Journal of General Microbiology (1 987), 133, 3229-3236.
Printed in Great Britain
3229
Iron Uptake by the Yeast Sacchavomyces ceveuisiae: Involvement of a
Reduction Step
By E . L E S U I S S E , * F . R A G U Z Z I A N D R . R . C R I C H T O N
Universitk Catholique de Louuain, Uniti de Biochimie. Place Louis Pasteur 1,
1348 Louvain-la- Neuoe, Belgium
(Receiiied 3 February 1987; rerised 15 June 1987)
Among several parameters affecting the rate and amount of iron uptake by Saccharomyces
cerevisiae, the oxidation state of iron appeared to be determinant. Iron presented as Fe(I1) was
taken up faster than Fe(II1) and the kinetic parameters were different. Iron was taken up by the
cells from different ferric chelates, at rates that did not depend on their stability constants, and
uptake was strongly inhibited by an iron(I1)-trapping reagent like ferrozine. Iron was
physiologically reduced by a transplasmamembrane redox system, which was induced in irondeficient conditions. We propose that iron must be reduced to be taken up by the cells in the
same way as other divalent cations.
INTRODUCTION
It is well established that iron is an essential element for the growth of yeast, but its uptake by
Sarcharomyces cerecisiae has not been described. Yeast cells take up many divalent cations, such
as Mg'+ (Conway & Beary, 1958), Zn' (White & Gadd, 1987), Co2+, Ni'+ (Fuhrmann &
Rothstein, 1968), Ca2+(Kovae, 1985), Cd'+ (Norris & Kelly, 1977) and Sr2+(Roomans et al.,
1979) and reciprocal interactions occur in the uptake of several of these cations (Borst-Pauwels,
198I ) ; cobalt can induce an iron-stress response, either in plants (Blaylock et al., 1985) or in yeast
(Light & Clegg, 1974), and interactions are observed between zinc and iron metabolism
(Lawford et al., 1980). It thus seems theoretically possible that iron could be taken up as Fez+in
the same way as other divalent cations, involving perhaps a common mechanism for the uptake
of several different ions. Kovalev et al. (1985) showed that the intracellular iron content in S .
cerecisiae was determined by the concentration of iron(I1) in the medium. However, in aerobic
media, iron generally exists as its insoluble ferric form. It can become available to the cell
principally by two inducible mechanisms. A variety of bacteria, fungi and monocotyledonous
plants excrete specific iron-chelating compounds (siderophores or phytosiderophores) which
remove iron from insoluble ferric forms, and therefore facilitate its transport into the cells
(Raymond et al., 1984). Another common strategy, predominant in dicotyledonous and some
monocotyledonous plants, consists in maintaining iron in a soluble form by excreting protons
and/or reducing agents (Olsen & Brown, 1980).Iron is then taken up either as ferric chelate or as
Fez+after reduction by a transmembrane electron transport system with cytosolic NAD(P)H as
electron donor (Bienfait, 1985).
Plasma-membrane-bound redox systems have been detected in several eukaryotic cells
(Goldenberg, 1982); in S. cerecisiae such a system, reducing extracellular ferricyanide, has been
described but the physiological electron acceptor has not been identified (Crane et al., 1982;
Yamashoji & Kajimoto, 1986).
Abhretiution: BPS, Bathophenanthroline sulphonate (4,7-diphenyl-l, 10-phenanthrolindisulphonic acid,
disodium salt).
0001-3981 0 1987 SGM
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3230
E . LESUISSE, F . R A G U Z Z I A N D R . R . CRICHTON
In this paper, we describe iron uptake by S . cereuisiae. We show that a reduction step is
involved in the uptake mechanism, and propose that iron is a natural electron acceptor of the
plasma membrane redox system.
METHODS
Growth conditions. Saccharornyces cereuisiae D261 was grown aerobically at 30 "C in liquid nutrient medium.
Complete medium contained (in 1 litre of water): 20 g yeast extract, 2 g KH2P04,2 g (NH,)2S04 and either 30 g
glucose or 30 g ethanol plus 1 g glucose or 30 g pyruvate plus 1 g glucose. Synthetic medium was as described by
Van Steveninck & Booij (1964); for cultures in iron-deficient conditions, iron was removed from synthetic medium
as described by Nicholas (1957). Controls were done with the same iron-deficient medium containing ferric citrate
(0.1 mM or 1 mM), to ensure that any change was due to iron stress. Culture vessels were previously washed with
nitric acid.
