Siderophore uptake and use by the yeast Saccharomyces cerevisiae
Emmanuel Lesuisse1,
Pierre-Louis Blaiseau1,
Andrew Dancis2 and
Jean-Michel Camadro1
Laboratoire dIngénierie des Protéines et Contrôle Métabolique, Institut Jacques Monod, Tour 43, Université Paris 7/Paris 6, 2 place Jussieu, 75251 Paris cedex 05, France1
University of Pennsylvania, Department of Medicine, Division of Hematology/Oncology, BRBII Room 731, 431 Curie Blvd, Philadelphia, PA 19104, USA2
Author for correspondence: Emmanuel Lesuisse. Tel: +33 1 44 27 63 56. Fax: +33 1 44 27 57 16. e-mail: lesuisse{at}ijm.jussieu.fr
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ABSTRACT
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The non-reductive uptake of several siderophores (ferrioxamine B, ferrichrome, triacetylfusarinine C and ferricrocin) by various strains of Saccharomyces cerevisiae was studied. Several aspects of siderophore transport were examined, including specificity of transport, regulation of transport and intracellular localization of the ferri-siderophores. Ferrioxamine B was taken up preferentially via the products of the SIT1 gene and triacetylfusarinine C by the TAF1 gene product, but the specificity was not absolute. Ferrichrome and ferricrocin uptake was not dependent on a single major facilitator superfamily (MFS) gene product. The apparent specificity of transport was strongly dependent on the genetic background of the cells. Non-reductive uptake of siderophores was induced under more stringent conditions (of iron deprivation) than was the reductive uptake of ferric citrate. Regulation of transport depended on the transcriptional factors Aft1 and Tup1/Ssn6. Cells disrupted for the TUP1 or SSN6 genes were constitutively derepressed for the uptake of ferrichrome, ferricrocin or ferrioxamine B, but not for the uptake of triacetylfusarinine C. Cells bearing the AFT1up mutation accumulated large amounts of ferric siderophores. Intracellular decomplexation of the siderophores occurred when transcription of the AFT1up gene was repressed. Ferrioxamine B and ferrichrome seemed to accumulate in an endosomal compartment, as shown by biochemical studies and by confocal microscopy study of cells loaded with a fluorescent derivative of ferrichrome. Endocytosis was, however, not involved in the non-reductive uptake of siderophores.
Keywords: iron, siderophore, yeast, Saccharomyces cerevisiae
Abbreviations: BPS, bathophenanthrolinedisulfonate; FC, ferricrocin; FCH, ferrichrome; FOB, ferrioxamine B, mesylate form (Desferal); MFS, major facilitator superfamily; TAF, triacetylfusarinine C
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INTRODUCTION
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The yeast Saccharomyces cerevisiae can use two different high-affinity mechanisms (reductive and non-reductive) to take up iron from the extracellular medium. The reductive mechanism (Lesuisse et al., 1987
; Dancis et al., 1990
) involves the labilization of extracellular ferric chelates by reduction at the surface of the cells via the inducible plasma membrane reductases Fre1p and Fre2p (Dancis et al., 1992
; Georgatsou & Alexandraki, 1994
). A permeaseoxidase complex (Ftr1p and Fet3p) is then involved in translocating iron through the plasma membrane (Askwith et al., 1994
; Stearman et al., 1996
). The non-reductive mechanism requires that a ferric complex enters the cells prior to any reduction step. In 1989, we described a non-reductive mechanism for ferrioxamine B (FOB) uptake by S. cerevisiae (Lesuisse & Labbe, 1989
). This siderophore can enter cells via an FOB receptor present at the cell surface. We showed that the siderophore transport system of S. cerevisiae is specific, saturable, energy-dependent and has a high affinity for FOB (Lesuisse & Labbe, 1989
). We called this an opportunistic strategy of iron uptake because S. cerevisiae can use siderophores excreted by other micro-organisms although it does not produce any itself. The presence in a fungus of two different uptake systems, one reductive and another non-reductive, was first demonstrated by Ecker & Emery (1983)
for Ustilago sphaerogena.
