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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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FOB-dependent localization of Sit1-GFP.The sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium. Sit1-GFP synthesis was induced for 1 h by adding galactose to the medium with (+FOB) or without (−FOB) 10 μm FOB. A) Cells were visualized by fluorescence microscopy with a GFP filter set and B) processed for subcellular fractionation. Cells were lysed and protein extracts were fractionated on a 20–60% sucrose density gradient. Aliquots of the various fractions were analysed by Western immunoblotting for the presence of GFP, PGK (a cytosolic protein), plasma membrane ATPase 1 (Pma1), Vps10 (carboxypeptidase Y receptor, which cycles between the Golgi and late endosome compartments), Pep12 (SNARE protein involved in the fusion of vesicles with late endosomes), Vps55 (transmembrane protein of the late endosomes) and Vph1 (transmembrane subunit of the vacuolar ATPase).
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fig04: FOB-dependent localization of Sit1-GFP.The sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium. Sit1-GFP synthesis was induced for 1 h by adding galactose to the medium with (+FOB) or without (−FOB) 10 μm FOB. A) Cells were visualized by fluorescence microscopy with a GFP filter set and B) processed for subcellular fractionation. Cells were lysed and protein extracts were fractionated on a 20–60% sucrose density gradient. Aliquots of the various fractions were analysed by Western immunoblotting for the presence of GFP, PGK (a cytosolic protein), plasma membrane ATPase 1 (Pma1), Vps10 (carboxypeptidase Y receptor, which cycles between the Golgi and late endosome compartments), Pep12 (SNARE protein involved in the fusion of vesicles with late endosomes), Vps55 (transmembrane protein of the late endosomes) and Vph1 (transmembrane subunit of the vacuolar ATPase).

Mentions: For Arn1-GFP, a significant fraction of the cleaved GFP moiety appeared in the lumen of the vacuole (immunodetectable protein recovered as free GFP) (Figure 2A,B). However, under our experimental conditions, and in contrast to the results obtained by Yun et al. with an HA-tagged version of Arn1 (3), we observed two other types of fluorescent structures: small, intracellular, juxtavacuolar spots, probably corresponding to endosomes (Figure 2A, arrows), and the plasma membrane itself, which was slightly but clearly labelled in some cells, particularly under inducing conditions (low iron concentration; Figure 2A). Hence, most Arn1 is sorted to the vacuolar degradation pathway (19), whereas a small fraction of the protein is sorted to the plasma membrane and may be easier to observe by GFP fusion methods than by indirect immunofluorescence. Enb1-GFP was also observed under all growth conditions (Figure 2A). However, in this case, the fusion protein was systematically restricted to the plasma membrane and was not associated with any other compartment (Figure 2A). Consistent with the exclusive plasma membrane sorting of Enb1, we detected no free GFP by Western blotting (Figure 2B). Some Sit1-GFP was present in internal compartments (Figure 2A, arrows), probably endosomes, as confirmed by subcellular fractionation experiments (see below and Figure 4B). Clear vacuolar staining was observed and was the most prominent signal under noninducing conditions (Figure 2A). Under inducing conditions (low iron concentration), about 90% of the cells also displayed fluorescence associated with the plasma membrane (Figure 2A). We assumed that the GFP fusion proteins behaved similarly to the original proteins, as uptake activities were not affected by GFP tagging, as shown for Sit1 in Figure 2C. Thus, the various siderophore transport systems of S. cerevisiae undergo different trafficking processes. Under inducing conditions, Sit1 and Arn1 were detected both in internal compartments and at the plasma membrane; Enb1 was present only at the plasma membrane, and Taf1 was undetectable. We decided to compare the trafficking processes of detectable transporters with radically different patterns of behaviour: Sit1 and Enb1.


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)

FOB-dependent localization of Sit1-GFP.The sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium. Sit1-GFP synthesis was induced for 1 h by adding galactose to the medium with (+FOB) or without (−FOB) 10 μm FOB. A) Cells were visualized by fluorescence microscopy with a GFP filter set and B) processed for subcellular fractionation. Cells were lysed and protein extracts were fractionated on a 20–60% sucrose density gradient. Aliquots of the various fractions were analysed by Western immunoblotting for the presence of GFP, PGK (a cytosolic protein), plasma membrane ATPase 1 (Pma1), Vps10 (carboxypeptidase Y receptor, which cycles between the Golgi and late endosome compartments), Pep12 (SNARE protein involved in the fusion of vesicles with late endosomes), Vps55 (transmembrane protein of the late endosomes) and Vph1 (transmembrane subunit of the vacuolar ATPase).
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Related In: Results  -  Collection

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fig04: FOB-dependent localization of Sit1-GFP.The sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium. Sit1-GFP synthesis was induced for 1 h by adding galactose to the medium with (+FOB) or without (−FOB) 10 μm FOB. A) Cells were visualized by fluorescence microscopy with a GFP filter set and B) processed for subcellular fractionation. Cells were lysed and protein extracts were fractionated on a 20–60% sucrose density gradient. Aliquots of the various fractions were analysed by Western immunoblotting for the presence of GFP, PGK (a cytosolic protein), plasma membrane ATPase 1 (Pma1), Vps10 (carboxypeptidase Y receptor, which cycles between the Golgi and late endosome compartments), Pep12 (SNARE protein involved in the fusion of vesicles with late endosomes), Vps55 (transmembrane protein of the late endosomes) and Vph1 (transmembrane subunit of the vacuolar ATPase).
Mentions: For Arn1-GFP, a significant fraction of the cleaved GFP moiety appeared in the lumen of the vacuole (immunodetectable protein recovered as free GFP) (Figure 2A,B). However, under our experimental conditions, and in contrast to the results obtained by Yun et al. with an HA-tagged version of Arn1 (3), we observed two other types of fluorescent structures: small, intracellular, juxtavacuolar spots, probably corresponding to endosomes (Figure 2A, arrows), and the plasma membrane itself, which was slightly but clearly labelled in some cells, particularly under inducing conditions (low iron concentration; Figure 2A). Hence, most Arn1 is sorted to the vacuolar degradation pathway (19), whereas a small fraction of the protein is sorted to the plasma membrane and may be easier to observe by GFP fusion methods than by indirect immunofluorescence. Enb1-GFP was also observed under all growth conditions (Figure 2A). However, in this case, the fusion protein was systematically restricted to the plasma membrane and was not associated with any other compartment (Figure 2A). Consistent with the exclusive plasma membrane sorting of Enb1, we detected no free GFP by Western blotting (Figure 2B). Some Sit1-GFP was present in internal compartments (Figure 2A, arrows), probably endosomes, as confirmed by subcellular fractionation experiments (see below and Figure 4B). Clear vacuolar staining was observed and was the most prominent signal under noninducing conditions (Figure 2A). Under inducing conditions (low iron concentration), about 90% of the cells also displayed fluorescence associated with the plasma membrane (Figure 2A). We assumed that the GFP fusion proteins behaved similarly to the original proteins, as uptake activities were not affected by GFP tagging, as shown for Sit1 in Figure 2C. Thus, the various siderophore transport systems of S. cerevisiae undergo different trafficking processes. Under inducing conditions, Sit1 and Arn1 were detected both in internal compartments and at the plasma membrane; Enb1 was present only at the plasma membrane, and Taf1 was undetectable. We decided to compare the trafficking processes of detectable transporters with radically different patterns of behaviour: Sit1 and Enb1.

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