From Fachbereich Biologie/Parasitologie, Philipps-Universität Marburg, Karl-von-Frisch-Strasse, 35032 Marburg, Germany
Received for publication, October 17, 2000
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ABSTRACT |
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In searching for nuclear-encoded,
apicoplast-localized proteins we have cloned
ferredoxin-NADP+ reductase from Toxoplasma
gondii and a [2Fe-2S] ferredoxin from Plasmodium
falciparum. This chloroplast-localized redox system has been
extensively studied in photosynthetic organisms and is responsible for
the electron transfer from photosystem I to NADP+. Besides
this light-dependent reaction in nonphotosynthetic plastids (e.g. from roots), electrons can also flow in the reverse
direction, from NADPH to ferredoxin, which then serves as an important
reductant for various plastid-localized enzymes. These plastids possess related, but distinct, ferredoxin-NADP+ reductase and
ferredoxin isoforms for this purpose. We provide phylogenetic evidence
that the T. gondii reductase is similar to such
nonphotosynthetic isoforms. Both the P. falciparum
[2Fe-2S] ferredoxin and the T. gondii
ferredoxin-NADP+ reductase possess an N-terminal bipartite
transit peptide domain typical for apicoplast-localized proteins. The
recombinant proteins were obtained in active form, and antibodies
raised against the reductase recognized two bands on Western blots of
T. gondii tachyzoite lysates, indicative of the unprocessed
and native form, respectively. We propose that the role of this redox
system is to provide reduced ferredoxin, which might then be used for
fatty acid desaturation or other biosynthetic processes yet to be
defined. Thus, the interaction of these two proteins offers an
attractive target for drug intervention.
Apicomplexan parasites are a large phylum of unicellular, obligate
intracellular organisms, including such medically important members as
Plasmodium spp., the causative agent of malaria (1), and
Toxoplasma gondii, an opportunistic pathogen of
immunosuppressed individuals and a common cause of congenital disease
(2). The recent description and sequencing of a circular 35-kilobase
pair DNA element in several of these parasites, and its
localization to a plastid-like organelle (3-6), called the apicoplast,
has received great interest for at least two reasons. First, it
provides evidence for a secondary endosymbiotic event in apicomplexa
whereby their common ancestor engulfed an algal organism, giving rise to the organelle's four membranes visible by electron microscopy (for
review, see Ref. 3). Phylogenetic analysis of apicoplast-encoded genes
supports this hypothesis (5). Second, the plastid-like organelle offers
a new and promising target for rational drug design, because it is
assumed to contain a number of unique metabolic pathways not found in
the mammalian hosts (7). Recent reports have shown that at least the
rationale of this approach is valid and that new drugs, which are
urgently needed, especially for plasmodial infections, might be
developed in the near future based on these targets (7, 8).
The proteins encoded by the fully sequenced Plasmodium
falciparum and T. gondii plastid genomes are either
ribosomal proteins or proteins involved in the replication of the
episomal genome (3, 4), but are not sufficient to constitute the whole
organelle and to provide insights into its experimentally proven
essential function (9). It is, therefore, obvious that nuclear-encoded genes must exist that code for apicoplast localized proteins, similar
to the situation of chloroplasts and plants (10).
In search for such proteins we describe in this report the cloning and
functional expression of nuclear-encoded, but apicoplast-localized, ferredoxin-NADP+ reductase
(FNR)1 from T. gondii and ferredoxin (Fd) from P. falciparum. This
cellular redox system has been extensively studied for decades in
photosynthetic organisms (11) and is responsible for the electron
transfer from photosystem I to NADP+ for the provision of
reducing equivalents needed in the Calvin cycle for CO2
fixation according to the following reaction: 2Fdred + NADP+ + H+ In this reaction the small [2Fe-2S] protein ferredoxin donates its
electron to the FAD cofactor of FNR, which pairs two electrons for the
subsequent reduction of FNR-bound NADP+ to NADPH (11).
Thus, the presence of this redox pair in nonphotosynthetic apicomplexan
parasites was unexpected at first. However, besides the
light-dependent reaction, electron flow can also take place in the reverse direction in nonphotosynthetic plastids (e.g.
from roots) from NADPH to Fd, which then serves as an important
reductant for various plastid-localized enzymes like nitrite reductase, sulfite reductase, glutamate synthase, and lipid desaturases (12-14). These plastids possess related, but distinct, FNR and Fd isoforms for
this purpose (15). We provide phylogenetic data that imply that
T. gondii FNR and P. falciparum Fd are similar to
nonphotosynthetic isoforms and that reduced Fd is the likely
biosynthetic product of this interaction.
Parasites--
T. gondii tachyzoites of strain RH
were grown in human foreskin fibroblasts and purified by filtration
through 3-µm Isopore membrane filters (Milipore) as described
previously (16). P. falciparum (strain FCBR) trophozoite and
schizont stages were cultivated and enriched by Gelafundin floatation
as described previously (17).
Molecular Cloning Techniques--
DNA extraction from T. gondii was performed by acid phenol extraction in the presence of
LiCl as described previously (18). DNA and RNA from P. falciparum were obtained from mixed stages after saponin lysis
(17). Genomic P. falciparum DNA was isolated as
described previously (19). Total RNA from 109
trophozoites and schizonts each was isolated with TRI reagent (Sigma) according to the manufacturer's protocol. mRNA was
isolated from 100 µg of total RNA using the Poly(A)Tract
mRNA isolation kit from Promega. The final mRNA was dissolved
in 250 µl of water.
