©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Precursor of Pea Ferredoxin-NADP Reductase Synthesized in Escherichia coli Contains Bound FAD and Is Transported into Chloroplasts (*)

(Received for publication, February 24, 1995; and in revised form, April 10, 1995)

Esteban C. Serra(§)(¶) Adriana R. Krapp (**) Jorgelina Ottado(§)(**) Mario F. Feldman(§)(§§) Eduardo A. Ceccarelli (**) Néstor Carrillo(**)(¶¶)

From the Molecular Biology Section, Departamento de Ciencias Biológicas, Facultad de Ciencias Bioqu&ıacute;micas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000, Argentina

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The precursor of the chloroplast flavoprotein ferredoxin-NADP reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.


INTRODUCTION

Chloroplast ferredoxin-NADP oxidoreductases (FNRs, (^1)EC 1.18.1.2) are hydrophilic proteins of about 35 kDa that contain 1 mol of noncovalently bound FAD/monomer(1, 2) . They catalyze the reversible electron transfer between pyridine nucleotides and electron carrier proteins such as ferredoxin or flavodoxin. In chloroplasts and cyanobacteria the reaction is driven toward NADP reduction, providing the NADPH necessary for CO(2) fixation and other biosynthetic pathways(1, 2) . In addition to this physiological reaction, FNR is able to catalyze in vitro the oxidation of NADPH by suitable electron acceptors like potassium ferricyanide (diaphorase activity) or the ferredoxin-cytochrome c system (cytochrome c reductase). These two activities have been extensively used to study the FNR catalytic mechanism ( (1) and references therein).

Like most plastid proteins, FNR is nucleus-encoded (3) and synthesized in cytosolic ribosomes as a higher molecular mass precursor (pre-FNR), containing a transit peptide of 5 kDa at its amino terminus(3, 4) . This peptide extension allows the precursor protein to be targeted to and translocated across the plastid envelope. During or shortly after import, the transit peptide is cleaved off by a stromal protease, and the mature flavoprotein binds tightly to the outer surface of the thylakoid membrane(3, 4, 5) .

Although the mechanism of chloroplast protein import is being investigated extensively (for recent reviews see (6, 7, 8) ), it is not yet completely understood. From the available evidence it has been proposed that precursor proteins must be kept in a loosely folded state in order to be imported into plastids or mitochondria(9, 10) . This hypothesis is supported by a number of experimental observations (9, 10, 11, 12, 13, 14) , although the actual conformation of transport-competent precursors is far from understood. The degree of defined structure has been determined in some recombinant precursors solubilized from bacterial inclusion bodies by the use of chaotropic agents. The physiological meaning of the resulting conformations is, however, uncertain, and in any event, structural measurements on these solubilized precursors yielded rather conflicting results. For instance, a low content of defined secondary structure could be demonstrated for the small ferredoxin precursor(11, 12) , whereas a recombinant, import-competent pre-LHCP showed a folded conformation composed of both alpha-helix and beta-sheet(15) .

Other studies also suggest that some authentic precursors may adopt a folded conformation and still be competent for organelle import. Those of the chloroplast 5-enoylpyruvylshikimate-3-phosphate synthase (16) and of several mitochondrial proteins (17, 18, 19, 20) display enzymatic activity, suggesting an active site conformation similar to that of the mature enzyme. Some precursor proteins are able to assemble into active oligomeric structures(19, 20) , or to bind prosthetic groups or ligands prior to translocation(19, 21, 22, 23) .

In the case of plastid-targeted flavoproteins, the mechanisms of import and cofactor attachment are still poorly understood, and our goal is to study these processes using chloroplast FNR as a model. A FAD-containing FNR precursor has been synthesized in wheat germ extracts supplemented with FAD, but neither the import competence nor the structure of the precursor holoprotein was determined(24) . On the other hand, expression of a pea pre-FNR gene in Escherichia coli resulted in the accumulation of a nearly mature form of the reductase displaying catalytic and spectral properties that were very similar to those of the plant enzyme(25) . The transit peptide was removed by bacterial proteases in an apparently nonspecific reaction. The use of protease-deficient strains of E. coli largely prevented this processing, but the unprocessed precursor was insoluble (25) .

In this study we describe the preparation and properties of a recombinant pre-FNR that contained bound FAD but was largely devoid of activity. Limited proteolysis of the purified precursor resulted in activation of the reductase in the absence of exogenously added FAD. The purified precursor protein was imported into isolated chloroplasts and processed to mature size.