The sterilized medium was inoculated with 5 x lo6 cells ml-1 from 48 h precultures (in the same medium) and
the cells were harvested either in the exponential or stationary phase of growth by centrifugation. The cells were
washed three times with distilled water and resuspended at 10 mg ml-l in distilled water under aeration for 4 h
starvation at 30 "C. The washed cells were stored at 4 "C and used for experiments for about 5 d. When necessary,
the number of cells was estimated by microscopic observation on a numeration plate. All experiments were made
with non-growing cells. Yeast weight is expressed as wet weight of packed cells.
Iron compounds. 59Fe(III)4trate, 5sFe(III)Cl, and (NH,)2s9Fe(II)(S04)2were diluted with unlabelled iron to
give about 50 Bq (nmol iron)-'. Ferric chelates were prepared according to Chaney et al. (1972). Desferricompounds were obtained according to Wiebe & Winkelmann (1975).
Iron uptake. The washed yeast cells were suspended at 100 mg ml-' in 50 mM-trisodium citrate buffer, pH 6.5.
After a variable preincubation time at 30 "C with substrates and salts, radioactive iron was added; incubations
were done in the dark, in open glass flasks. At the indicated times, samples (1 ml) were withdrawn and added to
1 ml of an unlabelled iron solution [0-2%, w/v, Fe(I1) or Fe(III)]. The samples were centrifuged and washed twice
with 0.2% (w/v) Fe(I1) or FE(II1) (as ferrous ammonium sulphate or ferric citrate), four times with 2% (w/v)
EDTA in 50 mM-citrate buffer (pH 6.5) and twice with distilled water. The washings with EDTA could be
replaced by two washings with dithionite (100 mM). Iron uptake by the cells was measured by counting either the y
or p radioactivities of the washed samples.
Iron reduction. Iron reduction was followed with an Aminco DW-2A spectrophotometer, using the dual
wavelength mode. The formation of the Fe(I1)-(BPS), complex from Fe(1II)-citrate or Fe(II1)-EDTA plus BPS
was measured by following the changes in absorbance AA(A5,0 from ferrioxamine B or ferricrocin plus
BPS, it was measured by following AA(As,o - A430)or A L I ( A ~, ~A440), respectively; the formation of
ferrocyanide from ferricyanide was measured by following AA(A,oo - ASoo).The AE values were calculated in
each case from standard curves. The cells were suspended at 10 mg ml-1 in 50 mwsodium citrate buffer (pH 6.5)
and preincubated at 30 "C in thermostated quartz cells for the appropriate time with substrates and salts; Fe(II1)
complexes and BPS were then added simultaneously (or ferricyanide without chelator) and the absorbance values
were recorded on the spectrophotometer. Oxygen consumption by the cells was followed by using a Clark-type
oxygen electrode.
RESULTS A N D DISCUSSION
Iron uptake
If added to a suspension of S . cereuisiae preincubated with a suitable carbon source, iron was
taken up by the cells into a non-exchangeable pool, but several factors affected the amount and
rate of uptake (Table 1): the carbon source used for growth, the growth-phase in which the cells
were harvested, and the energy source supplied to non-growing cells. If iron was furnished
during growth, it was preferentially accumulated by glucose-grown cells : the amount of iron
accumulated from 0.36 mM Fe(II1)-citrate after 24 h of growth on glucose, ethanol or pyruvate,
was respectively 13-7, 0.63 or 0.1 3 mmol (kg wet wt)-] (mean of two experiments). However,
cells grown on pyruvate or ethanol (respiratory metabolism) had a higher potential capacity to
take up iron than glucose-grown cells (fermentative metabolism; Polakis et al., 1965), but
glucose was the best energy source for the transport of iron into the cells since fermentation of
glucose by ethanol- or pyruvate-grown cells produced the highest rates of iron uptake (Table 1).