The existence of two mechanisms for iron uptake makes the specific study of non-reductive uptake somewhat complicated. Uptake kinetics do not fit well with simple MichaelisMenten representations (Lesuisse & Labbe, 1989
). Reduction of siderophores at the cell surface is a low-affinity and low-specificity mechanism. At low extracellular concentrations (around 1 µM), uptake of the siderophore is essentially non-reductive. When the siderophore concentration is raised, the reductive mechanism becomes quantitatively more important as the non-reductive, high-affinity system approaches saturation (Lesuisse & Labbe, 1989
). Genetic studies are also not simple: a mutation that affects some step of non-reductive iron assimilation in S. cerevisiae will be difficult to detect because iron can still enter the cells by the reductive mechanism. Emery (1986)
used gallium analogues of siderophores, which cannot be reduced, to distinguish between reductive and non-reductive uptake of siderophores by U. sphaerogena. We used mutants of S. cerevisiae in which the FET3 and FET4 genes, encoding components of the high-affinity and low-affinity reductive uptake systems, respectively (Askwith et al., 1994
; Dix et al., 1994
), had been deleted. Such mutants are unable to grow on minimum medium (containing about 1 µM iron) unless a siderophore (or large amounts of a ferric salt) is added to the growth medium. This property allowed us to select mutants unable to take up FOB, and to identify a gene, SIT1 (Siderophore Iron Transport), whose product is probably responsible for the translocation of FOB through the plasmalemma (Lesuisse et al., 1998
). Sit1p belongs to the major facilitator superfamily (MFS) and has several homologues that may be expected to participate in the transport of other siderophores. This was recently demonstrated in three short studies (Heymann et al., 1999
, 2000a
, b
) in which the authors attributed the product of the YHL047c ORF to triacetylfusarinine C (TAF) transport, the product of the YHL040c ORF to the transport of a specific class of ferrichromes (FCHs), possessing anhydromevalonyl residues linked to N(
)-ornithine, and the product of the YOL158c ORF to the transport of enterobactin. They postulated that the product of any single gene of the SIT family was responsible for recognizing and transporting a single siderophore. This is not consistent with our own observations (present work) or with other recently published results (Yun et al., 2000b
). The present report re-examines some aspects of siderophore transport by S. cerevisiae, including the specificity of transport, energy requirements, the regulation of transport and the intracellular use of iron. To respect priority of names and to avoid unnecessary confusion, the following nomenclature is used throughout the paper: YEL065w is referred to as SIT1 (Lesuisse et al., 1998
), YHL047c as TAF1, YHL040c as ARN1 and YOL158c as ENB1 (Heymann et al., 1999
, 2000a
, b
).
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METHODS
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Strains, media and iron compounds.
The various strains of S. cerevisiae used in this study are described in Table 1
. Unless otherwise stated, cells were grown at 30 °C (in 50 ml Falcon tubes in a gyroshaker) in minimal YNB-glucose (without copper and iron) medium (Bio101) plus the required amino acids, 1 µM copper sulphate and various iron sources (siderophores or ferric citrate). For iron-deficient cultures, 100 µM BPS (bathophenanthrolinedisulfonate) was added to YNB-glucose medium without copper and iron. Desferri-compounds were obtained as described by Wiebe & Winkelmann (1975)
. When required, they were labelled with 55Fe [50 mCi mg-1 (1·85 GBq mg-1)]. Throughout this work, the name (des)ferrioxamine B refers to the commercially available mesylate derivative, Desferal (Novartis). The fluorescent derivative of FCH used was the glycine-based analogue B9 described by Berner et al. (1991)
.
Iron uptake assays.
Iron uptake was measured in micro-titration plates. Cells were grown overnight at 30 °C in various media (as specified above) to stationary phase. In the morning, cells were diluted 10-fold in the same medium and cultured for 6 h at 30 °C. Cells were then washed three times with distilled water and suspended in 50 mM citrate (tri-sodium) buffer (pH 6·5) containing 5% glucose to give an OD600 of 3. The cell suspension was distributed into the wells of a micro-titration plate (50 µl cells per well) at 0 °C. Iron was added (as a 55Fe chelate) to give a final concentration of 15 µM and the plate was incubated for 1560 min at 30 °C. The cells were collected with a cell harvester (Brandel) and washed on a filter.
