A Viable Hypomorphic Allele of the Essential IMP3 Gene Reveals Novel Protein Functions in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein complex required for processing of small ribosomal subunit RNA precursors. Mutation of the IMP3 termination codon to a sense codon resulted in a viable mutant allele producing a C-terminal elongated form of the Imp3 protein. A strain expressing the mutant allele displayed ribosome biogenesis defects equivalent to IMP3 depletion. This hypomorphic allele represented a unique opportunity to investigate and better understand the Imp3p functions. We demonstrated that the +1 frameshifting was increased in the mutant strain. Further characterizations revealed involvement of the Imp3 protein in DNA repair and telomere length control, pointing to a functional relationship between both pathways and ribosome biogenesis

Etude fonctionnelle des protéines Sup35 et Imp3 chez la levure Saccharomyces cerevisiae

Le domaine C-terminal très conservé de la protéine Sup35, impliquée dans la terminaison de la traduction, possède un site potentiel de phosphorylation par la PKA au niveau de la Thréonine341. Nous avons recherché si ce résidu était phosphorylé in vivo et s il était impliqué dans la régulation fonctionnelle de la protéine. Dans les conditions testées, aucune phosphorylation de Sup35p n a pu être mise en évidence in vivo mais nous avons montré que le résidu T341 était critique pour la fonctionnalité de la protéine et pourrait être impliqué dans des interactions fonctionnelles entre les domaines N et C terminaux. Le domaine N de Sup35p est responsable du phénotype prion [PSI+]. Jusqu à présent, les seules mutations caractérisées influençant les propriétés prion de cette protéine ont été localisées dans ce domaine N-terminal. Nous avons identifié une mutation dans le domaine C-terminal qui modifie les capacités d agrégation de la protéine. Cette observation apporte de nouveaux éléments pour la compréhension du mécanisme de conversion de Sup35p vers un état agrégé. IMP3 est un gène essentiel codant une protéine impliquée dans la biogenèse des ribosomes. Nous avons construit une souche capable d exprimer de façon endogène un allèle mutant hypomorphe du gène IMP3. Cette souche présente d importants défauts de biogenèse de la petite sous unité 40S du ribosome. Nous avons montré que la fidélité de la traduction est affectée dans cette souche : l efficacité de décalage du cadre lecture en +1 est augmentée. Nos expériences montrent que cette protéine pourrait être également impliquée dans la réparation de l ADN et le contrôle de la taille des télomères.All Sup35 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. We investigated whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein. In the conditions we tested, no phosphorylation of the Sup35 protein in vivo was detected. However our results point to a new critical residue involved in the translation termination activity of Sup35p and in functional interaction between the N- and C-domains of the protein. The N-terminal domain of Sup35p is required for prion propagation, driving the switch from the soluble, functional [psi-] state to the insoluble [PSI+] prion state. To date, all the critical elements for prion induction and propagation have been mapped to the N domain of the protein. Here we report for the first time a mutation in the C-terminal domain of Sup35p which alters the aggregation properties of Sup35p. This observation has important consequence for understanding the mechanism of prion conversion. The essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein required for processing of small subunit rRNA precursors. We constructed and analysed a mutant of the IMP3 gene able to sustain cell growth. A strain expressing this hypomorphic allele displayed ribosome biogenesis defects characteristic of a depletion in Imp3p. We demonstrated the +1 frameshifting was increased in the mutant strain. Our further characterization revealed involvement of the Imp3 protein in DNA repair and telomere length control, two pathways that are not directly related to ribosome biogenesis.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Electrogeneration of a Hydrophilic Cross-Linked Polypyrrole Film for Enzyme Electrode Fabrication. Application to the Amperometric Detection of Glucose

International audienc

New electropolymerizable amphiphilic viologens for the immobilization and electrical wiring of a nitrate reductase

International audienc

Electropolymerized biotinylated poly (pyrrole–viologen) film as platform for the development of reagentless impedimetric immunosensors

International audienc

ZnO nanoparticles as down-shifting material

8th-10th May 2014International audienceMost of the existing solar conversion plants are equipped with Si based PV cells. This technology will most certainly prevail for the coming decades. Therefore, any strategy that can help to increase the conversion efficiency, even slightly, is welcome; and all the more if it is cost-effective. One such possibility is to use a material that could convert the high energy radiation from the sun spectrum, namely UV and blue light, which are otherwise inefficiently absorbed by the Si based PV cells, and re-emit them at lower energy for which the conversion efficiency of the Si cell is optimum. The so-called “down-shifting” strategy belongs to the “add-on” technology. In technology, the aim is to fabricate a thin layer of the down-shifting material that could be added on top of existing cells at low cost. The criteria to fulfill are to design a material with as large a Stoke shift (discrepancy between absorption and emission energies) as possible with high luminescence external quantum efficiency (EQE). Furthermore, the material has to be environmental-friendly and cheap. Several attempts have been made to design such a material. CdS and CdSe nanoparticles embedded in polymers or silica have proved to be efficient but not necessarily cheap and environmental-friendly. Si nanoparticles in silica have been an alternative to chalcogenides. At the INL (institute of nanotechnology of Lyon), we develop down-shifting materials based on ZnO nanoparticles. ZnO is a low cost abundant and non-toxic material. It naturally absorbs the blue and UV radiations thanks to a band gap of about 3.37 eV at room temperature. ZnO can also emit visible light, from yellow to red, depending on the nature of the crystalline defects involved in the process. Even though the nature of these defects is still partially debated, it is established that reducing the size down to the nanoscale enhances their presence and efficiency. Therefore, we base our strategy on ZnO nanoparticles dispersed and embedded in a polymer matrix. In the present work, we will examine the efficiency of ZnO nanoparticles as down-shifting material. We will address the prominent issue of optimizing the EQE, in conjunction with the nano and mesotruscture. We will show how the chemical synthesis process affects the EQE and how we can use it to reach EQE larger than 20%. The possibility to disperse and embed the particles and polymers (PMMA for instance) will be discussed

A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability.

BACKGROUND: When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein. RESULTS: We detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein. CONCLUSION: Our results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast