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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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Siderophore-induced relocation of Sit1-GFP.The Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to midexponential growth phase in raffinose-containing medium. Sit1-GFP synthesis was induced by incubation with galactose for 60 min and was stopped by adding glucose and incubating for 15 min. The sorting of Sit1-GFP from the endosomes to the plasma membrane was assessed A) by fluorescence microscopy and B) by measuring FOB uptake after incubation with the indicated compound for 30 min. For uptake experiments, the cells were rapidly washed with water and filtered before resuspension in minimal glucose medium containing 1 μm55Fe-FOB. The iron content of the cells was determined after 10 min of incubation at 30°C. Uptake values (B) are expressed as means ± standard error of the mean for three experiments.
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fig05: Siderophore-induced relocation of Sit1-GFP.The Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to midexponential growth phase in raffinose-containing medium. Sit1-GFP synthesis was induced by incubation with galactose for 60 min and was stopped by adding glucose and incubating for 15 min. The sorting of Sit1-GFP from the endosomes to the plasma membrane was assessed A) by fluorescence microscopy and B) by measuring FOB uptake after incubation with the indicated compound for 30 min. For uptake experiments, the cells were rapidly washed with water and filtered before resuspension in minimal glucose medium containing 1 μm55Fe-FOB. The iron content of the cells was determined after 10 min of incubation at 30°C. Uptake values (B) are expressed as means ± standard error of the mean for three experiments.

Mentions: Thus, Sit1-GFP was mainly sorted to the endosomal/vacuolar pathway when synthesized in the absence of substrate, and mainly targeted to the plasma membrane when synthesized in the presence of substrate. We then checked whether Sit1-GFP initially sorted to internal compartments could subsequently be targeted to the plasma membrane after the addition of FOB or other siderophores. Experiments were performed on sit1Δ cells transformed with the pGAL-SIT1-GFP construct and grown overnight on a raffinose-based medium. The cells were incubated with galactose for 60 min for induction. Glucose was added and incubated with the medium for 10–15 min to stop transporter synthesis and a siderophore was then added. The location of the siderophore transporter was determined by assessing fluorescence 30 min after addition of the siderophore, whereas the rate of siderophore transport by the washed cells was measured in parallel experiments. Observations of GFP fluorescence showed that Sit1 was sorted to endosomes and vacuoles after the transient expression of SIT1-GFP(Figure 4A, left panel and Figure 5A, panel 1), but rapid, massive translocation to the plasma membrane was observed if the cells were briefly exposed to FOB, the principal substrate of Sit1 (Figure 5A, panels 2 and 3). Plasma membrane sorting from internal compartments was also observed when protein synthesis was inhibited by incubation with cycloheximide for 5 or 15 min before the addition of external FOB. Thus, the external substrate clearly alters the fate of presynthesized Sit1 (Figure S4). The movement of Sit1 to the plasma membrane was accompanied by an increase in the ability of cells to take up FOB (Figure 5B). The gallium analog of FOB [Ga-desferrioxamine (DFOB)] also promoted the movement of Sit1 to the plasma membrane (Figure 5A, panels 4 and 5), although less effectively than FOB itself. The presence of a strong GFP signal in the vacuole lumen indicated that a large proportion of the Sit1 present reached the vacuole for degradation. This effect was observed to a much lesser extent when FOB was the inducer (Figure 5A, panels 2 and 3). Thus, Ga-DFOB was also less effective than FOB at promoting FOB uptake by the washed cells (Figure 5B). Exposure of the cells to two other substrates of Sit1 – FCH and FC – also resulted in the rapid translocation of the protein to the plasma membrane (Figure 5A, panels 6 and 7). Remarkably, CG, which is not a substrate of Sit1 (see below), had the same effect (Figure 5A, panel 8), whereas ENB and TAF had no effect on the distribution of Sit1 (Figure 5A, panels 9 and 10). Other sources of iron, such as ferric citrate and hemin, were also unable to promote the plasma membrane targeting of Sit1 (Figure 5A, panels 11 and 12). The uptake of FOB by the cells was increased in all cases by prior incubation of the cells with a siderophore, promoting the movement of Sit1 to the plasma membrane (Figure 5B). Thus, FOB, FC, FCH and CG all caused Sit1 to move rapidly from internal compartments to the plasma membrane, and this effect occurred at all siderophore concentrations tested (within the range 1–100 μm). Thus, brief exposure of the cells to a siderophore (CG) that is not a substrate of a given transport system (Sit1) may induce this transport system by promoting plasma membrane sorting. This mechanism may have an important adaptive function for cells.


