Trafficking of Siderophore Transporters in Saccharomyces cerevisiae and Intracellular Fate of Ferrioxamine B Conjugates

We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1-mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB-nitrobenz-2-oxa-1,3-diazole, used as a Sit1-dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady-state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process

Structure and function of the vacuolar Ccc1/VIT1 family of iron transporters and its regulation in fungi

Iron is an essential micronutrient for most living beings since it participates as a redox active cofactor in many biological processes including cellular respiration, lipid biosynthesis, DNA replication and repair, and ribosome biogenesis and recycling. However, when present in excess, iron can participate in Fenton reactions and generate reactive oxygen species that damage cells at the level of proteins, lipids and nucleic acids. Organisms have developed different molecular strategies to protect themselves against the harmful effects of high concentrations of iron. In the case of fungi and plants, detoxification mainly occurs by importing cytosolic iron into the vacuole through the Ccc1/VIT1 iron transporter. New sequenced genomes and bioinformatic tools are facilitating the functional characterization, evolution and ecological relevance of metabolic pathways and homeostatic networks across the Tree of Life. Sequence analysis shows that Ccc1/VIT1 homologs are widely distributed among organisms with the exception of animals. The recent elucidation of the crystal structure of a Ccc1/VIT1 plant ortholog has enabled the identification of both conserved and species-specific motifs required for its metal transport mechanism. Moreover, recent studies in the yeast Saccharomyces cerevisiae have also revealed that multiple transcription factors including Yap5 and Msn2/Msn4 contribute to the expression of CCC1 in high-iron conditions. Interestingly, Malaysian S. cerevisiae strains express a partially functional Ccc1 protein that renders them sensitive to iron. Different regulatory mechanisms have been described for non-Saccharomycetaceae Ccc1 homologs. The characterization of Ccc1/VIT1 proteins is of high interest in the development of biofortified crops and the protection against microbial-derived diseases

Etude du stockage du fer chez la levure Saccharomyces cerevisiae

dissertn: Diss. Doct

Influence de la température d'irradiation et de l'intensité de dose sur la radiolyse d'amides: rendements en déshydrodimères en phases liquide et solide

dissertn: Diss. Doct

Iron uptake by the yeast Saccharomyces cerevisiae: involvement of a reduction step.

Among several parameters affecting the rate and amount of iron uptake by Saccharomyces cerevisiae, the oxidation state of iron appeared to be determinant. Iron presented as Fe(II) was taken up faster than Fe(III) and the kinetic parameters were different. Iron was taken up by the cells from different ferric chelates, at rates that did not depend on their stability constants, and uptake was strongly inhibited by an iron(II)-trapping reagent like ferrozine. Iron was physiologically reduced by a transplasmamembrane redox system, which was induced in iron-deficient conditions. We propose that iron must be reduced to be taken up by the cells in the same way as other divalent cations

Comparison of the effects of auranofin, heavy metals and retinoids on protein kinase C in vitro and on a protein kinase C mediated response in macrophages.

The effect of auranofin (AF), retinoic acid (RA), and three heavy metals reacting with thiol groups (Hg, Cd, Pb) has been compared on a PKC mediated response of intact macrophages (i.e. plasminogen activator (PA) induction) and on purified PKC activity. AF, cadmium chloride, and lead nitrate directly inhibit PKC and hence prevent the induction of PA activity in macrophages stimulated with PMA. In vitro, and in absence of chelators, mercuric chloride is also a potent inhibitor of PKC. However, at the cellular level, the PKC mediated response (PA induction) was not inhibited by non-cytotoxic concentrations of mercury possibly due to interference of the metal with additional cellular mechanisms such as calcium mobilisation. Direct inhibition of PKC is probably not the mechanism by which retinoids block the activation of macrophages

Induction of macrophage plasminogen activator by asbestos is independent of PKC activation.

This study was undertaken to assess whether plasminogen activator (PA) induction in macrophages exposed to chrysotile fibers is mediated by protein kinase C (PKC) activation. In PKC depleted J774 cells, PA induction could be elicited by chrysotile whereas, as expected, the response to phorbol myristate acetate (PMA) was abolished. The effect of PMA and chrysotile on the distribution of PKC activity in the J774 cell line was also compared by measuring the enzyme catalytic activity and phorbol dibutyrate (PDBu) binding sites. No redistribution of PKC was observed after stimulation with PA inducing doses of chrysotile, whereas a clear translocation was observed with PMA. It is concluded that the mechanism of PA induction by chrysotile in this macrophage-like cell line is independent of PKC activation

Purification et caract risation de la ferritine de levure (Saccharomyces Cerevisiae)

SIGLEBSE B224586X / UCL - Université Catholique de LouvainBEBelgiu