Iron Uptake by the Halotolerant Alga Dunaliella Is Mediated by a Plasma Membrane Transferrin*

Morly FisherDagger , Ada Zamir, and Uri Pick§

From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 71600, Israel

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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).

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

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%).

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.

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.

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.

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.

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.

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.

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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

We thank Dr. A. Katz, M. Weiss, and M. Schwarz for technical assistance and helpful discussions.

    FOOTNOTES

* This work was supported by grants from the Minerva Foundation (Munich, Germany) and the Soref Foundation (to A. Z.) and the Willstater Center for Photosythesis Research (to A. Z. and U. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Institute of Biological Research, P.O. Box 19, Nes-Ziona 74100, Israel.

§ Incumbent of the Charles and Louise Gartner Professorial Chair. To whom correspondence should be addressed. Tel.: 972-8-9342732; Fax: 972-8-9344118; E-mail: bcpick{at}weizmann.weizmann.ac.il.

1 The abbreviations used are: MES, 2-(N-morpholino)ethanesulfonic acid; SF-6847, 3,5-di-tert-butyl-4-hydroxybenzylidenemalonitrile; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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