The effect of CN- and of anaerobiosis (Table 1) showed that only a little of the energy used for
uptake could be derived from respiration, even though a respiratory type of metabolism implies
an increased need for iron. The maximal uptake activity was observed in cells harvested in
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323 1
Iron uptake by Succharomyces cererisiae
Table 1. EJhct of'dlJferentfactors on iron uptake .from 0.36 mM-Fe(IIfl-citrate bql S . cereuisiae
Cells growing on 3:< (w/v) glucose, ethanol or pyruvate were harvested either in exponential (E) or in
stationary (S) growth. The washed cells were preincubated at 100 mg wet wt ml-' at 30 'C with 5%
(w/v) glucose, ethanol or pyruvate, 30 min before the addition of iron. Incubations with iron were done
either in open glass flasks with magnetic stirring (aerobic conditions, AE I), in open glass flasks with
oxygen bubbling (aerobic conditions, AE 2) or in closed syringes, with the incubation medium
previously degassed (anaerobic conditions, ANA). When present, K C " and 2,4-dinitrophenol (DN P)
were added to the incubation medium just before iron. Results are the means of three experiments with
maximal deviations of 2004.
Growth phase
of cells
Energy source
for uptake
E
E
E
E
E
E
E
E
E
S
Glucose
Ethanol
Pyruvate
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
Substrate
added
AE 1
AE 1
AE 1
AE 1
AE 1
AE 1
AE 2
ANA
AE 1
AE 1
__
-
-
KCN (2mM)
KH2P0, ( 5 0 m ~ ) *
KH2POA (SO mM)t
.-
DNP (20pM)
-
* Present during preincubation only.
t Present during preincubation and during incubation
r
Iron uptake rate [pmol min-I
(kg wet wt)-']
by washed cells grown o n :
A
3
glucose
ethanol
pyruvate
0.6 1
0.04
0.02
0.67
2.95
0.30
0.29
0.69
0.56
0.15
2-52
0.29
0.10
1-75
3.94
1.59
2.23
247
2-41
1.26
2.2 1
0.17
0.20
1 -49
2.15
with iron.
exponential growth-phase (Table 1); the optimal pH was about 6-6.5, the rate of uptake
decreasing by about 55% at pH 4-5 (not shown).
Uptake was stimulated when cells were preincubated with glucose and phosphate (KH2PO,)
but washed free of residual phosphate before the addition of iron; in contrast, the presence of
phosphate during the incubation had an inhibitory effect (Table l), which could partly be
explained by the inhibitory effect of K + (not shown). Since phosphate had to be accumulated in
cells to stimulate iron uptake, it seems unlikely that it could act by way of a phosphate-iron
cotransport mechanism. The stimulatory effect could be due to the increase in the negative
charge of the cell membrane occurring when cells were loaded with phosphate (Borst-Pauwels &
Theuvenet, 1984), or to the involvement of a phosphorylation step in the iron transport process.
The iron uptake rate was also greatly dependent on the oxidation state of iron (Fig. 1). Uptake
from Fe(II1) was very low and required a lag period of more than 60 min to reach maximal rate.
In contrast, the kinetics of uptake from Fe(IT), which would not be oxidized at the low oxygen
content of the suspensions, were similar to the kinetics of uptake by S. cererisiae of other
divalent cations like Co2+,Ni2+and Zn2+(Fuhrmann & Rothstein, 1968). Below approximately
0-5 mM-Fe(III), the apparent values of K , and V,,,, were respectively 64 p~ and 3-6 kmol iron
min-I (kg wet wt)-*. For Fe(II), below 0.1 mM, these values were 274 PM and 150 pmol iron
min-' (kg wet wt)-'. The latter value is of the same order of magnitude as that [80 pmol min-I
(kg wet wt)-'] given by Fuhrmann & Rothstein (1968) for the uptake of Co2+.Nevertheless, our
buffer system (citrate) had chelating properties in order to maintain iron in solution so that, for a
real comparison of kinetic parameters, it would be necessary to introduce corrections according
to the stability constants of ferric and ferrous complexes in solution and at the yeast cell surface.