EXPERIMENTAL PROCEDURES

Plasmid Construction

FNR cDNA clones were kindly provided by Dr. J. C. Gray, Cambridge University, United Kingdom. Construction of the expression vector pGF202 is shown in Fig. 1. A PstI-EcoRI cDNA fragment (1.3 kilobase pairs), containing pea pre-FNR coding sequences and part of the 3`-noncoding region(3) , was inserted into the BamHI and EcoRI sites of pGEX-3X (26) to generate recombinant plasmid pGF202. Two complementary synthetic oligonucleotides (Biodynamics SRL, Buenos Aires) were annealed to provide the BamHI-PstI adaptor.


Figure 1: Construction of the expression vector pGF202. A, a PstI-EcoRI cDNA fragment containing the pea pre-FNR coding region (3) was cloned into the BamHI and EcoRI sites of pGEX-3X. Insertion of the synthetic oligonucleotide duplex encoding Factor Xa recognition sequence provided the BamHI-PstI adaptor. B, linear map of pGF202 showing the GST-pre-FNR fusion scheme. The carboxyl-terminal residues of GST are linked in-frame to the FNR precursor through the Factor Xa target sequence (boxed), with the arrow indicating the predicted cleavage site for the protease. The initial methionines of GST and pre-FNR are numbered 1. Expression of the fusion protein is controlled by the inducible tac promoter (Ptac).



LINKER

Note that the 5`-end region of the adaptor does not regenerate a BamHI site upon ligation to its cohesive site in pGEX-3X. The 3`-end nucleotides of this linker encode the first four residues of pre-FNR (Met and three Ala residues), that were missing in the 1.3-kilobase pair cDNA fragment(3) . The four codon supstream of the initial ATG encode the recognition sequence for restriction protease Xa(26) , with the cleavage site placed immediately before methionine 1 of pea pre-FNR. After amplification in transformed E. coli cells (strain AD202; (27) ) and isolation of double-stranded plasmid DNA, recombinant clones were selected by the presence of the insert and the disappearance of the BamHI restriction site in the plasmids. The sequence at the adaptor-joining locus was determined by double-stranded DNA sequencing. Recombinant DNA techniques were carried out following established procedures(28) .

Expression and Purification of the Recombinant Ferredoxin-NADPReductase Precursor

Expression of pre-FNR in E. coli cells harboring plasmid pGF202, preparation of cell lysates, and purification of the GST-pre-FNR fusion were carried out using published methods(25, 26, 29) , with minor modifications. Briefly, bacterial crude extracts obtained from 400-800 ml of culture medium were applied to a 1.5 15-cm glutathione-agarose column (sulfur linkage; Sigma). With the aid of a peristaltic pump, the lysate was circulated through the agarose bead for 10 h at 6 °C in order to ensure maximum binding. The column was then washed with 5 volumes of 20 mM phosphate buffer, pH 7.3, 150 mM NaCl, and two volumes of 50 mM Tris-HCl, pH 8.0. The fusion product was finally eluted by 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, and exhaustively dialyzed against TS medium (50 mM Tris-HCl, pH 7.5, 75 mM NaCl). Following cleavage with restriction factor Xa (Boehringer Mannheim), the digest was passed through glutathione-agarose, applied to a DEAE-cellulose column equilibrated in TS medium, and washed with the same buffer. The first protein fraction eluting under these conditions contained electrophoretically pure FNR precursor.

To obtain S-labeled pre-FNR, E. coli cells harboring plasmid pGF202 were cultured (8 h at 37 °C, A = 0.6-0.8) in 0.25 LB broth (28) containing 0.1 mg/ml carbenicillin, and then supplemented with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside and 10 µCi/ml [S]methionine (1230 Ci/mmol) (DuPont). Following a 120-min incubation period under the same conditions, cells were harvested and disrupted, and the labeled pre-FNR was purified as described above. The specific activity of the purified precursor was estimated using a protein assay (30) and scintillation counting. Crude lysates, bacterial pellets, and purified samples were analyzed by SDS-PAGE in 12% gels according to Laemmli(31) .