PCR amplification of the partial FNR sequence from genomic DNA was
performed using Taq polymerase (Amersham Pharmacia Biotech) and primers 1 (5'-GGAATTCGCTTTATCGAGACAAAT-3') and 2 (5'-GGGGTACCTCAGTACACCTCAACGTGCCA-3'). Digoxigenin (Roche Molecular
Biochemicals) labeling of the mature cDNA FNR sequence (using
primers 3 (5'-CTTAATTAAGAATTCAGTACACCTCAACGTGCC-3') and 4 (5'-AGCATTAATATGCATTCCGTTGATGCCAAGGCCGA-3')) and subsequent Southern blot analysis were done as described previously (20). A
Construction of plasmid pTUK will be described
elsewhere.2 Briefly, it
consists of 6His-tagged yeast ubiquitin (6His-Ubi) and an additional
epitope tag inserted into T7 RNA polymerase promoter-driven pRSET
(Invitrogen) in which the ampicillin resistance gene was replaced by a
kanamycin cassette. For subcloning the mature form of TgFNR into the
expression vector pTUK, the cDNA coding for aa residues 148-497
was PCR-amplified using Pfu polymerase (Stratagene) with the
above primers 3 and 4 containing NsiI and EcoRI
restriction sites for insertion into the vector, resulting in a direct
fusion of FNR with 6His-Ubi (pTUK-TgFNR) to increase yield and
solubility of the recombinant protein (22). The same strategy was used for expression of P. falciparum Fd, starting from aa 108 (pTUK-PfFd). Its sequence was amplified from genomic P. falciparum DNA using primers 5 (5'-GCCCTGCAGTTATTTTATAATATAACATTAAG-3') and 6 (5'-CGAATTCTTAATTAATTACATGTCGTGTAGTTCGTC-3'), containing
PstI and EcoRI restriction sites for cloning into pTUK.
RT-PCR of P. falciparum mRNA was done as follows:
mRNA was primed using a mixture of 50 ng of random hexamers and 50 ng of oligo(dT) and elongated with 100 units of Superscript II reverse transcriptase (Life Technologies, Inc.) for 1.5 h at 42 °C in reaction buffer including 1 mM dNTP. The reaction was
heat-inactivated at 72 °C for 7 min. One reaction was treated with
RNase A (2 µg) before cDNA synthesis to serve as control for DNA
contamination in the subsequent PCR. Using either 5 µl each from the
above cDNA reaction or 50 ng of genomic P. falciparum DNA as template, Fd was amplified with primers 5 and 6, and the P. falciparum FNR-like cDNA with primers 7 (5'-AGTAAGACCAAATTCACCTAA-3') and 8 (5'-TCCTGTTGCAATAAAAATAAAGT-3') using the following conditions: denaturation at 94 °C, 1 min; annealing at 50 °C, 1 min; elongation at 60 °C, 2 min, for 35 cycles, with 1.25 units of Taq polymerase (Amersham
Pharmacia Biotech), 1.5 mM MgCl2, 200 µM dNTP, and 10 pmol of each primer in a final volume of
50 µl. 10 µl of each reaction were then analyzed on a 2% agarose gel.
For antibody production, the C-terminal part of FNR encoded by the
original EST clone (aa Asp432-Tyr497) was PCR
amplified using primers 9 (5'-GGAATTCGACGTGGTTTGGCAAGAAC-3') and 2, containing terminal EcoRI restriction sites and cloned into
the EcoRI site of plasmid pGEX2a, a derivative of pGEX2T (23), to yield a glutathione S-transferase (GST) fusion
construct. All constructs were verified by automated dideoxy sequencing.
Protein Expression, Purification, and Analysis--
For
functional expression of pTUK-TgFNR, pTUK-PfFd, and pET-FdI
encoding spinach Fd (24), Escherichia coli JM109(DE3)
(Promega) containing pLysS (Novagen) was used as host. In experiments
where fusion-free PfFd was required, bacteria also contained the yeast Ubp1 expressing plasmid pCG001, resulting in cotranslational cleavage of ubiquitin from PfFd (25). Bacteria were grown at 30 °C in LB
medium supplemented with kanamycin (50 µg/ml) and chloramphenicol (36 µg/ml), and expression was induced at an
A600 of 0.5 by the addition of IPTG to a
final concentration of 0.4 mM. Subsequent Ni2+-chelate affinity chromatography of 6His-Ubi-TgFNR
fusion protein was performed according to the instructions of the
manufacturer (Qiagen, Hilden, Germany). SDS-polyacrylamide gel
electrophoresis and Western blot analysis were conducted according to
Ref. 26. All antibodies were diluted in 5% nonfat dry milk. Goat
anti-rabbit secondary antibodies were either horseradish peroxidase
(Dianova, Hamburg, Germany) or alkaline phosphatase (Dako)
conjugated and detected by colorimetric staining with
3,3',5,5'-tetramethylbenzidine (horseradish peroxidase) or
p-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (alkaline phosphatase), respectively.
