From the Department of Biological Chemistry, Weizmann Institute of
Science, Rehovot 71600, Israel
A 150-kDa transferrin-like protein (Ttf) is
associated with the plasma membrane of the halotolerant unicellular
alga Dunaliella salina (Fisher, M., Gokhman, I., Pick, U.,
and Zamir, A. (1997) J. Biol. Chem. 272, 1565-1570).
The Ttf level rises with medium salinity or upon iron depletion.
Evidence that Ttf is involved in iron uptake by Dunaliella
is presented here. Algal iron uptake exhibits characteristics
resembling those of animal transferrins: high specificity and affinity
for Fe3+ ions, strict dependence on carbonate/bicarbonate
ions, and very low activity in acidic pH. Reducing the level of Ttf by
mild proteolysis of whole cells is accompanied by lowered uptake
activity. Conversely, accumulation of high levels of Ttf is correlated
with an enhancement of iron uptake. Kinetically, iron uptake consists
of two steps: an energy-independent binding of iron to the cell surface
and an energy-dependent internalization. Salinities as high
as 3.5 M NaCl do not inhibit iron uptake or decrease the
apparent affinity for Fe3+ ions, implying that Ttf activity
is not affected by high salt. These results indicate that transferrins,
hitherto identified only in animals, are present and function in iron
transport also in plant systems.
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INTRODUCTION |
Transferrins are the universal iron-carrier proteins in animal
cells and function in binding, mobilizing, and delivering ferric ions
(1). However, until recently no eukaryotic type transferrin has been
detected outside the animal kingdom. Typical animal transferrins, are
~80-kDa soluble proteins consisting of two internal repeats, each
forming an iron binding lobe. Serum transferrin transports Fe3+ ions into target cells via receptor-mediated
endocytosis.
Another class of transferrins, exemplified by human melanotransferrin,
p97, is attached to the plasma membrane via a glycosyl phosphatidylinositol anchor and plays a role in receptor-independent iron uptake (2).
Recently, we reported the identification of a transferrin-like protein
in the unicellular green alga Dunaliella salina (3, 4).
Algae belonging to the genus Dunaliella are outstanding in
their ability to proliferate in extremely varied salinities, up to
saturated NaCl. In a search for salt-induced proteins, we identified a
plasma membrane-associated 150-kDa protein, p150, whose level rises
with external salinity. Based on its cDNA-deduced sequence, p150 is
a transferrin-like protein distinct from other transferrins in
containing three rather than two internal repeats. Reflecting its
unique structure, p150 was designated Ttf, for triplicated
transferrin. That the function of Ttf could be
related to iron metabolism was first indicated by the accumulation of the protein in cells grown in iron-deficient media as well as the
in vivo labeling of the protein with 59Fe (4).
Based on these observations, we proposed that Ttf acted in algal iron
uptake and, furthermore, that its induction permitted the cells to
overcome a limitation in iron availability brought about by high
salinity.
The present study examines the role of Ttf in iron acquisition by
measurements of 59Fe uptake into D. salina
cells. The uptake characteristics exhibit features of iron binding to
transferrins in general, and of melanotransferrin-mediated uptake in
particular. These results are intriguing, since there have been no
previous indications for the presence of transferrins or their
involvement in iron acquisition in plants and algae.
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MATERIALS AND METHODS |
Algae and Growth Conditions--
The origin and growth
conditions of D. salina were as described (3-5). The media
contained 0.5 or 3.5 M NaCl. To deplete iron, the cells
were incubated in a medium without Fe3+-EDTA (4).
