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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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Sit1-dependent use of FOB-NBD by the cells.Wild-type (WT) or sit1Δ cells were plated on YPD-agar medium supplemented with 1 mm BPS (to prevent reductive iron uptake). Sterile filter paper disks containing siderophores (5 μL of 10 μm CG, FOB or FOB-NBD) were placed on the surface of the agar. Plates were incubated for 2 days at 30°C and photographed. Growing cells appeared as a white halo around the filter papers.
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fig07: Sit1-dependent use of FOB-NBD by the cells.Wild-type (WT) or sit1Δ cells were plated on YPD-agar medium supplemented with 1 mm BPS (to prevent reductive iron uptake). Sterile filter paper disks containing siderophores (5 μL of 10 μm CG, FOB or FOB-NBD) were placed on the surface of the agar. Plates were incubated for 2 days at 30°C and photographed. Growing cells appeared as a white halo around the filter papers.

Mentions: We investigated the intracellular fate of internalized FOB in S. cerevisiae, using the NBD fluorescent derivative of FOB (36). We first checked that the cells used the fluorescent derivative of FOB in the same way as native FOB. Desferrioxamine (DFOB)-NBD (7-nitrobenz-2-oxa-1,3-diazole-(D)FOB) (1 μm) fully restored the growth of a fet3Δfet4Δ strain cultured on minimal (YNB) medium, as did DFOB (1 μm). After 2 days of incubation in liquid YNB–glucose medium, the fet3Δfet4Δ strain had a cell density only 5% that of the wild-type strain, and normal growth was entirely restored when 1 μm DFOB-NBD or DFOB was added to the medium (data not shown). Thus, DFOB-NBD makes iron present in the medium available to cells deficient in reductive iron uptake. Moreover, both FOB and FOB-NBD, unlike CG, restored the growth of a wild-type strain, but not of a sit1Δ strain plated on complete agar medium containing a large excess (1 mm) of bathophenanthroline disulfonic acid (BPS) (which prevents reductive iron uptake by the cells; Figure 7). Thus, FOB-NBD is used as an iron source in a Sit1-dependent manner, as is native FOB.


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)

Sit1-dependent use of FOB-NBD by the cells.Wild-type (WT) or sit1Δ cells were plated on YPD-agar medium supplemented with 1 mm BPS (to prevent reductive iron uptake). Sterile filter paper disks containing siderophores (5 μL of 10 μm CG, FOB or FOB-NBD) were placed on the surface of the agar. Plates were incubated for 2 days at 30°C and photographed. Growing cells appeared as a white halo around the filter papers.
© Copyright Policy
Related In: Results  -  Collection

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

fig07: Sit1-dependent use of FOB-NBD by the cells.Wild-type (WT) or sit1Δ cells were plated on YPD-agar medium supplemented with 1 mm BPS (to prevent reductive iron uptake). Sterile filter paper disks containing siderophores (5 μL of 10 μm CG, FOB or FOB-NBD) were placed on the surface of the agar. Plates were incubated for 2 days at 30°C and photographed. Growing cells appeared as a white halo around the filter papers.
Mentions: We investigated the intracellular fate of internalized FOB in S. cerevisiae, using the NBD fluorescent derivative of FOB (36). We first checked that the cells used the fluorescent derivative of FOB in the same way as native FOB. Desferrioxamine (DFOB)-NBD (7-nitrobenz-2-oxa-1,3-diazole-(D)FOB) (1 μm) fully restored the growth of a fet3Δfet4Δ strain cultured on minimal (YNB) medium, as did DFOB (1 μm). After 2 days of incubation in liquid YNB–glucose medium, the fet3Δfet4Δ strain had a cell density only 5% that of the wild-type strain, and normal growth was entirely restored when 1 μm DFOB-NBD or DFOB was added to the medium (data not shown). Thus, DFOB-NBD makes iron present in the medium available to cells deficient in reductive iron uptake. Moreover, both FOB and FOB-NBD, unlike CG, restored the growth of a wild-type strain, but not of a sit1Δ strain plated on complete agar medium containing a large excess (1 mm) of bathophenanthroline disulfonic acid (BPS) (which prevents reductive iron uptake by the cells; Figure 7). Thus, FOB-NBD is used as an iron source in a Sit1-dependent manner, as is native FOB.

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