The GST C-terminal FNR fusion protein (GST-TgFNR) was purified from
transformed E. coli BL21(DE3) (Novagen) on immobilized glutathione (Amersham Pharmacia Biotech) using the protocol of (27).
For antibody production 200 µg of GST-TgFNR emulsified in "Antibody
Multiplier" adjuvant (Linaris GmbH, Bettingen, Germany) were
injected three times subcutaneously into a New Zealand rabbit at an
interval of 4 weeks.
NADPH-dependent diaphorase activity of FNR preparations was
determined by DCPIP reduction assay according to Ref. 28. It was set up
in a microtiter plate in a final volume of 100 µl as follows: 125 µM NADPH and 120 µM DCPIP were mixed in 50 mM Tris-HCl, pH 8.0, and the reaction started by the
addition of 20 pmol of purified 6His-Ubi-TgFNR. The change in
absorbance at 630 nm was followed over time in a microplate reader.
Computational Sequence Analysis--
Protein sequence alignments
were performed using CLUSTAL X (29) and optimized by visual inspection.
Phylogenetic tree construction was then performed using this optimized
alignment and either the maximum parsimony method (PROTPARS)
implemented in the PHYLIP 3.5 package (30), the neighbor
joining/distance matrix method from CLUSTAL X (with correction for
multiple substitutions), or the quartet puzzling method for maximum
likelihood tree building (31). The final trees were drawn using
TreeView (32). Signal sequence prediction was performed using
the SignalP server.3
The Lycopersicon esculentum cDNA FNR sequence was
assembled from EST sequences published in the dbest data
base3 using the Lasergene package (DNASTAR), yielding an
overlapping contig with no ambiguities in the consensus sequence
(data not shown). All other sequences were taken as published in the
data bases.3
Cloning and Genomic Organization of the T. gondii Gene Coding for
Ferredoxin-NADP+-reductase--
Searching the T. gondii EST data base for putative apicoplast localized proteins we
found a single entry among the approximately 10,000 sequences (EST
zy19d02.r1) showing significant homology to the C terminus of
exclusively chloroplast-localized plant-type FNR (33). To rule out the
possibility that this short sequence was a cloning artifact or
contamination its existence in the genome of the parasite was verified
by PCR with genomic DNA isolated from purified RH strain. A single
product of 400 bp was obtained, which was, however, 150 bp longer than
the reported EST sequence. To proof that the amplicon was indeed
related to EST zy19d02.r1 and that the additional bp reflected an
intron, the PCR product was sequenced. The resulting sequence proved it
to be the presumed EST containing exactly 150 bp of noncoding DNA, the
boundaries of it conforming to the usual exon-intron junctions of
eukaryotes (data not shown). This result confirmed that the single EST
zy19d02.r1 was of T. gondii origin and prompted us to
isolate the full-length cDNA.
Using digoxigenin-labeled EST zy19d02.r1 to screen a
To see if the isolated FNR gene existed as single copy in the genome of
T. gondii, Southern blot analysis under high stringency conditions was conducted, using a 1.1-kilobase pair fragment
from the coding region up to the stop codon as probe. As can be seen in
Fig. 2 restriction digests of purified
genomic DNA from strain RH showed distinct single bands, except for
PstI which yields two bands (and which cuts once in the
cDNA and once in the short known intron, releasing a fragment of
148 bp). The probe did not hybridize to genomic DNA from the host cells
(human foreskin fibroblasts). These results are in agreement with this
FNR sequence being a single copy gene.
FNR from T. gondii Contains a Transit Peptide Sequence Required to
Target It to the Apicoplast--
Alignment of the whole translated
protein sequence with all known plant-type FNR sequences revealed
significant differences in the N-terminal 150 aa compared with the
N-terminal precursor sequences of the plant enzymes (Fig. 1), which
would be in agreement with the assumption that the N terminus would
encode a putative transit peptide sequence (TPS) necessary to target
the protein to the apicoplast. Using the von Heijne algorithm (35) a
signal sequence at the N terminus was predicted, with a putative
cleavage site between Ser29 and Phe30. The
remaining 120 aa were then analyzed with respect to their physicochemical properties found in the three other nuclear-encoded, but apicoplast-localized, T. gondii proteins reported in the
literature (36). In accordance with the observations of Waller et
al. (36), we also found an overall net positive charge and a high
proportion of Ser and Thr residues (17.6%) in the ~120 aa predicted
to contain a TPS. Experimental evidence that the N-terminal 150-aa
sequence as well as the whole FNR sequence are able to target a
genetically fused fluorescent reporter protein into the plastid
organelle of the parasite has now been provided (58),
demonstrating that the putative TgFNR sequence is an apicoplast-located
protein containing a bipartite apicoplast targeting signal.