Cobalt acted as a competitive inhibitor in the uptake of Fe(I1) ( K , = 195 PM), but had no
inhibitory effect on the uptake from Fe(II1). Moreover, uptake from Fe(II1) showed deviations
from Michaelis-Menten kinetics [above 0.5 mM-Fe(III), the apparent K, and V,,, values were
840 p~ and 6.24 vmol min-' (kg wet wt)-')] and no competitive inhibition by Ga(II1) was
observed; in contrast, Ga(II1) decreased the apparent K , of uptake: above 0.5 mM-Fe(III) the
apparent K , and V,,, values in the presence of 1.43 mM-Ga(III) were 453 p~ and 6.24 pmol
min-' (kg wet wt)-l.
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3232
E . LESUISSE. F . R A G U Z Z I A N D R . R . C R I C H T O N
100
200
Time (min)
100
200
Time (min)
Fig. 1
Fig. 2
Fig. 1. Time course of iron uptake from Fe(I1) as ferrous ammonium sulphate (0)
or Fe(II1) as ferric
citrate ( 0 )by glucose-grown cells. Conditions of incubation: iron, 0.36 mM; glucose, 5% (w/v); 30 "C.
The cells were preincubated for 30 min with 5% (w/v) glucose.
Fig. 2. Uptake of ferrioxamine B by ethanol-grown cells. Preincubation (30 min) and incubation were
done at 30 "C with 5% (w/v) glucose. During incubation, iron (0.4 mM) was presented as 59Fe-labelled
ferrioxamine B or as IT-labelled ferrioxamine B. The uptake of 59Fe (0)
and of I4C ( 0 )was
measured.
Deviations from Michaelis-Menten kinetics could be explained by the decrease of the
negative surface potential accompanying the increase of cation concentration, since the
presence of negative groups near the cation binding sites of the cell surface results in an
accumulation of cations in that region (Borst-Pauwels, 1981). This effect was not observable on
the uptake of Fe(II), probably because of the low iron concentrations used in this case (10 to
1 0 0 p ~ )The
.
fact that Ga(II1) increased iron uptake rates from Fe(II1) could mean that the
Fe(II1) binding sites at the cell surface were not uptake sites : Ga(1II) could displace Fe(II1) from
non-specific binding sites and therefore increase the concentration of iron available for the
uptake. Thus, it seems unlikely that the yeast cell surface exhibits high affinity binding sites for
the transport of Fe(II1).
The cells were able to take up iron from several ferric chelates and in particular from
extremely stable complexes like ferricrocin and ferrioxamine B, two microbial siderophores
with stability constants of 1030-5and
respectively (Raymond et al., 1984). However, as
shown in Fig. 2 for ferrioxamine B, iron was taken up while the desferricompound did not enter
the cells. Moreover, when added in stoichiometric concentration, the iron(I1)-trapping reagent
ferrozine strongly inhibited iron uptake from ferrioxamine B (> 80%inhibition), as well as from
ferric citrate (>70% inhibition). Our results thus indicate that a reduction step is involved in
iron uptake, as in dicotyledonous plants (Bienfait, 1985). It seems unlikely that S . cereuisiae
possesses a specific uptake system for the transport of iron in the trivalent state; such a system
has never been described in S . cerevisiae, even though Kunst & Roomans (1985) showed that
chromium ions could be accumulated by the cells.
Reducing acticity of' the cells in basal conditions
Cells grown on complete media (basal conditions) showed an appreciable reducing activity
for Fe(II1) [about 20 pmol Fe(II1) reduced min-I (kg wet wt)-'] when incubated at 30 "C with a
ferric chelate, an iron(I1)-trapping reagent like BPS and a metabolizable substrate like glucose.
Reduction rates reached maximal values at pH 6 and were decreased by about 55% at pH 4.5.
Like iron uptake, the role of respiration in iron reduction was not obvious: 1 mhl-cyanide or
arsenite did not inhibit reduction; the rate of ferric citrate reduction by starved cells [3.6 pmol
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3233
Iron uptake by Sacchhromyces cerevisiae
Table 2. Rates of' iron reduction and uptake by S . cerevisiae from diflerent iron chelates
Ethanol-grown cells were preincubated at 10 mg wet wt ml-l (for reduction assays) or at 100 mg wet wt
ml-' (for uptake assays) with 5 % (w/v) glucose at 30 "C. Iron (0.36 mM) was added after either 3 min of
preincubation (for reduction assays) or 30 min (for uptake assays). When present, ferrozine was at
1 mM. Iron reduction was followed spectrophotometrically after the addition of 1 mM-BPS. Iron uptake
was followed by measuring the accumulation of 59Fe within the cells. Results are the mean of three
experiments with maximal deviation of 25%.