Protease Sensitivity Assays

Protease digestions were carried out at 25 °C in 50 mM HEPES-KOH, pH 7.5, 5 mM CaCl(2), and 0.1 mg/ml pre-FNR. The reactions were started by the addition of protease to a final concentration of 10 µg/ml. At the indicated times, 50-µl aliquots were withdrawn into ice-cold tubes containing 5 µl of the corresponding protease inhibitor: 10 mM EDTA (thermolysin), soybean trypsin inhibitor (trypsin), or 100 µM phenylmethylsulfonyl fluoride (chymotrypsin). A 10-µl aliquot was taken using a Hamilton syringe and immediately assayed for FNR activity. The rest of the sample was mixed with 0.25 volumes of 10 mM EDTA, 2.5% (v/v) beta-mercaptoethanol, 10% (w/v) SDS, boiled for 3 min, and subjected to SDS-PAGE.

Chloroplast Import

Intact chloroplasts were isolated from 10-14-day-old pea seedlings (Pisum sativum L., cv. Cuarentona Enana) using percoll gradient centrifugation (32) and suspended in import buffer (50 mM HEPES-KOH, pH 8.0, 330 mM sorbitol) at about 2 mg chlorophyll/ml. Plastid numbers per microliter of suspension were estimated by counting on a modified Neubauer camera. A standard import reaction contained the radiolabeled precursor (5 10^5 dpm at a pre-FNR concentration of about 0.4 µM), 5 10^7 chloroplasts, and (unless otherwise stated) 2.2 mM MgATP in import buffer. Pea leaf extracts, prepared according to Waegemann et al.(13) , were added at a final protein concentration of 0.3 mg/ml. The samples were incubated at 25 °C for 30 min. At the end of the import reaction, thermolysin was added to the suspension to 100 µg/ml and incubated on ice for 30 min. The protease treatment was stopped by the addition of EDTA to 10 mM, and chloroplasts were reisolated by centrifugation through silicone oil layers (AR 200) according to Robinson and Walker(33) . Aliquots from labeled precursor and import reactions were analyzed by SDS-PAGE(31) . After electrophoresis, the gels were treated for fluorography using 20% (w/v) diphenyloxazole in dimethyl sulfoxide (34) and exposed to Kodak XAR x-ray films. The radioactive bands were excised using the developed fluorograms as a guide and quantitated by liquid scintillation counting. Import efficiency (the fraction of added protein imported by the chloroplasts in a 30-min assay) was calculated assuming that all methionine residues in pre-FNR were equally labeled. Thus the precursor contains 12 labeled residues, and the mature protein contains 11(3) .

Preparation of stromal processing extracts and in vitro processing of purified pre-FNR were carried out essentially as described by Archer and Keegstra(35) .

Analytical Procedures

Amino terminal sequence analysis was performed using an Applied Biosystems 477/A protein sequencer. Total protein was determined by a dye-binding assay(30) , using bovine serum albumin or purified FNR as standards. Glutathione S-transferase, FNR diaphorase, and cytochrome c reductase activities were measured spectrophotometrically as described previously(29) . FAD content of recombinant FNR was determined using high performance liquid chromatography and fluorescence detection(24) . Absorption and fluorescence spectra were recorded in a Hitachi 150-20 spectrophotometer and in a Jasco FP-770 spectrofluorometer, respectively.


RESULTS

Expression and Purification of the Ferredoxin-NADPReductase Precursor

Expression vector pGF202 (Fig. 1) contains the entire coding sequence of pre-FNR fused in-frame to the 3`-end of the Schistozoma japonicum glutathione S-transferase gene cloned in plasmid pGEX-3X (26) . A target sequence for protease Xa was introduced with the oligonucleotide adaptor (Fig. 1). The new cleavage site is placed immediately prior to methionine 1 of pea pre-FNR. The construct is under the control of the tac promoter(26) , providing high level synthesis of the GST-pre-FNR fusion upon induction by isopropyl-1-thio-beta-D-galactopyranoside.

The presence of pea FNR polypeptides in E. coli cells was studied by SDS-PAGE and immunoblotting(28) . An immunoreactive peptide with an apparent molecular mass of 65 kDa was detected in the soluble fraction of disrupted bacteria (not shown). This size agrees fairly well with that expected for the fusion protein between GST (27 kDa; (26) ) and pre-FNR (40 kDa; (3) ). As already reported for the GST-FNR fusion expressed in E. coli(29) , several reactive bands of smaller size were also evident (data not shown, but see below). The appearance of both the full-length and shorter reactive peptides was strictly dependent on isopropyl-1-thio-beta-D-galactopyranoside induction.