The TgFNR Protein Contains Unique Sequence Features Compared with
Its Plant Counterparts--
The alignment in Fig. 1 revealed a
significant homology of the T. gondii sequence with its
plant and algal counterparts over the entire sequence (not encompassing
the putative TPS) but, not surprisingly, the most conserved residues
being the binding sites for cofactors FAD and NADP(H). However, there
are several differences with possibly functional consequences. Most
obviously, TgFNR contains a large insertion of 31 aa (with regard to
Anabaena variabilis FNR) at position 233, located
immediately N-terminal to one FAD binding motif and flanked by two
adjacent prolines (Fig. 1). This extension might influence the dynamics
and/or accessibility of cofactor binding as well as electron transfer
rates. Interestingly, the root isoforms and the green algal FNRs
contain 3 to 4 aa insertions at the very same position. There are 4 additional smaller insertions in TgFNR (between 3 and 8 aa in length),
one of them being again conserved in the root and green algal FNRs,
which would, however, not be expected to significantly influence the
overall structure of TgFNR or its function.
Another significant difference related to the 31-aa insertion point in
TgFNR is the exchange of a positively charged lysine (Lys75
in the A. variabilis mature sequence, equivalent to position 212 of the whole A. variabilis sequence shown in Fig. 1),
which is conserved in all species except T. gondii, the
green algal and the root isoforms, against either negatively charged or
uncharged residues (e.g. Ala232 in the whole
T. gondii sequence in Fig. 1). Lys75 was shown
by site-directed mutagenesis to be essential for Fd binding (37). In
addition, there is perfect conservation of the preceding A. variabilis Lys72 (residue 209 in Fig. 1), which is
also implicated in Fd contact, between all other FNRs (Fig. 1) except
T. gondii, which displays again an alanine at that position.
It is, however, important to note that all of these studies were done
with photosynthetic active FNR and Fd (see "Discussion").
Nevertheless, these differences let us assume that differences in
catalysis and/or binding of Fd might exist.
Functional Expression of TgFNR and the Size of the Native
Protein--
To prove that the TgFNR sequence encoded a functional
enzyme, the cDNA was expressed in E. coli as a fusion
protein with 6His-tagged yeast ubiquitin. Because the exact cleavage
site of the transitpeptide sequence from TgFNR is at present unknown
(but see below), the start of the mature enzyme at residue 148 was
somewhat arbitrarily chosen, taking the cleavage site of mature
A. variabilis FNR as reference. It is known that the N
terminus of plant FNRs is not critical for activity (38). The resulting
fusion protein was found to be mostly soluble upon induction. It could
be purified under native conditions by means of
Ni2+-chelate affinity chromatography (Fig.
3A).
NADPH-dependent diaphorase activity of the purified fusion
protein by DCPIP reduction assays were conducted and showed the
expected activity (Fig. 3C), which could be partially
blocked by diphenylene iodonium chloride and, to a lesser extent, by
iodoniumdiphenyl HCl, two known inhibitors of FAD-dependent
enzymes (39), but not by N-ethylmaleimide, a
sulfhydryl-modifying agent. This inhibition profile is consistent with
results reported for Solanum tuberosum FNR (40). In other experiments the TgFNR fusion was also shown to possess
Fd-dependent cytochrome c reduction
capability.4 Together these results confirmed
that the T. gondii protein is an active FNR-type enzyme.
The purified recombinant protein was recognized by an antiserum
directed against the C-terminal part of TgFNR, produced by immunization
with a GST fusion protein (see "Experimental Procedures"), as well
as by a polyclonal antiserum raised against purified spinach leaf FNR
(asterisks in Fig. 3B). The reactivity was
specific, because another unrelated ubiquitin fusion protein
(6His-Ubi-PfFd) was not detected by both sera (open
arrowheads in Fig. 3B). At present the nature of the
reactive bands of higher molecular masses is unclear, but presumably
they are adducts of the monomeric form. Despite considerable
differences in primary amino acid sequences, TgFNR is sufficiently
conserved to react with an antibody raised against the plant enzyme.
To investigate the size of the native protein in whole lysates from RH
strain tachyzoites separated by nonreducing SDS-polyacrylamide gel
electrophoresis, the polyclonal anti-GST-FNR serum was used in a
Western blot analysis. While no specific signals were detectable with
the preimmune serum, two faint bands of approximately 55 and 50 kDa
were observed with the anti-GST-FNR serum (Fig. 3D). The
slower migrating band at 55 kDa is in good agreement with it being the
unprocessed FNR still containing the uncleaved transitpeptide, while
the more prominent, faster migrating band is probably the mature form.
Detection of unprocessed and processed forms of apicoplast-targeted enzymes has been described previously (33, 42). Because at present the exact cleavage site for the mature form is not known, and
no N-glycosylation sites are present in the primary sequence, a size of
50 kDa would mean that the mature TgFNR is unusually large compared
with all other known FNRs.
The P. falciparum Genome Contains a Transcribed Coding Sequence for
a Typical [2Fe-2S] Ferredoxin--
The presence of FNR in T. gondii infers that the intimate redox partner, ferredoxin, must be
present in the parasite and would also be expected to be localized in
the apicoplast. Therefore the different Plasmodium genome
data bases were searched for such a homologue (currently there is no
advanced T. gondii genome project). Indeed, a genomic
fragment encoding an open reading frame of 194 aa (contained within
contig MAL13_05317, obtained from The Sanger Center website, see
"Experimental Procedures") with strong homology to plant-type Fds
could be identified (starting at aa 97, corresponding to residue 1 in
Fig. 4). It shows a predicted signal
peptide cleavage site between Thr19 and Tyr20
and contains 16% of Ser (9) and Thr (3) residues and 18% of basic aa
in its putative 76-aa transit peptide (data not shown). All four
cysteines required for the typical Fd [2Fe-2S] cluster formation are
conserved (41) as well as most of the residues implicated in the direct
contact with plant or cyanobacterial FNR (42, 43). RT-PCR with
PfFd-specific primers proved that it is transcribed in the parasite
(see Fig. 6B). PfFd starting from aa 108 was expressed in
the same way as TgFNR as a fusion with 6His-ubiquitin, however, in the
presence of Ubp1 from yeast, which leads to cotranslational cleavage of
the ubiquitin moiety (25), resulting in the release of mature PfFd.
