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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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Intracellular location of FOB-NBD in wild-type (WT) and mutant cells.A) Wild-type and mutant cells were cultured in glucose-containing medium to midexponential growth phase and were then incubated in the presence of 10 μm Ga-DFOB-NBD for 3 h. The cells were then washed with water and examined for Ga-DFOB-NBD fluorescence using the GFP filter set and with Nomarski optics. B) The sit1Δ cells transformed with pGAL-SIT1-GFP were grown overnight in galactose-defined medium supplemented with 100 μm FOB. The whole organellar fraction was collected after protoplast lysis by moderate osmotic shock (Materials and Methods). Intact organelles were separated on a discontinuous Ficoll gradient and collected as nine separate fractions. The absorbance of each fraction was measured at 420 nm (maximum absorbance of FOB) after solubilization with 0.1% SDS. The fractions were analysed by Western blotting with an anti-Vph1 (vacuolar marker) and an anti-GFP antibody (free GFP only, corresponding to the cleaved form of Sit1-GFP, present in the vacuolar fractions).
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fig08: Intracellular location of FOB-NBD in wild-type (WT) and mutant cells.A) Wild-type and mutant cells were cultured in glucose-containing medium to midexponential growth phase and were then incubated in the presence of 10 μm Ga-DFOB-NBD for 3 h. The cells were then washed with water and examined for Ga-DFOB-NBD fluorescence using the GFP filter set and with Nomarski optics. B) The sit1Δ cells transformed with pGAL-SIT1-GFP were grown overnight in galactose-defined medium supplemented with 100 μm FOB. The whole organellar fraction was collected after protoplast lysis by moderate osmotic shock (Materials and Methods). Intact organelles were separated on a discontinuous Ficoll gradient and collected as nine separate fractions. The absorbance of each fraction was measured at 420 nm (maximum absorbance of FOB) after solubilization with 0.1% SDS. The fractions were analysed by Western blotting with an anti-Vph1 (vacuolar marker) and an anti-GFP antibody (free GFP only, corresponding to the cleaved form of Sit1-GFP, present in the vacuolar fractions).

Mentions: After 1 h of synthesis in the absence of FOB, Sit1-GFP was detected mostly as small dots (−FOB, Figure 4A). These dots may correspond to the Golgi apparatus/endosomal compartments, consistent with cofractionation of internal Sit1-GFP with the Golgi marker Vps10 and the endosomal markers Pep12 and Vps55 (Figure 4B). Faint vacuolar staining was also observed. Accordingly, free GFP was detected by western immunoblotting in light fractions, corresponding to both cytosolic proteins [phosphoglycerate kinase (PGK)] and soluble vacuolar lumen proteins released by cell disruption with glass beads (Figure 4B). After prolonged growth in the presence of FOB and the preparation of intact vacuoles from protoplasts, free GFP was found to colocalize with the vacuolar membrane-bound Vph1 (see Figure 8B). No Sit1-GFP plasma membrane staining was observed in these experimental conditions, but we cannot exclude the possibility that Sit1-GFP was present in trace amounts at the plasma membrane, as a faint signal was detected in subcellular fractions colocalising with the plasma membrane marker plasma membrane ATPase 1 (Pma1) (Figure 4B). Moreover, the main Sit1-GFP signal detected corresponded to fraction 10 of the sucrose gradient (Figure 4B), whereas the main Golgi apparatus/endosomal signals were recovered in fractions 8–9. This observation suggests that Sit1-GFP was not strictly restricted to a ‘pure’ Golgi–endosomal pool. Instead, it may form two different pools: a major endosomal pool and a small plasma membrane pool resulting in a small shift of the main signal on the gradient (Figure 4B).


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)

Intracellular location of FOB-NBD in wild-type (WT) and mutant cells.A) Wild-type and mutant cells were cultured in glucose-containing medium to midexponential growth phase and were then incubated in the presence of 10 μm Ga-DFOB-NBD for 3 h. The cells were then washed with water and examined for Ga-DFOB-NBD fluorescence using the GFP filter set and with Nomarski optics. B) The sit1Δ cells transformed with pGAL-SIT1-GFP were grown overnight in galactose-defined medium supplemented with 100 μm FOB. The whole organellar fraction was collected after protoplast lysis by moderate osmotic shock (Materials and Methods). Intact organelles were separated on a discontinuous Ficoll gradient and collected as nine separate fractions. The absorbance of each fraction was measured at 420 nm (maximum absorbance of FOB) after solubilization with 0.1% SDS. The fractions were analysed by Western blotting with an anti-Vph1 (vacuolar marker) and an anti-GFP antibody (free GFP only, corresponding to the cleaved form of Sit1-GFP, present in the vacuolar fractions).
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Related In: Results  -  Collection

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fig08: Intracellular location of FOB-NBD in wild-type (WT) and mutant cells.A) Wild-type and mutant cells were cultured in glucose-containing medium to midexponential growth phase and were then incubated in the presence of 10 μm Ga-DFOB-NBD for 3 h. The cells were then washed with water and examined for Ga-DFOB-NBD fluorescence using the GFP filter set and with Nomarski optics. B) The sit1Δ cells transformed with pGAL-SIT1-GFP were grown overnight in galactose-defined medium supplemented with 100 μm FOB. The whole organellar fraction was collected after protoplast lysis by moderate osmotic shock (Materials and Methods). Intact organelles were separated on a discontinuous Ficoll gradient and collected as nine separate fractions. The absorbance of each fraction was measured at 420 nm (maximum absorbance of FOB) after solubilization with 0.1% SDS. The fractions were analysed by Western blotting with an anti-Vph1 (vacuolar marker) and an anti-GFP antibody (free GFP only, corresponding to the cleaved form of Sit1-GFP, present in the vacuolar fractions).
Mentions: After 1 h of synthesis in the absence of FOB, Sit1-GFP was detected mostly as small dots (−FOB, Figure 4A). These dots may correspond to the Golgi apparatus/endosomal compartments, consistent with cofractionation of internal Sit1-GFP with the Golgi marker Vps10 and the endosomal markers Pep12 and Vps55 (Figure 4B). Faint vacuolar staining was also observed. Accordingly, free GFP was detected by western immunoblotting in light fractions, corresponding to both cytosolic proteins [phosphoglycerate kinase (PGK)] and soluble vacuolar lumen proteins released by cell disruption with glass beads (Figure 4B). After prolonged growth in the presence of FOB and the preparation of intact vacuoles from protoplasts, free GFP was found to colocalize with the vacuolar membrane-bound Vph1 (see Figure 8B). No Sit1-GFP plasma membrane staining was observed in these experimental conditions, but we cannot exclude the possibility that Sit1-GFP was present in trace amounts at the plasma membrane, as a faint signal was detected in subcellular fractions colocalising with the plasma membrane marker plasma membrane ATPase 1 (Pma1) (Figure 4B). Moreover, the main Sit1-GFP signal detected corresponded to fraction 10 of the sucrose gradient (Figure 4B), whereas the main Golgi apparatus/endosomal signals were recovered in fractions 8–9. This observation suggests that Sit1-GFP was not strictly restricted to a ‘pure’ Golgi–endosomal pool. Instead, it may form two different pools: a major endosomal pool and a small plasma membrane pool resulting in a small shift of the main signal on the gradient (Figure 4B).

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