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)

Siderophore-induced relocation of Sit1-GFP.The Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to midexponential growth phase in raffinose-containing medium. Sit1-GFP synthesis was induced by incubation with galactose for 60 min and was stopped by adding glucose and incubating for 15 min. The sorting of Sit1-GFP from the endosomes to the plasma membrane was assessed A) by fluorescence microscopy and B) by measuring FOB uptake after incubation with the indicated compound for 30 min. For uptake experiments, the cells were rapidly washed with water and filtered before resuspension in minimal glucose medium containing 1 μm55Fe-FOB. The iron content of the cells was determined after 10 min of incubation at 30°C. Uptake values (B) are expressed as means ± standard error of the mean for three experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig05: Siderophore-induced relocation of Sit1-GFP.The Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to midexponential growth phase in raffinose-containing medium. Sit1-GFP synthesis was induced by incubation with galactose for 60 min and was stopped by adding glucose and incubating for 15 min. The sorting of Sit1-GFP from the endosomes to the plasma membrane was assessed A) by fluorescence microscopy and B) by measuring FOB uptake after incubation with the indicated compound for 30 min. For uptake experiments, the cells were rapidly washed with water and filtered before resuspension in minimal glucose medium containing 1 μm55Fe-FOB. The iron content of the cells was determined after 10 min of incubation at 30°C. Uptake values (B) are expressed as means ± standard error of the mean for three experiments.
Mentions: Thus, Sit1-GFP was mainly sorted to the endosomal/vacuolar pathway when synthesized in the absence of substrate, and mainly targeted to the plasma membrane when synthesized in the presence of substrate. We then checked whether Sit1-GFP initially sorted to internal compartments could subsequently be targeted to the plasma membrane after the addition of FOB or other siderophores. Experiments were performed on sit1Δ cells transformed with the pGAL-SIT1-GFP construct and grown overnight on a raffinose-based medium. The cells were incubated with galactose for 60 min for induction. Glucose was added and incubated with the medium for 10–15 min to stop transporter synthesis and a siderophore was then added. The location of the siderophore transporter was determined by assessing fluorescence 30 min after addition of the siderophore, whereas the rate of siderophore transport by the washed cells was measured in parallel experiments. Observations of GFP fluorescence showed that Sit1 was sorted to endosomes and vacuoles after the transient expression of SIT1-GFP(Figure 4A, left panel and Figure 5A, panel 1), but rapid, massive translocation to the plasma membrane was observed if the cells were briefly exposed to FOB, the principal substrate of Sit1 (Figure 5A, panels 2 and 3). Plasma membrane sorting from internal compartments was also observed when protein synthesis was inhibited by incubation with cycloheximide for 5 or 15 min before the addition of external FOB. Thus, the external substrate clearly alters the fate of presynthesized Sit1 (Figure S4). The movement of Sit1 to the plasma membrane was accompanied by an increase in the ability of cells to take up FOB (Figure 5B). The gallium analog of FOB [Ga-desferrioxamine (DFOB)] also promoted the movement of Sit1 to the plasma membrane (Figure 5A, panels 4 and 5), although less effectively than FOB itself. The presence of a strong GFP signal in the vacuole lumen indicated that a large proportion of the Sit1 present reached the vacuole for degradation. This effect was observed to a much lesser extent when FOB was the inducer (Figure 5A, panels 2 and 3). Thus, Ga-DFOB was also less effective than FOB at promoting FOB uptake by the washed cells (Figure 5B). Exposure of the cells to two other substrates of Sit1 – FCH and FC – also resulted in the rapid translocation of the protein to the plasma membrane (Figure 5A, panels 6 and 7). Remarkably, CG, which is not a substrate of Sit1 (see below), had the same effect (Figure 5A, panel 8), whereas ENB and TAF had no effect on the distribution of Sit1 (Figure 5A, panels 9 and 10). Other sources of iron, such as ferric citrate and hemin, were also unable to promote the plasma membrane targeting of Sit1 (Figure 5A, panels 11 and 12). The uptake of FOB by the cells was increased in all cases by prior incubation of the cells with a siderophore, promoting the movement of Sit1 to the plasma membrane (Figure 5B). Thus, FOB, FC, FCH and CG all caused Sit1 to move rapidly from internal compartments to the plasma membrane, and this effect occurred at all siderophore concentrations tested (within the range 1–100 μm). Thus, brief exposure of the cells to a siderophore (CG) that is not a substrate of a given transport system (Sit1) may induce this transport system by promoting plasma membrane sorting. This mechanism may have an important adaptive function for cells.

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