Search

We have recently identified DCT encoding dopachrome tautomerase (DCT) as the 8th gene for oculocutaneous albinism (OCA). Patients with loss of function of DCT suffer from eye hypopigmentation and retinal dystrophy. Here we investigate the eye phenotype in Dct-/- mice. We show that their retinal pigmented epithelium (RPE) is severely hypopigmented from early stages contrasting with the darker melanocytic tissues. Multimodal imaging reveals specific RPE cellular defects. Melanosomes are fewer with correct subcellular localization but disrupted melanisation. RPE cell size is globally increased and heterogeneous. P-cadherin labeling of Dct-/- newborn RPE reveals a defect in adherens junctions similar to what has been described in tyrosinase-deficient Tyrc/c embryos. The first intermediate of melanin biosynthesis, dihydroxyphenylalanine (L-Dopa), which is thought to control retinogenesis, is detected in substantial yet significantly reduced amounts in Dct-/- postnatal mouse eyecups. L-Dopa synthesis in the RPE alone remains to be evaluated during the critical period of retinogenesis. The Dct-/- mouse should prove useful in understanding the molecular regulation of retinal development and aging of the hypopigmented eye. This may guide therapeutic strategies to prevent vision deficits in patients with albinism.

We have recently identified

Significance The recent discovery of gasdermin-like proteins in fungi have brought to light that this family of pore-forming proteins controls cell death in two of the major eukaryotic kingdoms, fungi and mammals. Yet the regulation of cytotoxicity of the fungal gasdermins and their molecular pathways remain uncharacterized. Here, we describe the regulation through proteolytic cleavage of the fungal gasdermin HET-Q1 and uncover that a majority of fungal gasdermins are genomically clustered with protease-encoding genes. Some of these genes encode proteins with caspase-related domains and/or are members of a family of immune receptors in mammals and plants. Overall, this work contributes toward our understanding of the evolution of gasdermin-dependent cell death, enlightening multiple evolutionary parallels between signaling pathways in mammals and fungi.

International audience; Background The reported association of mTOR-inhibitor (mTORi) treatment with a lower incidence of cytomegalovirus (CMV) infection in kidney transplant recipients (KTR) who are CMV seropositive (R+) remains unexplained. Methods The incidence of CMV infection and T-cell profile was compared between KTRs treated with mTORis and mycophenolic acid (MPA), and in vitro mTORi effects on T-cell phenotype and functions were analyzed. Results In KTRs who were R+ and treated with MPA, both αβ and γδ T cells displayed a more dysfunctional phenotype (PD-1+, CD85j+) at day 0 of transplantation in the 16 KTRs with severe CMV infection, as compared with the 17 KTRs without or with spontaneously resolving CMV infection. In patients treated with mTORis ( n =27), the proportion of PD-1+ and CD85j+ αβ and γδ T cells decreased, when compared with patients treated with MPA ( n =44), as did the frequency and severity of CMV infections. mTORi treatment also led to higher proportions of late-differentiated and cytotoxic γδ T cells and IFN γ -producing and cytotoxic αβ T cells. In vitro , mTORis increased proliferation, viability, and CMV-induced IFN γ production of T cells and decreased PD-1 and CD85j expression in T cells, which shifted the T cells to a more efficient EOMES low Hobit high profile. In γδ T cells, the mTORi effect was related to increased TCR signaling. Conclusion Severe CMV replication is associated with a dysfunctional T-cell profile and mTORis improve T-cell fitness along with better control of CMV. A dysfunctional T-cell phenotype could serve as a new biomarker to predict post-transplantation infection and to stratify patients who should benefit from mTORi treatment. Clinical Trial registry name and registration number: Proportion of CMV Seropositive Kidney Transplant Recipients Who Will Develop a CMV Infection When Treated With an Immunosuppressive Regimen Including Everolimus and Reduced Dose of Cyclosporine Versus an Immunosuppressive Regimen With Mycophenolic Acid and Standard Dose of Cyclosporine A (EVERCMV), NCT02328963

International audience; 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.

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.

International audience; 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.

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., Some C. elegans strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Wormbase, which is supported by National Institutes of Health (NIH) grant U41HG002223. This work was supported by an AFM-Téléthon Trampoline grant (ref. 18313). RM is supported by AFM-Téléthon PhD scholarship (ref. 20450). RM stay in M. A-S. lab supported by a Short Term Scientific Mission financed by GENiE (COST Action BM1408). M.A-S and M.O are supported by the Ramón y Cajal program of the Spanish Ministerio de Economía y Competitividad, RYC-2010-06167 and RYC-2014-15551, respectively.

