Bertrand Daignan-Fornier

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

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.

Reversible cytoplasmic localization of the proteasome in quiescent yeast cells

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.

Metabolic status rather than cell cycle signals control quiescence entry and exit

The use of new candidate markers for yeast quiescence reveals that quiescence entry and exit primarily rely on cellular metabolic status and can be uncoupled from the cell cycle.

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.

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

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

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 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. Copyright

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.

Reactive oxygen species mediated regulation of mitochondrial biogenesis in the yeast Saccharomyces cerevisiae

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.

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.