Benoît Pinson

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.

Low Affinity Orthophosphate Carriers Regulate PHO Gene Expression Independently of Internal Orthophosphate Concentration in Saccharomyces cerevisiae

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 pho87Δ, pho90Δ, or pho91Δ mutations was neither correlated to impaired orthophosphate uptake capacity nor to a decrease of the intracellular orthophosphate or polyphosphate pools, as shown by 31P 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.

Co‐regulation of yeast purine and phosphate pathways in response to adenylic nucleotide variations

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.

Post-translational fate of CAN1 permease of Saccharomyces cerevisiae

To study the post‐translational fate of arginine permease (Can1p), the gene coding for this transport protein was placed behind a constitutive promoter of plasma membrane ATPase (PMA1) and furnished with a Myc tag. In exponential‐phase cells the amount of Can1p is constant, although turnover can be demonstrated. A rapid decrease in transport activity during the early stationary phase is paralleled by a corresponding net degradation of the protein. The amount of Can1p present in exponential cells grown on various nitrogen sources is the same, except in arginine‐grown cells, in which the amount of the protein is markedly lower. This occurs solely when arginine serves as nitrogen source but not as an immediate consequence of, for example, arginine addition to cells growing on other nitrogen sources. It was demonstrated that Can1p is phosphorylated. Since Can1p expression under the PMA1 promoter is glucose‐dependent, the amount of the permease expressed in high‐glucose‐grown cells is higher than in low‐glucose‐grown ones. Only a part of the Can1p overexpressed in high‐glucose‐grown cells is phosphorylated, while in low‐glucose‐grown cells the phosphorylated form probably represents the majority of Can1p. The permease phosphorylation or dephosphorylation is not related to transinhibition.

Characterization of the Saccharomyces cerevisiae cytosine transporter using energizable plasma membrane vesicles.

The purine-cytosine permease is a carrier localized in the plasma membrane of the yeast Saccharomyces cerevisiae. The energetics of cytosine transport catalyzed by this permease has been studied in an artificial system obtained by fusion between proteoliposomes containing beef heart cytochromec oxidase and plasma membrane-enriched fractions of aS. cerevisiae strain overexpressing the permease. Upon addition of an energy donor, a proton-motive force (inside alkaline and negative) is created in this system and promotes cytosine accumulation. By using different phospholipids, it is shown that cytosine uptake is dependent on the phospholipids surrounding the carrier. It was demonstrated that the purine-cytosine permease is able to catalyze a secondary active transport of cytosine. By using nigericin and valinomycin, the ΔpH component of the proton-motive force is shown to be the only force driving nucleobase accumulation. Moreover, transport measurements done at two pH values have shown that alkalinization of intravesicular pH leads to a significant increase in cytosine uptake rate. Finally, no specific role of K+ ions on cytosine transport could be demonstrated in this system.

Disruption of Nucleotide Homeostasis by the Antiproliferative Drug 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside Monophosphate (AICAR)

Background: AICAR is a potent anti-proliferative compound, but the basis of its cytotoxicity is poorly understood. Results: AICAR affects NTP homeostasis in a carbon source-dependent way, in both yeast and human cells. Conclusion: AICAR balance with nucleotides triphosphate is critical for its in vivo effects. Significance: AICAR is significantly more cytotoxic on glucose and thus potentially targets cells prone to Warburg effect. 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.

Adenylosuccinate Lyase activity in the Purine recycling pathway is essential for developmental timing, germline maintenance and muscle integrity in C. elegans.

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 the regulation of this biosynthesis network in metazoans. In humans, several diseases are linked to purine biosynthesis deficiencies through yet poorly understood etiologies. Particularly, the deficiency in Adenylosuccinate Lyase (ADSL), one 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 is required for developmental timing and germline stem cell maintenance, and muscle integrity. Our results allow to ascribe developmental and tissue specific phenotypes to separable steps of the purine metabolic network in an animal model. Particularly, the muscle, germline and developmental defects are linked specifically to the ADSL role in the purine recycling pathway.