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.

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.

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.

Purine homeostasis is ensured through a metabolic network widely conserved from prokaryotes to humans. Purines can either be synthesizedde 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 purinede novoand recycling pathways, causes severe muscular and neuronal symptoms. In order to address the mechanisms underlying this deficiency, we establishedCaenorhabditis elegansas a metazoan model organism to study purine metabolism, while focusing on ADSL. We show that the purine biosynthesis network is functionally conserved inC. 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.

Summary 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.

Two inorganic phosphate (Pi) uptake mechanisms operate in streptophytes and chlorophytes, the two lineages of green plants. PHOSPHATE TRANSPORTER B (PTB) proteins are hypothesized to be the Na(+) /Pi symporters catalysing Pi uptake in chlorophytes, whereas PHOSPHATE TRANSPORTER 1 (PHT1) proteins are the H(+) /Pi symporters that carry out Pi uptake in angiosperms. PHT1 proteins are present in all streptophyte lineages. However, Pi uptake in streptophyte algae and marine angiosperms requires Na(+) influx, suggesting that Na(+) /Pi symporters also function in some streptophytes. We tested the hypothesis that Na(+) /Pi symporters exist in streptophytes. We identified PTB sequences in streptophyte genomes. Core PTB proteins are present at the plasma membrane of the liverwort Marchantia polymorpha. The expression of M. polymorpha core PTB proteins in the Saccharomyces cerevisiae pho2 mutant defective in high-affinity Pi transport rescues growth in low-Pi environments. Moreover, levels of core PTB mRNAs of M. polymorpha and the streptophyte alga Coleochaete nitellarum are higher in low-Pi than in Pi-replete conditions, consistent with a role in Pi uptake from the environment. We conclude that land plants inherited two Pi uptake mechanisms - mediated by the PTB and PHT1 proteins, respectively - from their streptophyte algal ancestor. Both systems operate in parallel in extant early diverging land plants.

Summary Mitochondria are essential organelles producing most of the energy required for the cell. A selective autophagic process called mitophagy removes damaged mitochondria, which is critical for proper cellular homeostasis; dysfunctional mitochondria can generate excess reactive oxygen species that can further damage the organelle as well as other cellular components. Although proper cell physiology requires the maintenance of a healthy pool of mitochondria, little is known about the mechanism underlying the recognition and selection of damaged organelles. In this study, we investigated the cellular fate of mitochondria damaged by the action of respiratory inhibitors (antimycin A, myxothiazol, KCN) that act on mitochondrial respiratory complexes III and IV, but have different effects with regard to the production of reactive oxygen species and increased levels of reduced cytochromes. Antimycin A and potassium cyanide effectively induced nonspecific autophagy, but not mitophagy, in a wild-type strain of Saccharomyces cerevisiae; however, low or no autophagic activity was measured in strains deficient for genes that encode proteins involved in mitophagy, including ATG32, ATG11 and BCK1. These results provide evidence for a major role of specific mitophagy factors in the control of a general autophagic cellular response induced by mitochondrial alteration. Moreover, increased levels of reduced cytochrome b, one of the components of the respiratory chain, could be the first signal of this induction pathway.

Cell fate and proliferation are tightly linked to the regulation of the mitochondrial energy metabolism. Hence, mitochondrial biogenesis regulation, a complex process that requires a tight coordination in the expression of the nuclear and mitochondrial genomes, has a major impact on cell fate and is of high importance. Here, we studied the molecular mechanisms involved in the regulation of mitochondrial biogenesis through a nutrient-sensing pathway, the Ras-cAMP pathway. Activation of this pathway induces a decrease in the cellular phosphate potential that alleviates the redox pressure on the mitochondrial respiratory chain. One of the cellular consequences of this modulation of cellular phosphate potential is an increase in the cellular glutathione redox state. The redox state of the glutathione disulfide-glutathione couple is a well known important indicator of the cellular redox environment, which is itself tightly linked to mitochondrial activity, mitochondria being the main cellular producer of reactive oxygen species. The master regulator of mitochondrial biogenesis in yeast (i.e. the transcriptional co-activator Hap4p) is positively regulated by the cellular glutathione redox state. Using a strain that is unable to modulate its glutathione redox state (Δglr1), we pinpoint a positive feedback loop between this redox state and the control of mitochondrial biogenesis. This is the first time that control of mitochondrial biogenesis through glutathione redox state has been shown.

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.