The amount of recombinant protein produced in AD202 cells, estimated in a large number of assays by slot blot and immunoreaction(25) , ranged between 1 and 3% of the total cell protein (not shown). Following disruption of induced cells and fractionation of their contents, about 70% of the reactive material was recovered in the soluble fraction. The remainder was found aggregated into bacterial pellets and was largely represented by truncated species (not shown).

A protocol described for the purification of GST fusions (26, 29) was used to isolate GST-pre-FNR from transformed bacteria. The fusion protein was first bound to a glutathione-agarose matrix and then eluted with glutathione, together with the truncated immunoreactive products present in the crude lysates (Fig. 2A, lane1). The fraction eluted from the column displayed GST activity but little or no FNR-diaphorase (not shown). Incubation of this extract with factor Xa resulted in the disappearance of the fusion product, with accumulation of a 40-kDa protein (Fig. 2A, lanes 2-4). However, a peptide with electrophoretic mobility similar to that of mature FNR also accumulated as incubation progressed, indicating that some processing of the transit peptide might have occurred (Fig. 2A, lanes 2-4). Digestion of the prepiece was strictly dependent on the addition of Factor Xa, suggesting that degradation was caused by the coagulation factor itself or by traces of a contaminating protease. Absorption of a 15-min digest on glutathione-agarose removed most GST and undigested fusion protein, leaving a mixture of precursor and mature FNR (Fig. 2B, lane1). The two proteins could be separated by ion exchange chromatography on DEAE-cellulose, rendering an electrophoretically homogeneous precursor polypeptide (Fig. 2B, lanes4-6). The final yield is at present about 0.2 mg/precursor/liter of culture medium. Amino acid sequencing of the purified pre-FNR indicated that methionine 1 was the only significant residue at the amino terminus (not shown).


Figure 2: Expression and purification of the FNR precursor protein. Details of the purification scheme are given under ``Experimental Procedures.'' A, cleavage of the fraction eluted from glutathione-agarose with restriction protease Xa. The eluate (0.1 mg of protein) was incubated at 25 °C with 0.5 µg of Factor Xa in a medium containing 50 mM Tris-HCl, pH 7.5, 75 mM NaCl, and 1 mM CaCl(2). Aliquots (15 µg) were removed at 0 (lane1), 15 (lane2), 30 (lane3), and 60 min (lane4) and subjected to 12% SDS-PAGE followed by staining with Coomassie Brilliant Blue. Positions of mature FNR, precursor, and fusion protein are marked on the left. The arrowhead indicates truncated products of 28-30 kDa. B, chromatography of FNR precursor and mature protein on DEAE-cellulose. A glutathione-agarose eluate (1.1 mg of total protein) was cleaved for 15 min at 25 °C with 5 µg of factor Xa. Most GST and undigested fusion product was removed by a second passage through glutathione-agarose, and the run-through fraction (lane1) was applied onto a DEAE-cellulose column (see ``Experimental Procedures''). Lanes2-10 represent successive fractions (1.5 ml) obtained by washing the column with 50 mM Tris-HCl, pH 7.5, 75 mM NaCl. Aliquots (50 µl) were resolved by 12% SDS-PAGE and stained with Coomassie Brilliant Blue.



Properties of the Purified Precursor

The pre-FNR protein isolated from E. coli transformants displayed 1-5% of the specific diaphorase activity of mature reductase, which prompted us to investigate the spectral properties of the precursor. Purified pre-FNR indeed showed a flavoprotein absorption spectrum in the visible region. Fluorescence emission and excitation spectra also indicated the presence of FAD (not shown). Analysis of flavin content indicates that the purified cleaved precursor contains 0.87 ± 0.10 FAD/molecule of apoprotein, compared with 0.91 ± 0.08 in the plant holoenzyme assayed under the same conditions. The 275:456 spectral ratio of pre-FNR was 13.1, compared with 8.1 in mature FNR, and the extinction coefficients at 380 and 456 nm were 5-10% lower in the recombinant precursor (not shown). Taken together, the previous results suggest that although pre-FNR is able to bind FAD, the conformation of the flavin moiety may be different from that of the mature holoprotein, resulting in a nonfunctional preholoreductase.