Native gel electrophoresis of such cultures and subsequent staining
with iron-specific stains showed that PfFd contained bound iron,
indicating that it has an assembled iron-sulfur cluster (data not
shown).
Phylogenetic Relationship between TgFNR and Other Plant-type
Isoforms--
Phylogenetic analysis of all FNR sequences found in
various data bases corroborated the closer relationship of TgFNR to the green algal and root isozymes compared with the other FNRs from photosynthetic active plastids (termed ps and n-ps in the following sections for photosynthetic and
non-photosynthetic).
For construction of the phylogenetic trees shown in Fig.
5 the sequence alignment starting with aa
138 of T. gondii (see Fig. 1) was chosen, assuming that the
conserved block in the root isozymes denotes the processing site of the
mature form (15). However, similar results were obtained when the
sequences starting at the first FAD binding motif (Arg264
of T. gondii), leaving out the 31-aa insertion, were
compared (data not shown). From this analysis an interesting aspect
emerged. Although the topology of the inferred trees are somewhat
different with the three methods used, they all lead to the same
clustering of T. gondii, green algal and n-ps FNRs (Fig. 5,
shaded parts). Although it is not surprising that TgFNR is
most similar to green algal FNRs (Chlamydomonas and
Volvox), it is intriguing to note that TgFNR seems to be
closer related to n-ps FNRs from roots than to the cyanobacterial
proteins. Clustering of n-ps FNRs with the two green algae has been
noted earlier (33) but is substantiated here by including 13 new FNR
sequences, adding additional weight to the analysis. Likewise, the n-ps
plant isoforms are more similar to TgFNR than they are to their
respective counterparts from the photosynthetic active organelles. In
this respect it is also worthwhile to consider the above mentioned two
insertions common only to T. gondii, the n-ps and green
algal FNR as sequence signatures (44), being remnants of a common
ancestor. This notion is corroborated by the following findings.
Searching the data bases for a P. falciparum FNR homologue
revealed only two unfinished, overlapping DNA sequences, resulting in
the 213-aa-long open reading frame shown in Fig. 6A, covering the region of
TgFNR aa 145-373. It possesses significant differences compared with
the T. gondii enzyme, and even more so to the other FNRs,
but good conservation in the FAD and NADP(H) binding motifs present in
this partial sequence. More important, however, is the fact that it
shows perfect length conservation of the 31-aa insertion. Because other
FAD and NADP(H) binding enzymes could also have similar cofactor
binding motifs (with cytochrome P450 reductase and adrenodoxin
reductase the most relevant proteins in this respect), we searched the
data bases for these two and found very good matches for them, which
are, however, clearly distinct from the PfFNR-like sequence shown in
Fig. 6A, and without the 31-aa insertion at the relevant
sites (data not shown). While at present we do not know if this
FNR-like polypeptide belongs to a functional enzyme in P. falciparum, its gene is actively transcribed in the parasite as
shown in Fig. 6B. RT-PCR yielded a specific band of
identical size to the genomic product (460 bp, lane 6) in
the presence, but not in the absence, of mRNA (lanes 4 and 5). As control served Fd, which also gave a
mRNA-dependent signal (lanes 1 and
2) with respective primers being of the same size as the
genomic amplicon (310 bp, lane 3).
Phylogenetic analysis of PfFd and a subset of 34 plant-type Fds
representing all classes of organisms for which those sequences are
available indicated that PfFd, like TgFNR, showed a tendency to cluster
with n-ps proteins, albeit less significantly than in the case of TgFNR
(data not shown).
We have presented data that show that apicomplexan parasites
possess genes for plant-type FNR as well as for its intimate redox
partner, Fd. The rationale for the initial cloning of the gene
corresponding to a T. gondii EST with similarity to the C terminus of plant-type FNRs was that in plants this enzyme is found
exclusively in plastids. Thus, our assumption was that TgFNR should be
localized in the apicoplast. Indeed, both TgFNR and PfFd possess a
N-terminal bipartite targeting domain, a prerequisite for transporting
a protein to the apicoplast (36, 45), and which are sufficient to
target a GFP reporter into it (58).5 Both TPS
show an overall net positive charge and a high proportion of Ser and
Thr. Enrichment for these residues is a consistent finding within plant
TPS (46). However, in contrast to T. gondii, the plasmodial
proteins examined by Waller et al. (36) did not show a high
content of hydroxylated residues, and a recent study in P. falciparum confirmed that the only two Thr present in the TPS are
dispensable for proper targeting of a FabH TPS-GFP fusion to the
plastid of this parasite (45). This contrasts with the Fd TPS, having
16% of Ser (9) and Thr (3) residues and 18% of basic aa in its
putative 76-aa transit peptide. Thus, despite overall functional
similarity, which has been reported to allow experimental swapping of
plastid-targeting domains between T. gondii and P. falciparum (3), it is possible that "fine tuning" of the
targeting or processing requires different physico-chemical properties
of the transit peptide for different proteins in the two organisms.
