this regulatory sequence mediates the repression of transcription initiation. From our data, we conclude that GMP synthesis is downregulated upon nutrient limitation through an active mechanism. We show that this transcriptional shutoff abolishes any possibility of the induction of IMD2, even under drastic conditions of guanylic nucleotide limitation. Taken together, these results indicate that low levels of guanylic nucleotides could be required for proper entry into stationary phase.

Guanylic nucleotides are essential cellular players, and the critical enzyme in their tightly regulated synthesis in Saccharomyces cerevisiae is encoded by the IMD2 gene. The transcription of IMD2 is subject to general repression by nutrient limitation through the cis nutrient-sensing element. It is also subject to specific feedback regulation by the end products of the guanylic nucleotide synthesis pathway. The critical cis element for this latter mechanism is the guanine response element (GRE), a TATAATA sequence which is located 202 nucleotides upstream of the transcription initiation site and which functions as the IMD2 TATA box. We show that the GRE functions in conjunction with a 52-nucleotide stretch near the transcription start site. This very unusual promoter structure ensures low, basal expression of IMD2 and the recruitment of TFIID to the GRE in response to guanylic nucleotide limitation.

Background: The purine salvage enzyme inosine 5'-monophosphate (IMP)-specific 5'-nucleotidase catalyzes degradation of IMP to inosine. Although this enzymatic activity has been purified and characterized in Saccharomyces cerevisiae, the gene encoding IMP 5'-nucleotidase had not been identified., Results: Mass spectrometry analysis of several peptides of this enzyme purified from yeast allowed identification of the corresponding gene as YOR155c, an open reading frame of unknown function, renamed ISN1. The deduced Isn1p sequence was clearly not homologous to 5'-nucleotidases from other species. However, significant similarities to Isn1p were found in proteins of unknown function from Neurospora crassa, Plasmodium falciparum and several yeast species. Knock-out of ISN1 resulted in the total loss of IMP-specific 5'-nucleotidase activity, thus confirming that the ISN1 gene indeed encodes the enzymatic activity purified from yeast. In vivo studies revealed that, when IMP is overproduced through constitutive activation of the IMP de novo synthesis pathway, ISN1 is required for excretion of inosine and hypoxanthine in the medium., Conclusion: We have identified a new yeast gene, ISN1 (YOR155c), as encoding IMP-specific 5'-nucleotidase activity. The ISN1 gene defines a new type of 5'-nucleotidase which was demonstrated to be functional in vivo.

The immunosuppressive drug mycophenolic acid (MPA) is a potent and specific inhibitor of IMP dehydrogenase, the first committed step of GMP synthesis. A screen for yeast genes affecting MPA sensitivity, when overexpressed, allowed us to identify two genes, IMD2 and TPO1, encoding a homologue of IMP dehydrogenase and a vacuolar pump, respectively. In parallel, 4787 yeast strains, each carrying an identified knock-out mutation, were tested for growth in the presence of MPA, allowing identification of 100 new genes affecting MPA resistance when disrupted. Disturbance of several cellular processes, such as ergosterol biosynthesis, vacuole biogenesis, or glycosylation impaired the natural capacity of yeast to resist MPA, although most of the highly sensitive mutants affected the transcription machinery (19 mutants). Expression of TPO1 and/or IMD2 was strongly affected in 16 such transcription mutants suggesting that low expression of these genes could contribute to MPA sensitivity. Interestingly, the spt3, spt8, and spt20 mutants behaved differently than other Spt-Ada-Gcn5-acetyltransferase (SAGA) mutants. Indeed, in these three mutants, as in previously characterized transcription elongation mutants, IMD2 expression was only affected in the presence of MPA, thus suggesting a possible role for some SAGA subunits in transcription elongation.

Mycophenolic acid (MPA), one of the most promising immunosuppressive drugs recently developed, is a potent inhibitor of IMP dehydrogenase, the first committed step toward GMP synthesis. We found that all the drug effects on yeast cells were prevented by bypassing GMP synthesis, thus confirming the high specificity of MPA. Although the primary target of MPA is clearly identified, we aimed to further understand how GTP depletion leads to growth arrest and developed a new approach based on proteome analysis combined with overexpression studies. Essential proteins down-expressed in the presence of MPA were identified by protein two-dimensional gel analysis and subsequently overexpressed in yeast. Two such proteins, Cdc37p and Sup45p, when overexpressed allowed partial relief of MPA toxicity, strongly suggesting that their lower amount after MPA treatment significantly contributed to the MPA effect. These conserved proteins involved in cell cycle progression and translation are therefore important secondary targets for MPA. Our data establish that MPA effects occur through inhibition of a unique primary target resulting in guanine nucleotides depletion, thereby affecting multiple cellular processes.