(Received for publication, February 24, 1995; and in revised form, April 10, 1995)
From the
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.
Chloroplast ferredoxin-NADP oxidoreductases
(FNRs, (
)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
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
-helix and
-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.
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) .
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-
-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) .
Preparation of stromal processing extracts and in vitro processing of purified pre-FNR were carried out essentially as described by Archer and Keegstra(35) .
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--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. 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.
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
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
. 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.
, 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 () 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).
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).
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.