In contrast to P. falciparum Fd, which shows no unusual
sequence features compared with plant-type Fds, the TgFNR sequence is
distinct in several respects compared with all other known FNRs,
possibly with functional consequences. The most prominent difference is
the large insertion of 31 aa, located immediately N-terminal of the FAD
binding site, and the replacement of the positively charged
Lys75, which is conserved in all species except T. gondii, the green algal and the root isoforms. Lys75
was shown by site-directed mutagenesis to be essential for Fd binding
(37), whereby neutral residues showed a 50-100-fold increase in
Kd for Fd, and charge reversal led to total loss of
Fd binding. It is important to note that all of these studies were done
with FNR and Fd isoproteins involved in the photochemical pathway (i.e
oxidation of Fd) and that the isoforms from n-ps tissues also do not
possess this Lys residue. Only recently has there been interest in the
biochemical and structural differences between ps and n-ps FNR isoforms
(15, 47), and T. gondii, because of its ease of genetic
manipulation (16), might offer an attractive in vivo system
to study the functional consequences of this and other crucial FNR
residues by mutational analysis.
Although in most computational phylogenetic analyses amino acid
insertions or deletions (indels) are ignored, they can, on the other
hand, be very informative provided they are flanked by conserved
regions, thus ensuring that they are not alignment artifacts (for
review, see Ref. 44). Assuming that during evolution such "sequence
signatures" are introduced only once and are unlikely the result of
independent mutational events in already separated lineages, both the
31-aa insertion in TgFNR and the PfFNR-like protein, and the presumed
remnants thereof in the green algal and root FNRs, as well as the 3-aa
insertion at position 298 (Fig. 1) can be regarded as such sequence
signatures, being remnants of a common ancestor. The differences in
length for the 31-aa insertion between T. gondii (P. falciparum) and the others (31 versus 4 and 3 aa,
respectively) might reflect the shaping of this region over time in the
different organisms by adaptations of the enzymes to their
physiological roles in the different plastids.
Is the phylogenetic clustering of T. gondii, green algal,
and root FNRs helpful for inferring the events leading to the
diversification of ps and n-ps FNRs? It has been proposed that an
ancient duplication event of the FNR gene prior to the separation of
algal and plant groups, followed by the subsequent loss of the ps FNR
in the algal lineage, led to the observed clustering (33). Assuming
that TgFNR is a single copy gene (see Fig. 2), either the ancestor giving rise to the apicoplast was an alga that had already lost its ps
FNR gene, or ps FNR gene loss must have occurred twice independently.
Both scenarios are possible, as evidence for independent gene loss
during transfer of chloroplast genes to the nucleus has been provided
(10), and the debate about the nature of the secondary endosymbiont
(red or green alga) giving rise to the apicoplast is still ongoing (3,
48). In this regard cloning of more algal FNRs, especially from red
algae for which no sequences are available yet, could be very informative.
Plant-type FNRs/Fd are often referred to as the functional homologous
of animal adrenodoxin reductases (AdxR) and adrenodoxin (Adx) (33) and
can even substitute them in some in vitro assays, albeit
much less efficiently (49). The P. falciparum data bases contain two sequences with significant homology to AdxR and Adx (data
not shown). AdxRs are FAD-containing, NADPH-dependent
enzymes intimately connected to the mitochondrial cytochrome P450
electron transfer systems and, in mammals, are involved in the
biosynthesis of steroid hormones (50). Adxs are small Fd-related
iron-sulfur proteins functioning as an electron carrier between AdxR
and cytochromes P450 (51). Although the functionality of these two
proteins in P. falciparum has still to be determined
experimentally, it seems likely that in plants and apicomplexan
parasites FNR/Fd and AdxR/Adx fulfill different tasks. This assumption
is corroborated by the presence of homologues of both AdxR and Adx in
the genome of Arabidopsis and the recent description of a
yeast Adx (YAH1) being an essential component of the mitochondrial
iron-sulfur cluster assembling machinery (52), implying that AdxR/Adx
are vital proteins for all eukaryotes, but whose physiological function is distinct from that of FNR/Fd.