Iron
chelate
1% Kf
Fe(II1bitrate
Fe(II1)-citrate
ferrozine
Fe(II1tEDTA
Ferrioxamine B
Ferrioxamine B
ferrozine
Ferricrocin
Ferricyanide
Fe(I1)-ci tra te
+
+
Reduction rate
Uptake rate
[pmol min-' (kg wet wt)-']
11.8
20.4
2.9
25.1
30.5
15
14.1
0.87
0.88
2.27
30.4
13.4
143
3.08
0.37
2.52
42
min-' (kg wet wt)-' ; mean of three experiments] was increased by more than 450% after the
addition of 5 % (w/v) glucose, and by about 75% after the addition of 5% (w/v) ethanol or
pyruvate, whatever the carbon source used for the growth of cells; ethanol- and pyruvate-grown
cells had higher reducing activity than glucose-grown cells. In each case, maximal iron reduction
rates were observed from cells in exponential growth (not shown). On the other hand, the rate of
iron reduction depended on the nature of the ferric chelate used. In Table 2, we present some
values of iron reduction rates with the corresponding values for the uptake of different iron
complexes. The data should be considered as apparent rates since two factors should be taken
into account for the calculation of real rates: first, the influence of an iron(I1) chelator in
solution, which modifies the redox potential of Fe(II1) and therefore leads to an overestimation
of reduction rates; secondly, the presence of non-specific cation binding sites at the cell surface
(Van Steveninck & Booij, 1964), which could result in a decrease in the effective concentration
of cations available for transport into the cell (Borbolla & Pefia, 1980) and/or for reduction.
Nevertheless, iron uptake rates did not depend on the stability constants of the ferric chelates
used, while the stability constants of the ferrous chelates seemed to be more important (Table 2) :
iron uptake occurred from ferric complexes as stable as ferrioxamine B or ferricrocin but was
inhibited in the presence of ferrozine, a strong Fe(I1)-chelator, and EDTA, which chelates both
Fe(I1) (log K,= 14.3) and Fe(II1) (log K , = 25.1).
As in plants, the substrate specificity of the reduction system was low (Bienfait, 1985), but
while plant roots are unable to reduce ferrioxamine B (Bienfait et ul., 1983), yeast cells reduced
ferricrocin as well as ferrioxamine B (redox potentials of -412 and -468 mV respectively;
Raymond et al., 1984). Together with Fig. 2, this result indicates that, when used as iron source,
the iron-siderophore complex was dissociated by reduction outside the cells before uptake.
Nature of' the redox system ; inducibility in iron-de$cient conditions
The cells showed two distinct reducing activities, both of which were energy- and
temperature-dependent. Part of the iron(II1)-reducing activity of the cell suspension was
recovered in solution after the removal of cells by centrifugation (Fig. 3) and corresponded to the
excretion, as in certain plants (Brown & Ambler, 1973), of a reductant into the extracellular
medium. The excretion was stimulated by incubation of cells at 30°C with glucose, but it
became significant only after an incubation time of more than 30 min. Another part of the
reducing activity was bound to the cells and was maximally stimulated about 2 min after the
addition of glucose. It corresponded to the NAD(P)H-dependent plasma-membrane-bound
redox system previously described as ferricyanide reductase (Ramirez et al., 1984). Plasma
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3234
E . LESUISSE, F . RAGUZZI A N D R . R . CRICHTON
20
40
60
Time (min)
80
Fig. 3. Effect of incubation time with glucose on the initial reduction rates by whole cells ( 0 )or by
extracellular reductant (0).
Glucose-grown cells were washed three times and incubated with 5 % (w/v)
glucose. At the times indicated, the reductive activity of the whole suspension ( 0 )and of the
supernatant of centrifuged samples (0)
was measured on 0.36 mwferric citrate in the presence of
1 mM-BPS.
