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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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Synthesis and location of chromosomal GFP-tagged siderophore transporters.A) Cells expressing chromosomal GFP-tagged siderophore transporters were cultured to midexponential growth phase in complete medium (YPD) or in complete medium supplemented with 200 μm BPS for 4 h (YPD + BPS). Cells were then examined for GFP fluorescence and with Nomarski optics. Arrows indicate round structures possibly corresponding to endosomes. B) Total protein extracts were prepared and analysed by Western blotting for GFP [GFP-tagged transporter (T-GFP) and free vacuolar GFP] and for Gas1p, as a loading control. C) We determined FOB uptake by WT, sit1Δ and SIT1-GFP cells cultured to midexponential growth phase in complete medium supplemented with 200 μm BPS, 100 μm iron citrate (Fe) or 100 μm FOB (means ± standard error of the mean from three experiments). WT, wild type.
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fig02: Synthesis and location of chromosomal GFP-tagged siderophore transporters.A) Cells expressing chromosomal GFP-tagged siderophore transporters were cultured to midexponential growth phase in complete medium (YPD) or in complete medium supplemented with 200 μm BPS for 4 h (YPD + BPS). Cells were then examined for GFP fluorescence and with Nomarski optics. Arrows indicate round structures possibly corresponding to endosomes. B) Total protein extracts were prepared and analysed by Western blotting for GFP [GFP-tagged transporter (T-GFP) and free vacuolar GFP] and for Gas1p, as a loading control. C) We determined FOB uptake by WT, sit1Δ and SIT1-GFP cells cultured to midexponential growth phase in complete medium supplemented with 200 μm BPS, 100 μm iron citrate (Fe) or 100 μm FOB (means ± standard error of the mean from three experiments). WT, wild type.

Mentions: We checked whether the four siderophore transporters of S. cerevisiae(Arn1, Enb1, Sit1 and Taf1) displayed similar intracellular trafficking – sorting from the endosomes to plasma membrane triggered by a specific substrate. We first focused on the relative abundance of the various siderophore transporters and their subcellular distribution and trafficking as a function of growth conditions. We used GFP-tagged versions of the siderophore transporters as an experimental tool. The GFP tag was added to the C-terminus of the siderophore transporters, as previously reported for many plasma membrane transporters with a cytoplasm-oriented C-terminus (28,29). Cells producing chromosomal encoded GFP fusion proteins rather than the original siderophore transporters were used to evaluate the abundance and location of each transporter. We checked that the GFP fusion proteins were active by measuring the rate of iron uptake from FOB, FCH, ENB and TAF by cells expressing Sit1-GFP, Arn1-GFP, Enb1-GFP or Taf1-GFP and comparing these rates to those of cells with unmodified transporters (Figure 2 and S1). We also monitored the GFP-tagged proteins (and vacuolar free GFP) microscopically and by Western blotting (Figure 2). Taf1 is highly specific for TAF, but the Vmax of TAF transport is lower than that of other siderophores transported by Sit1 and Arn1 (30), probably because the concentration of Taf1 is lower than that of other siderophore transporters. Accordingly, the Taf1-GFP fusion protein was not detected by microscopy under any of the growth conditions tested (not shown). The Sit1-GFP, Arn1-GFP and Enb1-GFP transporters were detected in cells grown in both iron-rich (YPD) and iron-deficient (YPD + BPS) conditions (Figure 2A), although they were more abundant under iron-deficient growth conditions, as shown by fluorescence and western immunoblotting (Figure 2B).


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 and location of chromosomal GFP-tagged siderophore transporters.A) Cells expressing chromosomal GFP-tagged siderophore transporters were cultured to midexponential growth phase in complete medium (YPD) or in complete medium supplemented with 200 μm BPS for 4 h (YPD + BPS). Cells were then examined for GFP fluorescence and with Nomarski optics. Arrows indicate round structures possibly corresponding to endosomes. B) Total protein extracts were prepared and analysed by Western blotting for GFP [GFP-tagged transporter (T-GFP) and free vacuolar GFP] and for Gas1p, as a loading control. C) We determined FOB uptake by WT, sit1Δ and SIT1-GFP cells cultured to midexponential growth phase in complete medium supplemented with 200 μm BPS, 100 μm iron citrate (Fe) or 100 μm FOB (means ± standard error of the mean from three experiments). WT, wild type.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171038&req=5

fig02: Synthesis and location of chromosomal GFP-tagged siderophore transporters.A) Cells expressing chromosomal GFP-tagged siderophore transporters were cultured to midexponential growth phase in complete medium (YPD) or in complete medium supplemented with 200 μm BPS for 4 h (YPD + BPS). Cells were then examined for GFP fluorescence and with Nomarski optics. Arrows indicate round structures possibly corresponding to endosomes. B) Total protein extracts were prepared and analysed by Western blotting for GFP [GFP-tagged transporter (T-GFP) and free vacuolar GFP] and for Gas1p, as a loading control. C) We determined FOB uptake by WT, sit1Δ and SIT1-GFP cells cultured to midexponential growth phase in complete medium supplemented with 200 μm BPS, 100 μm iron citrate (Fe) or 100 μm FOB (means ± standard error of the mean from three experiments). WT, wild type.
Mentions: We checked whether the four siderophore transporters of S. cerevisiae(Arn1, Enb1, Sit1 and Taf1) displayed similar intracellular trafficking – sorting from the endosomes to plasma membrane triggered by a specific substrate. We first focused on the relative abundance of the various siderophore transporters and their subcellular distribution and trafficking as a function of growth conditions. We used GFP-tagged versions of the siderophore transporters as an experimental tool. The GFP tag was added to the C-terminus of the siderophore transporters, as previously reported for many plasma membrane transporters with a cytoplasm-oriented C-terminus (28,29). Cells producing chromosomal encoded GFP fusion proteins rather than the original siderophore transporters were used to evaluate the abundance and location of each transporter. We checked that the GFP fusion proteins were active by measuring the rate of iron uptake from FOB, FCH, ENB and TAF by cells expressing Sit1-GFP, Arn1-GFP, Enb1-GFP or Taf1-GFP and comparing these rates to those of cells with unmodified transporters (Figure 2 and S1). We also monitored the GFP-tagged proteins (and vacuolar free GFP) microscopically and by Western blotting (Figure 2). Taf1 is highly specific for TAF, but the Vmax of TAF transport is lower than that of other siderophores transported by Sit1 and Arn1 (30), probably because the concentration of Taf1 is lower than that of other siderophore transporters. Accordingly, the Taf1-GFP fusion protein was not detected by microscopy under any of the growth conditions tested (not shown). The Sit1-GFP, Arn1-GFP and Enb1-GFP transporters were detected in cells grown in both iron-rich (YPD) and iron-deficient (YPD + BPS) conditions (Figure 2A), although they were more abundant under iron-deficient growth conditions, as shown by fluorescence and western immunoblotting (Figure 2B).

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