Iron Uptake and Iron Binding--
D. salina cells
were grown to a density of 2-3 × 106 cells/ml and
harvested by centrifugation. The freshly harvested cells were suspended
in the same medium they were grown in, but without Fe-EDTA, and
preincubated in the light (40 W m
2) at 23 °C for
10-15 min. The iron uptake assay was initiated by the addition of
59Fe3+-citrate (prepared by mixing 10 mM sodium citrate with 30-100 µM
59FeCl3 in 0.1 N HCl, followed by
neutralization with Tris base) to a final concentration of 3-10
µM 59Fe3+ and 1 mM
sodium citrate. The final cell density in the assay was 108
cells/ml. At the indicated times, aliquots of 0.1 ml were removed into
2 ml of ice cold stop solution. The stop solution used for cells grown
in 0.5 M NaCl contained 0.5 M NaCl, 5 mM Na-EDTA and 20 mM
Na-MES,1 pH 5.5; the stop
solution for cells grown in 3.5 M NaCl contained 3.5 M NaCl (or 0.35 M NaCl together with 4.1 M glycerol), 10 mM Na-EDTA and 50 mM Na-MES, pH 6.0. The cells were collected by centrifugation for 3 min at 5000 × g, washed once in
stop solution, and counted in a
-counter. Iron binding was measured
under the same conditions except that the incubation was carried out in complete darkness at 1 °C, the cell aliquots removed contained 2 × 107 cells, and the stop solution contained no
Na-EDTA. (Uptake and binding assays were adapted from studies of
melanotransferrin-mediated iron uptake in animal cells (2)).
Pronase Treatment, Immunoblot Analysis, and ATP
Determination--
Cells were digested with 2 mg/ml Pronase for 1 h at 30 °C essentially as described (4). Immunoblot analysis with
anti-Ttf antibodies was carried out as described previously (3, 4). ATP
content was analyzed by the luciferase assay in a Lumac 3M photon
counter.
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RESULTS |
Kinetic Characteristics of Iron Uptake in Dunaliella--
Iron
uptake activity of D. salina cells was measured by following
the accumulation of 59Fe from
59Fe3+-citrate in standard growth media
containing different concentrations of NaCl. The time course and
temperature dependence of iron uptake at 0.5 M NaCl are
shown in Fig. 1A. At 24 °C,
iron accumulation proceeded for 2-3 h and reached a level of ~6
nmol/109 cells. Very little accumulation occurred at
1 °C, indicating that the uptake was
temperature-dependent. The accumulated iron was not lost
when the cells were treated with Pronase (see below) or when a large
excess of nonradioactive iron was included in the stop solution (data
not shown). These observations support the conclusion that the iron had
been internalized. To determine the dependence on Fe3+
concentration, iron-citrate in the medium was varied from 0.2 to 4 µM (Fig. 1B). iron uptake saturated at ~4
nmol/109 cells. The apparent Km for
iron-citrate, calculated from an Eadie-Hofstee plot (Fig.
1B, inset), is 0.6 µM. Analysis of
the effect of pH on iron uptake (Fig. 1C) indicated a broad pH optimum peaking at approximately pH 8.0 and sharply dropping below
pH 7.0.

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Fig. 1.
Effect of assay conditions on iron
uptake. A, time and temperature. Cells were incubated
with 59Fe3+ on ice or at room temperature for
the indicated times and analyzed for iron uptake as described under
"Materials and Methods." B, dependence on iron-citrate
concentration. Cells were incubated for 1 h with
Fe3+-citrate buffer consisting of 1 mM citrate
and 0.1-20 µM 59Fe3+
(corresponding to free Fe3+ concentrations 3.8 × 10 20 M to 7.6 × 10 18
M (15)). Inset, Eadie-Hofstee plot. The data
represent means of two measurements, (see also Table II). C,
pH dependence. Cells were analyzed for iron uptake in the presence of
50 mM Na-MES (pH 6.0), Na-HEPES (pH 7.8), or Na-Tricine (pH
9.0). Means of three measurements are shown (S.D. < 0.5 nmol/109 cells).
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The specificity of the uptake system for Fe3+ ions was
tested in a competition experiment, whereby Me2+ or
Me3+ ions in 100-fold excess over Fe3+ were
added as citrate complexes to the uptake medium (Table
I). None of the tested metal ions
effectively inhibited iron uptake, suggesting a very high selectivity
of the transport system for Fe3+ ions. The specificity for
Fe3+ over Fe2+ was indicated in uptake assays
conducted in the presence of a synthetic chelator preferably chelating
Fe2+ (bathophenantroline disulfate) or Fe3+
(synthetic trisoxamides). At 10 µM chelator, the uptake
was >90% inhibited by Fe3+ chelators but only 5%
inhibited by Fe2+ chelators (data not shown). These results
indicate a strong preference of the uptake system for Fe3+
over Fe2+ ions.