Cell fractionation.
Cell fractions were purified (Raguzzi et al., 1988
) after protoplast lysis (moderate osmotic shock) by centrifugation on a discontinuous Ficoll gradient.
Other.
Low-temperature spectra (-191 °C) of whole cells were prepared as described by Lesuisse & Labbe (1989)
. The optical path length was 1 mm and the reference was a single sheet of wet filter paper. Spectra were corrected for baseline shift. SDS-PAGE and subsequent transfer to nitrocellulose were done using standard procedures. The proteins on nitrocellulose were probed with monoclonal antibodies or with an antiserum specific for Hem15p that was purified in our laboratory (Camadro & Labbe, 1988
). Primary antibodies were detected with a horseradish-peroxidase-conjugated anti-mouse IgG and ECL chemiluminescence (Amersham). Fluorescence confocal microscopy was performed as described by Ardon et al. (1998)
. Cells bearing the AFT1up mutation were grown overnight on a minimum medium containing 10 µM of fluorescent FCH analogue and then examined for intracellular fluorescence. New mutants affected in siderophore transport were generated by insertion mutagenesis (Tn3) as described by Burns et al. (1994)
. Conditional expression of AFT1up was done by cloning the AFT1up ORF into the centromeric plasmid pRS316-Gal.
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RESULTS
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The specificity of Sit1 and homologues for various siderophores is not absolute
We previously showed (Lesuisse et al., 1998
) that FOB and ferricrocin (FC) are taken up by S. cerevisiae via an active transport system, and that Sit1p is responsible for the uptake of FOB. It was then predictable that gene products homologous to Sit1p should be involved in the uptake of other siderophores. We subsequently identified FCH and TAF as siderophores transported by an active, energy-dependent mechanism by S. cerevisiae. This has also been reported recently by others (Yun et al., 2000b
; Heymann et al., 1999
). We measured the rate of uptake of FOB, FC, FCH, TAF and ferric citrate by a wild-type strain and isogenic mutants disrupted for either the SIT1 gene, the TAF1 gene, the ARN1 gene or the ENB1 gene to evaluate the contribution of Sit1p and homologues to the transport of these various siderophores (Fig. 1
). These data indicate that TAF transport mainly depends on TAF1, as recently shown by Heymann et al. (1999)
. FCH transport was affected in strains in which the ARN1, TAF1 or SIT1 genes were disrupted (Fig. 1
). This confirms the recent results of Yun et al. (2000b)
showing that FCH uptake does not depend on a single MFS gene. Our data also indicate that the same is true for the FCH-type siderophore FC. Transport of that siderophore was affected in strains whose ARN1 or TAF1 genes were disrupted (Fig. 1
). Curiously, disruption of ARN1 seemed to facilitate the transport of FOB (Fig. 1
). Members of the Sit family are thus not completely specific for a single siderophore. SIT1 and TAF1 seem to encode the most specific transporters, for FOB and TAF, respectively. The relative non-specificity of the transporters was also indicated by the following observation. Constitutive overexpression of the SIT1 ORF in various strains led to an increase in the rate of uptake of FOB, but also of FC and FCH, and to a lesser extent of TAF (Table 2
).
Analysis of new mutants also indicated that each Sitp member is not completely specific for a single siderophore. Cells lacking the YFH1 gene are sensitive to iron (Babcock et al., 1997
), especially when it is presented as FOB, FC or FCH (see below). We used this property to select new mutants able to grow on plates enriched with 10 µM FCH. One of these mutants could not take up FOB, FCH or FC. The transport of these three siderophores by the mutant cells was restored when they were transformed with SIT1 on a multicopy plasmid (data not shown). Other mutants obtained in this way are being analysed. We previously showed that FC uptake is completely unaffected when the SIT1 gene is disrupted in the strain DEY1433 (Lesuisse et al., 1998
). We disrupted the same gene in strains of various genetic backgrounds and observed that the effect of SIT1 disruption on siderophore transport differed greatly from one strain to another (Table 3
). Disruption of SIT1 led to a specific defect in FOB uptake in cells of the DEY1433 background, but it led to a decrease in FOB, FC and FCH uptake in cells of the CM3260 background (Table 3
). SIT1 disruption affected FOB and FC uptake by cells of the BY4742 background, but not that of FCH (Fig. 1
, Table 2
). As cells of the CM3260 background are mutated in the GCN4 gene, we tested whether the expression of GCN4 interfered with siderophore transport. We found that overexpression of GCN4 on a multicopy plasmid in CM3260 and CM3260
sit1 cells did not significantly change the rate of iron uptake from FOB, FC, FCH or TAF (data not shown).
