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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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Kinetics of Enb1 and Sit1 targeting to the plasma membrane and/or in endosomes.A) Enb1Δ cells transformed with pGAL-ENB1-GFP, GAL-GFP-ENB1 cells and B) sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to midexponential growth phase with raffinose as the carbon source. Galactose was then added and cells were examined for GFP fluorescence every 30 min for 2 h and after incubation overnight. Sit1Δ cells transformed with pGAL-SIT1-GFP, grown in the presence of galactose for 1 h, were stained with CMAC (Materials and Methods) to visualize the vacuolar lumen (B).
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fig03: Kinetics of Enb1 and Sit1 targeting to the plasma membrane and/or in endosomes.A) Enb1Δ cells transformed with pGAL-ENB1-GFP, GAL-GFP-ENB1 cells and B) sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to midexponential growth phase with raffinose as the carbon source. Galactose was then added and cells were examined for GFP fluorescence every 30 min for 2 h and after incubation overnight. Sit1Δ cells transformed with pGAL-SIT1-GFP, grown in the presence of galactose for 1 h, were stained with CMAC (Materials and Methods) to visualize the vacuolar lumen (B).

Mentions: For monitoring of the fate of newly synthesized fusion proteins, genes encoding GFP fusions of Sit1 and Enb1 were inserted into a centromeric (CEN) plasmid (one to four copies/cell) under the control of the inducible GAL1 promoter. When this ‘Gal system’ was used to induce transcription in sit1Δ and enb1Δ cells, galactose was generally added for only short periods (60 min) to prevent the potential mislocalization of proteins because of prolonged overproduction. We checked, by Western blotting, that the level of Sit1 (as GFP fusion) after 1 h of galactose induction was not significantly different from that when the corresponding gene was expressed at the original locus with its endogenous promoter under conditions of iron deprivation (Figure S2). We monitored the intracellular fate of Enb1 and Sit1 and their distribution by fluorescence microscopy after the addition of galactose to cells grown in raffinose-containing medium. Enb1 was targeted exclusively to the plasma membrane, and newly synthesized protein was targeted to the bud (Figure 3A). We checked that the distribution of Enb1 was not affected by the GFP tag, by constructing a chromosome-encoded version of ENB1 tagged with GFP at the N-terminus and placed under the control of the GAL promoter. Plasma membrane targeting kinetics were similar for ENB1 tagged with GFP at the N-terminus and for ENB1 tagged at the C-terminus (Figure 3A).


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)

Kinetics of Enb1 and Sit1 targeting to the plasma membrane and/or in endosomes.A) Enb1Δ cells transformed with pGAL-ENB1-GFP, GAL-GFP-ENB1 cells and B) sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to midexponential growth phase with raffinose as the carbon source. Galactose was then added and cells were examined for GFP fluorescence every 30 min for 2 h and after incubation overnight. Sit1Δ cells transformed with pGAL-SIT1-GFP, grown in the presence of galactose for 1 h, were stained with CMAC (Materials and Methods) to visualize the vacuolar lumen (B).
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Related In: Results  -  Collection

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

fig03: Kinetics of Enb1 and Sit1 targeting to the plasma membrane and/or in endosomes.A) Enb1Δ cells transformed with pGAL-ENB1-GFP, GAL-GFP-ENB1 cells and B) sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to midexponential growth phase with raffinose as the carbon source. Galactose was then added and cells were examined for GFP fluorescence every 30 min for 2 h and after incubation overnight. Sit1Δ cells transformed with pGAL-SIT1-GFP, grown in the presence of galactose for 1 h, were stained with CMAC (Materials and Methods) to visualize the vacuolar lumen (B).
Mentions: For monitoring of the fate of newly synthesized fusion proteins, genes encoding GFP fusions of Sit1 and Enb1 were inserted into a centromeric (CEN) plasmid (one to four copies/cell) under the control of the inducible GAL1 promoter. When this ‘Gal system’ was used to induce transcription in sit1Δ and enb1Δ cells, galactose was generally added for only short periods (60 min) to prevent the potential mislocalization of proteins because of prolonged overproduction. We checked, by Western blotting, that the level of Sit1 (as GFP fusion) after 1 h of galactose induction was not significantly different from that when the corresponding gene was expressed at the original locus with its endogenous promoter under conditions of iron deprivation (Figure S2). We monitored the intracellular fate of Enb1 and Sit1 and their distribution by fluorescence microscopy after the addition of galactose to cells grown in raffinose-containing medium. Enb1 was targeted exclusively to the plasma membrane, and newly synthesized protein was targeted to the bud (Figure 3A). We checked that the distribution of Enb1 was not affected by the GFP tag, by constructing a chromosome-encoded version of ENB1 tagged with GFP at the N-terminus and placed under the control of the GAL promoter. Plasma membrane targeting kinetics were similar for ENB1 tagged with GFP at the N-terminus and for ENB1 tagged at the C-terminus (Figure 3A).

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