Since there is no reason to believe that T. gondii or other
apicomplexa have retained photosynthetic functions after acquiring the
plastid precursor (3), and because of the close relationship between
TgFNR and n-ps FNRs, it seems reasonable to suggest that the major
physiological role of FNR in the apicoplast lies in the provision of
reduced Fd for processes yet to be defined. In this regard
it is interesting to note that in addition to PfFd and the PfFNR-like
sequence, the Plasmodium genome data bases contain
additional sequences corresponding to proteins with functional relations to FNR/Fd: (i) a partial sequence for a In conclusion we have presented data that show that apicomplexa possess
a plant-type FNR/Fd redox system with an N-terminal apicoplast-targeting domain. The exact biochemical role of this prototype of a plant electron transport system in protists needs to be
determined. Nevertheless, it is reasonable to suggest, based on the
presented data, that its main task lies in the provision of reduced Fd,
whose most obvious function might be its involvement in fatty acid
desaturation by Fd-dependent desaturases (54, 55). This
would certainly be an essential function in the parasite, given the
great number of membranes it has to build up and to maintain under
different environmental conditions (vacuolar membrane, three-laminar
cell membrane, various organellar membranes, plus the four plastid
membranes), even considering only the tachyzoite stage. Because of its
absence in the host, the apicomplexan FNR/Fd system offers an
attractive target for the modeling of specific drugs against these
parasites. This goal is facilitated by the already known
three-dimensional structures of several plant FNRs and Fds (Ref. 56 and
Ref. 57 and references therein).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2Fdox + NADPH.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP II cDNA library made from strain RH mRNA was kindly
provided by J. Ajioka and screened using the digoxigenin-labeled
genomic FNR probe according to the manufacturer's instructions.
Positive clones were plaque-purified and the insert from the rescued
phagemid sequenced using standard techniques (21).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAP cDNA
library of strain RH, several clones were isolated, and the longest
clone, containing an insert of 2630 bp, was sequenced. Conceptual translation of the cDNA yielded the 497-amino acid sequence shown in Fig. 1, resulting in a
predicted protein of 54 kDa. The start codon was assumed to be at bp
372-375 of the cDNA sequence, because no other methionine in the
upstream sequence could be found in the same reading frame, and the DNA
sequence immediately upstream of the putative start codon conforms
reasonably well to the translation initiation consensus sequence of
T. gondii (34). Additional proof came from the protein
sequence analysis and multiple alignment (see below). As predicted, the
cDNA did not show the 150-bp insertion, confirming this to be a
small intron. The 3'-untranslated region of the cDNA clone consists
of 768 bp, followed by a poly(A) tract.
View larger version (83K):
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Fig. 1.
Multiple alignment of 26 plant-type FNR
sequences. Identical residues with TgFNR shared by more than 30%
of the sequences are shaded. FAD and NADP(H) binding motifs are
indicated by solid and broken boxes,
respectively. Signature sequences are marked by dark
shading. The position of the predicted signal peptide cleavage is
indicated with "V" and the position of the C-terminal intron by an
arrow. The following sequences (with their annotations) were
used: T. gondii, GenBankTM accession number
AJ242627; Volvox carteri, GenBankTM accession
number U22328; Chlamydomonas reinhardtii,
GenBankTM accession number U10545; Arabidopsis
thaliana nps-1, GenBankTM accession number AAF19753;
A. thaliana nps-2, SwissProt number CAB81081; Pisum
sativum root, SwissProt number Q41014; L. esculentum,
ESTs contig; Nicotiana tabacum root, SwissProt number
O04397; Oryza sativa root, GenBankTM accession
number D17410; O. sativa embryo, GenBankTM
accession number D87547; Zea mays root,
GenBankTM accession number U10418; A. variabilis, GenBankTM accession number X72394;
Spirulina sp., Protein Information Resource number RDSGXX;
Synechocystis sp., GenBankTM accession number
X94297; Synechococcus sp., GenBankTM accession
number M86234; Cyanophora paradoxa, GenBankTM
accession number X66372; O. sativa leaf,
GenBankTM accession number D17790; Z. mays
leaf-1, SwissProt number AB035644; Z. mays leaf-2, SwissProt
number AB035645; A. thaliana ps, GenBankTM
accession number AL161503; Mesembryanthemum crystallinum
leaf, GenBankTM accession number M25528; Spinacia
oleracea leaf, GenBankTM accession number X07981;
N. tabacum leaf, GenBankTM accession number
Y14032; Capsicum annum ps, GenBankTM accession
number AJ250378; P. sativum embryo, GenBankTM
accession number M21449; Vicia faba leaf
GenBankTM accession number U14956.
View larger version (31K):
[in a new window]
Fig. 2.
Southern blot analysis of genomic T. gondii DNA with FNR as probe. 50 µg of genomic DNA
was digested with either BamHI (B),
EcoRI (E), HindIII (H), or
PstI (P) to completion; electrophoresed through a
0.7% agarose gel; and blotted onto a nylon membrane. 100 pg of
supercoiled plasmid DNA (Pl.) containing EST zy19d02.r1 and
100 µg of human foreskin fibroblast DNA digested with
HindIII served as positive and negative control,
respectively. Molecular size marker was run in a separate lane.
View larger version (41K):
[in a new window]
Fig. 3.
Characterization of recombinant and native
TgFNR. A, Coomassie-stained 12% SDS-polyacrylamide gel
documenting the purification of 6His-Ubi-TgFNR by
Ni2+-chelate column chromatography. Lane 1,
total E. coli extract; lane 2, soluble lysate;
lane 3, column flow-through; lanes 4-6, eluate
fractions. * denotes the fusion protein of 55 kDa. B,
Western blot analysis of purified recombinant 6His-Ubi-TgFNR (5 µg,
lane 2) or 6His-Ubi-PfFd (10 µg, lane 1) with
rabbit antiserum directed against purified GST-TgFNR containing the
C-terminal part of TgFNR (1:2000 dilution) or rabbit antiserum directed
against purified spinach FNR (1:2000 dilution), respectively. The
secondary alkaline phosphatase-conjugated antibody was used at a 1:1500
dilution. * denotes the position of 6His-Ubi-TgFNR (55 kDa), > that of
6His-Ubi-PfFd (28 kDa). C, NADPH-dependent diaphorase
activity of 6His-Ubi-TgFNR with DCPIP as indicator. The decline in
A630 nm (i.e. reduction of
DCPIP) between "no FNR" reaction and
"untreated" is the maximal obtainable activity under
these conditions. Addition of the two inhibitors,
iodoniumdiphenyl HCl and diphenylene iodonium chloride, decreases the
activity (reflected by increased A compared with
untreated), whereas the controls do not (N-ethylmaleimide
(NEM) and Me2SO (DMSO)).