As previously shown for FOB and FC (Lesuisse et al., 1998
), there was competition between FOB and FCH, and between FCH and FC, in the DEY1433 strain (data not shown). TAF did not inhibit the uptake of FOB, FC or FCH (data not shown). Our kinetic parameters for the uptake of TAF and FCH were very similar to those published by Yun et al. (2000b
) (data not shown).
Differences in the regulation of siderophore transport and reductive iron uptake
In a previous study (Lesuisse et al., 1998
), we presented evidence that the transport of siderophore is regulated in an Aft1p-independent manner. Our conclusion arose from analysis of a mutant strain obtained by EMS mutagenesis. This strain was identified as an aft1 mutant by complementation with AFT1 on a centromeric plasmid (data not shown). However, a recent study (Yun et al., 2000b
) showed unambiguously that our conclusion was wrong: the SIT1 gene and homologues are actually regulated by Aft1p. We are currently trying to understand how a point mutation in AFT1 could lead to inactivation of the reductive pathway of iron uptake without affecting the non-reductive uptake of siderophores. The specific aspects of Aft regulation of siderophore transport will be discussed in another paper (P.-L. Blaiseau, E. Lesuisse & J.-M. Camadro, unpublished). We confirm here that deletion of AFT1 completely abolished the transport of FOB, FC, FCH and TAF by the cells (data not shown). But the reductive and non-reductive pathways of iron uptake in S. cerevisiae were not regulated identically. First, full induction of both siderophore uptake and ferrous iron uptake did not occur under the same growth conditions. We previously reported (Lesuisse et al., 1998
) that the induction of ferrous transport under iron deprivation was much higher than the induction of siderophore transport under the same conditions of growth in an iron-deficient medium. This was true when cells that had been cultured in a complete medium were washed and transferred to an iron-deficient medium for 6 h before measuring iron uptake from Fe2+ or FOB. The cell ferrireductase activity was then maximum and the rate of ferrous iron uptake was 10- to 30-fold higher than in cells grown in an iron-rich medium. Under the same conditions, the rate of FOB uptake was only increased fivefold (Lesuisse et al., 1998
). However, when cells were cultured in iron-deficient medium only, there was a much greater increase in siderophore uptake. Thus, the induction of non-reductive uptake of siderophore seems to require more stringent conditions than reductive uptake (Table 4
); the assumption that siderophore uptake is non-reductive is justified by our use of low (1 µM) siderophore concentrations (see Introduction). Table 4
shows that a long period of iron deprivation (culture and preculture) was needed for the full induction of FOB transport, while reductive uptake of ferric citrate was maximum after only 6 h of iron deprivation. We previously observed that cell ferrireductase activity seemed to depend more on the flux of iron entering the cells in exponential-growth phase than on the absolute iron content of the cells (Lesuisse & Labbe, 1989
). This could account for the high rate of reductive iron uptake by iron-replete cells subjected to iron-deprivation for short periods. Table 4
also shows that iron in the growth medium was more efficient at repressing reductive and non-reductive iron uptake when presented as ferric citrate than as FOB. This suggests that the intracellular processing of the two forms of iron is different.