Table 3 . Rates uf' iron reduction and uptake and uj'oxygen consumption (cyanide-insensitice)by
S . cerecisiae grown on sjxthetic medium ( 3o/o, w / u , glucose) with diflkrent iron concentrations
Cells were harvested in stationary phase, after 24 h of growth, starved for 4 h in distilled water and
washed three times with distilled water. Inhibition of growth was estimated by counting the cells just
before harvesting. Iron reduction was measured after the addition of 0.36 mM-Fe(III)--citrate plus
1 mM-BPS to a suspension of cells (10 mg wet wt ml-I) previously preincubated for 2 min with glucose
(5%, w/v). Oxygen consumption was followed after bubbling oxygen for 2 min through a suspension of
cells ( 5 mg wet wt ml-') in glucose ( 5 % , w/v) and 1 mM-KCN. Iron uptake was measured after the
addition of 0.36 mM-5~Fe(III)-citrateto a suspension of cells (100 mg wet wt ml-I) preincubated for 30
min with glucose ( 5 " / , w/v). All experiments were done at 30 "C.
(03
Iron
reduction
rate
[pmol m i r '
(kg wet wt)-'1
10
0
70
5
14
600
Ir
Iron (ferric citrate)
content of the medium
1 mM
0.1 r n M
Iron-deficient condition
Growth
inhibit ion
Oxygen
consumption
rate
[pmol min-'
(kg wet wt)-'1
13-2
5.8
0.22
Iron
uptake
rate
[pmol min-'
(kg wet wt)-'1
049
0.22
3.8
membranes were isolated from a whole homogenate of yeast protoplasts, as described by
Schibeci et al. (1973), and tested for their ability to reduce 0-3~ M - F ~ ( I I I ) - E D TinAthe presence
of 1 mM-BPS and 0-5 mM-NADH; while the specific activity of the membrane marker Mg2+ATPase was enriched 7-9-fold with respect to the whole homogenate, the specific activity of the
Fe(II1) NADH-dependent reductase was enriched 6-7-fold [from 2 nmol Fe(II1) to 13 nmol
Fe(II1) reduced min-' (mg protein)-' ; mean of two preparations].
Crane et al. (1982) proposed oxygen as the natural electron acceptor of this membrane-bound
redox system. They showed that the reduction of ferricyanide was accompanied by increased
release of protons by the cells and proposed that this activity could be coupled with the transport
of nutrients like amino acids. Our results indicate that iron could act as physiological electron
acceptor of the redox system, as shown by Bienfait (1985) for plants. Cells were cultured in
synthetic media with different iron concentrations, harvested after 24 h, in the stationary
growth phase, and tested for their ability to utilize iron(II1) or oxygen as electron acceptor and to
take up iron from ferric citrate. The ferrireductase activity of the washed cells was highly
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Iron uptake by Saccharomjws cerezisiae
3235
correlated with the iron content of the growth medium (Table 3); in iron-stressed cells, the
ferrireductase activity increased more than Wfold, to reach a maximum of about 600 pmol
ferric citrate reduced min-’ (kg wet wt)-’. On the other hand, the increase in the ferrireductase
activity was accompanied by a decrease in the ability of cells to utilize oxygen as electron
acceptor (cyanide-insensitive oxygen consumption, independent of mitochondria1 respiration),
and by an increase in the rate of iron uptake from Fe(II1) by the cells in non-growing conditions.
In contrast, the amount of reductant released by the cells into the extracellular medium was
not significantly affected by iron-deficient conditions (not shown) and thus its physiological
function remains conjectural : it could theoretically prevent reoxidation of Fe( I I ) or solubilize
Fe(III), or have nothing to do with iron metabolism.
From the data of Table 3, we conclude that there exists in yeast, as in plants (Bienfait, 1985),
an inducible transplasmamembrane redox system using ferric chelates as physiological electron
acceptors. Whether or not oxygen could act as a natural electron acceptor of such a system in
iron-sufficient conditions remains to be clarified. In any event, the iron status of the cells should
control the reductive capacity of the redox system, which itself appears to be one of the most
important features involved in the regulation of iron uptake by S . cerecisiae.
This work was supported by the Fonds National de la Recherche Scientifique (FNRS, Brussels. Belgium). We
thank Professor A. Goffeau and Professor M. Briquet for yeast strains, Professor G. Winkelmann for ferricrocin
and Dr H . K . Peter of CIBA-GEIGY (Basle, Switzerland) for desferal and “T-labelled desferal.
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