Limits...
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.

Show MeSH

Related in: MedlinePlus

Sit1-GFP location in wild-type (WT) and mutant cells.Wild-type and mutant cells, transformed with pGAL-SIT1-GFP, were cultured overnight in raffinose-containing medium to midexponential growth phase. Sit1-GFP synthesis was induced by incubating the cells for 1 h with galactose. Glucose was then added and the culture was incubated for 15 min to stop galactose induction, and GFP fluorescence was monitored either immediately (−FOB) or after a further 30-min incubation of the cells with 1 μm FOB (+FOB).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171038&req=5

fig06: Sit1-GFP location in wild-type (WT) and mutant cells.Wild-type and mutant cells, transformed with pGAL-SIT1-GFP, were cultured overnight in raffinose-containing medium to midexponential growth phase. Sit1-GFP synthesis was induced by incubating the cells for 1 h with galactose. Glucose was then added and the culture was incubated for 15 min to stop galactose induction, and GFP fluorescence was monitored either immediately (−FOB) or after a further 30-min incubation of the cells with 1 μm FOB (+FOB).

Mentions: After galactose induction followed by glucose repression and incubation for 30 min with (+FOB) or without (−FOB) substrate, the steady-state location of Sit1 varied according to the strain examined (Figure 6). As described above, in wild-type and sit1Δ cells, Sit1-GFP was targeted to endosomes and the vacuolar lumen in the absence of FOB (Figure 6, −FOB) and was targeted to the plasma membrane in the presence of FOB (Figure 6, +FOB). In the vps28Δ mutant, Sit1-GFP accumulated in large juxtavacuolar structures, the class E compartment, and at the vacuolar membrane. The cells also displayed faint plasma membrane staining (Figure 6, −FOB). Plasma membrane staining in the presence of FOB was clearly stronger than that in wild-type cells (Figure 6, +FOB). This observation is consistent with the higher level of FOB uptake observed in these mutant cells (31) and, more generally, with the larger amounts of many transporters at the plasma membrane in the steady state reported for various class E mutants, including vps28Δ(32,33). These properties are clearly explained by the sorting defect in these mutants. In vps28Δ cells, maturation of the late endosome is compromised and membrane proteins, such as transporters coming from the Golgi compartment or subjected to endocytosis from the plasma membrane, remain at the limiting membrane of a large MVB (known as the class E compartment), rather than being sorted to the internal vesicles of the MVB. This MVB sorting defect leads to the endosome-to-plasma membrane targeting of these proteins (32,34) and to an increase in their steady-state levels at the plasma membrane.


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)

Sit1-GFP location in wild-type (WT) and mutant cells.Wild-type and mutant cells, transformed with pGAL-SIT1-GFP, were cultured overnight in raffinose-containing medium to midexponential growth phase. Sit1-GFP synthesis was induced by incubating the cells for 1 h with galactose. Glucose was then added and the culture was incubated for 15 min to stop galactose induction, and GFP fluorescence was monitored either immediately (−FOB) or after a further 30-min incubation of the cells with 1 μm FOB (+FOB).
© Copyright Policy
Related In: Results  -  Collection

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

fig06: Sit1-GFP location in wild-type (WT) and mutant cells.Wild-type and mutant cells, transformed with pGAL-SIT1-GFP, were cultured overnight in raffinose-containing medium to midexponential growth phase. Sit1-GFP synthesis was induced by incubating the cells for 1 h with galactose. Glucose was then added and the culture was incubated for 15 min to stop galactose induction, and GFP fluorescence was monitored either immediately (−FOB) or after a further 30-min incubation of the cells with 1 μm FOB (+FOB).
Mentions: After galactose induction followed by glucose repression and incubation for 30 min with (+FOB) or without (−FOB) substrate, the steady-state location of Sit1 varied according to the strain examined (Figure 6). As described above, in wild-type and sit1Δ cells, Sit1-GFP was targeted to endosomes and the vacuolar lumen in the absence of FOB (Figure 6, −FOB) and was targeted to the plasma membrane in the presence of FOB (Figure 6, +FOB). In the vps28Δ mutant, Sit1-GFP accumulated in large juxtavacuolar structures, the class E compartment, and at the vacuolar membrane. The cells also displayed faint plasma membrane staining (Figure 6, −FOB). Plasma membrane staining in the presence of FOB was clearly stronger than that in wild-type cells (Figure 6, +FOB). This observation is consistent with the higher level of FOB uptake observed in these mutant cells (31) and, more generally, with the larger amounts of many transporters at the plasma membrane in the steady state reported for various class E mutants, including vps28Δ(32,33). These properties are clearly explained by the sorting defect in these mutants. In vps28Δ cells, maturation of the late endosome is compromised and membrane proteins, such as transporters coming from the Golgi compartment or subjected to endocytosis from the plasma membrane, remain at the limiting membrane of a large MVB (known as the class E compartment), rather than being sorted to the internal vesicles of the MVB. This MVB sorting defect leads to the endosome-to-plasma membrane targeting of these proteins (32,34) and to an increase in their steady-state levels at the plasma membrane.

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