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the "Crabtree effect." We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into "Crabtree-positive," where the Crabtree effect occurs, and "Crabtree-negative," where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

The fission yeast cytoskeleton is completely reorganized upon quiescence entry: the microtubule (MT) cytoskeleton is drastically reshaped as quiescent cells assemble a large spindle pole body–associated MT bundle composed of stable antiparallel MTs., Cells perpetually face the decision to proliferate or to stay quiescent. Here we show that upon quiescence establishment, Schizosaccharomyces pombe cells drastically rearrange both their actin and microtubule (MT) cytoskeletons and lose their polarity. Indeed, while polarity markers are lost from cell extremities, actin patches and cables are reorganized into actin bodies, which are stable actin filament–containing structures. Astonishingly, MTs are also stabilized and rearranged into a novel antiparallel bundle associated with the spindle pole body, named Q-MT bundle. We have identified proteins involved in this process and propose a molecular model for Q-MT bundle formation. Finally and importantly, we reveal that Q-MT bundle elongation is involved in polarity reestablishment upon quiescence exit and thereby the efficient return to the proliferative state. Our work demonstrates that quiescent S. pombe cells assemble specific cytoskeleton structures that improve the swiftness of the transition back to proliferation.

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.

Summary 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.

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.

Un projet de collaboration a reuni trois equipes, aux specialites complementaires, pour travailler sur l’identification de molecules capable d’induire une activite « HPRT-like » afin de suppleer au deficit de l’hypoxanthine phosphoribosyltransferase (HPRT) responsable de la maladie de Lesch-Nyhan (universite Paris Descartes/AP–HP, CNRS UMR5095, Bordeaux et Sanofi RD le laboratoire de Bordeaux a developpe un test sur levures deficientes en HPRT et le laboratoire de Necker un test biochimique sur les fibroblastes de patients Lesch-Nyhan. Ces tests complementaires ont ensuite ete utilises pour evaluer l’interet therapeutique potentiel de plusieurs milliers de molecules issues de librairies standard de Sanofi ou provenant d’une etude theorique de criblage virtuelle. Les equipes de Sanofi a Strasbourg ont alors miniaturise le test biochimique developpe precedemment pour qu’il puisse etre effectue par un automate et un criblage a haut debit de plus de 500 000 molecules a ete realise. Une famille de molecules a ete identifiee et evaluee sur plusieurs lignees cellulaires de patients Lesch-Nyhan presentant des mutations distinctes du gene hprt . Des etudes de validation sont prevues sur des modeles de neurones issus des iPS de patients Lesch-Nyhan.

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.

Control of the glutamate time course in the synapse is crucial for excitatory transmission. This process is mainly ensured by astrocytic transporters, high expression of which is essential to compensate for their slow transport cycle. Although molecular mechanisms regulating transporter intracellular trafficking have been identified, the relationship between surface transporter dynamics and synaptic function remains unexplored. We found that GLT-1 transporters were highly mobile on rat astrocytes. Surface diffusion of GLT-1 was sensitive to neuronal and glial activities and was strongly reduced in the vicinity of glutamatergic synapses, favoring transporter retention. Notably, glutamate uncaging at synaptic sites increased GLT-1 diffusion, displacing transporters away from this compartment. Functionally, impairing GLT-1 membrane diffusion through cross-linking in vitro and in vivo slowed the kinetics of excitatory postsynaptic currents, indicative of a prolonged time course of synaptic glutamate. These data provide, to the best of our knowledge, the first evidence for a physiological role of GLT-1 surface diffusion in shaping synaptic transmission.

International audience; 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.

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.

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.

Summary 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, DNAbinding 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.

International audience; Adenosine is an endogenous molecule that regulates many physiological processes via the activation of four specific G-protein-coupled ADORA receptors. Extracellular adenosine may originate either from the hydrolysis of released ATP by the ectonucleotidases or from cellular exit via the equilibrative nucleoside transporters (SLC29A). Adenosine extracellular concentration is also regulated by its successive hydrolysis into uric acid by membrane-bound enzymes or by cell influx via the concentrative nucleoside transporters (SLC28A). All of these members constitute the adenosine signaling pathway and regulate adenosine functions. Although the roles of this pathway are quite well understood in adults, little is known regarding its functions during vertebrate embryogenesis. We have used Xenopus laevis as a model system to provide a comparative expression map of the different members of this pathway during vertebrate development. We report the characterization of the different enzymes, receptors, and nucleoside transporters in both X. laevis and X. tropicalis, and we demonstrate by phylogenetic analyses the high level of conservation of these members between amphibians and mammals. A thorough expression analysis of these members during development and in the adult frog reveals that each member displays distinct specific expression patterns. These data suggest potentially different developmental roles for these proteins and therefore for extracellular adenosine. In addition, we show that adenosine levels during amphibian embryogenesis are very low, confirming that they must be tightly controlled for normal development.