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Table I
Competition of different metal ions with Fe3+
Metal ions were added as citrate complexes to final concentrations of
100 µM metal and 1 mM citrate. Fe3+
concentration in all samples was 1 µM
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Dependence on Bicarbonate/Carbonate Ions--
Binding of
Fe3+ ions to animal transferrins requires, and occurs
synergistically with, binding of bicarbonate/carbonate ions. If Ttf
plays a role in iron uptake in Dunaliella, one expects the
uptake activity to depend on carbonate/bicarbonate ions. To examine
this possibility, the uptake medium was first extensively depleted of
CO2 and then supplemented with varying concentrations of
bicarbonate. The results (Fig.
2A) clearly indicate a strict dependence of iron uptake on added bicarbonate ions. The specificity for bicarbonate/carbonate was tested by comparison with several other
anions. Only oxalate was found to partially substitute for bicarbonate/carbonate in stimulating iron uptake (Fig.
2B).

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Fig. 2.
Dependence of iron uptake on
carbonate/bicarbonate concentration. A, assay media
without bicarbonate were adjusted to pH 3.0 with HCl and degassed for
1 h to remove dissolved CO2. The solutions were
readjusted to pH 7.0 with NaOH and supplemented with the indicated
bicarbonate concentrations (Ci). After adding
59Fe3+-citrate, the bicarbonate-free cell
suspensions were sealed in 1-ml Teflon-capped glass vials and assayed
for iron uptake. Means of three measurements are shown (S.D. < 0.65 nmol of Fe/109 cells). B, anion specificity.
Iron uptake was analyzed as in A in the absence or presence
of 50 mM of the indicated anions added as sodium salts.
Control, activity in the absence of added anion. Means of three
measurements are shown (S.D. < 30%).
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Fe3+ Binding and Internalization--
In order to gain
further insights into the uptake mechanism, a distinction was made
between iron bound to the cell surface and intracellular iron. The
total amount of 59Fe in both compartments was estimated by
stopping the uptake assay with an isotonic solution devoid of EDTA. The
difference between this value and the internalized (EDTA-resistant)
radioactivity provided an estimate of the externally bound iron.
An analysis of the effect of temperature was performed with cells
incubated with Fe3+ for 50 min at 1 °C in the dark and
subsequently transferred to 24 °C in the light (Fig.
3A). The results indicate a
relatively rapid binding of 59Fe in the cold, which
remained practically unaltered after transfer to 24 °C. A rather
different course was observed for iron internalization; it was barely
evident in the cold and started only after transfer to 24 °C,
finally reaching a level severalfold higher than the amount bound at
any given time.

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Fig. 3.
Iron binding and internalization.
A, effect of temperature on iron binding and internalization
kinetics. Cells were incubated with
59Fe3+-citrate in standard uptake medium on ice
in the dark and after 1 h were transferred to room temperature and
illuminated. Total, total cell-associated iron
(EDTA-resistant plus EDTA-sensitive); internalized,
EDTA-resistant iron; bound, EDTA-sensitive iron.
B, internalization of prebound iron. Cells were preincubated
for 1 h with 59Fe3+-citrate in standard
medium on ice, washed twice in EDTA-free stop solution to eliminate
unbound iron, and resuspended in a fresh 59Fe-free uptake
assay medium. The cells were either kept on ice in the dark and washed
with EDTA-free stop solution (total cell-associated) or illuminated at
23 °C or 1 °C and washed in EDTA stop solution (internalized).
Means of two measurements (S.D. < 0.1 nmol of iron/109
cells). C, effects of metabolic inhibitors on iron binding
and internalization. Cells were preincubated for 40 min in the light in
the absence or presence of 5 µM SF-6847 or of 5 mM iodoacetamide (IAA) together with 5 mM NaCN (CN). Cell samples were analyzed for ATP
content and iron uptake (1 h at 23 °C), iron binding (1 h at
1 °C), or iron internalization (1 h at 1 °C followed by 1 h
at 23 °C with or without inhibitors). Means of two measurements are
shown (S.D. < 15%). Control values were as follows: intracellular ATP
concentration, 3 mM; iron uptake, 3.6 nmol/109
cells/h; iron bound, 280 pmol/109 cells; iron internalized,
220 pmol/109 cells.
