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Trafficking of siderophore transporters in Saccharomyces cerevisiae and intracellular fate of ferrioxamine B conjugates.

Froissard M, Belgareh-Touzé N, Dias M, Buisson N, Camadro JM, Haguenauer-Tsapis R, Lesuisse E - Traffic (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Trafic intracellulaire des protéines dans la levure, Département de biologie Cellulaire, Institut Jacques Monod, Unité Mixte de Recherche 7592 CNRS-Universités Paris 6 et 7, France.

ABSTRACT
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.

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Related in: MedlinePlus

Synthesis of conjugate AF-DFOB. Reagents and conditions: (i) AF, pentafluorophenol; DCC, Dicyclohexylcarbodiimide; THF, tetrafuran and (ii) desferal, 2, Et3N, THF, 50°C.
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fig01: Synthesis of conjugate AF-DFOB. Reagents and conditions: (i) AF, pentafluorophenol; DCC, Dicyclohexylcarbodiimide; THF, tetrafuran and (ii) desferal, 2, Et3N, THF, 50°C.

Mentions: We also studied the intracellular fate of the siderophore FOB in S. cerevisiae, using siderophore analogs. We tried to develop a simple assay for monitoring siderophore fate, using canonical FOB or FOB covalently coupled to the fluorescent moiety nitrobenz-2-oxa-1,3-diazole (NBD). We also investigated the fate of FOB covalently coupled to acifluorfen (AF) (Figure 1), a diphenylether that strongly inhibits protoporphyrinogen oxidase, an essential mitochondrial enzyme (27), to determine whether it would be feasible to use AF coupled to siderophores and taken up by yeast cells as a means of developing new antifungal compounds.


Trafficking of siderophore transporters in Saccharomyces cerevisiae and intracellular fate of ferrioxamine B conjugates.

Froissard M, Belgareh-Touzé N, Dias M, Buisson N, Camadro JM, Haguenauer-Tsapis R, Lesuisse E - Traffic (2007)

Synthesis of conjugate AF-DFOB. Reagents and conditions: (i) AF, pentafluorophenol; DCC, Dicyclohexylcarbodiimide; THF, tetrafuran and (ii) desferal, 2, Et3N, THF, 50°C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171038&req=5

fig01: Synthesis of conjugate AF-DFOB. Reagents and conditions: (i) AF, pentafluorophenol; DCC, Dicyclohexylcarbodiimide; THF, tetrafuran and (ii) desferal, 2, Et3N, THF, 50°C.
Mentions: We also studied the intracellular fate of the siderophore FOB in S. cerevisiae, using siderophore analogs. We tried to develop a simple assay for monitoring siderophore fate, using canonical FOB or FOB covalently coupled to the fluorescent moiety nitrobenz-2-oxa-1,3-diazole (NBD). We also investigated the fate of FOB covalently coupled to acifluorfen (AF) (Figure 1), a diphenylether that strongly inhibits protoporphyrinogen oxidase, an essential mitochondrial enzyme (27), to determine whether it would be feasible to use AF coupled to siderophores and taken up by yeast cells as a means of developing new antifungal compounds.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Trafic intracellulaire des protéines dans la levure, Département de biologie Cellulaire, Institut Jacques Monod, Unité Mixte de Recherche 7592 CNRS-Universités Paris 6 et 7, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus