Trafficking of siderophore transporters in Saccharomyces cerevisiae and intracellular fate of ferrioxamine B conjugates

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.

Figure 1

Synthesis of conjugate AF-DFOB. Reagents and conditions: (i) AF, pentafluorophenol; DCC, Dicyclohexylcarbodiimide; THF, tetrafuran and (ii) desferal, 2, Et₃N, THF, 50°C.

Figure 2

Synthesis and location of chromosomal GFP-tagged siderophore transporters.A) Cells expressing chromosomal GFP-tagged siderophore transporters were cultured to midexponential growth phase in complete medium (YPD) or in complete medium supplemented with 200 μm BPS for 4 h (YPD + BPS). Cells were then examined for GFP fluorescence and with Nomarski optics. Arrows indicate round structures possibly corresponding to endosomes. B) Total protein extracts were prepared and analysed by Western blotting for GFP [GFP-tagged transporter (T-GFP) and free vacuolar GFP] and for Gas1p, as a loading control. C) We determined FOB uptake by WT, sit1Δ and SIT1-GFP cells cultured to midexponential growth phase in complete medium supplemented with 200 μm BPS, 100 μm iron citrate (Fe) or 100 μm FOB (means ± standard error of the mean from three experiments). WT, wild type.

Figure 4

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).

Figure 3

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).

Figure 8

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).

Figure 5

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.

Figure 6

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).

Figure 7

Sit1-dependent use of FOB-NBD by the cells.Wild-type (WT) or sit1Δ cells were plated on YPD-agar medium supplemented with 1 mm BPS (to prevent reductive iron uptake). Sterile filter paper disks containing siderophores (5 μL of 10 μm CG, FOB or FOB-NBD) were placed on the surface of the agar. Plates were incubated for 2 days at 30°C and photographed. Growing cells appeared as a white halo around the filter papers.

Figure 9

The AF-FOB conjugate does not reach its target in vivo.A) The IC₅₀ values of free AF (○), DFOB-AF (•) and FOB-AF (▪) for protopor. oxidase activity were determined in vitro on whole cell extracts. B) Wild-type S. cerevisiae cells were plated as a lawn on YPGly agar. Wells were created in the agar with a hole punch, into which 50 μL of 1 mm FOB-AF or 25 μL FOB 2 mm + 25 μL AF 2 mm was dispensed. Cell growth on the plate was examined after 3 days at 30 seconds. It was inhibited only with FOB + AF (inhibition visible as a halo around the bottom well). C) Accumulation of FOB and FOB conjugates in cells overexpressing SIT1. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in raffinose-containing medium to midexponential growth phase. We then added glucose (2%) or galactose (2%) and cultured the cells for a further 4 h. ⁵⁵Fe-labelled FOB, FOB-NBD or FOB-AF was then added, and the amount of ⁵⁵Fe accumulated by the cells was determined after 15 min of incubation. Results are expressed as means ± standard error of the mean for six experiments. D) Accumulated FOB-AF inhibits protopor. oxidase in vitro. Sit1Δ cells transformed with pGAL-SIT1-GFP were cultured overnight in galactose-containing medium in the presence of 100 μm FOB (□) or 100 μm FOB-AF (▪). The cells were then washed and disrupted with glass beads. The activity of protopor. oxidase was determined on whole cell extracts at various protein concentrations. Protopor., protoporphyrinogen.