Volume 8 issue 11 November 2007
Trafficking of siderophore transporters in Saccharomyces cerevisiae and intracellular fate of ferrioxamine B conjugates
M Froissard, N Belgareh-Touzé, M Dias, N Buisson, J-M Camadro, R Haguenauer-Tsapis and E Lesuisse

Supplemental Figure 1: Siderophore uptake by cells expressing native transporters (WT) and by cells expressing chromosome-encoded GFP-fusion transporters (SIT1-GFP, ARN1-GFP, TAF1-GFP, ENB1-GFP). The indicated strains were first cultured overnight in YPD medium. Cultures were then diluted 1:10 in the same medium supplemented with 200 µM BPS and incubated for 6 h. The cells were washed with water, resuspended in fresh YPD medium and iron uptake was measured with 1 µM of FOB, FCH, TAF or ENB. Results are expressed as the rate of uptake per A600 unit. Means ± SE from 3 experiments are shown.

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Supplemental Figure 2: Comparison of Sit1-GFP levels. Cells expressing chromosome-encoded Sit1-GFP were cultured to mid-exponential growth phase in complete medium (YPD). Sit1Δ cells bearing pGAL-SIT1-GFP were grown to mid-exponential growth phase in raffinose-containing medium. Galactose was then added to the medium and the cultures incubated for 1 or 2 hours to induce Sit1-GFP synthesis. Protein extracts were prepared from both strains and analyzed by western blotting for GFP, using a monoclonal anti-GFP antibody

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Supplemental Figure 3: Colocalization of Sit1-GFP with CHC1-RFP and Snf7-RFP. CHC1-RFP and SNF7-RFP strains transformed with pGAL-SIT1-GFP were grown overnight in raffinose-containing medium. Galactose was then added and the culture incubated for one hour to induce Sit1-GFP expression. The distributions of Sit1-GFP, CHC1-RFP and Snf7-RFP were compared, using the GFP and rhodamine filter sets. Arrows indicate colocalized structures and arrowheads, structures that were not colocalized. Snf7-RFP expression leads to the accumulation of a class E compartment (large late endosome apposed to the vacuole), in which Sit1-GFP is trapped.

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Supplemental Figure 4: Siderophore-induced relocation of Sit1-GFP independent of protein synthesis. sit1Δ cells transformed with pGAL-SIT1-GFP were cultured to mid-exponential growth phase in raffinose-containing medium. Sit1-GFP synthesis was induced by incubation with galactose for 60 min. Protein synthesis was blocked by adding cycloheximide (CHX, 10µg/ml) to the medium either together with galactose (pannel 1; control to ensure that protein synthesis was immediately blocked with this concentration of cycloheximide) or after the galactose incubation period (panel 2-3). In the latter case, cells were incubated with CHX for 5 min (panel 2) or 15 min (panel 3) before adding FOB (1 µM) and incubating the cells for a further 15 min. Sit1-GFP sorting was assessed by fluorescence microscopy, using the GFP filter set.

Figure 4 (.jpg)

Supplemental Figure 5: Constitutive targeting of CaSit1/CaArn1 from C. albicans to the plasma membrane. Cells with chromosomal GFP tags were cultured to mid-exponential growth phase in raffinose minimal medium. CaSit1/CaArn1-GFP synthesis was induced by incubation with galactose for 90 min. GFP fluorescence was assessed by fluorescence microscopy, using the GFP filter set.

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