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Table 4. Rates of iron uptake from ferric citrate or FOB by S. cerevisiae cells precultured and cultured under various conditions
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The Ssn6-Tup1 repressor forms one of the largest and most important gene-regulatory circuits in yeast. In previous unpublished experiments, we observed an effect of TUP1 deletion on cell ferrireductase activity. We checked if TUP1/SSN6 expression could influence reductive and/or non-reductive uptake of iron. The relative dependence of the two pathways on these transcription factors indicated differences in the regulation of reductive and non-reductive iron uptake pathways in S. cerevisiae. The reductive uptake of ferric citrate was decreased in strains with disrupted TUP1 or SSN6 genes (Fig. 2
). The opposite was true for the non-reductive uptake of siderophores, especially for that of the FCH-type siderophores (Fig. 2
). The reductive uptake of ferric citrate by
tup1 or
ssn6 strains was decreased under iron-sufficient conditions and unchanged by iron deprivation (compared to the wild-type grown under iron-sufficient conditions). Under the same conditions, the non-reductive uptake of FC and FCH was constitutively activated (Fig. 2
). TAF uptake did not respond the same way as FC/FCH uptake to TUP1/SSN6 disruption. The uptake of TAF was completely repressed in
tup1 or
ssn6 mutant cells grown in iron-rich medium, and was significantly decreased (compared to the wild-type) in the same mutants grown in iron-deficient medium (Fig. 2
).

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Fig. 2. Effect of disrupting TUP1 and SSN6 genes on reductive uptake of ferric citrate and non-reductive uptake of siderophores in S. cerevisiae. Wild-type, tup1 and ssn6 cells were cultured overnight in iron-deficient medium (-Fe; without iron but with 1 µM copper), or in iron-rich medium (+Fe, same medium plus 10 µM ferric citrate). In the morning, cells were diluted 10-fold in the same medium and cultured for 6 h before iron uptake (1 µM) was measured as described in Methods. , Wild-type+Fe; , wild-type-Fe; , tup1+Fe; , tup1-Fe; , ssn6+Fe; , ssn6-Fe. Representative data from one of six independent experiments are shown.
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It has been suggested that members of the Sit family function as H+/drug antiports (Goffeau et al., 1997
). We therefore compared the effect of extracellular pH on the non-reductive uptake of siderophores (FCH and FC) and the reductive uptake of ferric citrate (Fig. 3
). The effect of pH on reductive and non-reductive uptakes was different. Reductive uptake was much more efficient at low pH, while non-reductive uptake was maximal at pH 67 (Fig. 3
). This result is not conclusive, but it is compatible with the hypothesis that Sit family proteins act as siderophorein/
antiports.
Intracellular fate of iron
The gain-of-function AFT1up mutation causes cells to be constitutively activated for the reductive transport of iron (Yamaguchi-Iwai et al., 1995
). It had the same effect on the non-reductive uptake of FC, FCH, FOB and TAF (data not shown). AFT1up mutants seemed to accumulate siderophores inside the cells as the undissociated ferric complex (Fig. 4
). AFT1up cells grown on a FOB-containing plate had the colour of the siderophore itself. The same observation was previously made with haem-less mutants (Lesuisse & Labbe, 1989
). There are two possible explanations for these observations. One is that the siderophore itself acts as an iron-storage form, as shown for the fungus Neurospora crassa (Matzanke et al., 1987
). The cells would then accumulate large amounts of the ferric siderophore as a direct consequence of the deregulation of transport. The other is that AFT1up cells are defective at some step in the intracellular decomplexation of siderophores. A haem component could be involved in this dissociation step, which would explain why the same result was obtained with haem-less mutants (Lesuisse & Labbe, 1989
). We transformed a
aft1 strain with a plasmid bearing the AFT1up ORF under the inducible promoter of GAL4. The cells were grown on a galactose medium containing a large excess of FOB (100 µM), harvested, washed with water and placed in an iron-deficient medium with galactose or glucose as the carbon source for 24 h. We then recorded the low-temperature spectra of the washed cells (Fig. 5
). Cells placed in the glucose medium (AFT1up expression repressed) showed a spectrum similar to that of the wild-type, indicating that most of the intracellular FOB was dissociated. In contrast, cells placed in galactose medium still showed a high absorbance peak in the range 420440 nm, indicating that the FOB remained undissociated inside the cells (Fig. 5