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To test whether iron binding precedes uptake, cells were first
incubated with 59Fe at 1 °C in the dark for 1 h,
and unbound iron was removed by washing with an isotonic solution
devoid of Na-EDTA. The cell suspension was subsequently incubated at
24 °C in the light. The results (Fig. 3B) indicate that
all of the 59Fe bound in the first step is internalized in
the second step. No internalization is evident when the suspension is
left in the cold.
Further evidence that iron binding is part of the uptake pathway was
provided by comparing the effect of several parameters on binding and
uptake. The results (Table II) indicate
that both activities show a similar requirement for
carbonate/bicarbonate and respond similarly to changes in pH. Periodate
anions, previously reported to inhibit iron binding to transferrins
(6), also inhibited both iron binding and uptake in
Dunaliella. Binding and uptake also show similar
Km values for iron-citrate.
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Table II
Characteristics of iron binding and iron uptake
Iron binding and iron uptake were measured as described under
"Materials and Methods" at the indicated conditions. KIO4
was added at a concentration of 500 µM.
Km for iron-citrate was determined as in Fig.
1B. Control activities were 380 pmol/109 cells and
3.2 nmol/109 cells/h for iron binding and iron uptake,
respectively.
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Energy Requirements--
The results described above pointed to an
obligatory energy requirement of the internalization but not the
binding step. To further examine this issue, we tested the effect of
metabolic inhibitors on the binding and internalization steps.
Treatment of cells with the protonophore SF-6847 or with iodoacetamide
and cyanide (glycolytic and respiratory inhibitors, respectively), which drastically decrease the cellular ATP level, inhibited uptake and
internalization but not binding of Fe3+ ions (Fig.
3C). Similarly, incubation in the dark lowered both the ATP
level and internalization by 20-60% (not shown). These results
support the conclusion that Fe3+ internalization, but not
binding, is energy-dependent.
Correlation between Iron Uptake Activity and the Level of
Ttf--
Iron uptake and binding activities were compared in cells
containing variable levels of Ttf. Cells were induced to accumulate the
protein by high salinity or iron depletion (3, 4). As shown in Fig.
4, cells grown in the absence of added
iron in a medium with 0.5 M NaCl, or in 3.5 M
NaCl in the presence of Fe-EDTA were 50% or 100% more active,
respectively, in iron uptake than cells grown in 0.5 M NaCl
in the presence of iron (Fig. 4, top). The corresponding
levels of Ttf increased by about 2- or 3-fold, respectively, compared
with control cells (bottom). Iron binding activity at
1 °C increased by 5-6-fold in the high salt-grown and iron-depleted
cells compared with the control cells (Fig. 5). These results indicated a general
correspondence between the increase in Ttf level and iron uptake and
binding activities.

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Fig. 4.
Correlation between level of Ttf and iron
uptake. Cells were cultured either in 3.5 M NaCl or in
0.5 M NaCl in iron-sufficient (control) or iron-deficient
growth media and analyzed for iron uptake (top) or the level
of Ttf by immunoblotting (bottom). Relative levels of Ttf
from densitometric analysis are 100, 202, and 316 for control,
iron-deficient, and high NaCl cells, respectively. Each iron uptake
value is an average of two measurements (S.D. < 18%).
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Fig. 5.
Effect of growth in high salinity and in
iron-deficient medium on iron binding. Cell cultures in
iron-sufficient (C) or iron-deficient 0.5 M NaCl
media ( Fe) or in a 3.5 M NaCl medium were
analyzed for iron binding. Means of two ( Fe; 3.5 M NaCl, S.D. < 0.6 nmol/109 cells) or four
(control, S.D. < 0.1 nmol/109 cells) measurements are
shown.