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.

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

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

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. ? 1998 John Wiley & Sons, Ltd.

The purine-cytosine permease (PCP) is an active transporter located in the plasma membrane of the yeast Saccharomyces cerevisiae This protein mediates purine (adenine, guanine, and hypoxanthine) and cytosine accumulation in the cell by using an electrochemical potential difference in proton as the energy source. Various mutant strains, with altered Kt(app) (apparent Michaelis constant of transport) of uptake for one or several bases, have already been selected. Their cloning and sequencing revealed that three of them presented substitutions in the same region of the putative sequence of the PCP: this region might correspond to the hydrophilic segment 371-377 (I-A-N-N-I-P-N). Two mutants displayed single mutations, resulting in only one amino acid residue change (N377I and N374I, respectively), and the other displayed three amino acid substitutions (I371V, I375V, and N377G). Therefore, to analyze the contribution of individual amino acid changes to the phenotype of the complex mutant, single (N377G) and double (I371V,I375V) mutants were constructed by site-directed mutagenesis. The influence of single mutations in this region was studied by measuring, for adenine, hypoxanthine, and cytosine, the uptake constants on cells and equilibrium binding parameters on plasma membrane-enriched fractions. Uptake and binding constant determinations showed that all the variations observed for the Kt(app) of uptake were correlated with variations of the binding Kd(app) for the corresponding solutes. Thus, our results emphasize the role of the two asparagine residues, located at positions 374 and 377, respectively, in the binding of the bases. In addition, the sole substitution of the 377 asparagine residue by glycine is responsible for the phenotype of the triple mutant. The effect of pH on the apparent hypoxanthine binding dissociation constant showed that the effects of N377G and N377I changes were, at least partially, due to a shift of the pKa of an ionizable amino acid residue of the unliganded permease. These two amino acid residue changes induced a shift of the pKa of this group in the unliganded, deprotonated permease about two units toward acidic pH. This result suggests that the 371-377 segment might play a key role in the proper three-dimensional structure of the active purine-cytosine permease.

International audience; 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.

Cell fate and proliferation are tightly linked to the regulation of the mitochondrial energy metabolism. Hence, mitochondrial biogenesis regulation, a complex process that requires a tight coordination in the expression of the nuclear and mitochondrial genomes, has a major impact on cell fate and is of high importance. Here, we studied the molecular mechanisms involved in the regulation of mitochondrial biogenesis through a nutrient-sensing pathway, the Ras-cAMP pathway. Activation of this pathway induces a decrease in the cellular phosphate potential that alleviates the redox pressure on the mitochondrial respiratory chain. One of the cellular consequences of this modulation of cellular phosphate potential is an increase in the cellular glutathione redox state. The redox state of the glutathione disulfide-glutathione couple is a well known important indicator of the cellular redox environment, which is itself tightly linked to mitochondrial activity, mitochondria being the main cellular producer of reactive oxygen species. The master regulator of mitochondrial biogenesis in yeast (i.e. the transcriptional co-activator Hap4p) is positively regulated by the cellular glutathione redox state. Using a strain that is unable to modulate its glutathione redox state (Δglr1), we pinpoint a positive feedback loop between this redox state and the control of mitochondrial biogenesis. This is the first time that control of mitochondrial biogenesis through glutathione redox state has been shown.

International audience; 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.

Dynamin-related GTPase proteins (DRPs) are main players in membrane remodelling. Conserved DRPs called mitofusins (Mfn1/Mfn2/Fzo1) mediate the fusion of mitochondrial outer membranes (OM). OM fusion depends on self-assembly and GTPase activity of mitofusins as well as on two other proteins, Ugo1 and Mdm30. Here, we define distinct steps of the OM fusion cycle using in vitro and in vivo approaches. We demonstrate that yeast Fzo1 assembles into homo-dimers, depending on Ugo1 and on GTP binding to Fzo1. Fzo1 homo-dimers further associate upon formation of mitochondrial contacts, allowing membrane tethering. Subsequent GTP hydrolysis is required for Fzo1 ubiquitylation by the F-box protein Mdm30. Finally, Mdm30-dependent degradation of Fzo1 completes Fzo1 function in OM fusion. Our results thus unravel functions of Ugo1 and Mdm30 at distinct steps during OM fusion and suggest that protein clearance confers a non-cycling mechanism to mitofusins, which is distinct from other cellular membrane fusion events.