The strength of the interaction between FAD and pre-FNR was not determined, but the precursor could be precipitated in 30% saturation ammonium sulfate and redissolved without apparent loss of bound flavin. The FNR precursor was stable at -20 °C for several months, but repetitive freezing and thawing resulted in the disappearance of the 40-kDa band, with formation of a mature sized immunoreactive product (not shown). We thus stored the purified pre-FNR in aliquots at -70 °C.

To evaluate if the precursor holoprotein can be cleaved by the chloroplast processing protease, we exposed pre-FNR to a stromal lysate containing processing activity(35) . Fig. 3shows that pre-FNR was processed to mature size upon incubation with the extracts. Over a period of 45 min, the amounts of mature FNR increased as the precursor declined, indicating that binding of FAD did not affect the accessibility of the transit peptidase target site.


Figure 3: Plastid-free processing of FNR precursor protein. A, radiolabeled pre-FNR (1 10^6 dpm, 0.8 µg of precursor) was incubated with a soluble stromal extract (20 µg of total protein) containing processing activity. Reactions were carried out at 25 °C in import buffer containing 1 mM MgCl(2). Samples were taken over a period of 45 min and analyzed by 12% SDS-PAGE and fluorography. Lane1, recombinant FNR precursor. Products of plastid-free processing obtained after 2.5, 5, 10, 15, 30, and 45 min of incubation are shown in lanes2-7. B, quantitation of pre-FNR processing. The datapoints represent the mean of three experiments similar to that shown in panelA. bullet, FNR precursor; , mature sized FNR.



In order to get an indication of the overall chain packing of the precursor polypeptide, we performed protease sensitivity experiments. Spinach FNR is known to possess a protease-resistant core that begins at lysine 35 (lysine 29 in the pea reductase) and extends far beyond into the carboxyl-terminal domain(36) . Removal of the protease-sensitive regions had no effect on diaphorase activity, while cytochrome c reduction was inactivated due to an impairment of ferredoxin binding(36) . Protease accessibility assays had not been performed on the pea enzyme, but the sequence conservation between the two species (3, 37) predicted a similar behavior. In fact, digestion products obtained after limited proteolysis of mature pea FNR with thermolysin (Fig. 4A), trypsin, or chymotrypsin (not shown) were very similar to those reported for the spinach enzyme(36) . Activity measurements confirmed inhibition of cytochrome c reduction and little effect on diaphorase (Fig. 4C).


Figure 4: Limited proteolysis of pea FNR mature and precursor proteins by thermolysin. Experimental conditions are given under ``Experimental Procedures.'' The time course of proteolysis was analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. A, mature FNR; B, FNR precursor. Lanes1-9 show the protein patterns of the digest after 0, 1, 3, 5, 10, 15, 20, 30, and 60 min of incubation, respectively. The final positions and molecular masses of protein standards are indicated on the left. C and D, effect of thermolysin digestion on diaphorase (bullet) and cytochrome c reductase () activities of mature FNR (C) or pre-FNR (D). The inset in panelD shows diaphorase activity data plotted on an expanded time scale. One activity unit (U) is defined as the amount of enzyme capable of catalyzing the conversion of 1 µmol of substrate/min under the conditions of the reactions(29) .



When pre-FNR was assayed under the same conditions, the digestion products did not differ significantly between the mature reductase and the precursor (Fig. 4, A and B), suggesting that the preholoprotein is packed to a certain extent. Fig. 4B shows a time course for thermolysin proteolysis of pre-FNR. Under our experimental conditions there is a rapid degradation to a product of 33 kDa in the first 1-3 min. Further digestion occurred at a much lower rate.

Interestingly enough, the diaphorase activity of purified pre-FNR increased abruptly upon partial thermolysin digestion. The time course of activation showed a good correlation with the appearance of the 33-kDa product and was followed by a slow inactivation, which paralleled that of the mature reductase under the same conditions of incubation time and protease concentration (Fig. 4D). Similar results were obtained by using trypsin or chymotrypsin (not shown). Also, incubation of the FNR precursor with stromal extracts (Fig. 3) resulted in small variable increases in diaphorase activity, but the extent of the activation could not be evaluated due to the high endogenous activity of the chloroplast lysates (3 diaphorase units/mg of total protein).