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To further test the correlation between Ttf and iron uptake, cells were
subjected to proteolytic digestion under conditions that removed Ttf
without disrupting cell integrity. Preincubation with Pronase prior to
the uptake assay brought about partial digestion of Ttf and a
concomitant reduction in uptake activity (Fig.
6A). Conversely, when Pronase
treatment followed the uptake assay, no reduction in internalized iron
level was evident (Fig. 6B).

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Fig. 6.
Effect of proteolytic digestion on iron
uptake and Ttf level. D. salina cells were treated with
Pronase prior to (A) or following (B) the iron
uptake assay. Top, iron uptake activity, means of two
measurements (S.D. < 15%). Bottom, immunoblot analysis
with anti-Ttf antibodies. Densitometric quantitation of residual Ttf
level after Pronase treatment equals 58 and 11% in A and
B, respectively.
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Effect of Salt on Accumulation and Activity of Ttf--
We
previously proposed that Ttf accumulated in high salinity in response
to restricted iron availability under these conditions (4). Iron
limitation could arise, for example, from salt inhibition of
Ttf-mediated iron uptake or from a reduction in Fe3+ ion
solubility in high salt.
In order to examine whether high salt directly limited iron
availability, a medium with 3.5 M NaCl was supplemented
with 100 µM Fe3+-citrate. Cells grown in this
medium were compared with cells grown with in the same salinity but
with the standard 2 µM Fe-EDTA. Cells grown in the
iron-enriched medium contained a considerably lower level of Ttf as
compared with cells grown in standard medium or a medium supplemented
only with citrate (Fig. 7). These results imply that high salinity per se does not trigger Ttf
accumulation but exerts its effect indirectly by lowering iron
availability.

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Fig. 7.
Effect of iron enrichment on the level of Ttf
in high salt-grown D. salina. Cells were cultured in
3.5 M NaCl medium supplemented either with 1 mM
sodium citrate or with 1 mM citrate plus 0.1 mM
FeCl3 (100 µM Fe3+-citrate).
Aliquots of 106 cells were extracted and resolved by
SDS-PAGE. A, immunoblot; B, Coomassie
Blue-stained. Densitometric quantitation of Ttf level in A
equals 0.5, 1.0, and 1.0 in iron-citrate, citrate, and control cells,
respectively.
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A direct examination of the effect of salt on iron uptake/binding was
carried out by varying the salt concentration in the assay medium. The
analysis was performed with cells grown in 3.5 M NaCl in
standard medium. The assays were conducted in media where the NaCl was
gradually replaced by osmotically equivalent glycerol. The isoosmotic
mixtures compared varied in salinity between 0.35 M NaCl
(with 4.1 M glycerol) and 3.5 M NaCl (no
glycerol). The kinetics of iron binding and uptake in four different
NaCl/glycerol mixtures shows that salt reduces the rate of iron binding
at low temperature but does not significantly decrease its extent (Fig. 8A). Conversely, the rate of
iron uptake at 24 °C is not affected at all by high salt (Fig.
8B). The affinity for iron ions is also hardly affected by
high salt. The apparent Km for iron-citrate in 3.5 M NaCl salt is 0.19 µM compared with 0.23 µM in 0.35 M NaCl. The corresponding
Vmax values are also quite similar (11.7 and 9.8 nmol Fe/109 cells/h, respectively; not shown).

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Fig. 8.
Effect of high salt on the rate of iron
binding and uptake. Cells grown in 3.5 M NaCl were
suspended in isoosmotic mixtures of NaCl and glycerol equivalent to 3.5 M NaCl, containing 0.35-3.5 M NaCl as
indicated. A, iron binding; B, iron uptake.
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These results show that high salt does not interfere with the binding
and internalization of Fe3+ ions by Dunaliella
cells. Hence, the activity of Ttf is probably insensitive to high salt
concentrations.
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DISCUSSION |
Iron uptake in Dunaliella exhibits kinetic and
specificity characteristics typical to transferrins, in general, and to
melanotransferrin, in particular. The apparent Km
for iron-citrate, 0.2-0.6 µM, corresponds to a
calculated free Fe3+ concentration of 2.3 × 10
19 M: [Fe3+]free = 3.8 × 10
13 [Fe]total at pH 7.4 (7).