). The total iron content of the cells were identical (not shown). This suggests that Aft1p is involved in the intracellular decomplexation of siderophores. Another observation supports the hypothesis that ferric siderophores are normally not accumulated inside the cells: in cells deleted for the YFH1 gene, reductive iron uptake is constitutively increased, iron accumulates into the mitochondria and is toxic (Babcock et al., 1997
). The non-reductive uptake of FOB or FCH (but interestingly not of TAF) is also constitutively increased in
yfh1 mutants (data not shown). If FOB/FCH were mainly accumulated inside the cells as the undissociated form, one would expect these siderophores to be less toxic than ferric citrate. But the opposite was true (Fig. 6
), which could be due to the fact that the uptake rate for FCH was higher than that for ferric citrate. We used this property to select new mutants whose siderophore uptake/accumulation was affected. Aft1p is known as a transcriptional activator, not as a repressor (Yamaguchi-Iwai et al., 1995
). Thus, we needed to know how overexpression of AFT1up could lead to a defect in siderophore use by the cells. One explanation is that Aft1p activates a gene whose product is involved in the intracellular storage of siderophores in a compartment where decomplexation of the molecule cannot occur. We investigated the intracellular storage of siderophores using fluorescent derivatives of FCH, as previously described for Ustilago maydis (Ardon et al., 1998
). Wild-type cells took up very little of the fluorescent probes, so that we could not see any signal by confocal fluorescence microscopy (data not shown). But an AFT1up strain took up fluorescent material, which was seen in peri-vacuolar structures (Fig. 7
). Fractionation on a Ficoll gradient showed that most of the 55Fe-labelled FOB taken up into AFT1up cells was in the same fraction as the late endosome marker Pep12 (Fig. 8
). Unexpectedly, much less iron was associated with the vacuolar fraction, while a significant peak of iron co-migrated with the mitochondrial fraction (Fig. 8
). Yun et al. (2000a)
recently showed that Sit1p was mostly associated with an internal compartment (probably late endosome), unlike the Fet3pFtr1p complex (components of the reductive uptake system), which was mostly associated with the plasma membrane fraction. Therefore, we tested the possibility that the uptake of siderophore depended on endocytosis. We tested the capacity of a thermosensitive act1 strain, unable to carry out endocytosis at 37 °C (Kubler & Riezman, 1993
), to reductively take up iron from ferric citrate, or non-reductively from FOB (each 1 µM), after growing in various media (complete medium, minimum iron-deficient medium, minimum iron-rich medium) and incubation at 28 or 37 °C in the presence of 55Fe. The reductive uptake of ferric citrate by the act1 strain was somewhat lower than that of the wild-type, but its non-reductive uptake of FOB seemed to be unaffected. Data from a typical experiment were as follows: cells of the wild-type strain and of the act1 mutant were cultured for 6 h at 28 °C in iron-deficient medium and than shifted to 37 °C for 30 min before iron uptake was measured (at 37 °C). The uptake rate of ferric citrate was 9·4±1·1 pmol min-1 (OD600 unit)-1 for the wild-type and 6·9±0·8 pmol min-1 (OD600 unit)-1 for the act1 mutant. These values were, respectively, 2·0±0·15 and 2·8±0·2 pmol min-1 (OD600 unit)-1 for the uptake of FOB (mean±SE from three experiments). The transport of TAF, FC and FCH was similarly unaffected in the act1 mutant at the non-permissive temperature (data not shown). We conclude that siderophore uptake does not require Act1-mediated endocytosis.

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Fig. 4. Accumulation of FOB inside AFT1up mutant cells of S. cerevisiae. AFT1up (a) and wild-type (b) cells were plated on minimum agar medium containing 10 µM FOB and grown for 3 d at 30 °C.
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Fig. 5. Low-temperature spectra of S. cerevisiae cells loaded with FOB and induced (A) or repressed (B) for the expression of the AFT1up gene. aft1 cells were transformed with a multicopy plasmid bearing the AFT1up ORF under the GAL4 promoter. Cells were grown for 24 h in a YNB-galactose medium containing 100 µM FOB, washed with distilled water and transferred to an iron-deficient medium (without iron and copper) with either galactose or glucose as the carbon source and grown for 24 h. Cells were then washed and low-temperature spectra were recorded.
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Fig. 7. Confocal micrographs of S. cerevisiae M2p. Cells were grown for 24 h with 10 µM of a B9 fluorescent analogue of FCH (Ardon et al., 1998 ). Fluorescence is concentrated in vesicles within the cells. Superimposition of the pictures recorded by phase-contrast microscopy (a) and by confocal fluorescence microscopy (b) shows that the fluorescent material lies around the vacuole (c). Bars, 5 µm (bar in a also applies to b).