The previous data suggest that the rapid phase of proteolysis caused cleavage of the transit peptide and probably of a portion of the amino-terminal mature sequence as it occurs in spinach(36) . Removal of the transit peptide region apparently induced the protease-resistant core to adopt a functional conformation with development of diaphorase activity, suggesting that the bound FAD changes into a configuration that favors electron transfer between the isoalloxazine ring and its redox partners. The resistant core of pre-FNR did not display cytochrome c reductase activity (Fig. 4D).

Chloroplast Import of Ferredoxin-NADPReductase Precursor

The ability of the folded FNR precursor to be transported into isolated chloroplasts was tested in an in vitro import assay. Induced AD202/pGF202 cells were labeled with [S]methionine, and the FNR precursor was purified to homogeneity (Fig. 5, laneP). This pre-FNR had a specific activity of 1-2 10^6 dpm/µg and appeared as one band on protein-stained SDS-PAGE. The precursor was readily translocated into washed intact chloroplasts and processed to a mature sized FNR, which was protected against externally added protease after the import reaction (Fig. 5). Import was strictly dependent on the presence of ATP (Fig. 5, lanes1 and 2). Purified pre-FNR showed by itself a certain import competence (Fig. 5, lanes3 and 4) that was strongly stimulated by the addition of soluble leaf extracts to the medium (Fig. 5, lanes5 and 6). Preincubation of the purified precursor with leaf extract (10 min at 25 °C) did not further increase the subsequent import rates (not shown). Under optimal conditions, the import efficiency was about 10%. Pretreatment of the leaf extract with thermolysin (38) eliminated its ability to stimulate transport, whereas dithiothreitol had no detectable effect on pre-FNR translocation (not shown).


Figure 5: Import of recombinant FNR precursor into intact pea chloroplasts. Import assays were conducted as described under ``Experimental Procedures,'' and the samples were analyzed by SDS-PAGE and fluorography. MgATP (2.2 mM) and leaf extracts were added or omitted in the import reaction as shown above the panels. Protease (+ or -) indicates whether the chloroplast suspensions were treated or untreated with 100 µg/ml thermolysin after import. LaneP, purified FNR precursor (20% of the amount of pre-FNR added to each import reaction).




DISCUSSION

The pea FNR precursor could be expressed in E. coli cells in a soluble form, fused to the carboxyl-terminal region of S. japonicum GST ( Fig. 1and Fig. 2). The presence of a folded protein at the amino terminus largely prevented both transit peptide processing and pre-FNR precipitation. The recombinant precursor was able to assemble the prosthetic group FAD, which requires the formation of a specific three-dimensional motif at the flavin binding domain(2, 39) . Also, limited proteolysis of pre-FNR resulted in rapid removal of the transit peptide sequence, with accumulation of a protease-resistant core similar to that of mature FNR (Fig. 4). The previous results suggest that the FNR precursor has a folded structure, with protease-sensitive sequences limited to the transit peptide and amino-terminal regions.

The conformations of precursor and mature FNR are probably similar but not identical, as judged by the different shapes of the visible absorption spectra (not shown) and the low levels of activity displayed by the precursor (Fig. 4C). Most of the diaphorase activity could be regained upon removal of the transit sequence (Fig. 4C). The ferredoxin-dependent cytochrome c reductase activity was not recovered by proteolysis, presumably due to digestion of the amino-terminal region of the mature protein, involved in ferredoxin binding(36) . Indeed, the corresponding activity of mature FNR was rapidly lost upon incubation with thermolysin (Fig. 4D).

Heijne and Nishikawa (40) have proposed that transit peptide sequences are perfect random coils designed to interact with a succession of different molecular chaperones on the plastid import pathway. But in the recombinant FNR precursor, we observed also a structural effect. The particular design of the presequence apparently imposes severe structural constraints on the flavoprotein that, without precluding FAD binding, effectively prevent the acquisition of a functional conformation. It is possible that this conformational effect plays a role in the subsequent membrane translocation of the precursor. Recent studies have shown that secretion of a recombinant FNR from yeast cells, mediated by a yeast-specific secretion signal sequence, was strongly enhanced by introducing the FNR transit peptide between the secretion signal and the mature protein(41) .