D, Western blot analysis of T. gondii tachyzoite
whole cell lysate (107 parasites/lane) with either rabbit
preimmune serum (lane 1) or rabbit antiserum directed
against purified GST-TgFNR (lane 2), both at a 1:250
dilution. The secondary horseradish peroxidase-conjugated antibody was
used at a 1:5000 dilution.
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[in a new window]
Fig. 4.
Sequence alignment of P. falciparum [2Fe-2S] ferredoxin with a subset of other
plant-type Fds. The C-terminal 97 aa of an open reading
frame of 194 aa contained within contig Pf MAL13_05317 with
strong homology to plant-type Fds (extracted from the protein
superfamily 00029 of the PROTFAM data base).3 The four
Cys required for [2Fe-2S] cluster formation are boxed.
Residues implicated in the direct contact with plant FNR are marked by
asterisks.
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[in a new window]
Fig. 5.
Phylogenetic analysis of all 26 FNR
sequences shown in Fig. 1. The trees were obtained either by
distance matrix/neighbor joining (DM), maximum parsimony
(MP), or maximum likelihood/quartetpuzzling (ML)
methods. Clustering of TgFNR with algal and n-ps FNRs is
shaded.
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[in a new window]
Fig. 6.
Sequence comparison and RT-PCR of a P. falciparum FNR-like gene. A, alignment of a
partial P. falciparum FNR-like sequence with the respective
segments of T. gondii and C. reinhardtii FNR. The
31-aa insertion is overlined. B, aliquots of a
RT-PCR performed on RNA from P. falciparum were run on a 2%
agarose gel (PfFd (lanes 1-3) and PfFNR-like (lanes
4-6)). Specific bands of identical size to the predicted genomic
products (310 bp for Fd (lane 3) and 460 bp for FNR-like
(lane 6)) are detected in the presence (lanes 1 and 4), but not in the absence (lanes 2 and
5), of purified mRNA, proving that the signals
in lanes 1 and 4 are not due to genomic DNA
contamination.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9-desaturase, although sequence information from the N terminus is not available yet
to determine whether it contains a putative TPS, and Fd (as opposed to
NADPH) dependence is hard to infer from sequence data alone (13), and
(ii) a complete sequence for glutamate synthase (GenBankTM accession number Y17045). However, the
encoded protein is clearly of the NADPH-dependent type,
characterized by the lack of a 17-aa insertion typical for
ferredoxin-dependent isoforms (53), and it also contains no
obvious TPS (data not shown). At present it is not known if an
apicoplast-localized Fd-dependent glutamate synthase exists.
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ACKNOWLEDGEMENTS |
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We thank the following individuals: J. Ajioka for the cDNA library, A. Aliverti and G. Zanetti for plasmid pET-FdI and the spinach FNR anti-serum and for fruitful discussions, B. Striepen and D. Roos for sharing results, R. Baker for plasmid pCG001, U. G. Mayer and S. Zauner for initial sequencing, B. Beuerle for technical assistance, L. Kremp for antibody production, K. Lingelbach for continuos support and discussions, and S. Baumeister, A. Aliverti, and G. Zanetti for critical comments on the manuscript. Sequence data for P. falciparum chromosome 9 and 13 were obtained from The Sanger Center website3.
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FOOTNOTES |
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* This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (to F. S.) (Se622-3/2). Sequencing of P. falciparum chromosomes 9 and 13 was accomplished as part of the Malaria Genome Project with support by The Wellcome Trust.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ242627 (for T. gondii FNR).
To whom correspondence should be addressed. Tel.: 49-6421-2826596;
Fax: 49-6421-2821531; E-mail
seeber@mailer.uni-marburg.de.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M009452200
2 F. Seeber and N. Thomsen, manuscript in preparation.
5 B. Striepen, personal communication.
3 Please contact the corresponding author for further information.
4 A. Aliverti and G. Zanetti, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: FNR, ferredoxin-NADP+ reductase (EC 1.18.1.2); 6His-Ubi, 6-histidine-tagged yeast ubiquitin; aa amino acid(s), Adx, adrenodoxin; AdxR, adrenodoxin reductase; DCPIP, 2,6-dichlorophenolindophenol; EST, expressed sequence tag; Fd, ferredoxin; GST, glutathione S-transferase; n-ps, nonphotosynthetic; ps, photosynthetic; PfFd, P. falciparum ferredoxin; TgFNR, T. gondii FNR; TPS, transit peptide sequence; RT-PCR, reverse transcripton polymerase chain reaction; bp, base pair(s).
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