Such high affinity combined with a high selectivity for
Fe3+ ions over Fe2+ and other transition metal
ions is characteristic for soluble animal transferrins (1). The
calculated Vmax and Km (for
iron-citrate) for iron uptake in Dunaliella, 15,000-30,000 iron ions/cell/min and 0.6 µM, respectively, are
comparable with corresponding values of 60,000 Fe/cell/min and 2.6 µM for melanotransferrin-mediated uptake measured in
Chinese hamster ovary cells heterologously expressing melanotransferrin
(2).
Strict dependence on the presence of carbonate/bicarbonate is a
hallmark of iron binding to transferrins. The selectivity for these
anions exhibited by the algal uptake system also closely resembles that
of mammalian Tfs (8). The sharp drop in iron binding/uptake at acidic
pH and the inhibition of iron binding and uptake by periodate are also
characteristic of mammalian Tfs (6, 9). Moreover, algal iron binding
and uptake activities are correlated with the cellular level of Ttf.
These results clearly point to the involvement of Ttf in iron uptake by
Dunaliella.
The Ttf-mediated uptake of iron is initiated by its binding to
extracellular sites, most likely on Ttf, followed by internalization. The sensitivity of iron internalization to temperature and to metabolic
inhibitors indicates that the internalization of iron ions is an
energy-requiring process. Internalization is the rate-limiting step in
iron uptake; even at 1 °C, iron binding is faster than internalization. A similar general pathway has been proposed for melanotransferrin (2).
The observation that an increase in available iron supply diminishes
the accumulation of Ttf in high salt-grown cells complements the
earlier observation of Ttf accumulation in iron-deficient cells.
Together these results further support the conclusion that high
salinity limits iron availability to the cells. The mechanism by which
high salt interferes with iron availability in standard media is still
unclear. We have previously proposed that salt may decrease iron
solubility or affect the activity of the uptake machinery (4). The
latter possibility is largely eliminated by the present results
indicating that high salt affects neither the affinity nor the rate of
iron uptake. Hence, high salt does not significantly restrict iron
association with Ttf, except for the minor decrease in the rate of iron
binding at low temperature. The observation that salt affects iron
binding but not iron uptake is consistent with the idea that the
binding step is not a rate-limiting step in iron uptake.
These results bear several important implications concerning the role
of Ttf particularly under iron-limiting conditions. Under such
conditions, the extremely high affinity for iron ions and the high
accumulation capacity (~3 nmol of iron/109 cells,
equivalent to ~2 × 106 iron ions/cell) ensure
efficient iron binding and acquisition even at extremely low iron
concentrations.
The fact that iron binding and uptake in Dunaliella are
hardly affected by high salt is of crucial importance for an organism thriving over practically the entire range of salt concentrations. In
its apparent salt tolerance, Ttf differs from animal transferrins, in
which salt alters the properties of iron binding (11-13).
Proteins similar to Ttf may exist and function in iron uptake in other
algae. A detailed kinetic characterization of iron uptake in two
coastal phytoplanktons (10) revealed similar affinity, specificity, and
energetic requirements to those of Ttf-catalyzed iron uptake in
Dunaliella. Moreover, screening with anti-Ttf antibodies revealed a cross-reacting protein of ~120 kDa in the microalga Nanochloropsis and a ~100-kDa cross-reacting protein in
the macroalga Ulva (data not shown). Thus, transferrin-like
proteins may participate in a ubiquitous mechanism of iron uptake in
algae.
The results presented here bear special significance in view of the
fact that iron availability is a critical limiting factor for marine
phytoplankton photosynthesis in oceans and hence global biomass
productivity (14, 15). Furthermore, the intriguing similarity between
Ttf and melanotransferrin-mediated iron uptake in animal cells suggests
the operation of an evolutionarily conserved pathway. The elucidation
of the algal mechanism may therefore shed light on the mechanism of
action of membrane-anchored transferrins in general.
We thank Dr. A. Katz, M. Weiss, and M. Schwarz for technical assistance and helpful discussions.