The precursor holoprotein could be translocated into isolated chloroplasts and processed to mature size, both in organello (Fig. 5) or in a plastid-free medium containing stromal extracts (Fig. 3). The highest import rates were obtained when the medium was supplemented with a soluble leaf extract (Fig. 5). Previously, a number of different precursor proteins synthesized in E. coli were shown to be import-competent without the addition of cytosolic factors, depending only on the presence of ATP (Refs. 11, 12, 14, 15, 42, and 43; for a contrasting observation see also (13) ). The different behavior of pre-FNR may be related to the fact that all of the precursors mentioned above accumulated in the bacterial host as insoluble inclusion bodies. They were made transport-competent only after solubilization in 8 M urea, which is known to cause extensive protein unfolding. To the best of our knowledge, pre-FNR is the first chloroplast precursor isolated in soluble form after expression in E. coli. It is folded and contains bound FAD and may require interaction with cytosolic factors to attain full import competence. Further work will be necessary to determine if this interaction is related to the unfolding of the FNR precursor.

This leads to another important question related to pre-FNR import, namely the site of FAD attachment. The FNR precursor holoprotein is transported into isolated chloroplasts, but the fate of bound FAD during import is unknown. If the effect of the leaf extracts is to mediate transition of pre-FNR to a loosely folded, transport-competent state, this in turn might result in FAD release. Work is now in progress to address this question.

Many flavoproteins, including FNR, are transported into organelles after in vitro translation(4, 44, 45) , which has been assumed to imply import of apoproteins, even when translation systems may contain trace amounts of FAD as demonstrated for rabbit reticulocyte lysates(45) . Results obtained with flavoprotein precursors imported into rat and yeast mitochondria suggested that incorporation of FAD ligand occurs inside the organelle after the mature protein is produced(44, 45) . Furthermore, FAD appeared to be taken up by mitochondria in its free form independent of the presence of an apoflavoprotein(44) . Comparable information is lacking for plastids.

The largest portion of a precursor corresponds to the sequence of the mature protein, which contains most of the information needed for protein folding. Then, at least some domains of the precursor should be able to adopt folding pathways similar to those of the mature protein and eventually bind prosthetic groups. This proposal is supported by our results and those of several other groups(16, 17, 18, 19, 20, 21, 22, 23) . However, the strong structuring induced by incorporation of FAD into the FNR precursor (2, 39) would certainly challenge its possibilities of transport into plastids. Our hypothesis is that the transit peptide largely prevents this effect of the prosthetic group by generation of a ``molten globule state,'' which stabilizes the protein and makes it able to enter the import pathway. Then, transit peptides are proposed to play a double role in precursor import: recognition of specific receptors and chaperones of the import machinery, and maintenance of a precursor conformation compatible with membrane translocation. Experiments are in progress to check these hypotheses.

In conclusion, the finding reported here that a recombinant FNR precursor can be isolated as a transportable holoprotein opens a spectrum of experimental possibilities, providing a tool to study the mechanisms of import and assembly of this flavoenzyme under defined conditions.


FOOTNOTES

*
This work was supported by Grants A-12830/1-000021 and A-13015/1-000018 from Fundación Antorchas (Buenos Aires, Argentina), Grant C/1354-2 from the International Foundation for Science (Stockholm, Sweden), and Grant CRP/AR688-15 from the International Centre for Genetic Engineering and Biotechnology (ICGEB/UNIDO) (Trieste, Italy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors are fellows of the Consejo Nacional de Investigaciones Cient&ıacute;ficas y Técnicas (CONICET), Argentina.

Present address: Centre d'Immunologie et de Biologie Parasitaire, Institut Pasteur, Lille 59800, France.

**
These authors are staff members of the CONICET.

§§
Present address: IQUIFIB, Facultad de Farmacia y Bioqu&ıacute;mica y Farmacia, Universidad de Buenos Aires, Jun&ıacute;n 956, Buenos Aires 1113, Argentina.

¶¶
To whom correspondence should be addressed. Fax: 54-41-240010 or 54-41-300309; cecca{at}unrobi.edu.ar.

(^1)
The abbreviations used are: FNR, ferredoxin-NADP oxidoreductase (EC 1.18.1.2); GST, glutathione S-transferase (EC 2.5.1.18); LHCP, light-harvesting chlorophyll a/b binding protein; pre-FNR, FNR precursor; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. J. C. Gray (Cambridge University) for the generous gift of the original FNR cDNA clone and Dr. T. Saito (Chiba University, Japan), who kindly provided the E. coli AD202 strain. We also acknowledge Dr. A.M. Viale (University of Rosario, Argentina) for critical reading of the manuscript.


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