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Fig. 8. Intracellular distribution of iron in an AFT1up strain of S. cerevisiae loaded with 55Fe-labelled FOB. Cells of the strain M2p were grown overnight in YNB-raffinose medium containing 100 µM FOB, lysed and the cell lysate separated on a discontinuous Ficoll gradient. The total amount of iron in each fraction (numbered 1 to 8 from top to bottom) was measured. Samples of the same fractions were used for Western blotting experiments, in which markers of mitochondria (ferrochelatase, A), of the vacuole (vacuolar ATPase, B) and of the late endosome (pep12p, C) were revealed by specific antibodies.
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DISCUSSION
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S. cerevisiae has proven a very good model for studying reductive iron uptake in eukaryotes (reviewed by Askwith & Kaplan, 1998
) and it will probably help us to better understand non-reductive iron uptake as well, even though it does not itself produce any siderophores. We previously showed that disruption of SIT1 blocked FOB uptake by S. cerevisiae (strain DEY1433) without affecting the transport of FC (Lesuisse et al., 1998
). However, there is competition for the transport of the two siderophores. This suggested that the uptake of a given siderophore is very specific and that some step common to several siderophores are upstream or downstream of the transport system itself (Lesuisse et al., 1998
). Here again, we found that there is competition between FOB, FC and FCH. Like Yun et al. (2000b
), we found that siderophore transport by S. cerevisiae is not strictly specific. While FOB and TAF are each taken up via a single transporter (Sit1p and Taf1, respectively), several transporters (especially Sit1p and Arn1p) contribute to the uptake of FCH and FC. The apparent specificity is also strongly strain-dependent. Thus, it is not clear whether or not a single protein is responsible for both recognition and transport of siderophores by S. cerevisiae. We are examining this question with in vitro binding experiments on isolated plasma membranes. The fact that the uptake of several siderophores (FOB, FC, FCH) is competitive implies that other proteins are involved in their transport. We are currently trying to identify these proteins by analysing new mutant strains affected in siderophore uptake/use. The present study suggests that the siderophore FOB is associated with an endosomal compartment when it accumulates inside the cells. Another study (Yun et al., 2000a
) showed that Sit1p is also associated with the late endosome compartment. We used AFT1up mutant cells, which accumulate large amounts of siderophores. The intracellular processing of siderophores is probably blocked in AFT1up cells. This would explain why iron was released from FOB within the cells when the AFT1up gene on a plasmid was turned off in a
aft1 strain loaded with FOB. Yun et al. (2000a
) proposed that Sit1p is involved downstream of the uptake system for FOB, which itself could involve endocytosis. However, this possibility can be ruled out since a mutant completely blocked for endocytosis showed no defect in siderophore uptake. In addition, involvement of Sit1p downstream of the uptake system would not explain why disruption of SIT1 completely abolished FOB uptake in some strains. Sit1p is more likely to act as a transporter at the cell surface and be rapidly recycled by internalization. Nothing is known about the intracellular processing of siderophores in S. cerevisae. Preliminary studies suggest that a haemoprotein is involved in the reductive decomplexation of siderophores inside the cells (Lesuisse & Labbe, 1989
). This could involve one of the Fre1p/Fre2p homologues. Many other aspects of siderophore metabolism in S. cerevisiae remain to be clarified, including the energetics of transport, the relationship between recognition and transport, and its regulation. These questions are under investigation in our laboratory.
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ACKNOWLEDGEMENTS
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We thank Dr G. Winkelmann for kindly donating ferricrocin, Dr A. Shanzer for providing the fluorescent ferrichrome analogues and Dr H. Haas for donating triacetylfusarinine C. This work was supported by grants from CNRS and Ministère de la Recherche et de lEnseignement Supérieur (Programme de Recherches Fondamentales en Microbiologie et Maladies Infectieuses). We thank G. Géraud for his help in confocal microscopy studies and Dr O. Parkes for checking the English text.
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Received 18 July 2000;
revised 26 October 2000;
accepted 3 November 2000.